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Sample records for human plasma kallikrein

  1. Calibrated kallikrein generation in human plasma

    DEFF Research Database (Denmark)

    Biltoft, D; Sidelmann, J J; Olsen, L F

    2016-01-01

    generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s(-1)), and kallikrein generation could be measured in undiluted plasma when silica was added as activator. Disturbing effects, including substrate depletion and the inner......-filter effect, however, affected the signal. These problems were corrected for by external calibration with α2-macroglobulin-kallikrein complexes. Selectivity studies of the substrate, experiments with FXII and PK depleted plasmas, and plasma with high or low complement C1-esterase inhibitor activity indicated...

  2. Immunochemical Studies of Plasma Kallikrein

    Science.gov (United States)

    Bagdasarian, Andranik; Lahiri, Biswajit; Talamo, Richard C.; Wong, Pat; Colman, Robert W.

    1974-01-01

    A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion. Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XIIA, while large activator and the low molecular weight prekallikrein activators are less inhibited. With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and α2-macroglobulin, or α1-antitrypsin, although the concentration of free kallikrein antigen decreases after interaction with the former inhibitor. By radial immunodiffusion, plasma from healthy individuals contained 103±13 μg/ml prekallikrein antigen. Although in mild liver disease, functional and immunologic kallikrein are proportionally depressed, the levels of prekallikrein antigen in plasma samples from patients with severe liver disease remains 40% of normal, while

  3. Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

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    Gozzo A.J.

    2003-01-01

    Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates reduced (1.2 to 3.0 times the catalytic efficiency of kallikrein (in a nanomolar range on the hydrolysis of plasminogen (0.3 to 1.8 µM and increased (1.9 to 7.7 times the enzyme efficiency in factor XII (0.1 to 10 µM activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times kallikrein inhibition by antithrombin (1.4 µM, while chondroitin 4- and 6-sulfates reduced it (1.3 times. Heparin and heparan sulfate increased (1.4 times the enzyme inhibition by the C1-inhibitor (150 nM.

  4. Radioimmunoassay of glandular kallikrein in human plasma after partial purification by immunoaffinity column

    International Nuclear Information System (INIS)

    Nishimura, Kazutaka; Inoue, Yoshikazu; Kokubu, Tatsuo

    1987-01-01

    Glandular kallikrein in human plasma was partially purified by immunoaffinity column and was measured by a radioimmunoassay (RIA). Plasma was diluted with an equal volume of 10 mmol/l sodium phosphate buffer, pH 7.4, containing 0.9% NaCl (PBS) and was applied to an immunoaffinity column from which glandular kallikrein was eluted with 3 mol/l NaSCN (20 ml). The enzymic fraction was concentrated with an Amicon PM 10 filter and dialyzed against PBS. The final recovery of the enzyme was 51.6 ± 1.6%, determined by using [ 125 I]kallikrein. The usable range of the standard curve covered 2.5-100 ng/tube. The coefficient of variation within the series was 5.9%, and the coefficient of variation between the series was 7.6%. In healthy controls, the plasma content of glandular kallikrein was 1.36 ± 0.39 ng/ml. In patients with acute pancreatitis, the plasma concentration was 8.02 ± 6.15 ng/ml, significantly different from the control group. (Auth.)

  5. Radioimmunoassay of human urinary kallikrein

    International Nuclear Information System (INIS)

    Goering, W.

    1980-01-01

    Using a human urinary kallikrein, purified by means of Trasylol sepharose, it has been possible to develop a radioimmunoassay of kallikrein capable of detecting the substance down to a concentration of 0.5 ng/ml. The specific activity of the tracer labelled with 125-iodine using the Chloramine-T method was 30-70 nCi/ng of kallikrein. The antiserum titres for the antikallikrein serum were 1:20.000 up to 1:50.000. Human urine, submandibular and parotid salivae as well as pancreatic secretion in this RIA reacted in the same manner as the kallikrein standard solution. The kallikrein content in urine, as determined by the RIA was between 0 and 300 ng/ml, in the saliva between 400 and 2.000 ng/ml, and in the pancreatic juice between 300 and 12.000 ng/ml. Using human serum, only an incomplete immunological cross-reaction could be achieved. In human liquor as well as in animal preparation, no cross-reacting substances could be detected. (orig.) [de

  6. Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

    OpenAIRE

    Motta, Guacyara; Tersariol, Ivarne L. S.

    2017-01-01

    Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis a...

  7. Structure and chromosomal localization of the human renal kallikrein gene

    International Nuclear Information System (INIS)

    Evans, B.A.; Yun, Z.X.; Close, J.A.

    1988-01-01

    Glandular kallikreins are a family of proteases encoded by a variable number of genes in different mammalian species. In all species examined, however, one particular kallikrein is functionally conserved in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. This kallikrein is found in the kidney, pancreas, and salivary gland, showing a unique pattern of tissue-specific expression relative to other members of the family. The authors have isolated a genomic clone carrying the human renal kallikrein gene and compared the nucleotide sequence of its promoter region with those of the mouse renal kallikrein gene and another mouse kallikrein gene expressed in a distinct cell type. They find four sequence elements conserved between renal kallikrein genes from the two species. They have also shown that the human gene is localized to 19q13, a position analogous to that of the kallikrein gene family on mouse chromosome 7

  8. Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

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    Guacyara Motta

    2017-07-01

    Full Text Available Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis. The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

  9. Lack of plasma kallikrein-kinin system cascade in teleosts.

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    Marty Kwok-Shing Wong

    Full Text Available The kallikrein-kinin system (KKS consists of two major cascades in mammals: "plasma KKS" consisting of high molecular-weight (HMW kininogen (KNG, plasma kallikrein (KLKB1, and bradykinin (BK; and "tissue KKS" consisting of low molecular-weight (LMW KNG, tissue kallikreins (KLKs, and [Lys(0]-BK. Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg(0]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade.

  10. Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP.

    Science.gov (United States)

    Ottaiano, Tatiana F; Andrade, Sheila S; de Oliveira, Cleide; Silva, Mariana C C; Buri, Marcus V; Juliano, Maria A; Girão, Manoel J B C; Sampaio, Misako U; Schmaier, Alvin H; Wlodawer, Alexander; Maffei, Francisco H A; Oliva, Maria Luiza V

    2017-04-01

    Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin α IIb β 3 through interactions with the KGD/KGE sequence motif in huPK. Integrin α IIb β 3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS 473 , ERK1/2, and p38 MAPK, and to Ca 2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and α IIb β 3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Crystallization and preliminary crystallographic studies of human kallikrein 7, a serine protease of the multigene kallikrein family

    Energy Technology Data Exchange (ETDEWEB)

    Fernández, Israel S. [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Ständker, Ludger [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Forssmann, Wolf-Georg [Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Giménez-Gallego, Guillermo; Romero, Antonio, E-mail: romero@cib.csic.es [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2007-08-01

    The cloning, expression, purification and crystallization of recombinant human kallikrein 7, directly synthesized in the active form in E. coli, is described. Diffraction data were collected to 2.8 Å resolution from native crystals. Human kallikreins are a group of serine proteases of high sequence homology whose genes are grouped as a single cluster at chromosome 19. Although the physiological roles of kallikreins are generally still unknown, members of the kallikrein family have been clearly implicated in pathological situations such as cancer and psoriasis. Human kallikrein 7 (hK7) has been shown to be involved in pathological keratinization, psoriasis and ovarian cancer. In order to gain insight into the molecular structure of this protein, hK7 was crystallized after recombinant production in its folded and active form using a periplasmic secretion vector in Escherichia coli. The crystals belonged to the rhombohedral space group H32 and diffracted to 2.8 Å. The phase problem was solved by molecular replacement using the mouse kallikrein-related protein neuropsin. Completion of the model and structure refinement are under way.

  12. Effect of focal irradiation on plasma kallikrein activity in tumor bearing rats

    International Nuclear Information System (INIS)

    Makoyo, P.O.Z.R.; West, W.L.

    1977-01-01

    The activated plasma kallikrein from tumor bearing rats before and after focal irradiation of the hind limbs was measured by the hydrolysis of 0.015M N-(p-toluene sulfonyl)-L arginine methyl ester (TAME). Blood was collected from abdominal aorta of rats anesthetized with diethyl ether into plastic tubes containing 1 volume of 3.8 percent sodium citrate for each 9 volumes. Plasma was isolated by centrifugation (2000 g) at 4 0 C. Small pieces of minced tumor tissue were inserted in a trochar and inoculated in the hind limbs of the rats. Morris hepatomas 7777 and 3924A were transplanted subcutaneously over the hind legs while Walker 256 tumor was transplanted intramuscularly in the hind limbs. Plasma kallikrein (μm TAME utilized/ml/hr) was decreased in three different groups of tumor bearing rats: Walker tumor from 207 +- 34 to 114 +- 30 Hepatoma 7777 from 219 +- 13 to 106 +- 3 and Hepatoma 3924A from 227 +- 7 to 133 +- 5. Two hours after focal irradiation (1000 R) plasma kallikrein decreased further in Walker tumor and hepatoma 7777 to 55 +- 5 and 96 +- 3.6 respectively. The plasma of tumor bearing rats becomes deficient in kallikrein at about the time metastases may be identified. The acute fall in plasma kallikrein is consistent with the overall stress mechanism

  13. Discovery of novel transcripts of the human tissue kallikrein (KLK1) and kallikrein-related peptidase 2 (KLK2) in human cancer cells, exploiting Next-Generation Sequencing technology.

    Science.gov (United States)

    Adamopoulos, Panagiotis G; Kontos, Christos K; Scorilas, Andreas

    2018-03-31

    Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer. Copyright © 2018. Published by Elsevier Inc.

  14. Improving virtual screening predictive accuracy of Human kallikrein 5 inhibitors using machine learning models.

    Science.gov (United States)

    Fang, Xingang; Bagui, Sikha; Bagui, Subhash

    2017-08-01

    The readily available high throughput screening (HTS) data from the PubChem database provides an opportunity for mining of small molecules in a variety of biological systems using machine learning techniques. From the thousands of available molecular descriptors developed to encode useful chemical information representing the characteristics of molecules, descriptor selection is an essential step in building an optimal quantitative structural-activity relationship (QSAR) model. For the development of a systematic descriptor selection strategy, we need the understanding of the relationship between: (i) the descriptor selection; (ii) the choice of the machine learning model; and (iii) the characteristics of the target bio-molecule. In this work, we employed the Signature descriptor to generate a dataset on the Human kallikrein 5 (hK 5) inhibition confirmatory assay data and compared multiple classification models including logistic regression, support vector machine, random forest and k-nearest neighbor. Under optimal conditions, the logistic regression model provided extremely high overall accuracy (98%) and precision (90%), with good sensitivity (65%) in the cross validation test. In testing the primary HTS screening data with more than 200K molecular structures, the logistic regression model exhibited the capability of eliminating more than 99.9% of the inactive structures. As part of our exploration of the descriptor-model-target relationship, the excellent predictive performance of the combination of the Signature descriptor and the logistic regression model on the assay data of the Human kallikrein 5 (hK 5) target suggested a feasible descriptor/model selection strategy on similar targets. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Preclinical characterization of recombinant human tissue kallikrein-1 as a novel treatment for type 2 diabetes mellitus.

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    Tadeusz Kolodka

    Full Text Available Modulation of the kallikrein-kinin system (KKS has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D. The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1, as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D.

  16. Rapid elimination kinetics of free PSA or human kallikrein-related peptidase 2 after initiation of gonadotropin-releasing hormone-antagonist treatment of prostate cancer

    DEFF Research Database (Denmark)

    Ulmert, David; Vickers, Andrew J; Scher, Howard I

    2012-01-01

    The utility of conventional prostate-specific antigen (PSA) measurements in blood for monitoring rapid responses to treatment for prostate cancer is limited because of its slow elimination rate. Prior studies have shown that free PSA (fPSA), intact PSA (iPSA) and human kallikrein-related peptidase...... of tPSA, fPSA, iPSA and hK2 after rapid induction of castration with degarelix (Firmagon(®)), a novel GnRH antagonist....

  17. Inhibition of plasma kallikrein-kinin system to alleviate renal injury and arthritis symptoms in rats with adjuvant-induced arthritis.

    Science.gov (United States)

    Zhu, Jie; Wang, Hui; Chen, Jingyu; Wei, Wei

    2018-04-01

    Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Impairment of kidney functions in RA was observed. However, the mechanism of kidney injury of RA has not been clear. Plasma kallikrein-kinin system (KKS) was involved in inflammatory processes in kidney disease. This study aimed to explore the role of plasma KKS in immune reactions and kidney injury of RA. The paw of AA rats appeared to be swelling and redness, the arthritis index was significantly increased on the 18, 21 and 24 d after injection and secondary inflammation in multi-sites was observed. Kidney dysfunction accompanied with inflammatory cell infiltration, tubular epithelial cell mitochondrial swelling and vacuolar degeneration, renal glomerular foot process fusions and glomerular basement membrane thickening were observed in AA rats. The expressions of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (Kim-1) in kidney of AA rats were increased. In addition, expressions of BK, PK, B1R and B2R in the renal tissue of AA rats were up-regulated. Pro-inflammatory cytokines IL-2, IFN-γ and TNF-α were increased and anti-inflammatory cytokines IL-4 and IL-10 were low in kidney. Plasma kallikrein (PK) inhibitor PKSI-527 attenuated arthritis signs and renal damage, and inhibited BK, PK, B1R and B2R expressions. The protein expressions of P38, p-P38 and p-JNK and IFN-γ and TNF-α were inhibited by PKSI-527. These findings demonstrate that plasma KKS activation contributed to the renal injury of AA rats through MAPK signaling pathway. Plasma KKS might be a potential target for RA therapy.

  18. Enhancement of lymphocyte proliferation by mouse glandular kallikrein.

    Science.gov (United States)

    Hu, Z Q; Murakami, K; Ikigai, H; Shimamura, T

    1992-03-01

    Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

  19. The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models.

    Science.gov (United States)

    Brito, Marlon V; de Oliveira, Cleide; Salu, Bruno R; Andrade, Sonia A; Malloy, Paula M D; Sato, Ana C; Vicente, Cristina P; Sampaio, Misako U; Maffei, Francisco H A; Oliva, Maria Luiza V

    2014-05-01

    The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (Kiapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

    Science.gov (United States)

    Lee, Sang Wook; Hosokawa, Kazuo; Kim, Soyoun; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas; Maeda, Mizuo

    2015-05-22

    Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

  1. The Long-Term Outcome Comparison of Different Time-Delayed Kallikrein Treatments in a Mouse Cerebral Ischemic Model

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    Yaohui Ni

    2018-01-01

    Full Text Available Delayed administration of kallikrein after cerebral infarction can improve neurological function. However, the appropriate kallkrein treatment time after ischemic stroke has not been illuminated. In this study, we compared the long-term outcome among three kallikrein therapeutic regimens starting at different time points following mouse cerebral ischemia. Furthermore, the protective mechanisms involving neurogenesis, angiogenesis, and AKT-GSK3β-VEGF signaling pathway were analyzed. Human tissue kallikrein was injected through the tail vein daily starting at 8 h, 24 h, or 36 h after right middle cerebral artery occlusion (MCAO until the 28th day. Three therapeutic regimens all protected against neurological dysfunction, but kallikrein treatment starting at 8 h after MCAO had the best efficacy. Additionally, kallikrein treatment at 8 h after MCAO significantly enhanced cell proliferation including neural stem cell and induced differentiation of neural stem cell into mature neuron. Kallikrein treatment starting at 8 h also promoted more angiogenesis than other two treatment regimens, which was associated with AKT-GSK3β-VEGF signaling pathway. Thus, we confirm that three delayed kallikrein treatments provide protection against cerebral infarction and furthermore suggest that kallikrein treatment starting at 8 h had a better effect than that at 24 h and 36 h. These findings provide the experimental data contributing to better clinical application of exogenous kallikrein.

  2. Kallikrein-like amidase activity in renal ischemia and reperfusion

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    M.D. Carattino

    2000-05-01

    Full Text Available We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 µg of Evans blue/g dry tissue in the basal condition and 1750 µg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage

  3. Expression of kallikrein-related peptidase 7 is decreased in prostate cancer

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    Chong-Yu Zhang

    2015-02-01

    Full Text Available Recent evidence suggests that the human kallikrein 7 (KLK7 is differentially regulated in a variety of tumors. The aim of this study was to determine the expression of kallikrein-related peptidase 7 and KLK7 in our large collection of prostate samples. Between August 2000 and December 2012, 116 patients with histologically confirmed prostate cancer (PCa and 92 with benign prostate hyperplasia (BPH were recruited into the study. Using immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-PCR and western blot, kallikrein-related peptidase 7 expression in BPH and PCa tissues was determined at the mRNA and protein levels. The relationships between kallikrein-related peptidase 7 mRNA expression and clinicopathological features were analyzed. A total of 64 of 92 (69.57% benign cases showed positive staining for KLK7 and 23 of 116 (19.83% malignant cases showed positive, the difference of KLK7 expression between PCa and BPH was statistically significant (P < 0.001. The expression level of kallikrein-related peptidase 7 mRNA was significantly decreased in PCa tissues compared with that in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 mRNA exhibited different expression patterns in terms of localization depending on pathological category of PCa. Similarly, our western immunoblot analyses demonstrated that the protein expression levels of KLK7 was lower in PCa than in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 and KLK7 expression are down-regulated in PCa and lower expression of kallikrein-related peptidase 7 closely correlates with higher Gleason score and higher prostate-specific antigen level.

  4. Skin barrier disruption by sodium lauryl sulfate-exposure alters the expressions of involucrin, transglutaminase 1, profilaggrin, and kallikreins during the repair phase in human skin in vivo.

    Science.gov (United States)

    Törmä, Hans; Lindberg, Magnus; Berne, Berit

    2008-05-01

    Detergents are skin irritants affecting keratinocytes. In this study, healthy volunteers were exposed to water (vehicle) and 1% sodium lauryl sulfate (SLS) under occlusive patch tests for 24 hours. The messenger RNA (mRNA) expression of keratinocyte differentiation markers and of enzymes involved in corneodesmosome degradation was examined in skin biopsies (n=8) during the repair phase (6 hours to 7 days postexposure) using real-time reverse-transcription PCR. It was found that the expression of involucrin was increased at 6 hours, but then rapidly normalized. The expression of transglutaminase 1 exhibited a twofold increase after 24 hours in the SLS-exposed skin. Profilaggrin was decreased after 6 hours. Later (4-7 days), the expression in SLS-exposed areas was >50% above than in control areas. An increased and altered immunofluorescence pattern of involucrin, transglutaminase 1, and filaggrin was also found (n=4). At 6 hours post-SLS exposure, the mRNA expression of kallikrein-7 (KLK-7) and kallikrein-5 (KLK-5) was decreased by 50 and 75%, respectively, as compared with control and water-exposed areas. Thereafter, the expression pattern of KLK-7 and KLK-5 was normalized. Changes in protein expression of KLK-5 were also found. In conclusion, SLS-induced skin barrier defects induce altered mRNA expression of keratinocyte differentiation markers and enzymes degrading corneodesmosomes.

  5. The Kallikrein-Kinin System in Bartter's Syndrome and Its Response to Prostaglandin Synthetase Inhibition

    Science.gov (United States)

    Vinci, Joseph M.; Gill, John R.; Bowden, Robert E.; Pisano, John J.; Izzo, Joseph L.; Radfar, Nazam; Taylor, Addison A.; Zusman, Randall M.; Bartter, Frederic C.; Keiser, Harry R.

    1978-01-01

    The kallikrein-kinin system was characterized in seven patients with Bartter's syndrome on constant metabolic regimens before, during, and after treatment with prostaglandin synthetase inhibitors. Patients with Bartter's syndrome had high values for plasma bradykinin, plasma renin activity (PRA), urinary kallikrein, urinary immunoreactive prostaglandin E excretion, and urinary aldosterone; urinary kinins were subnormal and plasma prekallikrein was normal. Treatment with indomethacin or ibuprofen which decreased urinary immunoreactive prostaglandin E excretion by 67%, decreased mean PRA (patients recumbent) from 17.3±5.3 (S.E.M.) ng/ml per h to 3.3±1.1 ng/ml per h, mean plasma bradykinin (patients recumbent) from 15.4±4.4 ng/ml to 3.9±0.9 ng/ml, mean urinary kallikrein excretion from 24.8±3.2 tosyl-arginine-methyl ester units (TU)/day to 12.4±2.0 TU/day, but increased mean urinary kinin excretion from 3.8±1.3 μg/day to 8.5±2.5 μg/day. Plasma prekallikrein remained unchanged at 1.4 TU/ml. Thus, with prostaglandin synthetase inhibition, values for urinary kallikrein and kinin and plasma bradykinin returned to normal pari passu with changes in PRA, in aldosterone, and in prostaglandin E. The results suggest that, in Bartter's syndrome, prostaglandins mediate the low urinary kinins and the high plasma bradykinin, and that urinary kallikrein, which is aldosterone dependent, does not control kinin excretion. The high plasma bradykinin may be a cause of the pressor hyporesponsiveness to angiotensin II which characterizes the syndrome. PMID:96139

  6. Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice

    DEFF Research Database (Denmark)

    List, K; Jensen, Ole Nørregaard; Bugge, T H

    2000-01-01

    )-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations....

  7. The involvement of proteoglycans in the human plasma prekallikrein interaction with the cell surface.

    Directory of Open Access Journals (Sweden)

    Camila Lopes Veronez

    Full Text Available INTRODUCTION: The aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen (H-kininogen, influence this interaction. METHODS: We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type and CHO-745 (deficient in proteoglycans. Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule. RESULTS: At 37°C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4±0.010 and 1,070.3±0.001 pixels/cell, respectively, for ECV304 and 1,319.1±0.003 and 631.3±0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein colocalized with LysoTracker in the absence and presence of exogenous H-kininogen at levels of 76.0% and 88.5%, respectively, for ECV304 and at levels of 40.7% and 57.0%, respectively, for CHO-K1. After assembly on the cell surface, a plasma kallikrein fragment of 53 kDa was predominant in the incubation buffer of all the cell lines studied, indicating specific proteolysis; plasma kallikrein fragments of 48-44 kDa and 34-32 kDa were also detected in the incubation buffer, indicating non-specific cleavage. Bradykinin free H-kininogen internalization was not detected in CHO-K1 or CHO-745 cells at 37°C. CONCLUSION: The prekallikrein interaction with the cell surface is temperature-dependent and independent of exogenously applied H-kininogen, which results in prekallikrein endocytosis promoted by proteoglycans. Prekallikrein proteolysis/activation is influenced by H-kininogen/glycosaminoglycans assembly and controls plasma kallikrein

  8. Human seminal proteinase and prostate-specific antigen are the ...

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/033/02/0195-0207. Keywords. Kallikrein; prostate cancer biomarker; proteinase activity; seminal plasma; tumour proliferation and metastasis; therapeutic target. Abstract. Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and ...

  9. Critical role of tissue kallikrein in vessel formation and maturation : Implications for therapeutic revascularization

    NARCIS (Netherlands)

    Stone, O.A.; Richer, C.; Emanueli, C.; Weel, V. van; Quax, P.H.A.; Katare, R.; Kraenkel, N.; Campagnolo, P.; Barcelos, L.S.; Siragusa, M.; Sala-Newby, G.B.; Baldessari, D.; Mione, M.; Vincent, M.P.; Benest, A.V.; Al Haj Zen, A.; Gonzalez, J.; Bates, D.O.; Alhenc-Gelas, F.; Madeddu, P.

    2009-01-01

    OBJECTIVE : Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function

  10. Development of a radioimmunoassay for pig pancreatic kallikrein and its application in physiological studies

    International Nuclear Information System (INIS)

    Fink, E.; Guettel, C.; Seifert, J.

    1978-01-01

    A RIA for pig pancreatic kallikrein has been developed. Purified kallikrein was labelled with 125 I. The use of radioimmunoassays in investigating the intestinal absorption of pig pancreas kallikrein is dealt with. (VJ) [de

  11. Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

    Science.gov (United States)

    Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

    2013-03-01

    In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies

  12. Development of a radioimmunoassay for pig pancreatic kallikrein

    Energy Technology Data Exchange (ETDEWEB)

    Fink, E; Guettel, C [Muenchen Univ. (Germany, F.R.). Chirurgische Klinik

    1978-07-01

    A radioimmunoassay for the determination of pig pancreatic kallikrein was developed. The chloramine-T method was employed for the labelling of the antigen with /sup 125/I. The assay allows the determination of kallikrein in concentrations as low as 0.4 ..mu..g/l. Pig urinary and pig submandibular kallikreins are indistinguishable from pig pancreatic kallikrein by the assay. No cross reactivity was observed for bovine trypsin and chymotrypsin, porcine trypsin and kallikreins of guinea pig submandibular glands and guinea pig coagulation glands. Because of the high specificity of the assay, which is not attainable with conventional assays based on the enzymatic activity, the radioimmunoassay is highly suited for investigations into the physiological role and the pharmacological mechanism of action of pig glandular kallikreins.

  13. Identification, purification, and localization of tissue kallikrein in rat heart.

    OpenAIRE

    Xiong, W; Chen, L M; Woodley-Miller, C; Simson, J A; Chao, J

    1990-01-01

    A tissue kallikrein has been isolated from rat heart extracts by DEAE-Sepharose and aprotinin-affinity column chromatography. The purified cardiac enzyme has both N-tosyl-L-arginine methyl ester esterolytic and kinin-releasing activities, and displays parallelism with standard curves in a kallikrein radioimmunoassay, indicating it to have immunological identity with tissue kallikrein. The enzyme is inhibited by aprotinin, antipain, leupeptin and by high concentrations of soybean trypsin inhib...

  14. Reactivity of alpha 1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement, and fibrinolytic systems

    NARCIS (Netherlands)

    Patston, P.A.; Roodi, N.; Schifferli, J.A.; Bischoff, Rainer; Courtney, M.; Schapira, M.

    1990-01-01

    Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the

  15. Characterization of elements of the kallikrein-kinin system in rainbow trout (Salmo gairdneri)

    International Nuclear Information System (INIS)

    Lipke, D.W.

    1988-01-01

    Elements of kallikrein-kinin system (KKS) are found in anamniotic vertebrates, however, research has failed to substantiate the presence of a KKS in these animals. This research was conducted to verify the presence of elements of the KKS in rainbow trout. Trout branchial angiotensin-converting enzyme (ACE, kininase II) was similar to mammalian ACE in many physical, chemical and kinetic parameters. Angiotensin-converting enzyme-like activity (ACELA) was also demonstrated in the most primitive vertebrates. ACELA was consistently found in vertebrate respiratory tissues and was prevalent in salt and water exchanging organs. 3 H-bradykinin, perfused through the trout gill was not metabolized, as demonstrated by high voltage paper electrophoresis. However, trout gills extracted and retained approximately 40% of the 3 H-bradykinin perfused into the branchial vasculature. Gill tissue homogenates metabolized both 3 H-bradykinin and the vasoactive substance produced by incubating trout plasma with glandular kallikrein

  16. Kallikrein and Renin in the Membrane Fractions of the Rat Kidney.

    Science.gov (United States)

    1980-05-23

    fraction. PM-kallikrein re- leased a kinin, cleaved the peptide substrate, S-2266 and a radio-labeled arginine ester. The ester was also hydrolyzed by renal...more by activators of kallikrein, e.g. by phospholipase A/, lyso- lecithin and melittin. Low sodium diet increased the activity of kallikrein in the...peptide substrate, S-2266 and a radio- labeled arginine ester. The ester was also hydrolyzed by renal enzymes other than kallikrein. PM-kallikrein was

  17. Human ovolution and plasma oxytocin

    International Nuclear Information System (INIS)

    Kumaresan, Perianna; Kumaresan, Malathi; Hossini, Mahmood; Arellano, Carolina; Vasicka, Alois

    1983-01-01

    Plasma oxytocin (OT) concentrations were measured by radioimmunoassay (RIA) method without extracting plasma in 11 normal menstruating women. Mean plasma OT level began to increase steadily from the 7th day of the menstrual cycle and this level rose up to 20+-5 μU/ml (Mean+-S.E.) on the 10th day of the cycle. OT level declined to 13+-6 μU/ml on the day of the LH peak and continuously declined for another 2 days - then rose. The OT level was higher during the follicular phase than during the luteal phase. In 1 individual OT measured in 2 cycles a year apart showed the highest level of OT coincided with LH and FSH peak and abruptly declined. When there was the highest level of progesterone, the OT level was measurable 1 out of 11 cycles. From this study, we conclude that OT may have a role in human ovulation either synergistically or alone with other ovulatory mechanisms and ovarian estradiol and progesterone control the secretion of OT and also suggests that OT may play some role in the regulation of the luteolysis and the menstrual cycle in women. (author)

  18. A radioimmunoassay for neurotensin in human plasma

    International Nuclear Information System (INIS)

    Blackburn, A.M.; Bloom, S.R.

    1979-01-01

    A radioimmunoassay was developed for detecting the neurotensin peptide in human plasma. The plasma was specific for neurotensin as no cross-reaction was found with any of the other gut hormones tested. Changes of 5 pmol/l could be detected with 95% confidence. Neurotensin was unstable in both blood and plasma but considerable protection was afforded by addition of aprotinin, rapid separation of plasma and immediate deep freezing. Neurotensin-like immunoreactivity was detected in human plasma in both a small and large molecular form. The mean fasting level of plasma neurotensin-like immunoreactivity in 36 healthy volunteers was 29 +- 3 pmol/l. A significant increment of 27 +- 8 pmol/l plasma neurotensin immunoreactivity was detected after a large meal in nine healthy men. In view of the present results in man and also of neurotensin's potent pharmacological actions in experimental animals, neurotensin appears to fulfil some of the criteria needed for a hormone. (UK)

  19. Radioimmunoassay of sodium cromoglycate in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K; Gardner, J J; Lockley, W J.S.; Preston, J R; Wilkinson, D J [Fisons plc, Loughborough (UK). Pharmaceutical Div.

    1983-01-01

    A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0.93 nmol/l when 0.1 ml plasma samples are analysed. The range of the method is limited; both 0.01 and 0.1 ml volumes of plasma must be analysed to encompass the concentration range 0.93-139 nmol/l which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

  20. Is the renal kallikrein-kinin system a factor that modulates hypercalciuria?

    Directory of Open Access Journals (Sweden)

    Armando Luis Negri

    2017-01-01

    Full Text Available Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK. Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%. These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics.

  1. A cytocidal tissue kallikrein isolated from mouse submandibular glands.

    Science.gov (United States)

    Murakami, K; Ikigai, H; Nagumo, N; Tomita, M; Shimamura, T

    1989-11-06

    A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.

  2. Prognostic significance of multiple kallikreins in high-grade astrocytoma

    International Nuclear Information System (INIS)

    Drucker, Kristen L.; Gianinni, Caterina; Decker, Paul A.; Diamandis, Eleftherios P.; Scarisbrick, Isobel A.

    2015-01-01

    Kallikreins have clinical value as prognostic markers in a subset of malignancies examined to date, including kallikrein 3 (prostate specific antigen) in prostate cancer. We previously demonstrated that kallikrein 6 is expressed at higher levels in grade IV compared to grade III astrocytoma and is associated with reduced survival of GBM patients. In this study we determined KLK1, KLK6, KLK7, KLK8, KLK9 and KLK10 protein expression in two independent tissue microarrays containing 60 grade IV and 8 grade III astrocytoma samples. Scores for staining intensity, percent of tumor stained and immunoreactivity scores (IR, product of intensity and percent) were determined and analyzed for correlation with patient survival. Grade IV glioma was associated with higher levels of kallikrein-immunostaining compared to grade III specimens. Univariable Cox proportional hazards regression analysis demonstrated that elevated KLK6- or KLK7-IR was associated with poor patient prognosis. In addition, an increased percent of tumor immunoreactive for KLK6 or KLK9 was associated with decreased survival in grade IV patients. Kaplan-Meier survival analysis indicated that patients with KLK6-IR < 10, KLK6 percent tumor core stained < 3, or KLK7-IR < 9 had a significantly improved survival. Multivariable analysis indicated that the significance of these parameters was maintained even after adjusting for gender and performance score. These data suggest that elevations in glioblastoma KLK6, KLK7 and KLK9 protein have utility as prognostic markers of patient survival. The online version of this article (doi:10.1186/s12885-015-1566-5) contains supplementary material, which is available to authorized users

  3. Radioimmunoassay study of neurophysins in human plasma

    International Nuclear Information System (INIS)

    Reinharz, A.C.; Tissot-Berthet, M.-C.; Vallotton, M.B.

    1978-01-01

    Using a homologous system we have developed a specific and sensitive radioimmunoassay for the measurement of one of the human neurophysins in unextracted human plasma. This neurophysin is specifically secreted in response to oestrogen and has therefore been referred to as human oestrogen-stimulated neurophysin (h-OeSN). The plasma concentration was 0.57 plus minus 0.17 ng/ml (SD) in females and 0.88 plus minus 0.76 ng/ml (SD) in males. This difference is not significant. In women on oral contraceptives, plasma h-OeSN was 2.0 plus minus 1.1 ng/ml. During pregnancy h-OeSN increased progressively to 3.7 plus minus 2.9 ng/ml at the end of the first trimester, and 5.2 plus minus 2.8 ng/ml at term. Plasma h-OeSN concentrations increased rapidly and markedly in men treated with ethinyl-oestradiol. We have also demonstrated the presence of h-OeSN in amniotic and cerebrospinal fluids. A second human neurophysin, which is stimulated by nicotine but not by oestrogen, was also measured. This neurophysin was monitored by a heterologous system using antiserum raised against bovine neurophysin II (b-NII), and b-NII as the standard and tracer. (author)

  4. Radioimmunoassay of human plasma protein C

    International Nuclear Information System (INIS)

    Wang Bocheng; Li Jinquan; Jing Jian; Zhang Manda

    1995-01-01

    A radioimmunoassay method for the measurement of human plasma protein C (PC) is established. PC was isolated and purified from human plasma. The antisera against PC was obtained by immunizing rabbits. Iodination of PC was carried out with chroramine-T. The sensitivity was 3.94% μg/L, and the assay covered 6.25∼1024 μg/L for PC. The intra-assay and inter-assay CV were 4.4% and 9.68% respectively, with a recovery rate of 104.28%. There was no cross reaction with factor II. The normal value was 3.84 +- 0.34 mg/L in 36 normal persons. Value of 1.03 +-0.41 mg/L was found in 16 patients with fulminating hepatitis complicated with coagulation disturbance. It is an effective approach for the diagnosis of hereditary or acquired PC deficiency and also for the study of thrombotic diseases

  5. Double Stranded RNA in Human Seminal Plasma

    Directory of Open Access Journals (Sweden)

    Maxim V. Zagoskin

    2017-10-01

    Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

  6. Studies on coagulation, fibrinolysis, kallikrein-kinin and complement activation in systemic and pulmonary circulation during hip arthroplasty with acrylic cement.

    Science.gov (United States)

    Dahl, O E; Molnar, I; Vinje, A; Rø, J S; Kierulf, P; Andersen, A B; Dalaker, K; Prydz, H

    1988-06-15

    This study was designed to evaluate the contribution of the plasma cascade systems to the cardiopulmonary complications, occasionally leading to sudden death during hip arthroplasty using acrylic cement. The intraoperative pattern following uneventful surgery was therefore investigated in 8 patients with osteoarthrosis with frequent sampling from the radial and pulmonary arteries. The following general findings emerged: A gradual consumption of coagulation factors (platelets, fibrinogen, factor VII, antithrombin III), fibrinolytic components (plasminogen, alpha-2-antiplasmin), kallikrein-kinin factors (prekallikrein, kallikrein inhibitor) and complement factors (C3c, C4) was observed. Some intrapulmonary proteolytic inhibition was noticed as evidenced by lower arterial than mixed venous blood levels of alpha-2-antiplasmin and kallikrein inhibitor. Insignificant changes occurred for factor VII-phospholipid complex. A rapid, massive and transient increase in fibrinopeptide A (FPA) values in the radial artery, as opposed to the moderate increase in the pulmonary artery, was found immediately after reaming and broaching of bone. This probably reflected intrapulmonary fibrinogen to fibrin conversion, reaching a maximum 15-20 minutes before the femoral implantation. As estimated from the FPA arterial peak values and the fall in fibrinogen concentrations, approximately 5-10% of the circulating fibrinogen molecules were devoided of FPA during this intraoperative phase. The marked a-v difference in FPA level supports earlier findings of intrapulmonary fibrin formation and deposition. This process, however preceded the critical period of cardiorespiratory collapse (CRC) by at least 15 minutes, and may thus predispose to this complication.

  7. Human plasma lecithin-cholesterol acyltransferase

    International Nuclear Information System (INIS)

    Jauhiainen, M.; Stevenson, K.J.; Dolphin, P.J.

    1988-01-01

    Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. The authors proposed a covalent catalytic mechanism of action for LCAT in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols they have probed the geometry of the catalytic site. They conclude that the two catalytic cysteine residues of LCAT (Cys 31 and Cys 184 ) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by teh bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue

  8. Bioassay of procoagulant albumin in human plasma.

    Science.gov (United States)

    Grosset, A; Liu, L; Parker, C J; Rodgers, G M

    1994-09-01

    Procoagulant albumin (P-Al) is present in normal human plasma and increases monocyte and endothelial cell expression of tissue factor activity. To develop a bioassay for P-Al, we partially purified plasma from healthy volunteers and several patient groups using BaCl2 and (NH4)2SO4 precipitation. The samples were assayed for tissue factor (TF) inducing activity, expressed as a percentage increase compared to a serum-free media control. Over six months, the assay was reproducible in stored samples and in serial samples from normal volunteers. The plasma P-Al activities of 35 volunteers averaged 141 +/- 8.2% (SEM). There was no diurnal variation. There was no difference in the P-Al activity after a 12 hour fast and 2 hours after a large meal in 4 healthy volunteers. There was no increase in activity (r = 0.16) with the subject's age. The average activity from 16 poorly-controlled diabetics was 131 +/- 11% (SEM). No alteration in activity was seen with samples from patients with uremia, liver dysfunction, hemophilia, thrombotic events, or adenocarcinoma. These results indicate that P-Al activity can be bioassayed in individual patient samples; however, pathologic states associated with abnormal P-Al-induced tissue factor activity presently remain unidentified.

  9. Activation of Membrane-Bound Kallikrein and Renin in the Kidney.

    Science.gov (United States)

    1980-05-23

    included repeated washings with hypotonic buffer. Kallikrein activity in the PM fraction (PM-kallikrein) averaged 1.81 nmol of S-2266 hydrolyzed per min...thousand Fig. 1 times more active than lysolecithin on a molar basis. Lecithin and arachidonic acid were active only at a much higher concentration...taglandin E2 (11), arachidonic acid or lecithin . However, melittin, on a molar basis, was about three orders of magnitude more potent than

  10. Excess antibody immunoassays for rat glandular kallikreins. Measurement of kallikrein from different organs in the presence of cross-reacting antigens

    International Nuclear Information System (INIS)

    Johansen, L.; Oerstavik, T.B.; Holck, M.; Nustad, K.

    1983-01-01

    An immunoradiometric assay has previously been developed for measurement of rat glandular kallikrein. In the present paper, further studies on the specificity and sensitivity of the method are described. Problems of interference of immunologically cross-reacting antigens were overcome by proper preabsorption of the antibody. A method was thus established in which enzymatic activity of the immunoreactive kallikrein could be measured even in the presence of enzymes sharing immunological determinants and substrate specificity with kallikrein. Two variants of the immunoradiometric assay have been evaluated. A simplified version with simultaneous addition of all reagents gave results equal to those obtained in the original assay. A further modification with delayed addition of the solid-phase antibody, gave considerable improvement in assay sensitivity. (Auth.)

  11. LC determination of praziquantel in human plasma.

    Science.gov (United States)

    Ridtitid, Wibool; Wongnawa, Malinee; Mahatthanatrakul, Werawath; Punyo, Jarurat; Sunbhanich, Methi

    2002-04-01

    A simple high-performance liquid chromatographic (HPLC) method for the determination of praziquantel in human plasma was developed and validated. The present method was described by adding drop-wise 0.2 M Zinc sulfate and acetonitrile to plasma sample for deproteinization. This method used a reversed-phase Spherisorb ODS 2 column (5 microm), 250 x 4.6 mm i.d. as a stationary phase with a mobile phase consisting of acetonitrile- methanol-water (36:10:54, v/v/v), a flow rate of 1.5 ml/min and UV detection wavelength of 217 nm. Diazepam was used as internal standard. The standard calibration curve was linear over the concentration range of 100-2000 ng/ml (r=0.999). The equation of a linear regression line was y=8.05E-04+7.25E-04x with slope and intercept values of 0.0007 and 0.0008, respectively. The limit of detection was 12.25 ng/ml and the limit of quantification was set at 100 ng/ml. The intra- and inter-day assay coefficients of variation (CV) were 3.0+/-1.7 and 6.3+/-1.9%, respectively. The percentage of recovery was 102.1+/-5.6. Therefore, the HPLC method described here was simple, rapid and reproducible since it did not require extraction and evaporation processes in sample preparation, which will reduce time-consuming or expensive sample preparation.

  12. Novel Parvovirus and Related Variant in Human Plasma

    Science.gov (United States)

    Fryer, Jacqueline F.; Kapoor, Amit; Minor, Philip D.; Delwart, Eric

    2006-01-01

    We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from 106 copies/mL plasma. PMID:16494735

  13. The link between high-fat meals and postprandial activation of blood coagulation factor VII possibly involves kallikrein

    DEFF Research Database (Denmark)

    Larsen, L F; Marckmann, P; Bladbjerg, Else-Marie

    2000-01-01

    Contrary to low-fat meals, high-fat meals are known to cause postprandial factor VII (FVII) activation, but the mechanism is unknown. To study the postprandial FVII activation in detail, 18 young men consumed in randomized order high-fat or low-fat test meals. Fasting and non-fasting blood samples...... that triglyceride-rich lipoproteins may activate prokallikrein. Neither plasma triglycerides nor kallikrein and activated FVII were statistically associated. This may suggest that additional factors are involved in the postprandial FVII activation. No clear evidence for a role of tissue factor expression...... by monocytes, factor XII or insulin in postprandial FVII activation was observed. Tissue factor pathway inhibitor and prothrombin fragment 1+2, a marker of thrombin generation, were not affected postprandially after either the high-fat or the low-fat meals. Our findings indicate that triglyceride...

  14. Endothelial cell permeability during hantavirus infection involves factor XII-dependent increased activation of the kallikrein-kinin system.

    Directory of Open Access Journals (Sweden)

    Shannon L Taylor

    Full Text Available Hemorrhagic fever with renal syndrome (HFRS and hantavirus pulmonary syndrome (HPS are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF. To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS. We show that incubation of factor XII (FXII, prekallikrein (PK, and high molecular weight kininogen (HK plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL and increased liberation of bradykinin (BK. Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS, we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation

  15. Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c.

    Science.gov (United States)

    Su, Jingjing; Tang, Yuping; Zhou, Houguang; Liu, Ling; Dong, Qiang

    2012-11-01

    Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Antioxidant capacity of plasma after red wine intake in Human

    Directory of Open Access Journals (Sweden)

    M. Gianmmanco

    2009-01-01

    Full Text Available Aim: Antioxidant effects after consumption of red wine have been investigated in several studies but results are contradictory and the difference in the plasma antioxidant capacity (AC after intake of red wine between women and men has never been studies. This work purpose is manifold: to ascertain whether red wine intake modifies the human plasma AC; to study the behaviour of plasma AC of women in comparison with men and finally to investigate on the plasma uric acid concentration and its relationships with the plasma AC after red wine intake.

  17. Radioimmunoassay of cholecystokinin in human plasma

    International Nuclear Information System (INIS)

    Byrnes, D.J.; Henderson, L.; Borody, T.; Rehfeld, J.F.

    1981-01-01

    A sensitive radioimmunoassay for cholecystokinin (CCK) has been developed. Porcine CCK-33 was labelled by conjugation with 125 I-hydroxyphenyl-propionic acid succinimide ester. Antibodies were raised against porcine CCK-33 covalently coupled to egg albumin. Plasma samples were extracted with 96% ethanol prior to assay. Free and bound hormone were separated by dextran-coated charcoal. The antibodies bound CCK-8 and CCK-33 with equimolar potency. The assay detection limit was 1 pmol/l plasma. Within and between assay coefficients of variation were +-12.7 and 13.0% at mean plasma CCK concentrations of 13.2 and 13.6 pmol/l. The concentration of CCK in 47 normal fasting subjects ranged from undetectable to 22 pmol/l. Ingestion of a mixed meal in 9 normal subjects increased the plasma concentration from 8.3 +- 2.5 S.E. to 24.4 +- 6.5 pmol/l. (Auth.)

  18. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  19. Bystander apoptosis in human cells mediated by irradiated blood plasma

    International Nuclear Information System (INIS)

    Vinnikov, Volodymyr; Lloyd, David; Finnon, Paul

    2012-01-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G 0 -stage lymphocytes. Plasma was collected from healthy donors’ blood irradiated in vitro to 0–40 Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 °C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 ± 1.8% in plasma-free cultures, 21.6 ± 1.1% in cultures treated with plasma from unirradiated blood, 20.2 ± 1.4% in cultures with plasma from blood given 2–4 Gy and 16.7 ± 3.2% in cultures with plasma from blood given 6–10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  20. Characterization of kallikrein-related peptidase 4 glycosylations.

    Science.gov (United States)

    Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

    2011-12-01

    Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

  1. Radioimmunoassay of arginine vasopressin in human plasma

    International Nuclear Information System (INIS)

    Uhlich, E.; Weber, P.; Groeschel-Stewart, U.; Roeschlau, T.; Wuerzburg Univ.

    1975-01-01

    Antibodies for the radioimmunoassay of arginine vasopressin (AVP) described here were produced in rabbits using synthetic AVP coupled to rabbit γ-globulin with carbodiimide. In three out of six rabbits, significant antibody titres were obtained. Using the best antisera produced, 40% of labelled AVP was bound at a final dilution of 1 : 50,000. After iodination of synthetic AVP with 125 I using the chloramin-T method, a gel filtration on Sephadex G-25 was performed to purify the iodinated AVP. For separation of antibody bound and free hormone, a second antibody precipitation was used. There was no crossreactivity with oxytocin. AVP was extracted from plasma after ammoniumsulfate precipitation of the proteins by adsorption to Florisil. The recovery of AVP added to plasma in amounts of 5-25 pg/ml was 60 +- 15% (n = 6). The minimum amount of AVP detectable was 1 pg per ml plasma. The plasma level in normal adults under standard conditions was 3.4 +- 2.2 pg/ml. This is in agreement with data recently published by other researchers. The applicability and reproducibility was further tested in measurements of samples taken hourly during the entire day under water diuresis and after hormonal stimulation of AVP. (orig.) [de

  2. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma

    International Nuclear Information System (INIS)

    Yandle, T.G.; Espiner, E.A.; Nicholls, M.G.; Duff, H.

    1986-01-01

    A RIA for alpha-human atrial natriuretic peptide (alpha hANP) in plasma was developed and used to study the immunoreactive components secreted by the heart and circulating in peripheral venous plasma. The assay used [125I]diiodotyrosyl-alpha hANP, purified by high pressure liquid chromatography (HPLC), and a C-terminal-specific antiserum purchased from Peninsula Laboratories. Serial dilution curves of coronary sinus plasma samples were parallel with the standard curve, but significant nonparallelism was found in peripheral plasma samples of low immunoreactivity. When plasma was extracted using C-18 Sep-Pak cartridges, serial dilution curves from both coronary sinus and peripheral plasma samples were parallel to the standard curve. Although values for plasma samples assayed before and after extraction agreed closely (r = 0.99; n = 76), immunoreactive ANP in unextracted plasma was consistently greater (70-79 pmol/liter) than in extracts of plasma, suggesting non-specific interference by a component in plasma when assayed without extraction. Mean plasma immunoreactive ANP in 19 normal subjects consuming a normal salt intake was 14 +/- 1 (+/- SE) pmol/liter. In 5 normal men, increasing dietary sodium intake from 10 to 200 mmol sodium/day was associated with a 2-fold increment in ANP levels, and similar changes accompanied acute sodium loading using iv saline. Elevated values were found in patients with congestive heart failure (mean, 58 pmol/liter; range, 0-200; n = 9), chronic renal failure (mean, 118 pmol/liter; range, 30-290; n = 8), and primary aldosteronism (range, 32-90 pmol/liter; n = 3). HPLC and gel chromatographic analysis of the immunoreactive material found in coronary sinus plasma extracts showed that a large amount of the material eluted in the position of alpha hANP

  3. Arginine-esterase activity of kallikrein in the sera of whole-body irradiated rats and guinea-pigs

    Energy Technology Data Exchange (ETDEWEB)

    Pouckova, P; Pospisil, J; Dienstbier, Z [Karlova Univ., Prague (Czechoslovakia). Biofyzikalni Ustav

    1977-09-01

    In whole-body irradiated rats (800 R=LDsub(50/30)) and guinea pigs (300 R=LDsub(50/30)) changes were investigated in the arginine esterase activity of kallikrein in native serum as well as in serum exposed to contact with a clay suspension. From the values obtained the activity of prekallikrein was calculated. While in the rat serum significant changes in the arginine esterase activity of kallikrein were found, in the guinea pig serum the kallikrein activity did not change markedly. The activity of prekallikrein immediately after irradiation assumes a similar course in both types of laboratory animals while during later intervals a reverse pattern was observed.

  4. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P03952) Plasma kallikrein precursor (EC 3.4.21.34) (Plasma prekallikrein...) (Kininogenin) (Fletcher factor) [Contains: Plasma kallikrein heavy chain; Plasma kallikrein light chain] KLKB1_HUMAN 2e-21 ...

  5. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P03952) Plasma kallikrein precursor (EC 3.4.21.34) (Plasma prekallikrein...) (Kininogenin) (Fletcher factor) [Contains: Plasma kallikrein heavy chain; Plasma kallikrein light chain] KLKB1_HUMAN 5e-21 ...

  6. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P03952) Plasma kallikrein precursor (EC 3.4.21.34) (Plasma prekallikrein...) (Kininogenin) (Fletcher factor) [Contains: Plasma kallikrein heavy chain; Plasma kallikrein light chain] KLKB1_HUMAN 4e-37 ...

  7. Informing the Human Plasma Protein Binding of ...

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

  8. Plasma urocortin in human systolic heart failure.

    Science.gov (United States)

    Ng, Leong L; Loke, Ian W; O'Brien, Russell J; Squire, Iain B; Davies, Joan E

    2004-04-01

    Urocortin (UCN), a member of the corticotrophin-releasing factor family, is expressed in heart, brain and gut. UCN has potent cardiostimulatory, cardioprotective, vasodilator and diuretic/natriuretic effects, and cardiac UCN expression is increased in heart failure (HF). In the present study, we investigated plasma levels of UCN in 119 patients with HF and 212 age- and gender-matched controls to clarify its relationship with gender and disease severity. UCN was elevated in HF [normal males, 19.5 (3.9-68.8) pmol/l and HF males, 50.2 (6.9-108.2) pmol/l, P fall in UCN levels with increasing NYHA class was reinforced by a significant correlation between UCN and ejection fraction ( r(s) = 0.45, P < 0.0005) in HF patients. Although receiver operating characteristic (ROC) curves for diagnosis of all HF cases yielded an area under the curve (AUC) of 0.76, ROC AUCs for patients with early HF (NYHA class I and II) were better (0.91). ROC AUCs for logistic models incorporating N-terminal probrain natriuretic peptide (N-BNP) and UCN were better than either peptide alone. In conclusion, plasma UCN is elevated in HF, especially in its early stages. Its decline with increasing HF severity may expedite disease progression due to diminished cardioprotective/anti-inflammatory effects. UCN measurement may also complement N-BNP in the diagnosis of early HF.

  9. Radioimmunoassay of human β-lipotropin in unextracted plasma

    International Nuclear Information System (INIS)

    Wiedemann, E.; Saito, T.; Linfoot, J.A.; Li, C.H.

    1977-01-01

    A sensitive and specific radioimmunoassay for human β-lipotropin (β/sub h/-LPH) in unextracted plasma was developed using pure β/sub h/-LPH as tracer and standard and an antiserum not cross-reacting with human β-MSH and hACTH. In healthy volunteers plasma β/sub h/-LPH ranged from <20 to 150 pg/ml at 8:00 a.m. and rose after metyrapone administration. β/sub h/-LPH was very low in panhypopituitarism, normal in most patients with untreated Cushing's disease, elevated in acromegaly and extremely high in Nelson's syndrome

  10. Endogenous pyrogen activity in human plasma after exercise.

    Science.gov (United States)

    Cannon, J G; Kluger, M J

    1983-05-06

    Plasma obtained from human subjects after exercise and injected intraperitoneally into rats elevated rat rectal temperature and depressed plasma iron and zinc concentrations. The pyrogenic component was heat-denaturable and had an apparent molecular weight of 14,000 daltons. Human mononuclear leukocytes obtained after exercise and incubated in vitro released a factor into the medium that also elevated body temperature in rats and reduced trace metal concentrations. These results suggest that endogenous pyrogen, a protein mediator of fever and trace metal metabolism during infection, is released during exercise.

  11. Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase

    Directory of Open Access Journals (Sweden)

    Seda Onder

    2018-02-01

    Full Text Available Human butyrylcholinesterase (BChE is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276. Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 μm filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.

  12. Human parvovirus PARV4 in plasma pools of Chinese origin.

    Science.gov (United States)

    Ma, Y-Y; Guo, Y; Zhao, X; Wang, Z; Lv, M-M; Yan, Q-P; Zhang, J-G

    2012-10-01

    Human parvovirus 4 (PARV4) is present in blood and blood products. As the presence and levels of PARV4 in Chinese source plasma pools have never been determined, we implemented real-time quantitative PCR to investigate the presence of PARV4 in source plasma pools in China. Results showed that 26·15% (51/195) of lots tested positive for PARV4. The amounts of DNA ranged from 2·83 × 10(3) copies/ml to 2·35×10(7) copies/ml plasma. The high level of PARV4 in plasma pools may pose a potential risk to recipients. Further studies on the pathogenesis of PARV4 are urgently required. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

  13. Radioimmunoassay of cholecystokinin in human tissue and plasma

    International Nuclear Information System (INIS)

    Jansen, J.B.M.J.; Lamers, C.B.H.W.

    1983-01-01

    A highly sensitive radioimmunoassay for cholecystokinin (CCK) without any cross-reactivity with gastrin is described. The antibody was raised in a rabbit by immunisation with 30% CCK and bound to all COOH-terminal CCK-peptides containing at least 14 amino acid residues. The affinity constant of the antibody was 59.4 x 10 10 l/mol. CCK 33 conjugated to [ 125 I]hydroxyphenylpropionic acid-succinimide ester was used as label. The binding between label and antibody was inhibited by 50% (ID 50 ) at a concentration of 2.8 pmol/l cholecystokinin 33. The detection limit of the assay was between 0.5 and 1.0 pmol/l plasma. Concentrations of CCK in aqueous acid extracts of human upper small intestine were 36.5 +- 9.8 pmol/g and of human cerebral cortex 28.2 +- 2.5 pmol/g tissue. Plasma samples were extracted in 96% ethanol prior to assay. No advantage was obtained by adding aprotinin to the tubes. When frozen at -20 0 C plasma CCK was stable for at least 6 months. Basal plasma CCK concentrations in 30 normal subjects were very low, 0.9 +- 0.1 pmol/l, range 0.5 to 3.1 pmol/l. Intraduodenal administration of fat induced significant increases in plasma CCK from 1.1 +- 0.1 to 8.2 +- 1.3 pmol/l (p = 0.01). Infusion of exogenous CCK, resulting in plasma CCK levels slightly lower than those measured during administration of fat, induced pancreatic enzyme secretion and gallbladder contraction. The reliability of this radioimmunoassay for measurements of CCK in human plasma was extensively evaluated. (Auth.)

  14. Posttranslational modifications in human plasma MBL and human recombinant MBL

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

    2007-01-01

    the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

  15. HPLC Method for Determination of Rifaximin in Human Plasma ...

    African Journals Online (AJOL)

    The present study was aimed at developing a simple, sensitive, and specific liquid chromatography–tandem mass spectrometry method for the quantification of rifaximin in human plasma using rifaximin D6 as internal standard. Chromatographic separation was performed on Zorbax SB C18, 4.6 x 75 mm, 3.5 μm column with ...

  16. Radioimmunoassay of follicle stimulating hornone in human plasma

    International Nuclear Information System (INIS)

    Akande, E.O.

    1976-01-01

    A radioimmunoassay method for Follicle Stimulating Hormone (fsh) in human plasma is described. The method proved to be reliable in determining fsh levels in normally menstruating women as well as women in varying clinical states. The patterns and levels of fsh obtained from 16 menstrual cycles in 12 normally menstruating women showed agreement with previous results of Franchimont, Faiman and Ryan, etc

  17. Lipidomics reveals a remarkable diversity of lipids in human plasma.

    Science.gov (United States)

    Quehenberger, Oswald; Armando, Aaron M; Brown, Alex H; Milne, Stephen B; Myers, David S; Merrill, Alfred H; Bandyopadhyay, Sibali; Jones, Kristin N; Kelly, Samuel; Shaner, Rebecca L; Sullards, Cameron M; Wang, Elaine; Murphy, Robert C; Barkley, Robert M; Leiker, Thomas J; Raetz, Christian R H; Guan, Ziqiang; Laird, Gregory M; Six, David A; Russell, David W; McDonald, Jeffrey G; Subramaniam, Shankar; Fahy, Eoin; Dennis, Edward A

    2010-11-01

    The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

  18. Increased Plasma Levels of Heme Oxygenase-1 in Human Brucellosis.

    Science.gov (United States)

    Chen, Zhe; Zhang, Yu-Xue; Fu, Dong-Wei; Gao, Qing-Feng; Ge, Feng-Xia; Liu, Wei-Hua

    2016-08-01

    Brucellosis is associated with inflammation and the oxidative stress response. Heme oxygenase-1 (HO-1) is a cytoprotective stress-responsive enzyme that has anti-inflammatory and anti-oxidant effects. Nevertheless, the role of HO-1 in human brucellosis has not yet been studied. The aim of this study was to examine the plasma levels of HO-1 in patients with brucellosis and to evaluate the ability of plasma HO-1 levels as an auxiliary diagnosis, a severity predictor, and a monitor for brucellosis treatments. A total of 75 patients with brucellosis were divided into the acute, subacute, chronic active, and chronic stable groups. An additional 20 volunteers were included as the healthy control group. The plasma HO-1 levels and other laboratory parameters were measured in all groups. Furthermore, the plasma levels of HO-1 in the acute group were compared before and after treatment. The plasma HO-1 levels were considerably increased in the acute (4.97 ± 3.55), subacute (4.98 ± 3.23), and chronic active groups (4.43 ± 3.00) with brucellosis compared to the healthy control group (1.03 ± 0.63) (p brucellosis (r = 0.707, p brucellosis status and may be used as a supplementary plasma marker for diagnosing brucellosis and monitoring its treatment.

  19. Site specific modification of the human plasma proteome by methylglyoxal

    International Nuclear Information System (INIS)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig S.; Monks, Terrence J.; Lau, Serrine S.

    2015-01-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  20. Site specific modification of the human plasma proteome by methylglyoxal

    Energy Technology Data Exchange (ETDEWEB)

    Kimzey, Michael J.; Kinsky, Owen R. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Yassine, Hussein N. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Tsaprailis, George [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Stump, Craig S. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Southern Arizona VA Health Care System, Tucson, AZ 85723 (United States); Monks, Terrence J. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Lau, Serrine S., E-mail: lau@pharmacy.arizona.edu [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States)

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  1. Evidence for radical-oxidation of plasma proteins in humans

    International Nuclear Information System (INIS)

    Wang, D.; Davies, M.; Dean, R.; Fu, S.; Taurins, A.; Sullivans, D.

    1998-01-01

    Oxidation of proteins by radicals has been implicated in many pathological processes. The hydroxyl radical is known to generate protein-bound hydroxylated derivatives of amino acids, for example hydroxyvaline (from Val), hydroxyleucine (from Leu), o-tyrosine (from Phe), and DOPA (from Tyr). In this study, we have investigated the occurrence of these oxidised amino acids in human plasma proteins from both normal subjects and dialysis patients. By employing previously established HPLC methods [Fu et al. Biochemical Journal, 330, 233-239, 1998], we have found that oxidised amino acids exist in normal human plasma proteins (n=32). The level of these oxidised amino acids is not correlated to age. Similar levels of oxidised amino acids are found in the plasma proteins of the dialysis patients (n=6), but a more detailed survey is underway. The relative abundance of the oxidised amino acids is similar to that resulting from oxidation of BSA by hydroxy radicals or Fenton systems [Fu et al. Biochemical Journal, 333, 519-525, 1998]. The results suggest that metal-ion catalysed oxyl-radical chemistry may be a key contributor to the oxidative damage in plasma proteins in vivo in humans

  2. A radioimmunoassay of gastric inhibitory polypeptide in human plasma

    International Nuclear Information System (INIS)

    Sarson, D.L.; Bryant, M.G.; Bloom, S.R.

    1980-01-01

    A sensitive radioimmunoassay for the measurement of human gastric inhibitory polypeptide (GIP), using pure porcine GIP, has been developed. Cross-reactivity of the antiserum with all available mammalian gut peptide preparations was negligible with the exception of glucagon when it was approximately 1%. Two major molecular forms of GIP were detectable in plasma and tissue extracts, one of large molecular size and the other corresponding to the elution coefficient of pure porcine standard. Concentrations of GIP in plasma from 50 normal subjects after overnight fasting were 9+-1.0(S.E.M.) pmol/1 rising to a peak of 34+-2.8 pmol/1 following the ingestion of a small mixed test meal. Ingestion of glucose or fat resulted in a similar rise of plasma GIP, whereas no change was observed after the ingestion of protein. (author)

  3. Radioimmunoassay for human plasma 8-arginine-vasopressin

    International Nuclear Information System (INIS)

    Conte-Devolx, B.; Rougon-Rapuzzi, G.; Millet, Y.

    1977-01-01

    A radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormone (ADH) level as low as 0.8pg/ml, was developed. The average plasma level of AVP after overnight water restriction was found to be 14.3pg/ml (sd=4.4pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2mg of nicotine. In 11 volunteer normal subjects this stimulation by nicotine provoked ADH hypersecretion which reached a maximum between 2nd and 15th minutes after injection. In 3 cases of diabetes insipidus, nicotine injection did not induce ADH hypersecretion: in 1 case of potomania this response was weak; in 2 cases of syndrome of inappropriate ADH secretion, AVP plasma levels were elevated and the response after nicotine stimulation was exaggerated [fr

  4. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

    1981-08-27

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

  5. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    International Nuclear Information System (INIS)

    Burnam, M.H.; Shell, W.E.

    1981-01-01

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

  6. Radioimmunoassay for human plasma 8-arginine-vasopressin

    Energy Technology Data Exchange (ETDEWEB)

    Conte-Devolx, B [Hopital de la Conception, 13 - Marseille (France); Rougon-Rapuzzi, G [Centre National de la Recherche Scientifique, 13 - Marseille (France); Millet, Y [Aix-Marseille-2 Univ., 13 - Marseille (France)

    1977-01-01

    A radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormone (ADH) level as low as 0.8pg/ml, was developed. The average plasma level of AVP after overnight water restriction was found to be 14.3pg/ml (sd=4.4pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2mg of nicotine. In 11 volunteer normal subjects this stimulation by nicotine provoked ADH hypersecretion which reached a maximum between 2nd and 15th minutes after injection. In 3 cases of diabetes insipidus, nicotine injection did not induce ADH hypersecretion: in 1 case of potomania this response was weak; in 2 cases of syndrome of inappropriate ADH secretion, AVP plasma levels were elevated and the response after nicotine stimulation was exaggerated.

  7. Plasma metabolism of apolipoprotein A-IV in humans

    International Nuclear Information System (INIS)

    Ghiselli, G.; Krishnan, S.; Beigel, Y.; Gotto, A.M. Jr.

    1986-01-01

    As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125 I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125 I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day

  8. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    Science.gov (United States)

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  9. Specific radioimmunoassay of human. beta. -endorphin in unextracted plasma

    Energy Technology Data Exchange (ETDEWEB)

    Wiedemann, E. (Univ. of California, Berkeley); Saito, T.; Linfoot, J.A.; Li, C.H.

    1979-09-01

    With an antiserum against human ..beta..-endorphin (..beta..-EP) crossreacting <2% with human ..beta..-lipotropin (..beta..-LPH) by weight we have developed a radioimmunoassay that can detect 1 pg ..beta..-EP in diluted raw plasma. In a.m. fasting plasma of 14 normal subjects ..beta..-EP ranged from <5 to 45 pg/ml. ..beta..-EP was elevated in untreated, but normal in successfully treated Cushing's disease; undetectable in a patient with adrenal adenoma; extremely high in Nelson's syndrome; and elevated in a patient with bronchogenic carcinoma before, but undetectable after tumor resection. In subjects with intact hypothalamic-pituitary-adrenal axis, ..beta..-EP was undectectable after dexamethasone and increased after metyrapone administration and insulin-induced hypoglycemia. ..beta..-EP concentration was considerably lower in serum than in simultaneously collected plasma, but increased in serum left unfrozen for several hours after clot removal. Thus, ..beta..-EP behaves like a hormone responding to the same stimuli as ACTH and ..beta..-LPH and blood appears to contain enzymes both generating and destroying immunoreactive ..beta..-EP.

  10. The Complex Exogenous RNA Spectra in Human Plasma: An Interface with Human Gut Biota?

    Science.gov (United States)

    Wang, Kai; Li, Hong; Yuan, Yue; Etheridge, Alton; Zhou, Yong; Huang, David; Wilmes, Paul; Galas, David

    2012-01-01

    Human plasma has long been a rich source for biomarker discovery. It has recently become clear that plasma RNA molecules, such as microRNA, in addition to proteins are common and can serve as biomarkers. Surveying human plasma for microRNA biomarkers using next generation sequencing technology, we observed that a significant fraction of the circulating RNA appear to originate from exogenous species. With careful analysis of sequence error statistics and other controls, we demonstrated that there is a wide range of RNA from many different organisms, including bacteria and fungi as well as from other species. These RNAs may be associated with protein, lipid or other molecules protecting them from RNase activity in plasma. Some of these RNAs are detected in intracellular complexes and may be able to influence cellular activities under in vitro conditions. These findings raise the possibility that plasma RNAs of exogenous origin may serve as signaling molecules mediating for example the human-microbiome interaction and may affect and/or indicate the state of human health. PMID:23251414

  11. Antibodies to lactalbumin interfere with its radioimmunoassay in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, U; Laurence, D J.R. [Royal Marsden Hospital, London (UK); Ormerod, M G [Institute of Cancer Research, Sutton (UK). Surrey Branch

    1978-01-01

    Two radioimmunoassays for human lactalbumin have been established using a rabbit antiserum. One assay uses a second antibody to separate bound from free label; the other uses polyethylene glycol to precipitate gamma globulin non-specifically. It is confirmed that about half the normal human population have a substance in their blood which inhibits the binding of lactalbumin to the rabbit antibody. Comparison of the two assays has demonstrated that this material is not lactalbumin but a naturally occurring antibody. It is shown that it is in the IgG fraction of human plasma and is probably a cross-reacting antibody to bovine lactalbumin. None out of fifteen males and fourteen out of fifty eight non-pregnant, non-lacatating females had low levels of lactalbumin in their blood (0.6-2.0 ng/ml). The assay could not detect a statistically significant difference between normal women and those with either benign breast disease or metastatic mammary carcinoma.

  12. New validated method for piracetam HPLC determination in human plasma.

    Science.gov (United States)

    Curticapean, Augustin; Imre, Silvia

    2007-01-10

    The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.

  13. Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates.

    Science.gov (United States)

    Moitinho-Silva, Lucas; Kondo, Marcia Y; Oliveira, Lilian C G; Okamoto, Debora N; Paes, Jéssica A; Machado, Mauricio F M; Veronez, Camila L; Motta, Guacyara; Andrade, Sheila S; Juliano, Maria A; Ferreira, Henrique B; Juliano, Luiz; Gouvea, Iuri E

    2013-05-03

    Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Obesity and Low-Grade Inflammation Increase Plasma Follistatin-Like 3 in Humans

    DEFF Research Database (Denmark)

    Brandt, Claus; Pedersen, Maria; Rinnov, Anders

    2014-01-01

    , plasma leptin, fasting insulin, and HOMA B and negatively with HOMA S. Furthermore plasma fstl3 correlated positively with plasma TNF-α and IL-6 levels. Infusion of LPS and TNF-α, but not IL-6 and insulin, increased plasma fstl3 in humans. CONCLUSION: Plasma fstl3 is increased in obese subjects......BACKGROUND: Rodent models suggest that follistatin-like 3 (fstl3) is associated with diabetes and obesity. In humans, plasma fstl3 is reduced with gestational diabetes. In vitro, TNF-α induces fstl3 secretion, which suggests a link to inflammation. OBJECTIVE: To elucidate the association between...... plasma fstl3 and obesity, insulin resistance, and low-grade inflammation in humans. STUDY DESIGN: Plasma fstl3 levels were determined in a cross-sectional study including three groups: patients with type 2 diabetes, impaired glucose tolerance, and healthy controls. In addition, lipopolysaccharide (LPS...

  15. Pre-stimulation of the kallikrein system in cisplatin-induced acute renal injury: An approach to renoprotection

    Energy Technology Data Exchange (ETDEWEB)

    Aburto, Andrés [Program of M.Sc., Faculty of Medicine, Universidad Austral de Chile, Valdivia (Chile); Barría, Agustín [School of Biochemistry, Faculty of Sciences, Universidad Austral de Chile, Valdivia (Chile); Cárdenas, Areli [Ph.D. Program, Faculty of Sciences, Universidad Austral de Chile, Valdivia (Chile); Carpio, Daniel; Figueroa, Carlos D. [Department of Anatomy, Histology and Pathology, Universidad Austral de Chile, Valdivia (Chile); Burgos, Maria E. [Department of Nephrology, Faculty of Medicine, Universidad Austral de Chile, Valdivia (Chile); Ardiles, Leopoldo, E-mail: leopoldoardiles@gmail.com [Department of Nephrology, Faculty of Medicine, Universidad Austral de Chile, Valdivia (Chile)

    2014-10-15

    Antineoplastic treatment with cisplatin is frequently complicated by nephrotoxicity. Although oxidative stress may be involved, the pathogenic mechanisms responsible for renal damage have not been completely clarified. In order to investigate the role of the renal kinin system in this condition, a group of rats was submitted to high potassium diet to stimulate the synthesis and excretion of tissue kallikrein 1 (rKLK1) previous to an intraperitoneal injection of 7 mg/kg cisplatin. A significant reduction in lipoperoxidation, evidenced by urinary excretion of malondialdehyde and renal immunostaining of hidroxy-nonenal, was accompanied by a decline in apoptosis. Coincident with these findings we observed a reduction in the expression of renal KIM-1 suggesting that renoprotection may be occurring. Stimulation or indemnity of the renal kinin system deserves to be evaluated as a complementary pharmacological measure to diminish cisplatin nephrotoxicity. - Highlights: • Mechanisms of cisplatin-induced-renal damage have not been completely clarified. • Cisplatin induces oxidative stress and apoptosis. • The renal kallikrein-kinin system is protective in experimental acute renal damage. • Kallikrein stimulation reduces oxidative stress and apoptosis induced by cisplatin. • Protection of the kallikrein-kinin system may reduce cisplatin toxicity.

  16. Study of the components of renin-angiotensinaldosterone system and KalliKrein -Kinin system in normal pregnancy

    International Nuclear Information System (INIS)

    Abreu Fagundes, V.G. de.

    1984-01-01

    The alterations in the renin-angiotensin-aldosterone system and Kallikrein-Kinin system were studied. The possible interferences of these systems on the arterial pressure and on the evolution of normal pregnancy were presented in the following situations: when the pregnant change from dorsal decumbency to left lateral decumbency and to orthostatic position. (M.A.C.) [pt

  17. Renal Kallikrein Activation and Renoprotection after Dual Blockade of Renin-Angiotensin System in Diet-Induced Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Xia Zou

    2015-01-01

    Full Text Available Purpose. The objective of this study is to investigate the effect of dual blockage of renin-angiotensin system (RAS on renal kallikrein expression and inflammatory response in diabetic nephropathy (DN. Methods. Rats were randomly divided into 5 groups with 10 rats in each group: normal control; DN model induced by high fat and high sucrose diets; and DN treated with either benazepril 10 mg/kg/d, irbesartan 30 mg/kg/d, or both. After 8-week treatment, we examined changes in the kidney histopathology, function and immunohistochemical stain of kallikrein, macrophage marker CD68, and profibrotic markers transforming growth factor- (TGF- β and α-smooth muscle action (SMA. Results. DN rats showed enlarged kidneys with glomerulosclerosis, interstitial chronic inflammation and fibrosis, and proteinuria. All the pathological damage and functional impairments were improved after the RAS blockades (all P<0.05. Compared with monotherapy, combined treatment further alleviated the kidney impairments in parallel to increased tubular immunoreactivity for kallikrein and decreased immunopositive cells for CD68, TGF-β, and α-SMA. Conclusion. The renoprotective effects of the dual RAS blockade in diabetic nephropathy may be attributed to improved tubular kallikrein expression and interstitial inflammatory response.

  18. Pre-stimulation of the kallikrein system in cisplatin-induced acute renal injury: An approach to renoprotection

    International Nuclear Information System (INIS)

    Aburto, Andrés; Barría, Agustín; Cárdenas, Areli; Carpio, Daniel; Figueroa, Carlos D.; Burgos, Maria E.; Ardiles, Leopoldo

    2014-01-01

    Antineoplastic treatment with cisplatin is frequently complicated by nephrotoxicity. Although oxidative stress may be involved, the pathogenic mechanisms responsible for renal damage have not been completely clarified. In order to investigate the role of the renal kinin system in this condition, a group of rats was submitted to high potassium diet to stimulate the synthesis and excretion of tissue kallikrein 1 (rKLK1) previous to an intraperitoneal injection of 7 mg/kg cisplatin. A significant reduction in lipoperoxidation, evidenced by urinary excretion of malondialdehyde and renal immunostaining of hidroxy-nonenal, was accompanied by a decline in apoptosis. Coincident with these findings we observed a reduction in the expression of renal KIM-1 suggesting that renoprotection may be occurring. Stimulation or indemnity of the renal kinin system deserves to be evaluated as a complementary pharmacological measure to diminish cisplatin nephrotoxicity. - Highlights: • Mechanisms of cisplatin-induced-renal damage have not been completely clarified. • Cisplatin induces oxidative stress and apoptosis. • The renal kallikrein-kinin system is protective in experimental acute renal damage. • Kallikrein stimulation reduces oxidative stress and apoptosis induced by cisplatin. • Protection of the kallikrein-kinin system may reduce cisplatin toxicity

  19. The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.

    Science.gov (United States)

    Schwenk, Jochen M; Omenn, Gilbert S; Sun, Zhi; Campbell, David S; Baker, Mark S; Overall, Christopher M; Aebersold, Ruedi; Moritz, Robert L; Deutsch, Eric W

    2017-12-01

    Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization's Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ∼43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely mapping peptides of nine amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ∼2000 proteins. Finally, we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.

  20. Identification of low abundance cyclophilins in human plasma.

    Science.gov (United States)

    Schumann, Michael; Ihling, Christian H; Prell, Erik; Schierhorn, Angelika; Sinz, Andrea; Fischer, Gunter; Schiene-Fischer, Cordelia; Malešević, Miroslav

    2016-11-01

    Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by N ε acetylation on residues Lys44, Lys133, Lys155, as well as N α  acetylation at the N-terminal Val residue. N α  acetylation of Ser2 residue was also found for Cyp40. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Plasma dihydroxyphenylalanine (DOPA) is independent of sympathetic activity in humans

    DEFF Research Database (Denmark)

    Eldrup, E; Christensen, N J; Andreasen, J

    1989-01-01

    in diabetic patients with autonomic neuropathy compared to diabetics without neuropathy, whereas baseline plasma DOPA concentrations were similar in the three groups investigated: 6.55 (5.03-7.26, median [interquartile range], n = 8) nmol l-1 in diabetics with neuropathy, 7.41 (5.79-7.97, n = 8) nmol l-1...... in diabetics without neuropathy, and 6.85 (5.58-7.36, n = 8) nmol l-1 in controls. No relationship was obtained between baseline values of plasma NE and plasma DOPA. Plasma DOPA did not change in the upright position, whereas plasma NE increased significantly. Our results indicate that plasma DOPA...... is not related to sympathetic activity and may be of non-neuronal origin....

  2. A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

    Science.gov (United States)

    Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins. PMID:22355656

  3. Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys?

    Directory of Open Access Journals (Sweden)

    Guanghou Shui

    Full Text Available BACKGROUND: Non-human primates (NHP are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS. We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA; (ii sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3. Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005 and plasmalogen PC species (p<0.0005, while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005 and GM(3 (p<0.0005, but higher level of Cer (p<0.0005 in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls. CONCLUSIONS: For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys

  4. Plasma norepinephrine in humans: limitations in assessment of whole body norepinephrine kinetics and plasma clearance

    DEFF Research Database (Denmark)

    Christensen, N J; Henriksen, Jens Henrik Sahl

    1989-01-01

    ]IP and 131I-hippurate, whole body clearance from plasma of [3H]NE, as obtained from infusion rate divided by plasma concentration of tracer [1.74 +/- 0.64 (SD) 1/min] was significantly higher than the value obtained by total tracer infusion divided by total plasma area of tracer (1.27 +/- 0.51, P less than 0...... irreversible removal of NE, is smaller than previously estimated due to recycling through the plasma space. Attention has been drawn to limitations of [3H]NE kinetics....

  5. Hormones and endocrine disruptors in human seminal plasma.

    Science.gov (United States)

    Hampl, R; Kubatova, J; Heracek, J; Sobotka, V; Starka, L

    2013-07-01

    Seminal plasma represents a unique environment for maturation, nutrition, and protection of male germ cells from damaging agents. It contains an array of organic as well as inorganic chemicals, encompassing a number of biologically and immunologically active compounds, including hormones. Seminal plasma contains also various pollutants transferred from outer environment known as endocrine disruptors. They interfere with hormones at the receptor level, act as inhibitors of their biosynthesis, and affect hormone regulation.In this minireview, the main groups of hormones detected in seminal plasma are summarized. Seminal gonadal steroids were investigated mostly with aim to use them as biomarkers of impaired spermatogenesis (sperm count, motility, morphology). Concentrations of hormones in the seminal plasma often differ considerably from the blood plasma levels in dependence on their origin. In some instances (dihydrotestosterone, estradiol), their informative value is higher than determination in blood.Out of peptide hormones detected in seminal plasma, peptides of transforming growth factor beta family, especially antimullerian hormone, and oligopeptides related to thyrotropin releasing hormone have the high informative value, while assessment of seminal gonadotropins and prolactin does not bring advantage over determination in blood.Though there is a large body of information about the endocrine disruptors' impact on male reproduction, especially with their potential role in decline of male reproductive functions within the last decades, there are only scarce reports on their presence in seminal plasma. Herein, the main groups of endocrine disruptors found in seminal plasma are reviewed, and the use of their determination for investigation of fertility disorders is discussed.

  6. A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

    NARCIS (Netherlands)

    Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

  7. A phospholipidomic analysis of all defined human plasma lipoproteins

    NARCIS (Netherlands)

    Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

  8. Plasma catecholamine responses to physiologic stimuli in normal human pregnancy.

    Science.gov (United States)

    Barron, W M; Mujais, S K; Zinaman, M; Bravo, E L; Lindheimer, M D

    1986-01-01

    The dynamic response of the sympathoadrenal system was evaluated during and after pregnancy in 13 healthy women with a protocol that compared cardiovascular parameters and plasma catecholamine levels during the basal state, after postural maneuvers, and following isometric exercise. Plasma epinephrine and norepinephrine levels were similar during and after gestation when the women rested on their sides, but heart rate was greater in pregnancy. Ten minutes of supine recumbency produced minimal changes, but attenuation of the anticipated increases in heart rate and plasma norepinephrine levels during standing and isometric exercise were observed during pregnancy. In contrast, alterations in plasma epinephrine appeared unaffected by gestation. Plasma renin activity and aldosterone levels were, as expected, greater during pregnancy; however, increments in response to upright posture were similar in pregnant and postpartum women. To the extent that circulating catecholamines may be considered indices of sympathoadrenal function, these data suggest that normal pregnancy alters cardiovascular and sympathetic nervous system responses to physiologic stimuli.

  9. Effect of pancreatic kallikrein combined with magnesium sulfate therapy on disease severity of patients with severe preeclampsia

    Directory of Open Access Journals (Sweden)

    Yi Yang

    2016-10-01

    Full Text Available Objective: To study the clinical effect of pancreatic kallikrein combined with magnesium sulfate therapy on severe preeclampsia. Methods: A total of 47 patients with severe preeclampsia treated in our hospital from May 2013 to October 2015 were retrospectively analyzed, patients who received pancreatic kallikrein combined with magnesium sulfate therapy were selected as observation group, patients who received magnesium sulfate therapy were selected as control group, and then blood pressure level, renal function, uterine artery and umbilical artery blood flow state, and hypoxia indexes in serum and placenta were compared between two groups. Results: After treatment, systolic pressure and diastolic pressure levels as well as serum CysC level and 24 h urine protein level of observation group were significantly lower than those of control group, the uterine artery and umbilical artery S/ D and PI were significantly lower than those of control group, PIGF and NO levels in serum as well as Xiap and Survivin levels in placenta were significantly higher than those of control group, and sVEGFR1 level in serum as well as Caspase-3 and Caspase-7 levels in placenta was significantly lower than those of control group. Conclusions: Pancreatic kallikrein combined with magnesium sulfate therapy can alleviate the disease severity, lower blood pressure and vascular resistance, and improve renal function and the apoptosis caused by placental hypoxia in patients with severe preeclampsia.

  10. Imbalance of kallikrein-kinin system in chronic pancreatitis combined with obesity and results of its comprehensive correction

    Directory of Open Access Journals (Sweden)

    L.S. Babinets

    2017-11-01

    Full Text Available Background. Chronic pancreatitis is one of the most common diseases in general therapeutic practice. Significant role in the onset and progression of chronic pancreatitis is played by the activation of the kallikrein-kinin system (KKS, which is involved in the development of inflammation, blood coagulation and fibrinolysis, immune responses, the formation of pain, etc. Objective: to investigate the dynamics of indicators of the kallikrein-kinin system when using metadoxine in the comprehensive therapy of patients with chronic biliary pancreatitis associated with obesity. Materials and methods. 75 patients with chronic biliary pancreatitis combined with obesity were examined. They were divided into 2 groups. The first group (30 people received the standard treatment. The second group (45 people received the standard treatment and metadoxine. Results. Analyzing the results, it has been found a reliable positive dynamics of KKS indices in both groups after the treatment. However, patients in the second group showed a more significant positive dynamics of KKS parameters: the level of proteolytic activity has decreased by 9.0 %, the kallikrein — by 20.0 %, a1-proteinase inhibitor — by 7.2 %, and prekallikrein has increased by 6.4 %, a2-macroglobulin — by 10.8 %, kininase II — by 10.7 % as compared to group 1. Conclusions. The use of methadoxine in the comprehensive therapy of patients with chronic biliary pancreatitis associated with obesity contributed to a significant increase in the effectiveness of conventional treatment.

  11. Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

    Science.gov (United States)

    Tsai, W B; Grunkemeier, J M; Horbett, T A

    1999-02-01

    The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogen's ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule. Copyright 1999 John Wiley & Sons, Inc.

  12. Differential Responses of Plasma Adropin Concentrations To Dietary Glucose or Fructose Consumption In Humans.

    Science.gov (United States)

    Butler, Andrew A; St-Onge, Marie-Pierre; Siebert, Emily A; Medici, Valentina; Stanhope, Kimber L; Havel, Peter J

    2015-10-05

    Adropin is a peptide hormone encoded by the Energy Homeostasis Associated (ENHO) gene whose physiological role in humans remains incompletely defined. Here we investigated the impact of dietary interventions that affect systemic glucose and lipid metabolism on plasma adropin concentrations in humans. Consumption of glucose or fructose as 25% of daily energy requirements (E) differentially affected plasma adropin concentrations (P Glucose consumption reduced plasma adropin from 3.55 ± 0.26 to 3.28 ± 0.23 ng/ml (N = 42). Fructose consumption increased plasma adropin from 3.63 ± 0.29 to 3.93 ± 0.34 ng/ml (N = 45). Consumption of high fructose corn syrup (HFCS) as 25% E had no effect (3.43 ± 0.32 versus 3.39 ± 0.24 ng/ml, N = 26). Overall, the effect of glucose, HFCS and fructose on circulating adropin concentrations were similar to those observed on postprandial plasma triglyceride concentrations. Furthermore, increases in plasma adropin levels with fructose intake were most robust in individuals exhibiting hypertriglyceridemia. Individuals with low plasma adropin concentrations also exhibited rapid increases in plasma levels following consumption of breakfasts supplemented with lipids. These are the first results linking plasma adropin levels with dietary sugar intake in humans, with the impact of fructose consumption linked to systemic triglyceride metabolism. In addition, dietary fat intake may also increase circulating adropin concentrations.

  13. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

  14. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma

    DEFF Research Database (Denmark)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine

    2016-01-01

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic...

  15. Contrast media effect on interleukin-2 levels in human plasma in vitro

    International Nuclear Information System (INIS)

    Napolov, Yu.K.; Borsukova, N.M.; Shimanovskij, N.L.

    1992-01-01

    As shown in the study of bilignost, iodamide and triombrast action on interleukin-2 (IL-2) level in human plasma in vitro, these contrast media (2.5x10 -2 -2.5x10 -4 M) elevate IL-2 content in blood plasma of sensitive to contrast media subjects in dose-dependent manner

  16. Inactivation of Zika virus by solvent/detergent treatment of human plasma and other plasma-derived products and pasteurization of human serum albumin.

    Science.gov (United States)

    Kühnel, Denis; Müller, Sebastian; Pichotta, Alexander; Radomski, Kai Uwe; Volk, Andreas; Schmidt, Torben

    2017-03-01

    In 2016 the World Health Organization declared the mosquito-borne Zika virus (ZIKV) a "public health emergency of international concern." ZIKV is a blood-borne pathogen, which therefore causes concerns regarding the safety of human plasma-derived products due to potential contamination of the blood supply. This study investigated the effectiveness of viral inactivation steps used during the routine manufacturing of various plasma-derived products to reduce ZIKV infectivity. Human plasma and intermediates from the production of various plasma-derived products were spiked with ZIKV and subjected to virus inactivation using the identical techniques (either solvent/detergent [S/D] treatment or pasteurization) and conditions used for the actual production of the respective products. Samples were taken and the viral loads measured before and after inactivation. After S/D treatment of spiked intermediates of the plasma-derived products Octaplas(LG), Octagam, and Octanate, the viral loads were below the limit of detection in all cases. The mean log reduction factor (LRF) was at least 6.78 log for Octaplas(LG), at least 7.00 log for Octagam, and at least 6.18 log for Octanate after 60, 240, and 480 minutes of S/D treatment, respectively. For 25% human serum albumin (HSA), the mean LRF for ZIKV was at least 7.48 log after pasteurization at 60°C for 120 minutes. These results demonstrate that the commonly used virus inactivation processes utilized during the production of human plasma and plasma-derived products, namely, S/D treatment or pasteurization, are effective for inactivation of ZIKV. © 2016 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

  17. In vivo plasma concentration for lindane after 6 hour exposure in human skin

    Data.gov (United States)

    U.S. Environmental Protection Agency — Dataset is a time course description of lindane disappearance in blood plasma after dermal exposure in human volunteers. This dataset is associated with the...

  18. Trypanocidal activity of human plasma on Trypanosoma evansi in mice Atividade tripanocida do plasma humano sobre Trypanosoma evansi em camundongos

    Directory of Open Access Journals (Sweden)

    Aleksandro Schafer Da Silva

    2012-03-01

    Full Text Available This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL-1 apolipoprotein L1 (APOL1 was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI, the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C and 80% (group D of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.Este estudo teve como objetivo testar um protocolo alternativo com plasma humano para controlar a infecção por Trypanosoma evansi em camundongos. O plasma foi oriundo de um homem aparentemente saudável, com idade entre 27 anos e tipo de sangue A+. Foi detectada uma concentração de 100 mg.dL -1 de apolipoproteína L1 (APOL1 no plasma. Quarenta camundongos foram divididos em quatro grupos, contendo dez animais cada. Grupo A, composto de animais não infectados. Os roedores dos grupos B, C e D foram inoculados intraperitonealmente com um isolado de T. evansi. O Grupo B foi usado como um controle positivo. Três dias pós-infecção (DPI, os camundongos foram tratados com plasma humano. Uma dose única de 0,2 mL de plasma foi administrada nos roedores do grupo C. Os ratos do grupo D receberam cinco

  19. Bioanalytical HPTLC Method for Estimation of Zolpidem Tartrate from Human Plasma

    OpenAIRE

    Abhay R. Shirode; Bharti G. Jadhav; Vilasrao J. Kadam

    2016-01-01

    A simple and selective high performance thin layer chromatographic (HPTLC) method was developed and validated for the estimation of zolpidem tartrate from human plasma using eperisone hydrochloride as an internal standard (IS). Analyte and IS were extracted from human plasma by liquid liquid extraction (LLE) technique. The Camag HPTLC system, employed with software winCATS (ver.1.4.1.8) was used for the proposed bioanalytical work. Planar chromatographic development was carried out with the h...

  20. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

  1. Identification and Characterization of Plasma Cells in Normal Human Bone Marrow by High-Resolution Flow Cytometry

    NARCIS (Netherlands)

    Terstappen, Leonardus Wendelinus Mathias Marie; Johnsen, Steen; Segers-Nolten, Gezina M.J.; Loken, Michael R.

    1990-01-01

    The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma

  2. Turnover of adenosine in plasma of human and dog blood

    International Nuclear Information System (INIS)

    Moeser, G.H.S.; Schrader, J.; Deussen, A.

    1989-01-01

    To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a ''stopping solution.'' Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [ 3 H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole

  3. Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

    Science.gov (United States)

    Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

    2014-01-01

    Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

  4. Kallikrein-related peptidase 7 is a potential target for the treatment of pancreatic cancer

    Science.gov (United States)

    Zheng, Jun; Zhang, Ding; Liu, Wei; Zheng, Wei Hong; Li, Xiao Song; Yao, Ru Cheng; Wang, Fangyu; Liu, Sen; Tan, Xiao

    2018-01-01

    Pancreatic cancer is one of the deadliest cancers with very poor prognosis, and the five-year survival rate of the patients is less than 5% after diagnosis. Kallikrein-related peptidases (KLKs) belong to a serine protease family with 15 members that play important roles in cellular physiological behavior and diseases. The high expression level of KLK7 in pancreatic cancer tissues is considered to be a marker for the poor prognosis of this disease. In this work, we set out to investigate whether KLK7 could be a target for the treatment of pancreatic cancer. Short hairpin RNAs (shRNAs) were designed and constructed in lentivirus to knock down KLK7 in pancreatic cancer cell line PANC-1, and the real time cellular analysis (RTCA) was used to evaluate cell proliferation, migration and invasion abilities. Small molecules inhibiting KLK7 were discovered by computer-aided drug screening and used to inhibit PANC-1 cells. Our results confirmed that KLK7 is significantly up-regulated in pancreatic cancer tissue, and knocking down or inhibiting KLK7 efficiently inhibited the proliferation, migration and invasion of pancreatic cancer cells. This study suggested that KLK7 could be a potential chemotherapy target for treatment of pancreatic cancer, which would provide us a novel strategy for the treatment of this disease. PMID:29560118

  5. Enhanced Detection of Human Plasma Proteins on Nanostructured Silver Surfaces

    Directory of Open Access Journals (Sweden)

    Zuzana Orságová Králová

    2013-08-01

    enhancement factor of 3.6×102 was achieved for a band with a Raman shift of 2104cm‐1 for globulin deposited onto silver nanostructured film on unpolished stainless steel substrate. The detection limit was 400g/mL. Plasma or serum could present a preferable material for non‐ invasive cancer disease diagnosis using the SERS method.

  6. Plasma dihydroxyphenylalanine (DOPA) is independent of sympathetic activity in humans

    DEFF Research Database (Denmark)

    Eldrup, E; Christensen, N J; Andreasen, J

    1989-01-01

    in diabetic patients with autonomic neuropathy compared to diabetics without neuropathy, whereas baseline plasma DOPA concentrations were similar in the three groups investigated: 6.55 (5.03-7.26, median [interquartile range], n = 8) nmol l-1 in diabetics with neuropathy, 7.41 (5.79-7.97, n = 8) nmol l-1...

  7. Inactivation of human pathogenic dermatophytes by non-thermal plasma

    Czech Academy of Sciences Publication Activity Database

    Scholtz, V.; Soušková, H.; Hubka, Vít; Švarcová, M.; Julák, J.

    2015-01-01

    Roč. 119, DEC 2015 (2015), s. 53-58 ISSN 0167-7012 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Corona discharge * Cometary discharge * Decontamination of surfaces Subject RIV: BL - Plasma and Gas Discharge Physics Impact factor: 1.857, year: 2015

  8. Radioimmunoassay of Human Thyrotropin - Part 1. Plasma TSH levels in various thyroid functions

    International Nuclear Information System (INIS)

    Koh, Chang Soon; Lee, Hong Kyu; Ro, Heung Kyu; Lee, Mun Ho

    1972-01-01

    The radioimmunoassay of human thyrotropin was performed in various thyroid states, utilizing the anti-h-T.S.H. antibody and purified human thyrotropin supplied from National Institute of Arthritis and Metabolic Diseases, Bethesda, Ma., U.S.A., and human thyrotropin standard-A obtained from National Institute for Biologic Standards, Mill Hill, London, England. 131 I labelled h-TSH was prepared after the Chloramine-T method of Greenwood et al. This double antibody system had a assay sensitivity of about l. 0 μU/ml of plasma HTS-A and could detect the plasma h-TSH level in the euthyroid patients. Plasma h-TSH level of the normal 26 Korean was l.1±0. 83 μU/ml, and that of the 8 hypothyroidisms were 8.3 to 67.5 μU/ml. In hyperthyroidisms, no cases showed the plasma h-TSH levels over l. 0 μU/ ml. Between the hypothyroidism and euthyroidism, no overlap is noticed on plasma h-TSH levels. A case of transient hypothyroid state identified by determination of plasma h-TSH level is presented. These results revealed that the radioimmunoassay of h-TSH in plasma could be a sensitive method to diagnose the hypothyroidism, if not caused by a pituitary disease.

  9. Alternative pathways of thromboplastin-dependent activation of human factor X in plasma

    International Nuclear Information System (INIS)

    Marlar, R.A.; Griffin, J.H.

    1981-01-01

    To determine the interrelationships of the major coagulation pathways, the activation of 3H-labeled factor X in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin and calcium were added to plasma samples containing 3H-factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin, the rate of factor X activation in plasmas deficient in factor VIII or factor IX was 10% of the activation rate of normal plasma or of factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, reconstituted normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these plasma experiments, it is inferred that the dilute thromboplastin-dependent activation of factor X requires factors VII, IX, and VIII. An alternative extrinsic pathway that involves factors IX and VIII may be the physiologic extrinsic pathway and hence help to explain the consistent clinical observations of bleeding diatheses in patients deficient in factors IX or VIII

  10. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    Directory of Open Access Journals (Sweden)

    Paola Brun

    Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

  11. [3H]cholesteryl ester labeling and transfer among human and honhuman primate plasma lipoproteins

    International Nuclear Information System (INIS)

    Thomas, M.S.; Rudel, L.L.

    1983-01-01

    Aliquots of human and nonhuman primate plasma containing 5,5'-dithiobis (2-nitrobenzoic acid) were incubated at 37 0 C in tubes previously coated with trace amounts of tritium-labeled cholesteryl oleate ([ 3 H]CO). Initially, cholesteryl esters were transferred at a rapid rate into plasma after which the rate slowed. During 24 h of incubation, an average of 55% of the [ 3 H]CO transferred from the side of the tube into African green monkey plasma, 44% into human plasma and 21% into rat plasma. Greater than 98% of the radioactive ester transferred into plasma was found to be associated with plasma lipoproteins that were then rapidly separated using vertical rotor density gradient ultracentrifugation. In very low density lipoprotein (VLDL)-poor plasma after 30 min incubations, high density lipoproteins (HDL) contained most of the [ 3 H]CO while 5- to 24-h incubations resulted in increased labeling of low density proteins (LDL). In VLDL-rich plasma, it was found that in addition to the labeling of HDL, VLDL contained about 25% of the labeled cholesteryl esters after 30-min incubations and, as above, the proportion in LDL subsequently increased. Compositional analyses showed that intermediate-sized LDL (ILDL) were accumulating cholesteryl ester mass while transfer occurred. LDL labeled using this method were injected intravenously into monkeys and their removal from plasma was found to be similar to that found for LDL labeled in vivo. It was concluded that this method of plasma lipoprotein cholesteryl ester labeling, presumably a result of cholesteryl ester transfer protein activity, was efficient, resulted in lipoproteins labeled only in the cholesteryl ester moiety, and induced minimal modification of lipoprotein particles that did not alter their biological activity

  12. Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    Björn, Erik; Nygren, Yvonne; Nguyen, Tam T. T. N.

    2007-01-01

    A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines w...

  13. Plasma bile acids are not associated with energy metabolism in humans

    NARCIS (Netherlands)

    Brufau, Gemma; Bahr, Matthias J.; Staels, Bart; Claudel, Thierry; Ockenga, Johann; Boker, Klaus H. W.; Murphy, Elizabeth J.; Prado, Kris; Stellaard, Frans; Manns, Michael P.; Kuipers, Folkert; Tietge, Uwe J. F.

    2010-01-01

    Bile acids (BA) have recently been shown to increase energy expenditure in mice, but this concept has not been tested in humans. Therefore, we investigated the relationship between plasma BA levels and energy expenditure in humans. Type 2 diabetic (T2DM) patients (n = 12) and gender, age and

  14. HIP2: An online database of human plasma proteins from healthy individuals

    Directory of Open Access Journals (Sweden)

    Shen Changyu

    2008-04-01

    Full Text Available Abstract Background With the introduction of increasingly powerful mass spectrometry (MS techniques for clinical research, several recent large-scale MS proteomics studies have sought to characterize the entire human plasma proteome with a general objective for identifying thousands of proteins leaked from tissues in the circulating blood. Understanding the basic constituents, diversity, and variability of the human plasma proteome is essential to the development of sensitive molecular diagnosis and treatment monitoring solutions for future biomedical applications. Biomedical researchers today, however, do not have an integrated online resource in which they can search for plasma proteins collected from different mass spectrometry platforms, experimental protocols, and search software for healthy individuals. The lack of such a resource for comparisons has made it difficult to interpret proteomics profile changes in patients' plasma and to design protein biomarker discovery experiments. Description To aid future protein biomarker studies of disease and health from human plasma, we developed an online database, HIP2 (Healthy Human Individual's Integrated Plasma Proteome. The current version contains 12,787 protein entries linked to 86,831 peptide entries identified using different MS platforms. Conclusion This web-based database will be useful to biomedical researchers involved in biomarker discovery research. This database has been developed to be the comprehensive collection of healthy human plasma proteins, and has protein data captured in a relational database schema built to contain mappings of supporting peptide evidence from several high-quality and high-throughput mass-spectrometry (MS experimental data sets. Users can search for plasma protein/peptide annotations, peptide/protein alignments, and experimental/sample conditions with options for filter-based retrieval to achieve greater analytical power for discovery and validation.

  15. High Performace Liquid Chromtographic Determination of Nicardipine Hydrochloride in Human Plasma

    Directory of Open Access Journals (Sweden)

    Y. S. R. Krishnaiah

    2004-01-01

    Full Text Available A sensitive high-performance liquid chromatographic method was developed for the estimation of nicardipine hydrochloride in human plasma. Varying amount of nicardipine hydrochloride (2.5 to 150 ng/0.5 mL and fixed quantity (100 ng/0.5 mL of nifedipine (internal standard was added to blank human plasma, and a single step extraction was carried out with ethyl acetate. The mixture was centrifuged, ethyl acetate layer separated, dried and reconstituted with 100 μL of acetonitrile. Twenty microliters of this solution was injected into a reverse phase C-18 column using a mobile phase consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate (pH 4.0 in the ratio of 60:40 v/v and the eluents were monitored at 239 nm. The method was validated for its linearity, precision and accuracy. The calibration curve was linear in the range of 5-150 ng/0.5 mL of plasma and the lower detection limit was 2.5 ng/0.5 mL of plasma. The intra- and inter-day variation was found to be less than 2.5% indicating that the method is highly precise. The mean recovery of nicardipine hydrochloride from plasma samples was 89.6±2.60%. The proposed HPLC method was applied for the estimation of nicardipine hydrochloride in human plasma after oral administration of an immediate release nicardipine hydrochloride capsule (dose 30 mg to 6 adult male volunteers. There was no interference of either the drug metabolites or other plasma components with the proposed HPLC method for the estimation of nicardipine hydrochloride in human plasma. Due to its simplicity, sensitivity, high precision and accuracy, the proposed HPLC method may be used for biopharmaceutical and pharmacokinetic evaluation of nicardipine hydrochloride and its formulations in humans

  16. The predominant cholecystokinin in human plasma and intestine is cholecystokinin-33

    DEFF Research Database (Denmark)

    Rehfeld, J F; Sun, G; Christensen, T

    2001-01-01

    Cholecystokinin (CCK) occurs in multiple molecular forms; the major ones are CCK-58, -33, -22, and -8. Their relative abundance in human plasma and intestine, however, is debated. To settle the issue, extracts of intestinal biopsies and plasma from 10 human subjects have been examined by chromato......Cholecystokinin (CCK) occurs in multiple molecular forms; the major ones are CCK-58, -33, -22, and -8. Their relative abundance in human plasma and intestine, however, is debated. To settle the issue, extracts of intestinal biopsies and plasma from 10 human subjects have been examined...... by chromatography, enzyme cleavages, and measurements using a library of sequence-specific RIAs. Plasma samples were drawn in the fasting state and at intervals after a meal. The abundance of the larger forms varied with the 8 C-terminal assays in the library, as 2 assays overestimated and 3 underestimated...... the amounts present. One assay, however, measured carboxyamidated and O:-sulfated CCKs with equimolar potency before and after tryptic cleavage. This assay showed that the predominant plasma form is CCK-33, both in the fasting state ( approximately 51%) and postprandially ( approximately 57%), whereas CCK-22...

  17. Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques

    Directory of Open Access Journals (Sweden)

    Elisabetta Zinellu

    2012-01-01

    Full Text Available Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS. The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P<0.01 in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

  18. Modulation of Human Plasma Fibronectin Levels Following Exercise,

    Science.gov (United States)

    1988-01-01

    forms of this large molecular weight (440 kilodaltons) glycoprotein,(17. While the tissue type is cell-associated and important to cell adhesion and...increased under conditions of pathology, such as in obesity (6). cancer (3). proteinuria (4). diabetic retinopathy (5). and preeclampsia (27). in the absence...Res. 1977: 22:709-716. 27. Stubbs. T.M.. Lazarchick. J.. and Horger. E.O. Plasma fibronectin levels in preeclampsia : A possible biochemical marker

  19. Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

    Science.gov (United States)

    Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

    2015-01-01

    Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

  20. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    Science.gov (United States)

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-05

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR search engines.

  1. Determination of flurbiprofen in human plasma by gas chromatography with mass spectrometry and its pharmacokinetics.

    Science.gov (United States)

    Yilmaz, Bilal; Sahin, Huseyin; Akba, Vedat; Erdem, Ali Fuat

    2014-01-01

    This paper describes a GCIMS method for the determination of flurbiprofen in human plasma. Flurbiprofen and internal standard ibuprofen were extracted from plasma by using a liquid-liquid extraction method. Derivatization was carried out using N-Methyl-N-(trimethylsilyl)trifluoroacetamide. The calibration curve was linear between the concentration range of 0.10 and 5.0 microg/mL. Intraday and interday precision values for flurbiprofen in plasma were less than 5.49%, and accuracy (relative error) was better than 5.33%. The extraction recoveries of flurbiprofen from human plasma were between 93.6 and 98.6%. The LOD and LOQ of flurbiprofen were 0.03 and 0.10 microg/mL, respectively. This assay was applied to determine the pharmacokinetic parameters of flurbiprofen in healthy Turkish volunteers who had been given 100 mg of flurbiprofen.

  2. Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

    International Nuclear Information System (INIS)

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-01

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  3. A high confidence, manually validated human blood plasma protein reference set

    DEFF Research Database (Denmark)

    Schenk, Susann; Schoenhals, Gary J; de Souza, Gustavo

    2008-01-01

    BACKGROUND: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list......-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two...... consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved. RESULTS: Herein we established a high confidence set of 697 blood plasma proteins...

  4. Human leukocyte antigen-G in the male reproductive system and in seminal plasma

    DEFF Research Database (Denmark)

    Horup Larsen, Margit; Bzorek, Michael; Pass, Malene B.

    2011-01-01

    -eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system.Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining...... was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA–G in hyperplastic prostatic...... tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma....

  5. Plasma bile acids are not associated with energy metabolism in humans

    Directory of Open Access Journals (Sweden)

    Brufau Gemma

    2010-09-01

    Full Text Available Abstract Bile acids (BA have recently been shown to increase energy expenditure in mice, but this concept has not been tested in humans. Therefore, we investigated the relationship between plasma BA levels and energy expenditure in humans. Type 2 diabetic (T2DM patients (n = 12 and gender, age and BMI-matched healthy controls (n = 12 were studied before and after 8 weeks of treatment with a BA sequestrant. In addition, patients with liver cirrhosis (n = 46 were investigated, since these display elevated plasma BA together with increased energy expenditure. This group was compared to gender-, age- and BMI-matched healthy controls (n = 20. Fasting plasma levels of total BA and individual BA species as well as resting energy expenditure were determined. In response to treatment with the BA sequestrant, plasma deoxycholic acid (DCA levels decreased in controls (-60%, p

  6. Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice.

    Science.gov (United States)

    Shimizu, Makiko; Suemizu, Hiroshi; Mitsui, Marina; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2017-10-01

    1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

  7. A Sulfur Amino Acid–Free Meal Increases Plasma Lipids in Humans123

    OpenAIRE

    Park, Youngja; Le, Ngoc-Anh; Yu, Tianwei; Strobel, Fred; Gletsu-Miller, Nana; Accardi, Carolyn J.; Lee, Kichun S.; Wu, Shaoxiong; Ziegler, Thomas R.; Jones, Dean P.

    2011-01-01

    The content of sulfur amino acid (SAA) in a meal affects postprandial plasma cysteine concentrations and the redox potential of cysteine/cystine. Because such changes can affect enzyme, transporter, and receptor activities, meal content of SAA could have unrecognized effects on metabolism during the postprandial period. This pilot study used proton NMR (1H-NMR) spectroscopy of human plasma to test the hypothesis that dietary SAA content changes macronutrient metabolism. Healthy participants (...

  8. Simultaneous extraction of. beta. -endorphin and leu- and met-enkephalins from human and rat plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bhathena, S.J.; Smith, P.M.; Kennedy, B.W. (Dept. of Agriculture, Beltsville, MD (USA)); Voyles, N.R.; Recant, L. (Diabetes Research Laboratory, Washington, DC (USA))

    1989-01-01

    A simple, rapid and reliable procedure is described to simultaneously concentrated and purify {beta}-endorphin, leu-and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C{sub 18} Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.

  9. PIXE elemental analysis of erythrocyte and blood plasma samples from human pregnancies

    International Nuclear Information System (INIS)

    Borbely-Kiss, I.; Koltay, E.; Laszlo, S.; Szabo, Gy.

    1984-01-01

    Elemental concentrations of P, S, Cl, K, Ca, Fe, Ni, Cu, Zn, Br, Rb have been determined in erythrocyte and blood plasma samples from normal and diabetic human pregnancies. Average values, the dependence of the concentrations on the time during gestation period, the correlation coefficients for pairs of elements as well as for the same elements in plasma and erythrocyte samples are given. A marked difference appeared in a number of cases between normal and diabetic pregnancies. (author)

  10. PIXE elemental analysis of erythrocyte and blood plasma samples from human pregnancies

    Energy Technology Data Exchange (ETDEWEB)

    Borbely-Kiss, I; Koltay, E; Laszlo, S; Szabo, Gy [Magyar Tudomanyos Akademia, Debrecen. Atommag Kutato Intezete; Goedeny, S [Orvostudomanyi Egyetem, Szeged (Hungary). Szueleszeti es Noegyogyaszati Klinika; Seif El-Nasr, S [Teachers' Coll. for Women, Samia (Kuwait)

    1984-07-01

    Elemental concentrations of P, S, Cl, K, Ca, Fe, Ni, Cu, Zn, Br, Rb have been determined in erythrocyte and blood plasma samples from normal and diabetic human pregnancies. Average values, the dependence of the concentrations on the time during gestation period, the correlation coefficients for pairs of elements as well as for the same elements in plasma and erythrocyte samples are given. A marked difference appeared in a number of cases between normal and diabetic pregnancies. 11 refs.

  11. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

    individuals and within an individual changes will also happen over time (e.g. after meal intake). Thus, the aim of the present study was to examine the inter-individual variability of plasma protein levels in humans after meal intake. Five subjects consumed three single meals in a randomised order separated...... by one-week interval. Blood samples were drawn before the meal intake and five times during 24 hours for proteome analysis. Plasma was fractionated by use of IgY-12 spin column depleting the 12 highly abundant proteins and further processed for two-dimensional gel electrophoresis. The plasma proteome...

  12. Plasma Dihydroceramides Are Diabetes Susceptibility Biomarker Candidates in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Leonore Wigger

    2017-02-01

    Full Text Available Summary: Plasma metabolite concentrations reflect the activity of tissue metabolic pathways and their quantitative determination may be informative about pathogenic conditions. We searched for plasma lipid species whose concentrations correlate with various parameters of glucose homeostasis and susceptibility to type 2 diabetes (T2D. Shotgun lipidomic analysis of the plasma of mice from different genetic backgrounds, which develop a pre-diabetic state at different rates when metabolically stressed, led to the identification of a group of sphingolipids correlated with glucose tolerance and insulin secretion. Quantitative analysis of these and closely related lipids in the plasma of individuals from two population-based prospective cohorts revealed that specific long-chain fatty-acid-containing dihydroceramides were significantly elevated in the plasma of individuals who will progress to diabetes up to 9 years before disease onset. These lipids may serve as early biomarkers of, and help identify, metabolic deregulation in the pathogenesis of T2D. : Wigger et al. find that several sphingolipids in mouse plasma correlate with glucose tolerance and insulin secretion. Quantitative analysis of these and closely related lipids in human plasma from two cohorts reveal that dihydroceramides are significantly elevated in individuals progressing to diabetes, up to 9 years before disease onset. Keywords: diabetes, T2D, ceramides, dihydroceramides, biomarkers, lipidomics, prognostic, mouse, human, high-fat diet, metabolic challenge, glucose intolerance, insulin sensitivity, prospective cohort

  13. Expression and localization of tissue factor pathway inhibitor-2 in normal and atherosclerotic human vessels

    NARCIS (Netherlands)

    Crawley, James T. B.; Goulding, David A.; Ferreira, Valérie; Severs, Nicholas J.; Lupu, Florea

    2002-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type, serine protease inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor:activated factor VII complex. In this study, we investigated TFPI-2

  14. Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

    International Nuclear Information System (INIS)

    Sakai, T.; Kisiel, W.

    1990-01-01

    Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which 125 I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity

  15. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation

    NARCIS (Netherlands)

    Wachtfogel, Y.T.; Hack, C.E.; Nuijens, J.H; Kettner, C.; Reilly, T.M.; Knabb, R.M.; Bischoff, Rainer; Tschesche, H.; Wenzel, H.; Kucich, U.

    1995-01-01

    Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during

  16. Endostatin concentration in plasma of healthy human volunteers

    International Nuclear Information System (INIS)

    Shah, I.; Malik, M.O.; Khan, M.J.; Fatima, S.; Habib, S.H.

    2017-01-01

    Angiogenesis is involved in many cardiovascular and cancerous diseases, including atherosclerosis and is controlled by a fine balance between angiogenic and angiostatic mediators. Endostatin is one of the main angiostatic mediators, and inhibits angiogenesis and prevents progression of atherosclerosis. The available literature shows a broad range of concentrations in relatively small samples of healthy controls and is calculated by using different techniques. This study was aimed to determine the basal endostatin concentration in plasma of healthy volunteers, to fully understand its physiological role. Methods: Fifty healthy adult volunteers were recruited to the study. Participants were advised not to participate in any physical activity on the day before the blood sampling. The volunteers' physical activity, height, weight, heart rate and blood pressure were recorded. The samples were analysed for plasma endostatin concentration, using ELISA. The participants were divided by gender and ethnic groups to calculate any difference. Results: Endostatin and other variables were normally distributed. Most of the participants had a moderate level of physical activity with no gender related difference (p=0.370). The mean value for plasma endostatin in all samples was 105+-12 ng/ml with range of 81-132 ng/ml. For males, it was 107+-13 ng/ml, while for females; 102+-12 ng/ml. There were no significant gender or ethnicity related differences in endostatin concentration. Moreover, endostatin was not significantly related with any anthropometric and physical variable. Conclusion: This study gives endostatin levels in normal healthy people and show no gender and ethnicity related differences in endostatin levels. Endostatin was not related with any anthropometric and physical variable. (author)

  17. Skin barrier homeostasis in atopic dermatitis: feedback regulation of kallikrein activity.

    Directory of Open Access Journals (Sweden)

    Reiko J Tanaka

    Full Text Available Atopic dermatitis (AD is a widely spread cutaneous chronic disease characterised by sensitive reactions (eg. eczema to normally innocuous elements. Although relatively little is understood about its underlying mechanisms due to its complexity, skin barrier dysfunction has been recognised as a key factor in the development of AD. Skin barrier homeostasis requires tight control of the activity of proteases, called kallikreins (KLKs, whose activity is regulated by a complex network of protein interactions that remains poorly understood despite its pathological importance. Characteristic symptoms of AD include the outbreak of inflammation triggered by external (eg. mechanical and chemical stimulus and the persistence and aggravation of inflammation even if the initial stimulus disappears. These characteristic symptoms, together with some experimental data, suggest the presence of positive feedback regulation for KLK activity by inflammatory signals. We developed simple mathematical models for the KLK activation system to study the effects of feedback loops and carried out bifurcation analysis to investigate the model behaviours corresponding to inflammation caused by external stimulus. The model analysis confirmed that the hypothesised core model mechanisms capture the essence of inflammation outbreak by a defective skin barrier. Our models predicted the outbreaks of inflammation at weaker stimulus and its longer persistence in AD patients compared to healthy control. We also proposed a novel quantitative indicator for inflammation level by applying principal component analysis to microarray data. The model analysis reproduced qualitative AD characteristics revealed by this indicator. Our results strongly implicate the presence and importance of feedback mechanisms in KLK activity regulation. We further proposed future experiments that may provide informative data to enhance the system-level understanding on the regulatory mechanisms of skin barrier

  18. Análise de ácidos graxos em plasma humano Analysis of fatty acids in human plasma

    Directory of Open Access Journals (Sweden)

    Jeane Eliete Laguila Visentainer

    2010-01-01

    Full Text Available Nesse fascículo da revista, o estudo de Morais et al. (2010 avaliou quatro metodologias clássicas de extração de lipídeos (métodos de Folch, Bligh-Dyer, Rose-Gottlieb e Gerber e uma técnica alternativa, com o objetivo de avaliar a eficiência da extração e a composição em ácidos graxos de plasma humano. O método alternativo proposto pelos autores usou o forno de micro-ondas como ferramenta e foi considerado muito rápido na extração lipídica e adequado na identificação de ácidos graxos, mas não em sua quantificação. O método de extração mais indicado para quantificação de ácidos graxos em plasma humano foi o método de Folch.In this issue of the journal, the study by Morais et al. (2010 evaluated four classical methodologies of lipid extraction (methods of Folch, Bligh-Dyer, Rose-Gottlieb and Gerber, and an alternative technique, in order to evaluate the efficiency of extraction and fatty acid composition of human plasma. The alternative method proposed by the authors used the microwave oven as a tool, and was considered very fast in lipid extraction and identification of fatty acids, but not in their quantification. The most suitable extraction method for quantification of fatty acids in human plasma was the method of Folch.

  19. Enhanced Chromatographic Determination of Nicotine in Human Plasma: Applied to Human Volunteers.

    Science.gov (United States)

    Ayoub, Bassam M; Mohamady, Samy; Hendy, Moataz S; Elmazar, Mohamed M

    2015-12-01

    Development of enhanced UPLC-UV method for determination of nicotine in human plasma was achieved on a Symmetry(®) C18 column (100 mm × 2.1 mm, 2.2 μm) applying isocratic elution based on Methanol: Acetonitrile: Phosphate Buffer (pH: 2.7) with the ratio (20:30:50, v/v/v) as a mobile phase. The ultraviolet detector was operated at 260 nm. The mobile phase was pumped through the column at a flow rate of 0.2 mL min(-1). The column temperature was adjusted to 50ºC and the injection volume was 2 μL. Quinine was selected as an internal standard (IS) due to its structure similarity to nicotine having basic pyridine ring to optimize the liquid liquid extraction procedure using diethyl ether coupled with vacuum evaporation at 40°C. Validation parameters for nicotine were found to be acceptable over the concentration range of 2.5-50 ng ml(-1). The application of the proposed method on four healthy human volunteers was approved by the ethical committee. The study was carried out under fasting conditions and the concerned subjects were informed about the objectives and possible risks involved in the study. The proposed method proved to be simple and fast which is a major advantage to analyze large number of samples per day using the accelerated vacuum evaporation technique. The method showed satisfactory data for all the parameters tested within the limits for bioanalytical assays. The lower limit of quantification (LLOQ) permits the application of the method for further pharmacological and clinical studies.

  20. DETECTION AND ISOLATION OF CD59 FROM HUMAN SEMINAL PLASMA

    Directory of Open Access Journals (Sweden)

    A REZAIE

    2002-06-01

    Full Text Available Introduction. CD59 is one of the complement regulatory proteins (CRPs which exert inhibitory function by blocking the formation of C5b-9 complex or Membrane Attacks complex (MAC. Regarding the therapeutic role of CD59 in treatment of pathological effects in uncontrolled activation of complements system and its efficiency to overcome the hyper-acute rejection, CD59 was suggested for maintenance of transplanted organ. In this study We determined and isolated CD59 from seminal plasma. Methods. Six normospermic sample according to WHO standards were chosen. Plasma of samples was separated and to remove the postasomes, the seminal plama was ultra centrifuged. CD59 was detected by Dot-Blot using CD59 mAb (MEM43. The molecular weight and purity of protein was detected by SOS-PAGE method follwed by Westerm Blot. Results. Protein was present in the 6.5 ml and 15ml of gel fitration fractions. Molecular weight based on marker size in these two fractions was 65 and 21KD respectively. Discussion. CD59 had previously beem purified by lysis of erythrocyte cell membrane. Because of use of detergent and preservative agents, this method decreased physiologic effects of the protein. In this study the isolation was performed from prostasome granules" without using of any detergent and preservative agents.

  1. Radioimmunoassay of human. beta. -lipotropin in unextracted plasma. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Wiedemann, E. (Univ. of California, Berkeley); Saito, T.; Linfoot, J.A.; Li, C.H.

    1977-11-01

    A sensitive and specific radioimmunoassay for human ..beta..-lipotropin (..beta../sub h/-LPH) in unextracted plasma was developed using pure ..beta../sub h/-LPH as tracer and standard and an antiserum not cross-reacting with human ..beta..-MSH and hACTH. In healthy volunteers plasma ..beta../sub h/-LPH ranged from <20 to 150 pg/ml at 8:00 a.m. and rose after metyrapone administration. ..beta../sub h/-LPH was very low in panhypopituitarism, normal in most patients with untreated Cushing's disease, elevated in acromegaly and extremely high in Nelson's syndrome.

  2. On the variability of the salting-out curves of proteins of normal human plasma and serum

    NARCIS (Netherlands)

    Steyn-Parvé, Elizabeth P.; Hout, A.J. van den

    1953-01-01

    Salting-out curves of proteins of normal human plasma reflect the influence of a number of other factors besides the protein composition: the manner of obtaining the blood, the nature of the anti-coagulant used, the non-protein components of the plasma. Diagrams of serum and plasma obtained from

  3. Autoradiographic research on cell proliferation of prenatal rat lung cells and their influence using the mitogen Kallikrein

    International Nuclear Information System (INIS)

    Bock-Lamberlin, P.R.

    1980-01-01

    In this work autoradiographic experiments were carried out on the kinetics of proliferation of four cell populations of the prenatal rat lung with the help of the determination of the 3 H-thymidine marker indices, with the following results: 1. The four studied cell populations exhibited variable proliferation rates on the twentieth or twenty-first day of development. 2. The strongest affect of the exogenously applied mitogen Kallikrein was demonstrated on the vessel wall cells, the next strongest on the bronchial epithelial cells, then the cartilage cells and finally the alveolar wall cells. 3. The mitogenic effect is dependent on dose. Higher doses significantly increased the 3 H-thymidine marker indices of the four cell populations tested in this work. 4. When the exposure time of the Kallikrein was extended by one hour this lead partially to stronger mitogenic effects than by the shorter exposure times at the same and higher dose levels of mitogen. 5. The 3 H-thymidine marker indices are dependent on the exposure time. 6. With increasing litter size, the 3 H indices as a rule decrease. (orig./MG) [de

  4. Digestion of atopic allergens with trypsin α-chymotrypsin and pancreatic kallikrein, and influence of the allergens upon the proteolytic and esterolytic activity of these enzymes

    NARCIS (Netherlands)

    Berrens, L.

    1968-01-01

    The action of bovine trypsin, α-chymotrypsin and pancreatic kallikrein upon a number of atopic allergens has been studied by pH-stat measurements during short-term incubation. Most atopic allergens proved chemically resistant towards these enzymes. Graphs of enzyme susceptibility vs. the ratio of

  5. Analysis of two human parvovirus PARV4 genotypes identified in human plasma for fractionation

    NARCIS (Netherlands)

    Fryer, Jacqueline F.; Delwart, Eric; Bernardin, Flavien; Tuke, Philip W.; Lukashov, Vladimir V.; Baylis, Sally A.

    2007-01-01

    The presence of the novel parvovirus PARV4 and a related variant, PARV5, was recently demonstrated in pooled plasma used in the manufacture of blood and plasma-derived medicinal products. DNA sequence analysis of nearly full-length genomes of four PARV4 and two PARV5 strains from manufacturing

  6. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans.

    Science.gov (United States)

    Douglas, Lois M; Konopka, James B

    2016-03-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

  7. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

    Science.gov (United States)

    Douglas, Lois M.; Konopka, James. B.

    2017-01-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

  8. Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.

    1987-01-01

    A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL [mean, 15 +/- 7 (+/- SD) pg/mL; n = 58]. Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term [1462 +/- 752 (+/- SD) pg/mL; n = 55], and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA

  9. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    International Nuclear Information System (INIS)

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

  10. Differential proteomics of human seminal plasma: A potential target for searching male infertility marker proteins.

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2012-04-01

    The clinical fertility tests, available in the market, fail to define the exact cause of male infertility in almost half of the cases and point toward a crucial need of developing better ways of infertility investigations. The protein biomarkers may help us toward better understanding of unknown cases of male infertility that, in turn, can guide us to find better therapeutic solutions. Many clinical attempts have been made to identify biomarkers of male infertility in sperm proteome but only few studies have targeted seminal plasma. Human seminal plasma is a rich source of proteins that are essentially required for development of sperm and successful fertilization. This viewpoint article highlights the importance of human seminal plasma proteome in reproductive physiology and suggests that differential proteomics integrated with functional analysis may help us in searching potential biomarkers of male infertility. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Determination of albendazole sulfoxide in human plasma by using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Saraner, Nihal; Özkan, Güler Yağmur; Güney, Berrak; Alkan, Erkin; Burul-Bozkurt, Nihan; Sağlam, Onursal; Fikirdeşici, Ezgi; Yıldırım, Mevlüt

    2016-06-01

    A rapid, simple and sensitive method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination of albendazole sulfoxide (ABZOX) in human plasma. The plasma samples were extracted by protein precipitation using albendazole sulfoxide-d3 as internal standard (IS). The chromatographic separation was performed on Waters Xbridge C18Column (100×4.6mm, 3.5μm) with a mobile phase consisting of ammonia solution, water and methanol at a flow rate of 0.70mL/min. ABZOX was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 3-1500ng/mL for ABZOX. This method was successfully applied to the bioequivalence study in human plasma samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Effects of chemosignals from sad tears and postprandial plasma on appetite and food intake in humans.

    Science.gov (United States)

    Oh, Tae Jung; Kim, Min Young; Park, Kyong Soo; Cho, Young Min

    2012-01-01

    Chemosignals from human body fluids may modulate biological functions in humans. The objective of this study was to examine whether chemosignals from human sad tears and postprandial plasma modulate appetite. We obtained fasting and postprandial plasma from male participants and sad tears and saline, which was trickled below the eyelids, from female volunteers. These samples were then randomly distributed to male participants to sniff with a band-aid containing 100 µl of each fluid on four consecutive days in a double-blind fashion. We checked appetite by a visual analogue scale (VAS) and food intake by measuring the consumption of a test meal. In addition, the serum levels of total testosterone and LH were measured. Twenty men (mean age 26.3±4.6 years) were enrolled in this study. They could not discriminate between the smell of fasting and postprandial plasma and the smell of sad tears and trickled saline. Appetite and the amount of food intake were not different between the groups. Although the VAS ratings of appetite correlated with the food intake upon sniffing fasting plasma, postprandial plasma, and trickled saline, there was no such correlation upon sniffing sad tears. In addition, the decrease in serum testosterone levels from the baseline was greater with sad tears than with the trickled saline (-28.6±3.3% vs. -14.0±5.2%; P = 0.019). These data suggest that chemosignals from human sad tears and postprandial plasma do not appear to reduce appetite and food intake. However, further studies are necessary to examine whether sad tears may alter the appetite-eating behavior relation.

  13. Time-related contact angle measurements with human plasma on biomaterial surfaces

    NARCIS (Netherlands)

    Rakhorst, G; Van der Mei, HC; van Oeveren, W; Spijker, HT; Busscher, HJ

    Axisymmetric drop shape analysis by profile (ADSA-P) was used to assess in time contact angle changes of human plasma drops placed on four different biomaterials. Results were related with conventional blood compatibility measurements: albumin adsorption, fibrinogen adsorption and platelet adhesion.

  14. Plasma proteome and metabolome characterization of an experimental human thyrotoxicosis model

    DEFF Research Database (Denmark)

    Pietzner, Maik; Engelmann, Beatrice; Kacprowski, Tim

    2017-01-01

    BACKGROUND: Determinations of thyrotropin (TSH) and free thyroxine (FT4) represent the gold standard in evaluation of thyroid function. To screen for novel peripheral biomarkers of thyroid function and to characterize FT4-associated physiological signatures in human plasma we used an untargeted O...

  15. Cold plasma inactivation of human pathogens on foods and regulatory status update

    Science.gov (United States)

    Contamination of foods with human pathogens such as Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, norovirus, and other pathogens is an ongoing challenge for growers and processors. In recent years, cold plasma has emerged as a promising antimicrobial treatment for fresh and fresh-cut...

  16. Proteomic biomarker discovery in 1000 human plasma samples with mass spectrometry

    DEFF Research Database (Denmark)

    Cominetti, Ornella; Núñez Galindo, Antonio; Corthésy, John

    2016-01-01

    automated proteomic biomarker discovery workflow. Herein, we have applied this approach to analyze 1000 plasma samples from the multicentered human dietary intervention study "DiOGenes". Study design, sample randomization, tracking, and logistics were the foundations of our large-scale study. We checked...

  17. Simple Quantification of Pentosidine in Human Urine and Plasma by High-Performance Liquid Chromatography

    Directory of Open Access Journals (Sweden)

    Ji Sang Lee

    2017-01-01

    Full Text Available Pentosidine is an advanced glycation end-product (AGE and fluorescent cross-link compound. A simple high-performance liquid chromatographic (HPLC method was developed for the detection and quantification of pentosidine in human urine and plasma. The mobile phase used a gradient system to improve separation of pentosidine from endogenous peaks, and chromatograms were monitored by fluorescent detector set at excitation and emission wavelengths of 328 and 378 nm, respectively. The retention time for pentosidine was 24.3 min and the lower limits of quantification (LLOQ in human urine and plasma were 1 nM. The intraday assay precisions (coefficients of variation were generally low and found to be in the range of 5.19–7.49% and 4.96–8.78% for human urine and plasma, respectively. The corresponding values of the interday assay precisions were 9.45% and 4.27%. Accuracies (relative errors ranged from 87.9% to 115%. Pentosidine was stable in a range of pH solutions, human urine, and plasma. In summary, this HPLC method can be applied in future preclinical and clinical evaluation of pentosidine in the diabetic patients.

  18. Lack of association between human plasma oxytocin and interpersonal trust in a Prisoner's Dilemma paradigm.

    Directory of Open Access Journals (Sweden)

    James C Christensen

    Full Text Available Expanding interest in oxytocin, particularly the role of endogenous oxytocin in human social behavior, has created a pressing need for replication of results and verification of assay methods. In this study, we sought to replicate and extend previous results correlating plasma oxytocin with trust and trustworthy behavior. As a necessary first step, the two most commonly used commercial assays were compared in human plasma via the addition of a known quantity of exogenous oxytocin, with and without sample extraction. Plasma sample extraction was found to be critical in obtaining repeatable concentrations of oxytocin. In the subsequent trust experiment, twelve samples in duplicate, from each of 82 participants, were collected over approximately six hours during the performance of a Prisoner's Dilemma task paradigm that stressed human interpersonal trust. We found no significant relationship between plasma oxytocin concentrations and trusting or trustworthy behavior. In light of these findings, previous published work that used oxytocin immunoassays without sample extraction should be reexamined and future research exploring links between endogenous human oxytocin and trust or social behavior should proceed with careful consideration of methods and appropriate biofluids for analysis.

  19. Amperometric Sensor for Heparin: Sensing Mechanism and Application in Human Blood Plasma Analysis

    Czech Academy of Sciences Publication Activity Database

    Langmaier, Jan; Olšák, J.; Samcová, E.; Samec, Zdeněk; Trojánek, Antonín

    2006-01-01

    Roč. 18, 13-14 (2006), s. 1329-1338 ISSN 1040-0397 R&D Projects: GA ČR GA203/04/0424 Institutional research plan: CEZ:AV0Z40400503 Keywords : heparin * amperometry * PVC membrane electrode * sensing mechanism * human blood plasma Subject RIV: CG - Electrochemistry Impact factor: 2.444, year: 2006

  20. LC determination of propylene glycol in human plasma after pre-column derivatization with benzoyl chloride

    NARCIS (Netherlands)

    Sinjewel, A.; Swart, E.L.; Lingeman, H.; Wilhelm, A.J.

    2007-01-01

    A simple high-performance liquid chromatographic method, using photodiode array detection was developed for the determination of propylene glycol in human plasma and in the fluid retreived after continuous veno-venous hemofiltration. The method entailed alkaline derivatization with benzoyl chloride

  1. Determination of telmisartan in human blood plasma: Part I: Immunoassay development

    NARCIS (Netherlands)

    Hempen, C.M.; Gläsle-Schwarz, Liane; Kunz, Ulrich; Karst, U.

    2006-01-01

    Telmisartan is an angiotensin II receptor antagonist and a known drug against high blood pressure. In this report, the development of a new and rapid analytical technique, an enzyme-linked immunosorbent assay (ELISA) for the determination of telmisartan in human blood plasma is described. The

  2. Determination of platinum drug release and liposome stability in human plasma by CE-ICP-MS

    DEFF Research Database (Denmark)

    Nguyen, Trinh Thi Nhu Tam; Ostergaard, Jesper; Stürup, Stefan

    2013-01-01

    An in vitro method for simultaneous assessment of platinum release and liposome stability of liposomal formulations in human plasma is demonstrated. The development and assessment of the method was performed on a PEGylated liposomal formulation containing cisplatin. Complete separation of free ci...

  3. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    International Nuclear Information System (INIS)

    Cao Hongbin; Banh, Alice; Kwok, Shirley; Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha; Bala, Rajeev; Pinsky, Benjamin A.; Colevas, Dimitrios; Pourmand, Nader; Koong, Albert C.; Kong, Christina S.; Le, Quynh-Thu

    2012-01-01

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16 INK4a staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using 18 F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  4. Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer

    International Nuclear Information System (INIS)

    Ramani, Vishnu C; Hennings, Leah; Haun, Randy S

    2008-01-01

    In a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells. The expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2. The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells. A reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion

  5. Kallikrein gene-modified EPCs induce angiogenesis in rats with ischemic hindlimb and correlate with integrin αvβ3 expression.

    Directory of Open Access Journals (Sweden)

    Shen Shen Fu

    Full Text Available BACKGROUND: Human tissue kallikrein (hTK plays an essential role in the physiological and pathological mechanisms of blood vessels. This study aimed to determine whether angiogenesis induced by endothelial progenitor cells (EPCs transduced with the adenovirus-mediated hTK gene could improve blood flow in rat hindlimb ischemia in vivo and to establish a promising mechanism in vitro. METHODS: EPCs transduced with adenovirus encoding hTK-162 (i.e., Ad/hTK-transduced EPCs or Ad/GFP-transduced EPCs were administered to Wister rats with hindlimb ischemia through therapeutic neovascularization. Muscular capillary density (MCD, blood flow (BF, and the number of myofibers were measured at days 7, 14, and 21 after treatment. Expressions of integrin αvβ3 and endothelial nitric oxide synthase (eNOS were detected on the surface of EPCs. RESULTS: MCD, BF, and the number of myofibers in rats with Ad/hTK-transduced EPCs remarkably increased at day 21 after treatment compared with rats with Ad/GFP-transduced EPCs or the control group (P<0.01. Expressions of integrin αvβ3 and eNOS protein on the surface of EPCs also increased in rats with Ad/hTK-transduced EPCs. The levels of integrin αvβ3 expression were reduced by PI3K and eNOS blockade, and the inhibitor of integrin αvβ3 abrogated the migration and adhesion of hTK-transduced EPCs (P<0.05. CONCLUSION: hTK gene delivery in vivo improves the natural angiogenic response to ischemia. The ability of hTK gene-transduced EPCs can be enhanced in vitro, in which integrin αvβ3 plays a role in the process.

  6. Very low levels of 6-keto-prostaglandin F 1/sub α/ in human plasma

    International Nuclear Information System (INIS)

    Siess, W.; Dray, F.

    1982-01-01

    Two stable derivatives of PGI 2 , its nonenzymatic hydrolysis product (6-keto-PGF 1 /sub α/) and an enzymatic metabolite (6, 15-diketo-PGF 1 /sub α/) were determined in human plasma and urine. These compounds were measured by RIA after separation on rp-HPLC. Previous purification of the samples on rp=HPLC markedly enhanced the specificity of the RIA determinations of those compounds in plasma and urine. The PGI 2 derivative 6-keto-PGF 1 /sub α/ was detected in both plasma (4.7 +/- 3.2 pg/ml, mean +/- S.D., n = 34) and urine (166 +/- 61 pg/ml, n = 9). No gender differences of the plasma or urinary levels of 6-keto-PGF 1 /sub α/ were found. The PGI 2 metabolite 6, 15-diketo-PGF 1 /sub α/ was not measurable in plasma or urine ( 2 in man. When [ 3 H]PGI 2 was added to citrated blood immediately after venipuncture, it was recovered entirely as [ 3 H]6-keto-PGF 1 /sub α/ after rp-HPLC. Therefore any circulating PGI 2 would be measured as 6-keto-PGF 1 /sub α/ by our method. The results obtained suggest that PGI 2 could be present in human venous blood under physiological conditions, but only in very low concentrations

  7. Egg beater as centrifuge: isolating human blood plasma from whole blood in resource-poor settings.

    Science.gov (United States)

    Wong, Amy P; Gupta, Malancha; Shevkoplyas, Sergey S; Whitesides, George M

    2008-12-01

    This paper demonstrates that a hand-powered egg beater can be modified to serve as a centrifuge for separating plasma from human whole blood. Immunoassays used to diagnose infectious diseases often require plasma from whole blood, and obtaining plasma typically requires electrically-powered centrifuges, which are not widely available in resource-limited settings. Human whole blood was loaded into polyethylene (PE) tubing, and the tubing was attached to the paddle of an egg beater. Spinning the paddle pelleted the blood cells to the distal end of the PE tubing; the plasma remained as the supernatant. A cholesterol assay (run on patterned paper) demonstrated the suitability of this plasma for use in diagnostic assays. The physics of the system was also analyzed as a guide for the selection of other rotating systems for use in centrifugation. Egg beaters, polyethylene tubing, and paper are readily available devices and supplies that can facilitate the use of point-of-care diagnostics at sites far from centralized laboratory facilities.

  8. Inhibition of phospholipase A2 from human plasma by sodium bisulfite

    International Nuclear Information System (INIS)

    Wiggins, C.W.; Franson, R.C.

    1987-01-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca ++ -dependent phospholipase A 2 (PLA 2 ), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca ++ -dependent PLA 2 from human plasma. Using [1- 14 C]oleate-labelled autoclaved E. coli as substrate, PLA 2 activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100μM bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA 2 inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA 2 was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA 2 activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA 2 , in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA 2 in vivo

  9. Human Plasma and Human Platelet-rich Plasma as a Substitute for Fetal Calf Serum during Long-term Cultivation of Mesenchymal Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Tereza Suchánková Kleplová

    2014-01-01

    Full Text Available Aims: Our aims were to isolate and cultivate mesenchymal dental pulp stem cells (DPSC in various media enriched with human blood components, and subsequently to investigate their basic biological properties. Methods: DPSC were cultivated in five different media based on α MEM containing different concentrations of human plasma (HP, platelet-rich plasma (PRP, or fetal calf serum (FCS. The DPSC biological properties were examined periodically. Results: We cultivated DPSC in the various cultivation media over 15 population doublings except for the medium supplemented with 10% HP. Our results showed that DPSC cultivated in medium supplemented with 10% PRP showed the shortest average population doubling time (DT (28.6 ± 4.6 hours, in contrast to DPSC cultivated in 10% HP which indicated the longest DT (156.2 ± 17.8 hours; hence this part of the experiment had been cancelled in the 6th passage. DPSC cultivated in media with 2% FCS+ITS (DT 47.3 ± 10.4 hours, 2% PRP (DT 40.1 ± 5.7 hours and 2% HP (DT 49.0 ± 15.2 hours showed almost the same proliferative activity. DPSC’s viability in the 9th passage was over 90% except for the DPSC cultivated in the 10% HP media. Conclusions: We proved that human blood components are suitable substitution for FCS in cultivation media for long-term DPSC cultivation.

  10. Radioimmunoassay of arginine-vasopressin in human plasma: Development and clinical application

    International Nuclear Information System (INIS)

    Hummerich, W.; Konrads, A.; Roesch, R.; Sofroniew, M.

    1983-01-01

    A sensitive and specific radioimmunoassay for the measurement of arg-vasopressin (AVP) in human plasma is described. Recovery of added AVP from plasma was about 65-70%. An acetone extraction step was necessary to prevent unspecific blank effects. Sensitivity of the assay in 0.5 pg AVP/ml plasma. In normally hydrated subjects AVP-concentration ranged from 0.7 pg/ml to 5.8 pg/ml and showed a good correlation with plasma osmolality. In patients with complete diabetes insipidus (D.i.) AVP-values were below the sensitivity limit of the method and they were subnormal when D.i. was incomplete. There was no increase of AVP-concentration during fluid restriction in patients with complete or incomplete D.i. In subjects with psychogenic polydipsia AVP-values were normal and dehydration produced adequate rises of plasma AVP. In patients with SIADH (Schwartz-Bartter-syndrome) AVP-values were greatly enhanced (> 10 pg/ml) when correlated to plasma osmolality ( 2 O). (orig.)

  11. Radioimmunoassay of arginine-vasopressin in human plasma: Development and clinical application

    Energy Technology Data Exchange (ETDEWEB)

    Hummerich, W.; Konrads, A.; Roesch, R.; Sofroniew, M.

    1983-02-15

    A sensitive and specific radioimmunoassay for the measurement of arg-vasopressin (AVP) in human plasma is described. Recovery of added AVP from plasma was about 65-70%. An acetone extraction step was necessary to prevent unspecific blank effects. Sensitivity of the assay in 0.5 pg AVP/ml plasma. In normally hydrated subjects AVP-concentration ranged from 0.7 pg/ml to 5.8 pg/ml and showed a good correlation with plasma osmolality. In patients with complete diabetes insipidus (D.i.) AVP-values were below the sensitivity limit of the method and they were subnormal when D.i. was incomplete. There was no increase of AVP-concentration during fluid restriction in patients with complete or incomplete D.i. In subjects with psychogenic polydipsia AVP-values were normal and dehydration produced adequate rises of plasma AVP. In patients with SIADH (Schwartz-Bartter-syndrome) AVP-values were greatly enhanced (> 10 pg/ml) when correlated to plasma osmolality (< 170 mosmol/kg H/sub 2/O).

  12. Microdose clinical trial: quantitative determination of nicardipine and prediction of metabolites in human plasma.

    Science.gov (United States)

    Yamane, Naoe; Takami, Tomonori; Tozuka, Zenzaburo; Sugiyama, Yuichi; Yamazaki, Akira; Kumagai, Yuji

    2009-01-01

    A sample treatment procedure and high-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantitative determination of nicardipine in human plasma were developed for a microdose clinical trial with nicardipine, a non-radioisotope labeled drug. The calibration curve was linear in the range of 1-500 pg/mL using 1 mL of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 0.2-100 ng/mL using 20 microL of plasma, was also conducted. Each method was successfully applied to making determinations in plasma using LC/MS/MS after administration of a microdose (100 microg) and clinical dose (20 mg) to each of six healthy volunteers. We tested new approaches in the search for metabolites in plasma after microdosing. In vitro metabolites of nicardipine were characterized using linear ion trap-fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) and the nine metabolites predicted to be in plasma were analyzed using LC/MS/MS. There is a strong possibility that analysis of metabolites by LC/MS/MS may advance to utilization in microdose clinical trials with non-radioisotope labeled drugs.

  13. Determination of N-methylsuccinimide and 2-hydroxy-N-methylsuccinimide in human urine and plasma.

    Science.gov (United States)

    Jönsson, B A; Akesson, B

    1997-12-19

    A method for determination of N-methylsuccinimide (MSI) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine and of MSI in human plasma was developed. MSI and 2-HMSI are metabolites of the widely used organic solvent N-methyl-2-pyrrolidone (NMP). MSI and 2-HMSI were purified from urine and plasma by C8 solid-phase extraction and analysed by gas chromatography-mass spectrometry in the negative-ion chemical ionisation mode. The intra-day precisions in urine were 2-6% for MSI (50 and 400 ng/ml) and 3-5% for 2-HMSI (1000 and 8000 ng/ml). For MSI in plasma it was 2% (60 and 1200 ng/ml). The between-day precisions in urine were 3-4% for MSI (100 and 1000 ng/ml) and 2-4% for 2-HMSI (10,000 and 18,000 ng/ml) and 3-4% for MSI in plasma (100 and 900 ng/ml). The recoveries from urine were 109-117% for MSI (50 and 400 ng/ml) and 81-89% for 2-HMSI (1000 and 8000 ng/ml). The recovery of MSI from plasma was 91-101% (50 and 500 ng/ml). The detection limits for MSI were 3 ng/ml in urine and 1 ng/ml in plasma and that of 2-HMSI in urine was 200 ng/ml. The method is applicable for analysis of urine and plasma samples from workers exposed to NMP.

  14. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    International Nuclear Information System (INIS)

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-01-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

  15. Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

    Science.gov (United States)

    Larsen, Margit Hørup; Bzorek, Michael; Pass, Malene B; Larsen, Lise Grupe; Nielsen, Mette Weidinger; Svendsen, Signe Goul; Lindhard, Anette; Hviid, Thomas Vauvert F

    2011-12-01

    One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

  16. A sulfur amino acid-free meal increases plasma lipids in humans.

    Science.gov (United States)

    Park, Youngja; Le, Ngoc-Anh; Yu, Tianwei; Strobel, Fred; Gletsu-Miller, Nana; Accardi, Carolyn J; Lee, Kichun S; Wu, Shaoxiong; Ziegler, Thomas R; Jones, Dean P

    2011-08-01

    The content of sulfur amino acid (SAA) in a meal affects postprandial plasma cysteine concentrations and the redox potential of cysteine/cystine. Because such changes can affect enzyme, transporter, and receptor activities, meal content of SAA could have unrecognized effects on metabolism during the postprandial period. This pilot study used proton NMR ((1)H-NMR) spectroscopy of human plasma to test the hypothesis that dietary SAA content changes macronutrient metabolism. Healthy participants (18-36 y, 5 males and 3 females) were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (depletion), and then fed isoenergetic, isonitrogenous meals containing 56 mg·kg(-1)·d(-1) SAA for 4.5 d (repletion). On the first and last day of consuming the chemically defined meals, a morning meal containing 60% of the daily food intake was given and plasma samples were collected over an 8-h postprandial time course for characterization of metabolic changes by (1)H-NMR spectroscopy. SAA-free food increased peak intensity in the plasma (1)H-NMR spectra in the postprandial period. Orthogonal signal correction/partial least squares-discriminant analysis showed changes in signals associated with lipids, some amino acids, and lactate, with notable increases in plasma lipid signals (TG, unsaturated lipid, cholesterol). Conventional lipid analyses confirmed higher plasma TG and showed an increase in plasma concentration of the lipoprotein lipase inhibitor, apoC-III. The results show that plasma (1)H-NMR spectra can provide useful macronutrient profiling following a meal challenge protocol and that a single meal with imbalanced SAA content alters postprandial lipid metabolism.

  17. Digitalis-like activity in human plasma: Relation to blood pressure and sodium balance

    Energy Technology Data Exchange (ETDEWEB)

    Goto, A.; Yamada, K.; Ishii, M.; Sugimoto, T. (Univ. of Tokyo (Japan))

    1990-10-01

    PURPOSE: On the assumption that renal tubular cells are more important as the target cells for a natriuretic factor than blood cells, we used a well-characterized cultured renal tubular cell line, Madin-Darby canine kidney (MDCK), cells to monitor the circulating digitalis-like factor in human plasma and examine its role in the regulation of blood pressure and sodium balance. SUBJECTS AND METHODS: We investigated the effects of plasma on binding of radioactive ouabain to monolayered MDCK cells in order to determine the level of a circulating digitalis-like factor. First, we measured specific 3H-ouabain binding to MDCK cells in the presence of plasma from 71 outpatients (34 normotensive subjects and 37 hypertensive patients) after incubation for 4 hours. Second, we measured specific 3H-ouabain binding after incubation of cells with plasma from 16 hospitalized subjects (eight normotensive subjects and eight hypertensive patients) receiving low and high sodium diets. RESULTS: In Study 1, ouabain binding was lower by 30% with plasma from hypertensive patients than with plasma from normotensive subjects (p less than 0.01). There was a significant negative correlation between individual subject's systolic or mean blood pressure and ouabain binding (r = -0.34, p less than 0.01 or r = -0.29, p less than 0.01). In Study 2, ouabain binding was also significantly reduced by 25% in the presence of plasma from hypertensive subjects as compared with plasma from normotensive subjects irrespective of sodium intake (p less than 0.01). A significant negative correlation was also found for all subjects between either systolic, diastolic, or mean blood pressure and ouabain binding (r = -0.58, p less than 0.01, r = -0.51, p less than 0.01, or r = -0.55, p less than 0.01, respectively).

  18. Simultaneous determination of cytosine arabinoside, daunorubicin and etoposide in human plasma

    DEFF Research Database (Denmark)

    Krogh-Madsen, Mikkel; Hansen, Steen Honoré; Honoré, Per Hartvig

    2010-01-01

    A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample....... The overall precision (% relative standard deviation) was within 0.2-13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at -20 degrees C. The method was successfully applied to quantification...

  19. Efficient discrimination and removal of phospholipids during electromembrane extraction from human plasma samples

    DEFF Research Database (Denmark)

    Vårdal, Linda; Gjelstad, Astrid; Huang, Chuixiu

    2017-01-01

    to be highly efficient for providing phospholipid-free extracts. CONCLUSION: Ultra-HPLC-MS/MS analysis of the donor solutions revealed that the phospholipids principally remained in the plasma samples. This proved that the phospholipids did not migrate in the electrical field and they were prevented from......AIM: For the first time, extracts obtained from human plasma samples by electromembrane extraction (EME) were investigated comprehensively with particular respect to phospholipids using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Thhe purpose...

  20. Parallel artificial liquid membrane extraction of acidic drugs from human plasma

    DEFF Research Database (Denmark)

    Roldan-Pijuan, Mercedes; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2015-01-01

    The new sample preparation concept “Parallel artificial liquid membrane extraction (PALME)” was evaluated for extraction of the acidic drugs ketoprofen, fenoprofen, diclofenac, flurbiprofen, ibuprofen, and gemfibrozil from human plasma samples. Plasma samples (250 μL) were loaded into individual......-performance liquid chromatography-ultraviolet detection of the individual acceptor solutions. Important PALME parameters including the chemical composition of the liquid membrane, extraction time, and sample pH were optimized, and the extraction performance was evaluated. Except for flurbiprofen, exhaustive...

  1. Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans

    Science.gov (United States)

    McGill, Mitchell R.; Cao, Mengde; Svetlov, Archie; Sharpe, Matthew R.; Williams, C. David; Curry, Steven C.; Farhood, Anwar; Jaeschke, Hartmut; Svetlov, Stanislav I.

    2014-01-01

    Context New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. Objective Characterize ASS in APAP hepatotoxicity. Methods ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. Results In mice, ASS increased before injury, peaked before ALT, and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. Conclusions : ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity. PMID:24597531

  2. High Performance Liquid Chromatographic Method for Determination of Dipyridamole in Human Plasma

    Directory of Open Access Journals (Sweden)

    DAVOOD BEIGI BAND ARAB ADI

    1999-08-01

    Full Text Available A simple, rapid and specific high-performance liquid chromatographic procedure is reported for"nquantitative determination of dipyridamole in human -plasma. The assay uses a reversed-phase"nhigh-performance liquid chromatographic (HPLC and UV detection at 280nm and has a limit"nof detection of approximately 5ng/mL. The mobile phase consists of MeOH-H20 (60:40"nadjusted to pH 3.3. Dipyridamole was extracted from plasma by back-extraction procedure, with"npropranolol as the internal standard. The reproducibility of the method is satisfactory

  3. Ontogeny of human IgE?expressing B cells and plasma cells

    OpenAIRE

    Ramadani, F.; Bowen, H.; Upton, N.; Hobson, P. S.; Chan, Y.?C.; Chen, J.?B.; Chang, T. W.; McDonnell, J. M.; Sutton, B. J.; Fear, D. J.; Gould, H. J.

    2016-01-01

    BACKGROUND: IgE-expressing (IgE+) plasma cells (PCs) provide a continuous source of allergen specific IgE that is central to allergic responses. The extreme sparsity of IgE+ cells in vivo has confined their study almost entirely to mouse models.OBJECTIVE: To characterise the development pathway of human IgE+ PCs and to determine the ontogeny of human IgE+ PCs.METHODS: To generate human IgE+ cells we cultured tonsil B cells with IL-4 and anti-CD40. Using FACS and RT-PCR we examined the phenoty...

  4. Flow Cytometry Assessment of In Vitro Generated CD138+ Human Plasma Cells

    Directory of Open Access Journals (Sweden)

    Rayelle Itoua Maïga

    2014-01-01

    Full Text Available The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs. As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98% with variable stain index (SI. The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+ cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25% while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%. DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138high and CD138lo cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

  5. Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC-QToF-MS Method.

    Directory of Open Access Journals (Sweden)

    Mariana Millan Fachi

    Full Text Available An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD 0.99, free of matrix and has no residual effects. The developed method was successfully applied to volunteers' plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents.

  6. Liver and Muscle Contribute Differently to the Plasma Acylcarnitine Pool During Fasting and Exercise in Humans

    DEFF Research Database (Denmark)

    Xu, G.; Hansen, J S; Zhao, Jian-xin

    2016-01-01

    BACKGROUND: Plasma acylcarnitine levels are elevated by physiological conditions such as fasting and exercise but also in states of insulin resistance and obesity. AIM: To elucidate the contribution of liver and skeletal muscle to plasma acylcarnitines in the fasting state and during exercise...... in humans. METHODS: In 2 independent studies, young healthy males were fasted overnight and performed an acute bout of exercise to investigate either acylcarnitines in skeletal muscle biopsies and arterial-to-venous plasma differences over the exercising and resting leg (n = 9) or the flux over the hepato......-splanchnic bed (n = 10). RESULTS: In the fasting state, a pronounced release of C2- and C3-carnitines from the hepato-splanchnic bed and an uptake of free carnitine by the legs were detected. Exercise further increased the release of C3-carnitine from the hepato-splanchnic bed and the uptake of free carnitine...

  7. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage

    Energy Technology Data Exchange (ETDEWEB)

    Frei, B. (Department of Nutrition, Harvard School of Public Health, Boston, MA (Unites States))

    1991-12-01

    The authors exposed human blood plasma and low-density lipoprotein (LDL) to many different oxidative challenges and followed the temporal consumption of endogenous antioxidants in relation to the initiation of oxidative damage. Under all types of oxidizing conditions, ascorbic acid completely protects lipids in plasma and LDL against detectable peroxidative damage as assessed by a specific and highly sensitive assay for lipid peroxidation. Ascorbic acid proved to be superior to the other water-soluble plasma antioxidants bilirubin, uric acid, and protein thiols as well as to the lipoprotein-associated antioxidants alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene. Although these antioxidants can lower the rate of detectable lipid peroxidation, they are not able to prevent its initiation. Only ascorbic acid is reactive enough to effectively intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids.

  8. Concentration of perfluorinated compounds and cotinine in human foetal organs, placenta, and maternal plasma

    DEFF Research Database (Denmark)

    Mamsen, Linn Salto; Jönsson, Bo A.G.; Lindh, Christian H.

    2017-01-01

    levels. Foetal exposure has previously been estimated from umbilical cord plasma, but the actual concentration in foetal organs has never been measured. Objectives The concentrations of 5 PFASs and cotinine – the primary metabolite of nicotine – were measured in human foetuses, placentas, and maternal...... plasma to evaluate to what extent these compounds were transferred from mother to foetus and to determine if the PFAS concentrations were associated with maternal cigarette smoking. Methods Thirty-nine Danish women who underwent legal termination of pregnancy before gestational week 12 were included; 24...... ratio for all five PFASs and cotinine. Smokers presented 99 ng/g cotinine in plasma, 108 ng/g in placenta, and 61 ng/g in foetal organs. No correlation between the maternal cotinine concentrations and PFAS concentrations was found. Conclusions PFASs were transferred from mother to foetus, however...

  9. Sterilization of Normal Human Plasma and Some of its Fractions by Means of Gamma Rays

    International Nuclear Information System (INIS)

    López Martínez de Alva, L.; Crespo, Y M.

    1967-01-01

    Owing to the frequency with which normal human plasma transmits hepatitis, various methods of sterilization have been tried. The method most used, but which has been shown to be ineffective, is sterilization of the liquid plasma with ultraviolet rays. The other method is the use of ß-propiolactone, but this has also been discontinued because of the changes it produces in the structure of plasma proteins. The object of the present study, which should be regarded as a preliminary report, is to present the results obtained by sterilizing, by means of gamma rays ( 60 Co, 1.3316 MeV) at doses of 2, 2. 5 and 3 Mrad, a substance which, like plasma, contains highly labile proteins easily able to undergo structural changes under irradiation. Pure fibrinogen, pure gamma globulin, and albumin of human origin were subjected to the same doses; the results were very satisfactory, in that no appreciable change could be demonstrated as regards the structure, solubility or chemical characteristics of the substances concerned. It was shown by means of simple coagulation tests that some of the proteins involved in this mechanism, such as prothrombin, die Power-Stuart factor and the Hageman factor, were practically unchanged. All the plasma samples and the various proteins were previously lyophilized to give a maximum moisture content of 0.034%, in order to avoid ionizing the water content into oxygenated water, which would modify and oxidize the proteins. It was shown that lyophilized plasmas initially contaminated with different strains and viruses remained sterile with doses as low as 2 Mrad. Finally, it was shown that this method is simple and practical, since sterilization can be checked in the final packaging. (author) [es

  10. Plasma PCSK9 levels are significantly modified by statins and fibrates in humans

    Directory of Open Access Journals (Sweden)

    Mbikay Majambu

    2008-06-01

    Full Text Available Abstract Background Proprotein convertase subtilisin kexin-like 9 (PCSK9 is a secreted glycoprotein that is transcriptionally regulated by cholesterol status. It modulates levels of circulating low density lipoprotein cholesterol (LDLC by negatively regulating low density lipoprotein receptor (LDLR levels. PCSK9 variants that result in 'gain of function' have been linked to autosomal dominant hypercholesterolemia, while significant protection from coronary artery disease has been documented in individuals who carry 'loss of function' PCSK9 variants. PCSK9 circulates in human plasma, and we previously reported that plasma PCSK9 is positively correlated with total cholesterol and LDLC in men. Results Herein, we report the effects of two lipid-modulating therapies, namely statins and fibrates, on PCSK9 plasma levels in human subjects. We also document their effects on endogenous PCSK9 and LDLR expression in a human hepatocyte cell line, HepG2, using immunoprecipitation and immunoblot analyses. Changes in plasma PCSK9 following fenofibrate or gemfibrozil treatments (fibric acid derivatives were inversely correlated with changes in LDLC levels (r = -0.558, p = 0.013. Atorvastatin administration (HMGCoA reductase inhibitor significantly increased plasma PCSK9 (7.40%, p = 0.033 and these changes were inversely correlated with changes in LDLC levels (r = -0.393, p = 0.012. Immunoblot analyses of endogenous PCSK9 and LDLR expression by HepG2 cells in response to statins and fibrates showed that LDLR is more upregulated than PCSK9 by simvastatin (2.6× vs 1.5×, respectively at 10 μM, while fenofibrate did not induce changes in either. Conclusion These results suggest that in vivo (1 statins directly increase PCSK9 expression while (2 fibrates affect PCSK9 expression indirectly through its modulation of cholesterol levels and (3 that these therapies could be improved by combination with a PCSK9 inhibitor, constituting a novel hypercholesterolemic therapy

  11. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

    Science.gov (United States)

    Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

    2017-08-01

    Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of 4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

  12. GMP-compliant radiosynthesis of [{sup 18}F]altanserin and human plasma metabolite studies

    Energy Technology Data Exchange (ETDEWEB)

    Hasler, F. [University Hospital of Psychiatry, Heffter Research Center, Zurich (Switzerland)], E-mail: fehasler@bli.uzh.ch; Kuznetsova, O.F.; Krasikova, R.N. [Institute of the Human Brain, Russian Academy of Science, St. Petersburg (Russian Federation); Cservenyak, T. [Center for Radiopharmaceutical Sciences of ETH, PSI and University Hospital Zurich (Switzerland); Quednow, B.B.; Vollenweider, F.X. [University Hospital of Psychiatry, Heffter Research Center, Zurich (Switzerland); Ametamey, S.M.; Westera, G. [Center for Radiopharmaceutical Sciences of ETH, PSI and University Hospital Zurich (Switzerland)

    2009-04-15

    [{sup 18}F]altanserin is the preferred radiotracer for in-vivo labeling of serotonin 2A receptors by positron emission tomography (PET). We report a modified synthesis procedure suited for reliable production of multi-GBq amounts of [{sup 18}F]altanserin useful for application in humans. We introduced thermal heating for drying of [{sup 18}F]fluoride as well as for the reaction instead of microwave heating. We furthermore describe solid phase extraction and HPLC procedures for quantitative determination of [{sup 18}F]altanserin and metabolites in plasma. The time course of arterial plasma activity with and without metabolite correction was determined. 90 min after bolus injection, 38.4% of total plasma activity derived from unchanged [{sup 18}F]altanserin. Statistical comparison of kinetic profiles of [{sup 18}F]altanserin metabolism in plasma samples collected in the course of two ongoing studies employing placebo, the serotonin releaser dexfenfluramine and the hallucinogen psilocybin, revealed the same tracer metabolism. We conclude that metabolite analysis for correction of individual plasma input functions used in tracer modeling is not necessary for [{sup 18}F]altanserin studies involving psilocybin or dexfenfluramine treatment.

  13. GMP-compliant radiosynthesis of [18F]altanserin and human plasma metabolite studies

    International Nuclear Information System (INIS)

    Hasler, F.; Kuznetsova, O.F.; Krasikova, R.N.; Cservenyak, T.; Quednow, B.B.; Vollenweider, F.X.; Ametamey, S.M.; Westera, G.

    2009-01-01

    [ 18 F]altanserin is the preferred radiotracer for in-vivo labeling of serotonin 2A receptors by positron emission tomography (PET). We report a modified synthesis procedure suited for reliable production of multi-GBq amounts of [ 18 F]altanserin useful for application in humans. We introduced thermal heating for drying of [ 18 F]fluoride as well as for the reaction instead of microwave heating. We furthermore describe solid phase extraction and HPLC procedures for quantitative determination of [ 18 F]altanserin and metabolites in plasma. The time course of arterial plasma activity with and without metabolite correction was determined. 90 min after bolus injection, 38.4% of total plasma activity derived from unchanged [ 18 F]altanserin. Statistical comparison of kinetic profiles of [ 18 F]altanserin metabolism in plasma samples collected in the course of two ongoing studies employing placebo, the serotonin releaser dexfenfluramine and the hallucinogen psilocybin, revealed the same tracer metabolism. We conclude that metabolite analysis for correction of individual plasma input functions used in tracer modeling is not necessary for [ 18 F]altanserin studies involving psilocybin or dexfenfluramine treatment

  14. Incorporation of deuterium-labeled trans- and cis-13-octadecenoic acids in human plasma lipids

    International Nuclear Information System (INIS)

    Emken, E.A.; Adlof, R.O.; Rohwedder, W.K.; Gulley, R.M.

    1983-01-01

    The absorption and distribution of deuterated trans- and cis-13-octadecenoic acid (13t-18:1 and 13c-18:1) in plasma lipids were compared to deuterated cis-9-octadecenoic acid (9c-18:1) in two young adult male subjects. A mixture of triglycerides was fed in a multiple-labeled experiment where each triglyceride contained a fatty acid labeled with a different number of deuterium atoms. Analysis of human plasma lipids by mass spectroscopy allowed the distribution of the two 13-octadecenoic acid isomers to be directly compared to cis-9-octadecenoic acid. Plasma lipids selectively excluded both the 13t-18:1 and 13c-18:1 isomers relative to 9c-18:1 in all neutral and phospholipid fractions. Discrimination against incorporation of the 13t-18:1 isomer into plasma cholesteryl ester and 2-acyl phosphatidylcholine was nearly absolute. The 1-acyl phosphatidylcholine fraction exhibited a large positive selectivity for the 13t-18:1 isomer. Differences in the relative distribution of the trans and cis 13-18:1 isomers vs. 9c-18:1 in the various lipoprotein lipid classes were found. Analysis of the chylomicron triglyceride component of the plasma lipids indicated all three fatty acids were equally well absorbed

  15. Zymosterol is located in the plasma membrane of cultured human fibroblasts

    International Nuclear Information System (INIS)

    Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

    1990-01-01

    Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

  16. Safflower oil consumption does not increase plasma conjugated linoleic acid concentrations in humans.

    Science.gov (United States)

    Herbel, B K; McGuire, M K; McGuire, M A; Shultz, T D

    1998-02-01

    Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (LA) with conjugated double bonds. CLA has anticarcinogenic properties and has been identified in human tissues, dairy products, meats, and certain vegetable oils. A variety of animal products are good sources of CLA, but plant oils contain much less. However, plant oils are a rich source of LA, which may be isomerized to CLA by intestinal microorganisms in humans. To investigate the effect of triacylglycerol-esterified LA consumption on plasma concentrations of esterified CLA in total lipids, a dietary intervention (6 wk) was conducted with six men and six women. During the intervention period a salad dressing containing 21 g safflower oil providing 16 g LA/d was added to the subjects' daily diets. Three-day diet records and fasting blood were obtained initially and during dietary and postdietary intervention periods. Although LA intake increased significantly during the dietary intervention, plasma CLA concentrations were not affected. Plasma total cholesterol and LDL-cholesterol concentrations were significantly lower after addition of safflower oil to the diet. In summary, consumption of triacylglycerol-esterified LA in safflower oil did not increase plasma concentrations of esterified CLA in total lipids.

  17. Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry.

    Science.gov (United States)

    Cheon, Dong Huey; Nam, Eun Ji; Park, Kyu Hyung; Woo, Se Joon; Lee, Hye Jin; Kim, Hee Cheol; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

    2016-01-04

    While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

  18. High-performance liquid chromatographic quantification of rifampicin in human plasma: method for Therapecutic drug monitoring

    International Nuclear Information System (INIS)

    Sameh, T.; Hanene, E.; Jebali, N.

    2013-01-01

    A high performance liquid chromatography (HPLC) method has been developed that allows quantification of Rifampicin in human plasma. The method is based on the precipitation of proteins in human plasma with methanol. Optimal assay conditions were found with a C18 column and a simple mobile phase consisting of 0.05 M dipotassic hydrogen phosphate buffer and acetonitrile (53/47, V/V) with 0.086 % diethylamin, pH = 4.46. The flow-rate was 0.6 ml /mm and the drug was monitored at 340 nm. Results from the HPLC analyses showed that the assay method is linear in the concentration range of 1-40 micro g/ml, (r2 >0.99). The limit of quantification and limit of detection of Rifampicin were 0.632 micro g/ml and 0.208 micro g/ml, respectively. Intraday and interday coefficient of variation and bias were below 10% for all samples, suggesting good precision and accuracy of the method. Recoveries were greater than 90% in a plasma sample volume of 100 micro l. The method is being successfully applied to therapeutic drug monitoring of Rifapicin in plasma samples of tuberculosis and staphylococcal infections patients. (author)

  19. Quantification of pharmaceutical peptides in human plasma by LC-ICP-MS sulfur detection

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Macherius, André; Hansen, Thomas Hesselhøj

    2016-01-01

    A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP-DRC-MS inst......A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP......-DRC-MS instrument and subsequent quantification by post-column isotope dilution (IDA). Plasma proteins were precipitated prior to analysis. Analytical figures of merit including linearity, precision, LOD, LOQ and accuracy were considered satisfactory for analysis of plasma samples. The selectivity of the developed...... method was demonstrated for five pharmaceutically relevant peptides: desmopressin, penetratin, substance P, PTH (1-34) and insulin. Preliminary experiments on an ICP-MS/MS system using oxygen to reduce the effect of organic solvents were also performed to compare sensitivity. The results of the study...

  20. New sensitive direct radioimmunoassay for human plasma renin and its clinical application

    International Nuclear Information System (INIS)

    Higaki, J.; Ogihara, T.; Imai, N.; Kumahara, Y.; Hontani, S.; Nishiura, M.; Ogawa, H.; Hirose, S.; Murakami, K.

    1984-01-01

    A new sensitive direct radioimmunoassay for human plasma renin has been developed. Renin was purified from Haas' preparation utilizing a pepstatin-C 6 -Sepharose affinity chromatography. Antiserum, prepared by immunizing rabbits with the purified renin, was used for the direct radioimmunoassay at a final dilution of 1:30,000. The antibody was specific for human renal and plasma renin, but did not cross-react with cathepsin D, trypsin, or renins of mouse, dog, and rat. Radioimmunoassay was performed by the double antibody technique using the delayed tracer addition method. In this method, a standard curve was obtained over a range from 0.2 to 8.0 ng/ml. The values from this assay correlated well with total renin activity measured as the generation rate of angiotensin I after trypsin activation, but correlated weakly with active renin activity. This finding disclosed that both active and inactive renin were detected by this method. In normal participants, plasma renin concentration determined by direct radioimmunoassay was increased by standing and furosemide injection. The plasma renin concentration determined by direct radioimmunoassay of patients with essential hypertension was not significantly different from values in normal controls. The values were higher in patients with renovascular hypertension, malignant hypertension and Bartter's syndrome, but lower in patients with primary aldosteronism than in normal controls. 20 references, 7 figures

  1. Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

    Directory of Open Access Journals (Sweden)

    Anita Muraglia

    2017-11-01

    Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

  2. High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

    Science.gov (United States)

    Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

    2012-05-01

    Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

  3. A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Lewis Fiona C

    2012-08-01

    Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

  4. Exhaustive and stable electromembrane extraction of acidic drugs from human plasma

    DEFF Research Database (Denmark)

    Huang, Chuixiu; Gjelstad, Astrid; Seip, Knut Fredrik

    2015-01-01

    The first part of the current work systematically described the screening of different types of organic solvents as the supported liquid membrane (SLM) for electromembrane extraction (EME) of acidic drugs, including different alcohols, ketones, and ethers. Seven acidic drugs with a wide logP rang......). With this SLM, exhaustive EME was performed from diluted human plasma, and the recoveries of five out of seven analytes were over 91% after 10min EME. This approach was evaluated using HPLC-UV, and the evaluation data were found to be satisfactory...... to increasing viscosity and decreasing α and π* values. The system-current during EME was found to be dependent on the type and the volume of the SLM. In contact with human plasma, an SLM of pure 1-heptanol was unstable, and to improve stability, 1-heptanol was mixed with 2-nitrophenyl octyl ether (NPOE...

  5. Predictive value of plasma β2-microglobulin on human body function and senescence.

    Science.gov (United States)

    Dong, X-M; Cai, R; Yang, F; Zhang, Y-Y; Wang, X-G; Fu, S-L; Zhang, J-R

    2016-06-01

    To explore the correlation between plasma β2-microglobulin (β2-MG) as senescence factor with age, heart, liver and kidney function as well as the predictive value of β2-MG in human metabolism function and senescence. 387 cases of healthy people of different ages were selected and the automatic biochemical analyzer was used to test β2-MG in plasma based on immunoturbidimetry and also all biochemical indexes. The correlation between β2-MG and age, gender and all biochemical indexes was analyzed. β2-MG was positively correlated to age, r = 0.373; and the difference was of statistical significance (p human body function and anti-senescence and have significant basic research and clinical guidance values.

  6. Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper

    2015-01-01

    Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject...... to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system...... in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator...

  7. Measurement of Ether Phospholipids in Human Plasma with HPLC-ELSD and LC/ESI-MS After Hydrolysis of Plasma with Phospholipase A1.

    Science.gov (United States)

    Mawatari, Shiro; Hazeyama, Seira; Fujino, Takehiko

    2016-08-01

    Ethanolamine ether phospholipid (eEtnGpl) and choline ether phospholipid (eChoGpl) are present in human plasma or serum, but the relative concentration of the ether phospholipids in plasma is very low as compared to those in other tissues. Nowadays, measurement of ether phospholipids in plasma depends on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is generally too expensive for usual clinical laboratories. Treatment of plasma with phospholipase A1 (PLA1) causes complete hydrolysis of diacylphospholipids, but ether phospholipids remain intact. After the treatment of plasma with PLA1, both eEtnGpl and eChoGpl are detected as independent peaks by high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The same sample used for HPLC-ELSD can be applied to detect eEtnGpl and eChoGpl with electrospray ionization mass spectrometry. Presence of alkylacylphospholipids in both eChoGpl and eEtnGpl in human plasma was indicated by sequential hydrolysis of plasma with PLA1 and hydrochloric acid.

  8. Mass-spectrometric identification of a novel angiotensin peptide in human plasma

    DEFF Research Database (Denmark)

    Jankowski, Vera; Vanholder, Raymond; van der Giet, Markus

    2007-01-01

    Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may eli...... elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients....

  9. Lack of Association between Human Plasma Oxytocin and Interpersonal Trust in a Prisoners Dilemma Paradigm

    Science.gov (United States)

    2014-12-30

    from data using rodent models. Zhang et al. [17] used two- dimensional liquid chromatography -tandem mass spectrometry (2D LC-MS/MS) to demonstrate that...DM, Lin Z, Steenwyk R (2011) Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography ... Handbook of motivation and cognition: Foundations of social behavior, 1, 96–121. 42. Feldman R, Weller A, Zagoory-Sharon O, Levine A (2007) Evidence for

  10. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

  11. Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum

    NARCIS (Netherlands)

    Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de; Wied, D. de

    1979-01-01

    β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [125I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At

  12. Purification and preliminary X-ray crystallographic studies of β-microseminoprotein from human seminal plasma

    International Nuclear Information System (INIS)

    Kumar, Vijay; Roske, Yvette; Singh, Nagendra; Heinemann, Udo; Singh, Tej P.; Yadav, Savita

    2009-01-01

    The preliminary X-ray data of β-microseminoprotein isolated from human seminal plasma at 2.4 Å resolution are reported. β-Microseminoprotein (β-MSP) is a small cysteine-rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino-acid sequences of β-MSP proteins suggests that the protein is a rapidly evolving protein. The function of β-MSP is poorly understood. Furthermore, no crystal structure has been reported of any β-MSP; therefore, determination of the crystal structure of β-MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X-ray diffraction analysis of β-MSP from human seminal plasma are described. The protein was purified using anion-exchange and size-exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4 3 22 and contained three β-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 Å resolution

  13. Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Wishu, S; Varma, D P

    2005-12-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. (c) 2005 John Wiley & Sons, Ltd.

  14. Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo; Heibeck, Tyler H.; Wang, Jun; Qian, Weijun; Lin, Yuehe

    2012-04-20

    Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.

  15. Biological stimulation of the Human skin applying health promoting light and plasma sources

    Energy Technology Data Exchange (ETDEWEB)

    Awakowicz, P.; Bibinov, N. [Center for Plasma Science and Technology, Ruhr-University, Bochum (Germany); Born, M.; Niemann, U. [Philips Research, Aachen (Germany); Busse, B. [Zell-Kontakt GmbH, Noerten-Hardenberg (Germany); Gesche, R.; Kuehn, S.; Porteanu, H.E. [Ferdinand-Braun-Institut fuer Hoechstfrequenztechnik, Berlin (Germany); Helmke, A. [University of Applied Sciences and Arts, Goettingen (Germany); Kaemling, A.; Wandke, D. [CINOGY GmbH, Duderstadt (Germany); Kolb-Bachofen, V.; Liebmann, J. [Institute for Immunobiology, Heinrich-Heine University, Duesseldorf (Germany); Kovacs, R.; Mertens, N.; Scherer, J. [Aurion Anlagentechnik GmbH, Seligenstadt (Germany); Oplaender, C.; Suschek, C. [Clinic for Plastic Surgery, University Clinic, Aachen (Germany); Vioel, W. [Laser-Laboratorium, Goettingen (Germany); University of Applied Sciences and Arts, Goettingen (Germany)

    2009-10-15

    In the frame of BMBF project ''BioLiP'', new physical treatment techniques aiming at medical treatment of the human skin have been developed. The acronym BioLiP stands for ''Desinfektion, Entkeimung und biologische Stimulation der Haut durch gesundheitsfoerdernde Licht- und Plasmaquellen'' (Disinfection, germ reduction and biological stimulation of the human skin by health promoting light and plasma sources). A source applying a low-temperature dielectric barrier discharge plasma (DBD) has been investigated on its effectiveness for skin disinfection and stimulation of biological material. Alternatively an atmospheric plasma source consisting of a microwave resonator combined with a solid state power oscillator has been examined. This concept which allows for a compact and efficient design avoiding external microwave power supply and matching units has been optimized with respect to nitrogen monoxide (NO) production in high yields. In both cases various application possibilities in the medical and biological domain are opened up. Light sources in the visible spectral range have been investigated with respect to the proliferation of human cell types. Intensive highly selective blue light sources based on LED technology can slow down proliferation rates without inducing toxic effects which offers new opportunities for treatments of so-called hyperproliferative skin conditions (e.g. with psoriasis or in wound healing) using UV-free light. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  16. Development and validation of analytical method for Naftopidil in human plasma by LC–MS/MS

    Directory of Open Access Journals (Sweden)

    Pritam S. Jain

    2015-09-01

    Full Text Available A highly sensitive and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS-MS assay is developed and validated for the quantification of Naftopidil in human plasma. Naftopidil is extracted from human plasma by methyl tertiary butyl ether and analyzed using a reversed-phase gradient elution on a discovery C 18 5 μ (50 × 4.6 column. A methanol: 2 mM ammonium formate (90:10 as mobile phase, is used and detection was performed by MS using electrospray ionization in positive mode. Propranolol is used as the internal standard. The lower limits of quantification are 0.495 ng/mL. The calibration curves are linear over the concentration range of 0.495–200.577 ng/mL of plasma for each analyte. This novel LC–MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in humans.

  17. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  18. Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard.

    Science.gov (United States)

    Mamidi, Rao N V S; Benjamin, Biju; Ramesh, Mullangi; Srinivas, Nuggehally R

    2003-09-01

    To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were 80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS. Copyright 2003 John Wiley & Sons, Ltd.

  19. Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

    International Nuclear Information System (INIS)

    Wun, T.C.; Capuano, A.

    1987-01-01

    A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above

  20. Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis

    Energy Technology Data Exchange (ETDEWEB)

    Wun, T.C.; Capuano, A.

    1987-05-01

    A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

  1. Analysis of human blood plasma proteome from ten healthy volunteers from Indian population.

    Directory of Open Access Journals (Sweden)

    Poonam Gautam

    Full Text Available Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used 'reference plasma sample', a pool of plasma from 10 healthy individuals from Indian population in the age group of 25-60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a without prefractionation, b after prefractionation at peptide level by strong cation exchange chromatography and c after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by ≥ 2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

  2. Development of biomonitoring equivalents for barium in urine and plasma for interpreting human biomonitoring data.

    Science.gov (United States)

    Poddalgoda, Devika; Macey, Kristin; Assad, Henry; Krishnan, Kannan

    2017-06-01

    The objectives of the present work were: (1) to assemble population-level biomonitoring data to identify the concentrations of urinary and plasma barium across the general population; and (2) to derive biomonitoring equivalents (BEs) for barium in urine and plasma in order to facilitate the interpretation of barium concentrations in the biological matrices. In population level biomonitoring studies, barium has been measured in urine in the U.S. (NHANES study), but no such data on plasma barium levels were identified. The BE values for plasma and urine were derived from U.S. EPA's reference dose (RfD) of 0.2 mg/kg bw/d, based on a lower confidence limit on the benchmark dose (BMDL 05 ) of 63 mg/kg bw/d. The plasma BE (9 μg Ba/L) was derived by regression analysis of the near-steady-state plasma concentrations associated with the administered doses in animals exposed to barium chloride dihydrate in drinking water for 2-years in a NTP study. Using a human urinary excretion fraction of 0.023, a BE for urinary barium (0.19 mg/L or 0.25 mg/g creatinine) was derived for US EPA's RfD. The median and the 95 th percentile barium urine concentrations of the general population in U.S. are below the BE determined in this study, indicating that the population exposure to inorganic barium is expected to be below the exposure guidance value of 0.2 mg/kg bw/d. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. GLUT4 expression at the plasma membrane is related to fibre volume in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Gaster, M; Vach, W; Beck-Nielsen, H

    2002-01-01

    In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane...

  4. A quantitative assay of cortisol in human plasma by high performance liquid chromatography using a selective chemically bonded stationary phase

    NARCIS (Netherlands)

    van den Berg, J.H.M.; Mol, C.R.; Deelder, R.S.; Thijssen, J.H.H.

    1977-01-01

    The extraction and subsequent liquid chromatographic analysis of human plasma samples for cortisol is described. Extraction and chromatography are optimized, resulting in a recovery for cortisol of 96% and a detection limit of 1 microgram cortisol in 100 ml plasma. The application of two chemically

  5. Selective enrichment of n-3 fatty acids in human plasma lipid motifs following intake of marine fish

    Science.gov (United States)

    Plasma levels of n-3 long chain polyunsaturated fatty acids (LCPUFA) are associated with a reduction in risk of cardiovascular disease and other chronic, age-related diseases like Alzheimer’s disease. In this work, we tested the hypothesis that n-3 LCPUFA fatty acids in human plasma are incorporated...

  6. The effect of fluoride on the scavenging of organophosphates by human butyrylcholinesterase in buffer solutions and human plasma

    International Nuclear Information System (INIS)

    Ashani, Yacov; Segev, Omri; Balan, Ayala

    2004-01-01

    Fluoride ion is a reversible inhibitor of human butyrylcholinesterase (HuBChE) that is a viable drug candidate against organophosphates (OPs) toxicity. Since large numbers of communities in many countries are occasionally exposed to relatively high amount of fluoride, its effect on the kinetics of inhibition of HuBChE by OPs was investigated. In saline phosphate, pH 7.4, fluoride in the lower millimolar range significantly slowed the inhibition of HuBChE by paraoxon, DFP, echothiophate, soman, sarin, and VX. The kinetics of the inhibition was found consistent with the formation of a reversible fluoride-HuBChE complex that is at least 25-fold less active towards phosphorylation or phosphonylation than the free enzyme. Heat inactivation experiments indicate that the binding of fluoride to HuBChE probably involves enhanced cross-domain interaction via hydrogen bonds formation that may decrease enzyme activity. In spite of distinct structural differences among the OP used, the dissociation constants of the fluoride-HuBChE reversible complex varied over a narrow range (K F , 0.31-0.70 mM); however, K F in human plasma increased to 2.75-3.40 mM. 19 F-NMR spectroscopy revealed that fluoride ion is complexed to plasma components, an observation that explains in part the apparent increase in K F . Results suggest that an estimate of the relative decrease in the rate of OPs sequestration in presence of fluoride can be obtained from the fraction of the free HuBChE (1 + [F]/K F ) -1 . Considering K F values in human plasma, it is concluded that the scavenging efficacy of OPs by HuBChE is not compromised by the normal concentration range of circulating fluoride ions

  7. Determination of GABA and vigabatrin in human plasma by a rapid and simple HPLC method: correlation between clinical response to vigabatrin and increase in plasma GABA.

    Science.gov (United States)

    Löscher, W; Fassbender, C P; Gram, L; Gramer, M; Hörstermann, D; Zahner, B; Stefan, H

    1993-03-01

    The novel antiepileptic drug vigabatrin (Sabril) acts by inhibiting degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), increasing the GABA concentrations in the brain. Because the GABA degrading enzyme GABA aminotransferase (GABA-T) is also present in peripheral tissues, including blood platelets, measurement of plasma GABA levels might be a useful indication of the pharmacological response to vigabatrin during therapeutic monitoring. However, because of the very low concentrations of GABA in plasma, the few methods available for plasma GABA analysis are time-consuming, difficult to perform and/or not selective enough because of potential interference with other plasma constituents. In the present study, a rapid, selective and sensitive amino acid analysis HPLC method has been developed for plasma GABA determination with fluorescence detection, using o-phthaldialdehyde as a precolumn derivatizing agent. By employing a 3 microns particle size reversed-phase column and a multi-step gradient system of two solvents, the very low endogenous concentration of GABA in human plasma could be reproducibly quantitated without interference of other endogenous compounds. Incubation of human plasma samples with GABA degrading enzyme(s) resulted in an almost total loss of the GABA peak, thus demonstrating the specificity of the method for GABA analysis. In addition to GABA and other endogenous amino acids, the HPLC method could be used to quantitate plasma levels of vigabatrin. Thus, this improved HPLC amino acid assay might be used to examine whether concomitant monitoring of plasma GABA and vigabatrin is useful for clinical purposes. This was examined in 20 epileptic patients undergoing chronic treatment with vigabatrin. The average plasma GABA level of these 20 patients did not differ significantly from non-epileptic controls. However, when epileptic patients were subdivided according to their clinical response to vigabatrin, vigabatrin responders

  8. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

  9. Simultaneous Estimation and Validation for Determination of Lamivudine and Zidovudine in Human Plasma By LCMS/MS Method

    Directory of Open Access Journals (Sweden)

    C. Parthiban

    2012-01-01

    Full Text Available A high performance liquid chromatographic method with mass detection was developed for determination of Lamivudine and Zidovudine in human plasma. Lamivudine and Zidovudine were extracted from an aliquot of human plasma using Solid Phase Extraction technique, and then injected into a liquid chromatograph equipped with a tandem mass spectrometry detector; quantitation was done by peak area ratio method. A weighted (1/Conc2 linear regression was performed to determine the concentration of analyte. This method demonstrates acceptable performance and is suitable for the determination of Lamivudine and Zidovudine in human plasma over the range of 25.291 to 4046.621 ng/mL and 23.357 to 4057.l4lng/mL respectively using Solid Phase Extraction Procedure. As all the values obtained were within the limits as specified, the method has been successfully used to analyse the human plasma containing Lamivudine and Zidovudine with good recoveries and proved to be robust.

  10. Quantitation of Metformin in Human Plasma and Urine by Hydrophilic Interaction Liquid Chromatography and Application to a Pharmacokinetic Study

    DEFF Research Database (Denmark)

    Nielsen, Flemming; Hougaard Christensen, Mette Marie; Brøsen, Kim

    2014-01-01

    : We describe an analytical method for the quantification of the widely used antihyperglycemic agent, metformin, in human plasma and urine. The separation was performed using isocratic hydrophilic interaction liquid chromatography on a Luna hydrophilic interaction liquid chromatography column (125...

  11. Metabolism of oxycodone in human hepatocytes from different age groups and prediction of hepatic plasma clearance

    Directory of Open Access Journals (Sweden)

    Timo eKorjamo

    2012-01-01

    Full Text Available Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1 and 10 µM was incubated with hepatocytes for 4 hours, and 1 µM oxycodone also with CYP3A inhibitor ketoconazole (1 µM. Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography-mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. In general, this in vitro to in vivo extrapolation method provides valuable information on the maturation of oxycodone metabolism that can be utilized in the design of clinical pharmacokinetic studies in infants and young children.

  12. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  13. Effect of storage duration on cytokine stability in human serum and plasma.

    Science.gov (United States)

    Vincent, Fabien B; Nim, Hieu T; Lee, Jacinta P W; Morand, Eric F; Harris, James

    2018-06-14

    Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology.

    Science.gov (United States)

    Ibsen, Stuart; Sonnenberg, Avery; Schutt, Carolyn; Mukthavaram, Rajesh; Yeh, Yasan; Ortac, Inanc; Manouchehri, Sareh; Kesari, Santosh; Esener, Sadik; Heller, Michael J

    2015-10-01

    The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Susceptibility of Mice to Trypanosoma evansi Treated with Human Plasma Containing Different Concentrations of Apolipoprotein L-1

    Science.gov (United States)

    Fanfa, Vinicius R.; Otto, Mateus A.; Gressler, Lucas T.; Tavares, Kaio C.S.; Lazzarotto, Cícera R.; Tonin, Alexandre A.; Miletti, Luiz C.; Duarte, Marta M.M.F.; Monteiro, Silvia G.

    2011-01-01

    The aim of this study was to test the susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1 (APOL1). For this experiment, a strain of T. evansi and human plasma (plasmas 1, 2, and 3) from 3 adult males clinically healthy were used. In vivo test used 50 mice divided in 5 groups (A to E) with 10 animals in each group. Animals of groups B to E were infected, and then treated with 0.2 ml of human plasma in the following outline: negative control (A), positive control (B), treatment with plasma 1 (C), treatment with plasma 2 (D), and treatment with plasma 3 (E). Mice treated with human plasma showed an increase in longevity of 40.9±0.3 (C), 20±9.0 (D) and 35.6±9.3 (E) days compared to the control group (B) which was 4.3±0.5 days. The number of surviving mice and free of the parasite (blood smear and PCR negative) at the end of the experiment was 90%, 0%, and 60% for groups C, D, and E, respectively. The quantification of APOL1 was performed due to the large difference in the treatments that differed in the source plasma. In plasmas 1, 2, and 3 was detected the concentration of 194, 99, and 115 mg/dl of APOL1, respectively. However, we believe that this difference in the treatment efficiency is related to the level of APOL1 in plasmas. PMID:22355213

  16. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; Hickman, S; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  17. The preparation of albumin as a biological drug from human plasma by fiber filtration

    Directory of Open Access Journals (Sweden)

    Mousavi Hosseini K

    2011-08-01

    Full Text Available "nBackground: In recent years, consumption of whole-blood for the treatment of patients has decreased but use of biological plasma-derived medicines such as albumin, immunoglobulin and coagulation factors have increased instead. Paying attention to albumin molecular structure is important for its isolation from human plasma. Albumin is a single-chain protein consisting of about 585 amino acids and a molecular weight of 66500 Daltons. Albumin is a stable molecule and it is spherical in shape. There are different methods for human albumin preparation. Considering the large consumption of this biological drug in clinical settings, methods with fewer steps in production line are of big advantage in saving time and manufacturing more products."n "nMethods: In this project, we prepared human albumin using hollow fiber cartridges in order to omit the rework on fraction V+VI. Human albumin is usually produced by the application of cold ethanol method, where albumin is obtained from fraction V by doing a rework on fraction V+VI to separate fraction V."n "nResults: In the current work, human albumin was prepared from fraction V+VI by the help of hollow fiber cartridges. With a concentration of 20%, the obtained albumin had 96.5% of monomer and 3.5% of polymer and polymer aggregate."n "nConclusion: Comparing the obtained human albumin with a number of commercial human albumin samples by the use of SDS-page, the results were satisfactory regarding the 3.5 percent polymer and aggregate rate for the prepared albumin.

  18. HPLC/Fluorometric Detection of Carvedilol in Real Human Plasma Samples Using Liquid-Liquid Extraction.

    Science.gov (United States)

    Yilmaz, Bilal; Arslan, Sakir

    2016-03-01

    A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify carvedilol in human plasma using an isocratic system with fluorescence detection. The method included a single-step liquid-liquid extraction with diethylether and ethylacetate mixture (3 : 1, v/v). HPLC separation was carried out by reversed-phase chromatography with a mobile phase composed of 20 mM phosphate buffer (pH 7)-acetonitrile (65 : 35, v/v), pumped at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 240 nm (excitation) and 330 nm (emission). The calibration curve for carvedilol was linear from 10 to 250 ng/mL. Intra- and interday precision values for carvedilol in human plasma were plasma averaged out to 91.8%. The limits of detection and quantification of carvedilol were 3.0 and 10 ng/mL, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 25 mg carvedilol. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma

    Directory of Open Access Journals (Sweden)

    Beata Olas

    2016-01-01

    Full Text Available Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation and the generation of superoxide anion (O2-∙ in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2 or H2O2/Fe (a donor of hydroxyl radicals. The tested fraction of H. rhamnoides (0.5– 50 µg/mL; the incubation time: 15 and 60 min inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2-∙ in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL. The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases.

  20. Proteomic Biomarker Discovery in 1000 Human Plasma Samples with Mass Spectrometry.

    Science.gov (United States)

    Cominetti, Ornella; Núñez Galindo, Antonio; Corthésy, John; Oller Moreno, Sergio; Irincheeva, Irina; Valsesia, Armand; Astrup, Arne; Saris, Wim H M; Hager, Jörg; Kussmann, Martin; Dayon, Loïc

    2016-02-05

    The overall impact of proteomics on clinical research and its translation has lagged behind expectations. One recognized caveat is the limited size (subject numbers) of (pre)clinical studies performed at the discovery stage, the findings of which fail to be replicated in larger verification/validation trials. Compromised study designs and insufficient statistical power are consequences of the to-date still limited capacity of mass spectrometry (MS)-based workflows to handle large numbers of samples in a realistic time frame, while delivering comprehensive proteome coverages. We developed a highly automated proteomic biomarker discovery workflow. Herein, we have applied this approach to analyze 1000 plasma samples from the multicentered human dietary intervention study "DiOGenes". Study design, sample randomization, tracking, and logistics were the foundations of our large-scale study. We checked the quality of the MS data and provided descriptive statistics. The data set was interrogated for proteins with most stable expression levels in that set of plasma samples. We evaluated standard clinical variables that typically impact forthcoming results and assessed body mass index-associated and gender-specific proteins at two time points. We demonstrate that analyzing a large number of human plasma samples for biomarker discovery with MS using isobaric tagging is feasible, providing robust and consistent biological results.

  1. Determination of Flurbiprofen in Human Plasma by High-Performance Liquid Chromatography.

    Science.gov (United States)

    Yilmaz, Bilal; Erdem, Ali Fuat

    2015-10-01

    A simple high-performance liquid chromatography method has been developed for determination of flurbiprofen in human plasma. The method was validated on an Ace C18 column using UV detection. The mobile phase was acetonitrile-0.05 M potassium dihydrogen phosphate solution (60:40, v/v) adjusted to pH 3.5 with phosphoric acid. The calibration curve was linear between the concentration range of 0.10-5.0 μg/mL. Intra- and inter-day precision values for flurbiprofen in plasma were flurbiprofen from human plasma were between 93.0 and 98.9%. The limits of detection and quantification of flurbiprofen were 0.03 and 0.10 μg/mL, respectively. In addition, this assay was applied to determine the pharmacokinetic parameters of flurbiprofen in six healthy Turkish volunteers who had been given 100 mg flurbiprofen. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  3. Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry.

    Science.gov (United States)

    Din, Li; Li, Limin; Tao, Ping; Yang, Jin; Zhang, Zhengxing

    2002-02-05

    A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.

  4. A quick and easy high performance liquid chromatography method for evaluation of cefixime in human plasma

    Directory of Open Access Journals (Sweden)

    Hossein Danafar

    2015-12-01

    Full Text Available Cefixime is essential member of orally energetic third generation cephalosporin and has tremendous activity aligned with many pathogens. The virtual bioavailability of a newly industrial dispersible tablet as compared with a recognized identified formulation.  A simple and available reversed-phase HPLC method with UV detection has been urbanized and validate for cefixime evaluate in human plasma using a C18 analytical column and a mobile phase of tetrabutylammonium hydroxide (pH 6.5-acetonitrile (3:1 v/v. The detection wavelength was 280 nm. To method observed major linear response-concentration association all through the cefixime concentration range of 15-100 ng/ml, with the average accuracy within-run and between-run values of 97.29% and 99.27%. The average drug recovery from plasma was 98.2% throughout the linear concentration range. The limits of detection (LOD and quantitation (LOQ of the method were 5 and 15 ng/ml, respectively. The method is quick, easy, very steady and precise for the partition, assignment, evaluation of cefixime in human plasma.

  5. Large-scale production and properties of human plasma-derived activated Factor VII concentrate.

    Science.gov (United States)

    Tomokiyo, K; Yano, H; Imamura, M; Nakano, Y; Nakagaki, T; Ogata, Y; Terano, T; Miyamoto, S; Funatsu, A

    2003-01-01

    An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large-scale manufacturing process to obtain plasma-derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. FVII was purified from human cryoprecipitate-poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca2+-dependent anti-FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane-15 nm was partially converted to FVIIa by autoactivation on an anion-exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca2+ for 18 h at 10 degrees C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 mm citrate, pH 6.9, containing 13 mm glycine and 240 mm NaCl, the FVIIa preparation was supplemented with 2.5% human albumin (which was first pasteurized at 60 degrees C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 degrees C for 96 h in a water bath. Total recovery of FVII from 15 000 l of plasma was approximately 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIabeta and FVIIagamma). The specific activity of the FVIIa was approximately 40 U/ micro g. Furthermore, virus-spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry-heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. We established a large-scale manufacturing process of human plasma

  6. Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

    Science.gov (United States)

    Poulsen, Ebbe Toftgaard; Pedersen, Kata Wolff; Marzeda, Anna Maria; Enghild, Jan J

    2017-02-14

    The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca 2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

  7. Effective method for the detection of piroxicam in human plasma using HPLC.

    Science.gov (United States)

    Calvo, Adriana Maria; Prado, Mariel Tavares de Oliveira; Dionísio, Thiago José; Marques, Maria Paula; Brozoski, Daniel Thomas; Lanchote, Vera Lúcia; Faria, Flávio Augusto Cardoso; Santos, Carlos Ferreira

    2016-05-20

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5'-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC) after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo - USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg) and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5'-hydroxypiroxicam was only detected in 2 out of 10 volunteers.

  8. Direct radioimmunoassay of proinsulin and insulin in human plasma by the chromatographic technique

    Energy Technology Data Exchange (ETDEWEB)

    Megahed, Y M; Abdel-Wahab, M F; El-Shawarbie, K; Sadek, S; Amer, M S [Atomic Energy Establishment, Cairo (Egypt). Radioisotope Department; Ain Shams Univ., Cairo (Egypt). Faculty of Medicine)

    1976-04-01

    Specific method for direct radioimmunoassay of IRP and IRI separately in human plasma has been described. The method is used for extraction of total insulin and separation of IRP from IRI by paper chromatography to be assayed separately. The separation of the two components is identified and confirmed by column chromatography, paper chromatography and ultraviolet spectral analysis in comparison with the standard compounds. 134 plasma samples of different cases were investigated for the determination of IRI, IRP and IRT. Out of these 39 were normals, 16 normal obes, 21 juvinil diabetes, 18 overt adult diabetes, 10 recent adult diabetes, 12 hypothyroidism and 18 bilharzial hepatosplenomegaly. They were used to evaluate the test levels in comparison with blood sugar concentration.

  9. Culture of normal human blood cells in a diffusion chamber system II. Lymphocyte and plasma cell kinetics

    International Nuclear Information System (INIS)

    Chikkappa, G.; Carsten, A.L.; Chanana, A.D.; Cronkite, E.P.

    1979-01-01

    Normal human blood leukocytes were cultured in Millipore diffusion chambers implanted into the peritoneal cavities of irradiated mice. The evaluation of survival and proliferation kinetics of cells in lymphyocytic series suggested that the lymphoid cells are formed from transition of small and/or large lymphocytes, and the lymphoblasts from the lymphoid cells. There was also evidence indicating that some of the cells in these two compartments are formed by proliferation. The evaluation of plasmacytic series suggested that the plasma cells are formed from plasmacytoid-lymphocytes by transition, and the latter from the transition of lymphocytes. In addition, relatively a small fraction of cells in these two compartments are formed by proliferation. mature plasma cells do not and immature plasma cells do proliferate. Estimation of magnitude of plasma cells formed in the cultures at day 18 indicated that at least one plasma cell is formed for every 6 normal human blood lymphocytes introduced into the culture

  10. The effects of therapeutic concentrations ofamisulpride andrisperidone on human plasma lipid peroxidation – invitro studies

    Directory of Open Access Journals (Sweden)

    Anna Dietrich-Muszalska

    2011-09-01

    Full Text Available Introduction: Antipsychotics may in different ways affect the oxidative stress measured by plasma lipid peroxidation. Probably some of them may intensify the oxidative balance disturbances occurring in schizophrenia. The effects of amisulpride and risperidone on redox processes are not known sufficiently yet. Aim of the study: Establishment of the effects of amisulpride and risperidone on human plasma lipid peroxidation measured by determination of the level of thiobarbituric acid-reactive substances (TBARS, in vitro. Material and methods: Blood for the studies was collected from healthy volunteers (aged 24-26 years for ACD solution. Active substances of the examined drugs were dissolved in 0.01% dimethylsulfoxide (DMSO to the final concentrations (of amisulpride 578 ng/ml and risperidone 64 ng/ml and incubated with plasma for 1 and 24 hours at 37ºC. For each experiment the control samples of plasma with DMSO (without the drug were performed. The lipid peroxidation level was measured in plasma by determining the TBARS concentration, using the spectrophotometric method (acc. to Rice-Evans, 1991. The results were analysed using the following statistical methods: the paired Student t-test and ANOVA II variance analysis and NIR test (StatSoft Inc., Statistica v. 6.0. Results: The ANOVA II variance analysis indicated significant differences in the effects of both drugs on TBARS level (F=4.26; df=2, p0.05. Conclusion: Amisulpride and risperidone in concentrations corresponding to doses recommended for treatment of acute episode of schizophrenia do not induce oxidative stress measured by lipid peroxidation. Unlike risperidone, amisulpride exhibits antioxidative effects.

  11. Long term effects on human plasma lipoproteins of a formulation enriched in butter milk polar lipid

    Directory of Open Access Journals (Sweden)

    Nilsson Åke

    2009-10-01

    Full Text Available Abstract Background Sphingolipids (SL, in particular sphingomyelin (SM are important components of milk fat polar lipids. Dietary SM inhibits cholesterol absorption in rats (Nyberg et al. J Nutr Biochem. 2000 and SLs decrease both cholesterol and TG concentrations in lipid- and cholesterol fed APOE*3Leiden mice (Duivenvoorden et al. Am J Clin Nutr. 2006. This human study examines effects of a butter milk formulation enriched in milk fat globule membrane material, and thereby in SLs, on blood lipids in healthy volunteers. In a four week parallel group study with 33 men and 15 women we examined the effects of an SL-enriched butter milk formulation (A and an equivalent control formulation (B on plasma lipid levels. Plasma concentrations of HDL and LDL cholesterol, triacylglycerols (TG, apolipoproteins AI and B, and lipoprotein (a were measured. The daily dose of SL in A was 975 mg of which 700 mg was SM. The participants registered food and drink intake four days before introducing the test formula and the last four days of the test period. Results A daily increase of SL intake did not significantly influence fasting plasma lipids or lipoproteins. In group B TG, cholesterol, LDL, HDL and apolipoprotein B concentrations increased, however, but not in group A after four weeks. The difference in LDL cholesterol was seen primarily in women and difference in TG primarily in men. No significant side effects were observed. Conclusion The study did not show any significant decrease on plasma lipids or lipoprotein levels of an SL-enriched formulation containing 2-3 times more SL than the normal dietary intake on cholesterol, other plasma lipids or on energy intake. The formulation A may, however, have counteracted the trend towards increased blood lipid concentrations caused by increased energy intake that was seen with the B formulation.

  12. Effects of propranolol and pindolol on plasma ANP levels in humans at rest and during exercise.

    Science.gov (United States)

    Bouissou, P; Galen, F X; Richalet, J P; Lartigue, M; Devaux, F; Dubray, C; Atlan, G

    1989-08-01

    In attempt to elucidate whether the beta-adrenoceptor is involved in the control of atrial natriuretic peptide (ANP) secretion, plasma immunoreactive ANP level was measured at rest, in recumbent and upright positions, and during graded maximal ergocycle exercise in nine healthy male subjects (23 +/- 0.5 years of age) treated for 3 days with nonselective beta-blockers propranolol (150 mg/day) or pindolol (15 mg/day) or with placebo. The effects of beta-blockers, which differ by their hemodynamic actions at rest because of the intrinsic sympathomimetic activity of pindolol, were compared. Maximal O2 consumption (VO2max) during beta-blockade was not significantly different from the placebo value. Resting heart rate was not affected by pindolol treatment but was decreased with propranolol (-10 beats/min). Both beta-blockers caused a reduction in heart rate at all the exercise intensities. Mean blood pressure was not affected by beta-blockade at rest but was significantly reduced during exercise. During placebo treatment, plasma ANP increased in response to exercise intensities greater than 65% of VO2max. At 100% VO2max plasma ANP was nearly doubled (101.5 +/- 14 pg/ml) compared with the basal value in upright position (56.6 +/- 15 pg/ml). beta-Blockade caused a marked elevation in plasma ANP at all the levels of activity. Despite different hemodynamic responses to pindolol and propranolol, both beta-blockers produced similar increases in the basal level of plasma ANP. These rises were maintained in the course of exercise tests, and no significant difference was found between propranolol and pindolol. We conclude that beta-adrenoceptor mechanisms are not directly responsible for tonic and exercise-induced ANP secretion in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Levels of contamination for various pollutants present in Belgian human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Wouwe, N. Van; Goeyens, L. [Scientific Inst. of Public Health, Brussels (Belgium); Covaci, A. [Toxicological Center, Univ. of Antwerp, Wilrijk (Belgium); Kannan, K. [Wadsworth Center, New York State Dept. of Health, Albany, NY (United States); Gordon, J.; Chu, A. [Xenobiotic Detection System Inc., Durham, NC (United States); Eppe, G.; Pauw, E. De [Center of Analysis of residues in Traces (CART), Univ. of Liege (Belgium)

    2004-09-15

    During the last century, numerous compounds, such as polychlorinated biphenyls (PCBs) or organochlorine pesticides (OCPs), were banned because of their bioaccumulative and toxic properties, while other compounds, such as polybrominated diphenyl ethers (PBDEs), appeared on the market and consequently in the environment. The experiences gained from accidents involving PBBs, PCBs or PCDD/Fs are useful to conduct scientific investigations focused on preventing similar catastrophies with the newly introduced compounds. Several studies have reported potential increase in the concentration of PBDEs in food and wildlife. Monitoring the levels of toxic chemicals is therefore useful to understand the exposure pathways, sources and trends. The aim of the paper is to present actual contamination's levels of various pollutants in human plasma from Belgium. Several classes of pollutants, such PCDD/Fs, PCBs and OCPs were determined in 20 human plasmas. In addition, perfluorooctanesulfonate (PFOS) and related fluorochemicals, which are of current concern, were measured. Although anticipated, concentrations of PBDEs in the same samples were not yet determined. Through this study, a good approximation of the contamination level in Belgian human is given, allowing thus comparison with concentrations observed in other countries.

  14. Plasma vs heart tissue concentration in humans - literature data analysis of drugs distribution.

    Science.gov (United States)

    Tylutki, Zofia; Polak, Sebastian

    2015-03-12

    Little is known about the uptake of drugs into the human heart, although it is of great importance nowadays, when science desires to predict tissue level behavior rather than to measure it. Although the drug concentration in cardiac tissue seems a better predictor for physiological and electrophysiological changes than its level in plasma, knowledge of this value is very limited. Tissue to plasma partition coefficients (Kp) come to rescue since they characterize the distribution of a drug among tissues as being one of the input parameters in physiologically based pharmacokinetic (PBPK) models. The article reviews cardiac surgery and forensic medical studies to provide a reference for drug concentrations in human cardiac tissue. Firstly, the focus is on whether a drug penetrates into heart tissue at a therapeutic level; the provided values refer to antibiotics, antifungals and anticancer drugs. Drugs that directly affect cardiomyocyte electrophysiology are another group of interest. Measured levels of amiodarone, digoxin, perhexiline and verapamil in different sites in human cardiac tissue where the compounds might meet ion channels, gives an insight into how these more lipophilic drugs penetrate the heart. Much data are derived from postmortem studies and they provide insight to the cardiac distribution of more than 200 drugs. The analysis depicts potential problems in defining the active concentration location, what may indirectly suggest multiple mechanisms involved in the drug distribution within the heart. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

    Science.gov (United States)

    Dicke, J M; Verges, D; Kelley, L K; Smith, C H

    1993-01-01

    Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

  16. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma

    Directory of Open Access Journals (Sweden)

    Watters CM

    2016-04-01

    Full Text Available Chase M Watters,1,2 Tarea Burton,1 Dickson K Kirui,1 Nancy J Millenbaugh1 1Maxillofacial Injury and Disease Department, Naval Medical Research Unit San Antonio, Joint Base San Antonio-Fort Sam Houston, TX, USA; 2Wound Infections Department, Naval Medical Research Center, Silver Spring, MD, USA Abstract: Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas a-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve

  17. Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

    Science.gov (United States)

    Barter, P J; Hopkins, G J; Gorjatschko, L

    1984-01-17

    A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

  18. Twenty-year Risk of Prostate Cancer Death by Midlife Prostate-specific Antigen and a Panel of Four Kallikrein Markers in a Large Population-based Cohort of Healthy Men.

    Science.gov (United States)

    Sjoberg, Daniel D; Vickers, Andrew J; Assel, Melissa; Dahlin, Anders; Poon, Bing Ying; Ulmert, David; Lilja, Hans

    2018-06-01

    Prostate-specific antigen (PSA) screening reduces prostate cancer deaths but leads to harm from overdiagnosis and overtreatment. To determine the long-term risk of prostate cancer mortality using kallikrein blood markers measured at baseline in a large population of healthy men to identify men with low risk for prostate cancer death. Study based on the Malmö Diet and Cancer cohort enrolling 11 506 unscreened men aged 45-73 yr during 1991-1996, providing cryopreserved blood at enrollment and followed without PSA screening to December 31, 2014. We measured four kallikrein markers in the blood of 1223 prostate cancer cases and 3028 controls. Prostate cancer death (n=317) by PSA and a prespecified statistical model based on the levels of four kallikrein markers. Baseline PSA predicted prostate cancer death with a concordance index of 0.86. In men with elevated PSA (≥2.0ng/ml), predictive accuracy was enhanced by the four-kallikrein panel compared with PSA (0.80 vs 0.73; improvement 0.07; 95% confidence interval 0.04, 0.10). Nearly half of men aged 60+ yr with elevated PSA had a four-kallikrein panel score of four-kallikrein panel score of ≥7.5% had a 13% risk of prostate cancer death at 15 yr. A prespecified statistical model based on four kallikrein markers (commercially available as the 4Kscore) reclassified many men with modestly elevated PSA, to have a low long-term risk of prostate cancer death. Men with elevated PSA but low scores from the four-kallikrein panel can be monitored rather than being subject to biopsy. Men with elevated prostate-specific antigen (PSA) are often referred for prostate biopsy. However, men with elevated PSA but low scores from the four-kallikrein panel can be monitored rather than being subject to biopsy. Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  19. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

    DEFF Research Database (Denmark)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J

    2017-01-01

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

  20. Quantitative determination of famotidine in human maternal plasma, umbilical cord plasma and urine using high-performance liquid chromatography - mass spectrometry

    Science.gov (United States)

    Wang, Xiaoming; Rytting, Erik; Abdelrahman, Doaa R.; Nanovskaya, Tatiana N.; Hankins, Gary D.V.; Ahmed, Mahmoud S.

    2013-01-01

    The liquid chromatography with electrospray ionization mass spectrometry for the quantitative determination of famotidine in human urine, maternal and umbilical cord plasma was developed and validated. The plasma samples were alkalized with ammonium hydroxide and extracted twice with ethyl acetate. The extraction recovery of famotidine in maternal and umbilical cord plasma ranged from 53% to 64% and 72% to 79%, respectively. Urine samples were directly diluted with the initial mobile phase then injected into the HPLC system. Chromatographic separation of famotidine was achieved by using a Phenomenex Synergi™ Hydro-RP™ column with a gradient elution of acetonitrile and 10 mM ammonium acetate aqueous solution (pH 8.3, adjusted with ammonium hydroxide). Mass Spectrometric detection of famotidine was set in the positive mode and used a selected ion monitoring method. Carbon-13-labeled famotidine was used as internal standard. The calibration curves were linear (r2> 0.99) in the concentration ranges of 0.631-252 ng/mL for umbilical and maternal plasma samples, and of 0.075-30.0 μg/mL for urine samples. The relative deviation of method was less than 14% for intra- and inter-day assays, and the accuracy ranged between 93% and 110%. The matrix effect of famotidine in human urine, maternal and umbilical cord plasma is less than 17%. PMID:23401067

  1. Determination of moxifloxacin in human plasma, plasma ultrafiltrate, and cerebrospinal fluid by a rapid and simple liquid chromatography- tandem mass spectrometry method.

    Science.gov (United States)

    Pranger, Arianna D; Alffenaar, Jan-Willem C; Wessels, A Mireille A; Greijdanus, Ben; Uges, Donald R A

    2010-04-01

    Moxifloxacin (MFX) is a useful agent in the treatment of multi-drug-resistant tuberculosis (MDR-TB). At Tuberculosis Centre Beatrixoord, a referral center for tuberculosis in the Netherlands, approximately 36% of the patients have received MFX as treatment. Based on the variability of MFX AUC, the variability of in vitro susceptibility to MFX of M. tuberculosis, and the variability of penetration into sanctuary sites, measuring the concentration of MFX in plasma and cerebrospinal fluid (CSF) could be recommended. Therefore, a rapid and validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyzing method with a simple pretreatment procedure was developed for therapeutic drug monitoring of MFX in human plasma and CSF. Because of the potential influence of protein binding on efficacy, we decided to determine both bound and unbound (ultrafiltrate) fraction of MFX. The calibration curves were linear in the therapeutic range of 0.05 to 5.0 mg/L plasma and CSF with CV in the range of -5.4% to 9.3%. MFX ultrafiltrate samples could be determined with the same method setup for analysis of MFX in CSF. The LC-MS-MS method developed in this study is suitable for monitoring MFX in human plasma, plasma ultrafiltrate, and CSF.

  2. Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Kravtsova, Oxana Yu; Paramonov, Sergey A; Vasilevich, Natalya I; Kazyulkin, Denis N; Vlasova, Ekaterina; Engsig, Michael

    2013-12-01

    A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high-performance liquid chromatography-tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative-ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C18(2) column (3 μM, 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10-5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precisions were flomoxef revealed that it could be successfully analyzed at 4 ºС over 24 h, but it was unstable in solutions at room temperature during short-term storage (4 h) and several freeze-thaw cycles. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Sunscreens in human plasma and urine after repeated whole-body topical application

    DEFF Research Database (Denmark)

    Janjua, N.R.; Kongshoj, B.; Andersson, A.M.

    2008-01-01

    . For all three compounds, only sporadic measurements of percutaneous absorption and excretion after topical application in humans have been described. Methods In this study, 32 healthy volunteers, 15 young males and 17 postmenopausal females, were exposed to daily whole-body topical application of 2 mg...... the first application, all three sunscreens were detectable in plasma. The maximum median plasma concentrations were 187 ng/mL BP-3, 16 ng/mL 4-MBC and 7 ng/mL OMC for females and 238 ng/mL BP-3, 18 ng/mL 4-MBC and 16 ng/mL OMC for men. In the females, urine levels of 44 ng/mL BP-3 and 4 ng/mL of 4-MBC...... and 6 ng/mL OMC were found, and in the males, urine levels of 81 ng/mL BP-3, 4 ng/mL of 4-MBC and OMC were found. In plasma, the 96-h median concentrations were higher compared with the 24-h concentrations for 4-MBC and OMC in men and for BP-3 and 4-MBC in females Udgivelsesdato: 2008/4...

  4. Human plasma lipid modulation in schistosomiasis mansoni depends on apolipoprotein E polymorphism.

    Directory of Open Access Journals (Sweden)

    Caíque Silveira Martins da Fonseca

    Full Text Available Schistosomiasis mansoni is a parasitic liver disease, which causes several metabolic disturbances. Here, we evaluate the influence of Apolipoprotein E (APOE gene polymorphism, a known modulator of lipid metabolism, on plasma lipid levels in patients with hepatosplenic schistosomiasis.Blood samples were used for APOE genotyping and to measure total cholesterol (TC, LDL-C, HDL-C and triglycerides. Schistosomiasis patients had reduced TC, LDL-C and triglycerides (25%, 38% and 32% lower, respectively; Pε3>ε4 was absent in patients (ε2 or ε4>ε3, and the increase in HDL-C of ε2 or ε4 patients compared to ε3 patients was not seen in the control groups.We confirm that human schistosomiasis causes dyslipidemia and report for the first time that certain changes in plasma lipid and lipoprotein levels depend on APOE gene polymorphism. Importantly, we also concluded that S. mansoni disrupts the expected regulation of plasma lipids by the different ApoE isoforms. This finding suggests ways to identify new metabolic pathways affected by schistosomiasis and also potential molecular targets to treat associated morbidities.

  5. Determination of linsidomine in human plasma by tandem LC-MS with ESI.

    Science.gov (United States)

    Sutherland, F C; de Jager, A D; Swart, K J; Hundt, H K; Scanes, T; Hundt, A F

    2000-04-01

    A sensitive method for the determination of linsidomine in plasma was developed, using high-performance liquid chromatographic (HPLC) separation with tandem mass spectrometric detection. Linsidomine was derivatised with propyl chloroformate and extracted with tert-butyl methyl ether/1,2-dichloroethane (55:45, v/v), back-extracted into HCl (0.01 M) followed by alkalinisation and back-extraction into ether; the final ether extract evaporated, reconstituted in mobile phase and then separated on a Phenomenex Luna C18 (2) 5 micron 2.1 x 150 mm column with a mobile phase consisting of methanol water formic acid (98/100%) (400:600:0.05, v/v/v) at a flow-rate of 0.4 ml min(-1). Detection was achieved by a Finnigan MAT mass spectrometer (LCQ) at unit resolution in the selected reaction monitoring (SRM) mode monitoring the transition of the protonated molecular ion m/z 257.0 to the product ion m/z 86.0. The mean recovery for linsidomine was 51% with a lower limit of quantification of 0.70 ng/ml using 1 ml plasma for extraction. This LC-MS/MS method for the determination of linsidomine in human plasma allows for better specificity and a higher sample throughput than the traditional LC-UV methods. It also demonstrates the profound effect that the composition of acidic modifiers and matrix constituents can have on the electrospray ionisation (ESI) of the analyte.

  6. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

    Directory of Open Access Journals (Sweden)

    Sylvie Faucher

    Full Text Available BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127 and common gamma (CD132 chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127 is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

  7. Identification of drug metabolites in human plasma or serum integrating metabolite prediction, LC-HRMS and untargeted data processing

    NARCIS (Netherlands)

    Jacobs, P.L.; Ridder, L.; Ruijken, M.; Rosing, H.; Jager, N.G.L.; Beijnen, J.H.; Bas, R.R.; Dongen, W.D. van

    2013-01-01

    Background: Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction,

  8. Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

    Science.gov (United States)

    Anitua, Eduardo; de la Fuente, María; Ferrando, Marcos; Quintana, Fernando; Larreategui, Zaloa; Matorras, Roberto; Orive, Gorka

    2016-11-01

    To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    Science.gov (United States)

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  10. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  11. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    Science.gov (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

  12. Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

    2015-11-27

    This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro

    International Nuclear Information System (INIS)

    Roberts, D.C.K.; Miller, N.E.; Price, S.G.L.; Crook, D.; Cortese, C.; Ville, A. La; Masana, L.; Lewis, B.

    1985-01-01

    A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4 0 C in human serum (d > 1.215) that had been prelabelled with [4- 14 C]cholesteryl oleate or [1,2- 3 H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. .pmol -1 (46d.p.m. .ng -1 ) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction. (author)

  14. The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI Attenuates Elastase-Induced Emphysema in Mice

    Directory of Open Access Journals (Sweden)

    Bruno Tadeu Martins-Olivera

    2016-01-01

    Full Text Available Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD. However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group or saline (SAL group and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups. At day 28, the following analyses were performed: (I lung mechanics, (II exhaled nitric oxide (ENO, (III bronchoalveolar lavage fluid (BALF, and (IV lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

  15. What have we learned about the kallikrein-kinin and renin-angiotensin systems in neurological disorders?

    Institute of Scientific and Technical Information of China (English)

    Maria; da; Graa; Naffah-Mazzacoratti; Telma; Luciana; Furtado; Gouveia; Priscila; Santos; Rodrigues; Simōes; Sandra; Regina; Perosa

    2014-01-01

    The kallikrein-kinin system(KKS) is an intricate endogenous pathway involved in several physiological and pathological cascades in the brain. Due to the pathological effects of kinins in blood vessels and tissues, their formation and degradation are tightly controlled. Their components have been related to several central nervous system diseases such as stroke, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy and others. Bradykinin and its receptors(B1R and B2R) may have a role in the pathophysiology of certain central nervous system diseases. It has been suggested that kinin B1R is up-regulated in pathological conditions and has a neurodegenerative pattern, while kinin B2R is constitutive and can act as a neuroprotective factor in many neurological conditions. The renin angiotensin system(RAS) is an important blood pressure regulator and controls both sodium and water intake. AngⅡ is a potent vasoconstrictor molecule and angiotensin converting enzyme is the major enzyme responsible for its release. AngⅡ acts mainly on the AT1 receptor, with involvement in several systemic and neurological disorders. Brain RAS has been associated with physiological pathways, but is also associated with brain disorders. This review describes topics relating to the involvement of both systems in several forms of brain dysfunction and indicates components of the KKS and RAS that have been used as targets in several pharmacological approaches.

  16. Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

    Science.gov (United States)

    Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

    2017-10-01

    The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  17. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Crosby, S.R.; Stewart, M.F.; Ratcliffe, J.G.; White, A.

    1988-01-01

    An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful

  18. Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

    Science.gov (United States)

    Reisner, P D; Brandt, P C; Vanaman, T C

    1997-01-01

    It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

  19. Ultra-performance liquid chromatographic determination of tocopherols and retinol in human plasma.

    Science.gov (United States)

    Bell, Edward C; John, Mathew; Hughes, Rodney J; Pham, Thu

    2014-10-01

    A rapid, selective and sensitive ultra-performance liquid chromatography method has been developed for the detection and quantification of tocopherols and retinol in human plasma. Alpha-tocopherol, gamma-tocopherol and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295/330 nm and 325/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed-phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl hybrid C18 column. The retention times for retinol, retinol acetate, gamma-tocopherol and alpha-tocopherol are 1.6, 1.8, 3.9 and 4.3 min, respectively. The limits of quantification for retinol, gamma-tocopherol and alpha-tocopherol were 0.02, 0.02 and 0.1 µg/mL, respectively. The assay method is suitable for the analysis of tocopherols and retinol in human plasma. The method may be applied following the ingestion of foods fortified with these fat-soluble vitamins. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Heterogeneity of human plasma insulin: techniques for separating immunoreactive components and their determination by radioimmunoassay

    International Nuclear Information System (INIS)

    Souza, Iracelia Torres de Toledo e

    1977-01-01

    When human plasma is filtered on Sephadex G-SO fine, insulin immunoreactivity is recovered in two peaks: 'big insulin', the higher molecular weight component and 'little insulin', the lower molecular component, having elution volumes that correspond to those of porcine proinsulin 125 I and porcine insulin 125 I respectively. The presence of another form of immunoreactive insulin 'big big insulin' was detected from an insuloma suspect and its elution pattern corresponding to serum albumin. The eluates correspondent to 'big' and 'little' insulin as well as 'big big' component were assayed by radioimmunoassay using crystalline human insulin as a standard, porcine insulin 125 tracer and anti insulin serum. The antibody, raised in guinea-pigs, was sensitive and potent being adequate for the assay. The reactivity of insulin and proinsulin was tested against the antibody. The relative proportions of several components of total immunoreactive insulin in plasma were studied in basal conditions in five normal subjects and in the patient JSC with pancreatic insulin-secreting tumor as well as after glucose stimuli in all tolbutamide in JSC. (author)

  1. A New Method to Simultaneously Quantify the Antioxidants: Carotenes, Xanthophylls, and Vitamin A in Human Plasma

    Directory of Open Access Journals (Sweden)

    Mariel Colmán-Martínez

    2016-01-01

    Full Text Available A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z-β-carotene isomers, and 1 fat-soluble vitamin (retinol were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and 330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight volunteers. Results. Limits of detection were between 0.1 μg/mL for lycopene and astaxanthin and 1.3 μg/mL for 15-Z-β-carotene. Recoveries were ranged between 89% and 113% for α-carotene and astaxanthin, respectively. Accuracy was between 90.7% and 112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy, precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies.

  2. Simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma using liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Gupta, V K; Jain, Rajeev; Lukram, Ojitkumar; Agarwal, Shilpi; Dwivedi, Ashish

    2011-01-15

    A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma. The solid-phase extraction technique was used for the extraction of ramipril, ramiprilat and telmisartan from human plasma. Trandolaprilat and hydrochlorothiazide were used as the internal standards (ISs). Chromatography was performed on a Hypurity C18, 5 μm, 50 mm × 4.6mm column, with the mobile phase consisting of ammonium acetate and acetonitrile (in a 20:80 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-nanogram levels. The method was validated and the lower limit of quantification for ramipril, ramiprilat and telmisartan was found to be 0.1 ng mL(-1), 0.1 ng mL(-1) and 2 ng mL(-1), respectively. The mean recovery for ramipril, ramiprilat and telmisartan ranged from 90.1 to 104.1%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of ramipril, ramiprilat and telmisartan using two different ISs in a single experiment for bioequivalence studies, with a chromatographic run time of 1.5 min only. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma

    Science.gov (United States)

    Omar, Mahmoud A.; Mohamed, Abdel-Maaboud I.; Derayea, Sayed M.; Hammad, Mohamed A.; Mohamed, Abobakr A.

    2018-04-01

    A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH 9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430 nm after excitation at 337 nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0-400.0) ng/mL. Detection and quantification limits were found to be (0.52-3.88) and (1.59-11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12 ± 1.34-100.66 ± 0.57, n = 3). A proposal for the reaction mechanism was presented.

  4. Surface modifying of microporous PTFE capillary for bilirubin removing from human plasma and its blood compatibility

    International Nuclear Information System (INIS)

    Jin Gu; Yao Qizhi; Zhang Shanzi; Zhang Lei

    2008-01-01

    In this study, human serum albumin (HSA) was covalently immobilized onto the inner surface of microporous poly(tetrafluoroethylene) (MPTFE) capillaries for direct bilirubin removal from human plasma. To obtain active binding sites for HSA, the MPTFE capillaries were chemically functionalized by using a coating of poly(vinyl alcohol) (PVA)-glycidyl methacrylate (GMA) copolymers. Characterization of grafted MPTFE capillaries was verified by XPS, Fourier transform infrared spectroscopy (FT-IR), scanning electronic microscopy (SEM). Non-specific adsorption on the PVA-GMA coated capillary remains low (< 0.38 mg bilirubin/g), and higher affinity adsorption capacity, of up to 73.6 mg bilirubin/g polymer was obtained after HSA is immobilized. Blood compatibility of the grafted MPTFE capillary was evaluated by SEM and platelet rich plasma (PRP) contacting experiments. The experimental data on blood compatibility indicated that PVA-coated and PVA-GMA-HSA coated PTFE capillary showed a sharp suppress on platelets adhesion. The proposed method has the potential of serving in bilirubin removal in clinical application

  5. Spectrofluorimetric protocol for antidepressant drugs in dosage forms and human plasma through derivatization with dansyl chloride

    Directory of Open Access Journals (Sweden)

    Mahmoud A. Omar

    2017-05-01

    Full Text Available A reliable, sensitive and selective spectrofluorimetric method has been developed for the determination of certain antidepressant drugs namely sertraline hydrochloride, fluoxetine hydrochloride, paroxetine hydrochloride, amineptine hydrochloride and bupropion hydrochloride in pure forms, pharmaceutical formulation and human plasma. The method is based on the reaction of investigated drugs with 5-(dimethylamino naphthalene-1-sulfonyl chloride (dansyl chloride in the presence of 0.5 M sodium carbonate to yield highly fluorescent derivatives, measured at 450 nm (excitation at 347 nm. The different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The calibration plots were constructed over the range of 0.02–0.14 μg mL−1. The proposed method was successfully applied for analysis of cited drugs in dosage forms. The high sensitivity of the proposed method allows the determination of investigated drugs in spiked and real human plasma. Statistical comparisons of the results with the reference methods show an excellent agreement and indicate no significant difference in accuracy and precision.

  6. Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

    Science.gov (United States)

    Tamboli, Vibha K; Bhalla, Nikhil; Jolly, Pawan; Bowen, Chris R; Taylor, John T; Bowen, Jenna L; Allender, Chris J; Estrela, Pedro

    2016-12-06

    The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

  7. A New Method to Simultaneously Quantify the Antioxidants: Carotenes, Xanthophylls, and Vitamin A in Human Plasma.

    Science.gov (United States)

    Colmán-Martínez, Mariel; Martínez-Huélamo, Miriam; Miralles, Esther; Estruch, Ramón; Lamuela-Raventós, Rosa M

    2015-01-01

    A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z-β-carotene isomers, and 1 fat-soluble vitamin (retinol) were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and 330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight volunteers. Results. Limits of detection were between 0.1 μg/mL for lycopene and astaxanthin and 1.3 μg/mL for 15-Z-β-carotene. Recoveries were ranged between 89% and 113% for α-carotene and astaxanthin, respectively. Accuracy was between 90.7% and 112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy, precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies.

  8. In vitro studies on the distribution of probucol among human plasma lipoproteins

    International Nuclear Information System (INIS)

    Urien, S.; Riant, P.; Albengres, E.; Brioude, R.; Tillement, J.P.

    1984-01-01

    The role of human plasma lipoproteins as carriers in the blood transport of the cholesterol-lowering and water-insoluble drug, probucol, was investigated in in vitro studies. [ 14 C]Probucol was incubated in whole human blood, a serum pool, individual diluted sera, and isolated protein and lipoprotein fractions. In whole blood, about 90% partitioned in plasma. Following ultracentrifugal fractionation of the serum, it was found that less than 5% distributed in the d greater than 1.20 protein fraction (albumin-rich fraction) and more than 95% in the lipoprotein fractions. The distribution of probucol in the lipoprotein fractions correlated with the lipoprotein total lipid volume under saturation conditions (incubation of isolated lipoprotein fractions) as well as nonsaturation conditions (fractionation of serum exposed to [ 14 C]probucol). Incubation of the albumin-rich fraction and of apolipoproteins originating from the isolated lipoprotein fractions showed that they account for a negligible part in the interaction of probucol with blood components. The probucol uptake of individual sera was shown to be correlated to the lipid content of the serum. When probucol was incubated in erythrocyte suspensions containing variable amounts of lipoproteins, probucol partitioned less in erythrocytes as the lipoprotein concentration increased in the suspension

  9. Effect of antigravity suit inflation on cardiovascular, PRA, and PVP responses in humans. [Plasma Renin Activity and Plasma VasoPressin

    Science.gov (United States)

    Kravik, S. E.; Keil, L. C.; Geelen, G.; Wade, C. E.; Barnes, P. R.

    1986-01-01

    The effects of lower body and abdominal pressure, produced by antigravity suit inflation, on blood pressure, pulse rate, fluid and electrolyte shift, plasma vasopressin and plasma renin activity in humans in upright postures were studied. Five men and two women stood upright for 3 hr with the suit being either inflated or uninflated. In the control tests, the suit was inflated only during the latter part of the trials. Monitoring was carried out with a sphygnomanometer, with sensors for pulse rates, and using a photometer and osmometer to measure blood serum characteristics. The tests confirmed earlier findings that the anti-g suit eliminates increases in plasma renin activity. Also, the headward redistribution of blood obtained in the tests commends the anti-g suit as an alternative to water immersion or bed rest for initial weightlessness studies.

  10. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  11. LC-MS/MS method for the determination of clodronate in human plasma.

    Science.gov (United States)

    Hasan, Mahmoud; Schumacher, Gitta; Seekamp, Anne; Taedken, Tobias; Siegmund, Werner; Oswald, Stefan

    2014-11-01

    Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma concentrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC-MS/MS assay for the quantification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid-liquid extraction using methyl tert-butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300μl/min on a reversed-phase column (Supelco Ascentis(®), C18) temporized at 50°C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5-800ng/ml, allowing an evaluation of the plasma concentration-time profiles of clodronate for approximately 7-8 half-life (∼24h). The method was validated according to current FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6-6.9% and 0.6-8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6-8.8% and 2.2-4.5%. The recovery of the analyte was low (2-3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3h (96.0±6%) and

  12. Histamine quantification in human plasma using high resolution accurate mass LC-MS technology.

    Science.gov (United States)

    Laurichesse, Mathieu; Gicquel, Thomas; Moreau, Caroline; Tribut, Olivier; Tarte, Karin; Morel, Isabelle; Bendavid, Claude; Amé-Thomas, Patricia

    2016-01-01

    Histamine (HA) is a small amine playing an important role in anaphylactic reactions. In order to identify and quantify HA in plasma matrix, different methods have been developed but present several disadvantages. Here, we developed an alternative method using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument, Q Exactive™ (Thermo Fisher) (LCHRMS). The method includes a protein precipitation of plasma samples spiked with HA-d4 as internal standard (IS). LC separation was performed on a C18 Accucore column (100∗2.1mm, 2.6μm) using a mobile phase containing nonafluoropentanoic acid (3nM) and acetonitrile with 0.1% (v/v) formic acid on gradient mode. Separation of analytes was obtained within 10min. Analysis was performed from full scan mode and targeted MS2 mode using a 5ppm mass window. Ion transitions monitored for targeted MS2 mode were 112.0869>95.0607m/z for HA and 116.1120>99.0855m/z for HA-d4. Calibration curves were obtained by adding standard calibration dilution at 1 to 180nM in TrisBSA. Elution of HA and IS occurred at 4.1min. The method was validated over a range of concentrations from 1nM to 100nM. The intra- and inter-run precisions were <15% for quality controls. Human plasma samples from 30 patients were analyzed by LCHRMS, and the results were highly correlated with those obtained using the gold standard radioimmunoassay (RIA) method. Overall, we demonstrate here that LCHRMS is a sensitive method for histamine quantification in biological human plasmas, suitable for routine use in medical laboratories. In addition, LCHRMS is less time-consuming than RIA, avoids the use of radioactivity, and could then be considered as an alternative quantitative method. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  13. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

    Science.gov (United States)

    Cunnion, Kenji M; Hair, Pamela S; Krishna, Neel K; Sass, Megan A; Enos, Clinton W; Whitley, Pamela H; Maes, Lanne Y; Goldberg, Corinne L

    2017-03-01

    The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone. © 2016 AABB.

  15. A four-kallikrein panel for the prediction of repeat prostate biopsy: data from the European Randomized Study of Prostate Cancer screening in Rotterdam, Netherlands.

    Science.gov (United States)

    Gupta, A; Roobol, M J; Savage, C J; Peltola, M; Pettersson, K; Scardino, P T; Vickers, A J; Schröder, F H; Lilja, H

    2010-08-24

    Most men with elevated levels of prostate-specific antigen (PSA) do not have prostate cancer, leading to a large number of unnecessary biopsies. A statistical model based on a panel of four kallikreins has been shown to predict the outcome of a first prostate biopsy. In this study, we apply the model to an independent data set of men with previous negative biopsy but persistently elevated PSA. The study cohort consisted of 925 men with a previous negative prostate biopsy and elevated PSA (>or=3 ng ml(-1)), with 110 prostate cancers detected (12%). A previously published statistical model was applied, with recalibration to reflect the lower positive biopsy rates on rebiopsy. The full-kallikrein panel had higher discriminative accuracy than PSA and DRE alone, with area under the curve (AUC) improving from 0.58 (95% confidence interval (CI): 0.52, 0.64) to 0.68 (95% CI: 0.62, 0.74), Por=7) at biopsy with AUC improving from 0.76 (95% CI: 0.64, 0.89) to 0.87 (95% CI: 0.81, 0.94), P=0.003). Application of the panel to 1000 men with persistently elevated PSA after initial negative biopsy, at a 15% risk threshold would reduce the number of biopsies by 712; would miss (or delay) the diagnosis of 53 cancers, of which only 3 would be Gleason 7 and the rest Gleason 6 or less. Our data constitute an external validation of a previously published model. The four-kallikrein panel predicts the result of repeat prostate biopsy in men with elevated PSA while dramatically decreasing unnecessary biopsies.

  16. Transcriptome reveals the overexpression of a kallikrein gene cluster (KLK1/3/7/8/12) in the Tibetans with high altitude-associated polycythemia.

    Science.gov (United States)

    Li, Kang; Gesang, Luobu; Dan, Zeng; Gusang, Lamu

    2017-02-01

    High altitude-associated polycythemia (HAPC) is a very common disease. However, it the disease is still unmanageable and the related molecular mechanisms remain largely unclear. In the present study, we aimed to explore the molecular mechanisms responsible for the development of HAPC using transcriptome analysis. Transcriptome analysis was conducted in 3 pairs of gastric mucosa tissues from patients with HAPC and healthy residents at a similar altitude. Endoscopy and histopathological analyses were used to examine the injury to gastric tissues. Molecular remodeling was performed for the interaction between different KLK members and cholesterol. HAPC was found to lead to morphological changes and pathological damage to the gastric mucosa of patients. A total of 10,304 differentially expressed genes (DEGs) were identified. Among these genes, 4,941 DEGs were upregulated, while 5,363 DEGs were downregulated in the patients with HAPC (fold change ≥2, P17-fold. All the members had high-score binding cholesterol, particularly for the polymers of KLK7. The kallikrein gene cluster (KLK1/3/7/8/12) is on chromosome 19q13.3-13.4. The elevated levels of KLK1, KLK3, KLK7, KLK8 and KLK12 may be closely associated with the hypertension, inflammation, obesity and other gastric injuries associated with polycythemia. The interaction of KLKs and cholesterol maybe play an important role in the development of hypertension. The findings of the present study revealed that HAPC induces gastric injury by upregulating the kallikrein gene cluster (KLK1/3/7/8/12), which can bind cholesterol and result in kallikrein hypertension. These findings provide some basic information for understanding the molecular mechanisms responsible for HAPC and HAPC-related diseases.

  17. Milk decreases urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans.

    Science.gov (United States)

    Mullen, William; Borges, Gina; Donovan, Jennifer L; Edwards, Christine A; Serafini, Mauro; Lean, Michael E J; Crozier, Alan

    2009-06-01

    Cocoa drinks containing flavan-3-ols are associated with many health benefits, and conflicting evidence exists as to whether milk adversely affects the bioavailability of flavan-3-ols. The objective was to determine the effect of milk on the bioavailability of cocoa flavan-3-ol metabolites. Nine human volunteers followed a low-flavonoid diet for 2 d before drinking 250 mL of a cocoa beverage, made with water or milk, that contained 45 micromol (-)-epicatechin and (-)-catechin. Plasma and urine samples were collected for 24 h, and flavan-3-ol metabolites were analyzed by HPLC with photodiode array and mass spectrometric detection. Milk affected neither gastric emptying nor the transit time through the small intestine. Two flavan-3-ol metabolites were detected in plasma and 4 in urine. Milk had only minor effects on the plasma pharmacokinetics of an (epi)catechin-O-sulfate and had no effect on an O-methyl-(epi)catechin-O-sulfate. However, milk significantly lowered the excretion of 4 urinary flavan-3-ol metabolites from 18.3% to 10.5% of the ingested dose (P = 0.016). Studies that showed protective effects of cocoa and those that showed no effect of milk on bioavailability used products that have a much higher flavan-3-ol content than does the commercial cocoa used in the present study. Most studies of the protective effects of cocoa have used drinks with a very high flavan-3-ol content. Whether similar protective effects are associated with the consumption of many commercial chocolate and cocoa products containing substantially lower amounts of flavan-3-ols, especially when absorption at lower doses is obstructed by milk, remains to be determined.

  18. Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

    Science.gov (United States)

    Helgeland, Erik; Breivik, Lars Ertesvåg; Vaudel, Marc; Svendsen, Øyvind Sverre; Garberg, Hilde; Nordrehaug, Jan Erik; Berven, Frode Steingrimsen; Jonassen, Anne Kristine

    2014-01-01

    Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where

  19. Plasma pharmacokinetics and synovial concentrations of S-flurbiprofen plaster in humans.

    Science.gov (United States)

    Yataba, Ikuko; Otsuka, Noboru; Matsushita, Isao; Kamezawa, Miho; Yamada, Ichimaro; Sasaki, Sigeru; Uebaba, Kazuo; Matsumoto, Hideo; Hoshino, Yuichi

    2016-01-01

    The purpose of this study is to investigate the pharmacokinetics and deep tissue penetration capability of the newly developed S-flurbiprofen plaster (SFPP) in humans. Study 1: SFPP tape-type patch (2-60 mg) was applied to the lower back for 24 h in healthy adult volunteers. S-flurbiprofen (SFP) plasma concentration was measured over time to examine SFP pharmacokinetics. Study 2: SFPP (20 mg) was applied for 12 h to the affected knee of osteoarthritis (OA) patients who were scheduled for total knee arthroplasty. Deep tissues (synovial tissue and synovial fluid) were collected during surgery to compare SFP concentrations after application of SFPP or a commercially available flurbiprofen (FP) gel-type patch. Study 1: The plasma concentration of SFP was sustained during 24-h topical application of the SFPP, showing a high percutaneous absorption ratio of 51.4-72.2 %. Cmax and AUC0-∞ were dose-proportional. Study 2: After application of the SFPP for 12 h, SFP concentrations in the synovial tissue and synovial fluid were 14.8-fold (p = 0.002) and 32.7-fold (p < 0.001) higher, respectively, than those achieved by the FP patch. Sustained plasma concentration of SFP and high percutaneous absorption ratio was observed after 24-h topical application of the SFPP. Compared to the FP patch, the SFPP showed superior percutaneous absorption and greater tissue penetration of SFP into the synovial tissue. Greater tissue penetration of the SFPP seemed to be primarily due to its formulation. Thus, SFPP is expected to show higher efficacy for the treatment of knee OA.

  20. Plasma cholesterol synthesis using deuterated water in humans: effect of short-term food restriction

    International Nuclear Information System (INIS)

    Jones, P.J.; Scanu, A.M.; Schoeller, D.A.

    1988-01-01

    Our purpose was to develop methods in humans to determine the fractional synthetic rate (FSR) of plasma pool free cholesterol using the rate of deuterium incorporation from body water. The sensitivity of this method was examined by measuring FSR after periods of fasting and feeding. Five healthy men with normal lipoprotein levels were given a prepared diet containing 40% of calories as fat and a polyunsaturated/saturated fatty acid ratio of 0.25 for 8 days, except for day 7 when they were given only drinking water. Beginning after the supper meal on day 6, they received no food until 8 AM on day 8 when they consumed meals as normal. Over days 7 and 8 the subjects were given prime and constant deuterium oxide orally to maintain body water deuterium enrichment at about 0.05 atom % excess. Plasma samples were obtained at 0 hours (day 7, 8 AM) and at 12, 24, 36, and 48 hours thereafter. Free cholesterol was extracted, purified by thin-layer chromatography, and combusted to water. The water was reduced to H 2 and analyzed for deuterium enrichment by isotope ratio mass spectrometry. Analytic precision of this system was determined as 3.5 0/00 (parts per mil) vs Standard Mean Ocean Water. Deuterium enrichment of plasma water for the group during the 48-hour deuterium oxide administration period was 3143 0/00 +/- 310 0/00 (mean +/- SEM). Cholesterol deuterium enrichment for the group during the 12-hour period of fasting (10.9 0/00 +/- 4.1 0/00) was not different from that during feeding (14.2 0/00 +/- 6.2 0/00)

  1. Plasma pharmacokinetics of catechin metabolite 4'-O-Me-EGC in healthy humans.

    Science.gov (United States)

    Renouf, Mathieu; Redeuil, Karine; Longet, Karin; Marmet, Cynthia; Dionisi, Fabiola; Kussmann, Martin; Williamson, Gary; Nagy, Kornél

    2011-10-01

    Tea is an infusion of the leaves of the Camellia sinensis plant and is the most widely consumed beverage in the world after water. Green tea contains significant amounts of polyphenol catechins and represents a promising dietary component to maintain health and well-being. Epidemiological studies indicate that polyphenol intake may have potential health benefits, such as, reducing the incidence of coronary heart disease, diabetes and cancer. While bioavailability of green tea bioactives is fairly well understood, some gaps still remain to be filled, especially the identification and quantification of conjugated metabolites in plasma, such as, sulphated, glucuronidated or methylated compounds. In the present study, we aimed to quantify the appearance of green tea catechins in plasma with particular emphasis on their methylated forms. After feeding 400 mL of green tea, 1.25% infusion to 9 healthy subjects, we found significant amounts of EC, EGC and EGCg in plasma as expected. EGC was the most bioavailable catechin, and its methylated form (4'-O-Me-EGC) was also present in quantifiable amounts. Its kinetics followed that of its parent compound. However, the relative amount of the methylated form of EGC was lower than that of the parent compound, an important aspect which, in the literature, has been controversial so far. The quantitative results presented in our study were confirmed by co-chromatography and accurate mass analysis of the respective standards. We show that the relative abundance of 4'-O-Me-EGC is ~40% compared to the parent EGC. 4'-O-Me-EGC is an important metabolite derived from catechin metabolism. Its presence in significant amounts should not be overlooked when assessing human bioavailability of green tea.

  2. Liquid Chromatography-Tandem Mass Spectrometric Assay for Determination of Stavudine in Human Plasma

    Directory of Open Access Journals (Sweden)

    Fengdan Jin

    2014-01-01

    Full Text Available A LC-MS/MS method for determination of stavudine in human plasma was established and validated, and it was applied to the pharmaceutical formulations bioequivalence study. 0.5 mL plasma sample was extracted by liquid-liquid extraction. Stavudine was detected by a LC-MS/MS system. The pharmacokinetic parameters of stavudine in different formulations were calculated by noncompartment model statistics. The method was linear over the concentration ranges 5.00–1000 ng/mL in plasma. The intra- and interassay relative standard deviation (RSD was <10%. The average accuracies for the assay at three concentrations (5.00, 80.0, and 900 ng/mL were from 100.2% to 102.5%. Pharmacokinetic parameters of stavudine reference formulation were obtained as follows: Tmax was 0.6±0.2 h, Cmax was 480.7±150.9 g/L, t1/2 was 1.7±0.4 h, and AUC0-t was 872.8±227.8 g·h/L, and pharmacokinetic parameters of stavudine test formulation were obtained as follows: Tmax was 0.5±0.2 h, Cmax was 537.5±178.5 g/L, t1/2 was 1.7±0.3 h, and AUC0-t was (914.1±284.5 g·h/L. Calculated with AUC0-t, the bioavailability of two formulations was 105.0%.

  3. Improved in vitro evaluation of novel antimicrobials: potential synergy between human plasma and antibacterial peptidomimetics, AMPs and antibiotics against human pathogenic bacteria

    DEFF Research Database (Denmark)

    Citterio, Linda; Franzyk, Henrik; Palarasah, Yaseelan

    2016-01-01

    was affected by conditions mimicking in vivo settings. Their activity was enhanced to an unexpected degree in the presence of human blood plasma for thirteen pathogenic Gram-positive and Gram-negative bacteria. MIC values typically decreased 2- to 16-fold in the presence of a human plasma concentration...... that alone did not damage the cell membrane. Hence, MIC and MBC data collected in these settings appear to represent a more appropriate basis for in vivo experiments preceding clinical trials. In fact, concentrations of peptidomimetics and peptide antibiotics (e.g. polymyxin B) required for in vivo...

  4. [Determination of 24 minerals in human milk by inductively coupled plasma mass spectrometry with microwave digestion].

    Science.gov (United States)

    Sun, Zhongqing; Yue, Bing; Yang, Zhenyu; Li, Xiaowei; Wu, Yongning; Yin, Shian

    2013-05-01

    To determine the levels of 24 minerals in human milk by inductively coupled plasma mass spectrometry with microwave digestion. The samples were digested by microwave. The contents of minerals were determined by inductively coupled plasma mass spectrometry. The standard reference minerals of 1849a and 1568a from National Institute of Science and Technology were used for quality control. The accuracy and reproduability for this method were evaluated with mix standards and 1849a and 1568a standard reference materials. The ranges of the levels of sodium, magnesium, phosphorus, potassium, calcium, aluminum, chromium, arsenic, selenium, iron, zinc, manganese, copper, molybdenum, vanadium, cobalt, nickel, gallium, cadmium, silver, strontium, cesium, barium, lead in human milk was 34.97-415.83 mg/kg, 19.00-39.52 mg/kg, 102.13-274.53 mg/kg, 351.19-713.99 mg/kg, 180.08-349.64 mg/kg, 0.06-0.44 mg/kg, 0.9-7.37 microg/kg, 0.92-2.72 microg/kg, 0.20-21.15 microg/kg, 0.10-0.70 mg/kg, 0.56-3.25 mg/kg, 3.00-16.12 micro.g/kg, 62.16-591.69 microg/kg, 0.02-6.91 microg/kg, 5.99-13.70 microg/kg, 0.07-2.11 microg/kg, 0.77-209.26 microg/kg, 0.005-0.28 microg/kg, 0.02-0.23 microg/kg, 0.02-0.71 microg/kg, 36.89-132.26 microg/kg, 0.01-4.72 microg/kg, 0.83-28.16 microg/kg, 2.5-5.3 microg/kg, respectively. The levels of minerals in human milk in present study were consisted with other similar studies. The experiment examined the levels of minerals in human milk satisfactorily. The method has high accuracy and good reproducibility, which could be used for understanding the levels of minerals in human milk.

  5. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    Science.gov (United States)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  6. "Determination of mycophenolic acid in human plasma by high-performance liquid chromatography"

    Directory of Open Access Journals (Sweden)

    "Mehdi Ahadi Barzoki

    2005-05-01

    Full Text Available A simple, sensitive and reproducible HPLC method is presented for determination of mycophenolic acid(MPA in human plasma. Samples were prepared after precipitation of the plasma protein by addition of acetonitrile and naproxen was used as internal standard (I.S.. Separation was performed by reversedphase HPLC, using a Hamilton PRP-C18 Column, 51% acetonitrile and 49% potassium phosphate buffer (20 mM at pH 3.0 as mobile phase, flow rate of 1.0 ml/min, and UV detection at 215 nm. MPA and I.S. had retention times of 7.5 and 11.35 min, respectively. The method showed an acceptable linearity in the range of 0.1µg/ml-40µg/ml with r2 of .9992. The concentration of 0.1µg/ml was determined as quantification limit. Mean absolute recovery was 94.8%. The mean intra- and inter-day reproducibility of method was 4.6 and 11.4% respectively.

  7. Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC-MS/MS method.

    Science.gov (United States)

    Pontarolo, Roberto; Gimenez, Ana Carolina; de Francisco, Thais Martins Guimarães; Ribeiro, Rômulo Pereira; Pontes, Flávia Lada Degaut; Gasparetto, João Cleverson

    2014-08-15

    The objective of this work was to develop and validate a HILIC-MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-μm particle size column maintained at 40°C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3mM ammonium formate. The flow rate was maintained at 400 μL min(-1). Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection vildagliptin in plasma volunteers who orally received a single dose of metformin (850 mg), vildagliptin (50mg) or drug association (metformin 850 mg+vildagliptin 50mg). The new method can thus also be used as a tool for the clinical monitoring of metformin and vildagliptin. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Effective method for the detection of piroxicam in human plasma using HPLC

    Directory of Open Access Journals (Sweden)

    Adriana Maria CALVO

    2016-01-01

    Full Text Available Abstract Non-steroidal anti-inflammatory drugs (NSAIDs are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5’-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo – USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5’-hydroxypiroxicam was only detected in 2 out of 10 volunteers.

  9. Simultaneous HPLC-F analysis of three recent antiepileptic drugs in human plasma.

    Science.gov (United States)

    Mercolini, Laura; Mandrioli, Roberto; Amore, Mario; Raggi, Maria Augusta

    2010-09-21

    An original high-performance liquid chromatographic method with fluorescence detection is presented for the simultaneous determination of the three antiepileptic drugs gabapentin, vigabatrin and topiramate in human plasma. After pre-column derivatisation with dansyl chloride, the analytes were separated on a Hydro-RP column with a mobile phase composed of phosphate buffer (55%) and acetonitrile (45%) and detected at lambda(em)=500 nm, exciting at 300 nm. An original pre-treatment procedure on biological samples, based on solid-phase extraction with MCX cartridges for gabapentin and vigabatrin, and with Plexa cartridges for topiramate, gave high extraction yields (>91%), satisfactory precision (RSDvigabatrin and in the 1.0-50.0 microg mL(-1) range for topiramate, with limits of detection (LODs) between 0.1 and 0.3 microg mL(-1). After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. Accuracy results were satisfactory (recovery >91%). Therefore, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients treated with gabapentin, vigabatrin and topiramate. Copyright 2010 Elsevier B.V. All rights reserved.

  10. [Determination of sulpride in human plasma by high performance liquid chromatography].

    Science.gov (United States)

    Yu, X; Luo, Z; Tang, J; Yu, P

    1997-11-01

    This paper describes a reliable method for the pharmacokinetic study of Sulpride in human plasma by reversed-phase high performance liquid chromatography. To compensate the loss of Sulpride during the extraction procedure we used an internal standard very similar in chemical structure and UV absorbance to those of Sulpride. The mobile phase was methanol-water-acetic acid (60:30:1) with a flow rate of 1.2 mL/min. A UV detector was used at 290 nm. The linear range was 5-100 mg/L and the detectable limit was 1.0 mg/L. The recovery and RSD were 97.95%-99.96% and 2.6%-5.1% respectively. The results showed that this method is a sensitive and accurate one which makes the pharmacokinetic study of Sulpride possible. If the concentration was too low to be detected by UV monitor, a fluorescence detector could be used with the excitation wavelength at 299 nm and emission at 342 nm. We analyzed the plasma samples from 30 day-treated psychotic patients and got the satisfactory results.

  11. A validated HPLC determination of the flavone aglycone diosmetin in human plasma.

    Science.gov (United States)

    Kanaze, Feras Imad; Bounartzi, Melpomeni I; Niopas, Ioannis

    2004-12-01

    Diosmetin, 3',5,7-trihydroxy-4'-methoxy flavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid-liquid extraction and separated on a C8 reversed-phase column with methanol-water-acetic acid (55:43:2, v/v/v) as the mobile phase at 43 degrees C. Peaks were monitored at 344 nm. The method was linear in the 10-300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%. Copyright 2004 John Wiley & Sons, Ltd.

  12. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    Science.gov (United States)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Monoclonal antibodies to human factor VII: production of immunodepleted plasma for VII:C assays.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    A high affinity monoclonal antibody to factor VII (RFF-VII/1), coupled to sepharose, was used to immunodeplete factor VII from normal plasma. The plasma could be used as a substrate in a one stage coagulation assay and performed as well as, or better than, commercially available factor VII deficient plasma or plasma from congenitally deficient factor VII patients. Plasma from normal donors (n = 20), patients with liver disease (n = 20), and patients receiving warfarin (n = 20), or congenitall...

  14. Predictive value of plasma human chorionic gonadotropin measured 14 days after Day-2 single embryo transfer

    DEFF Research Database (Denmark)

    Løssl, Kristine; Oldenburg, Anna; Toftager, Mette

    2017-01-01

    Introduction: Prediction of pregnancy outcome after in vitro fertilization is important for patients and clinicians. Early plasma human chorionic gonadotropin (p-hCG) levels are the best known predictor of pregnancy outcome, but no studies have been restricted to single embryo transfer (SET) of Day......-2 embryos. The aim of the present study was to investigate the predictive value of p-hCG measured exactly 14 days after the most commonly used Day-2 SET on pregnancy, delivery, and perinatal outcome. Material and methods: A retrospective analysis of prospectively collected data on 466 women who had...... p-hCG measured exactly 14 days after Day-2 SET during a randomized trial including 1050 unselected women (aged 18–40 years) undergoing their first in vitro fertilization/ intracytoplasmic sperm injection treatment. Results: The p-hCG predicted clinical pregnancy [area under the curve (AUC) 0.953; 95...

  15. Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

    International Nuclear Information System (INIS)

    Carter, M.G.; Shukla, S.D.

    1987-01-01

    Human platelet concentrate from the American Red Cross Blood Center was stored at 24 degree C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying 32 P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 x 10 -7 M PAF at 37 degree C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for 32 P-phosphoinositides. The percent stimulation of 32 P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage

  16. Determination of clarithromycin in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Jiang, Yao; Wang, Jiang; Li, Hao; Wang, Yingwu; Gu, Jingkai

    2007-03-12

    A rapid and sensitive method has been developed for the determination of clarithromycin in human plasma with liquid chromatography-tandem mass spectrometry. Clarithromycin and the internal standard, telmisartan were precipitated from the matrix (50 microl) with 200 microl acetonitrile and separated by HPLC using formic acid:10 mM ammonium acetate:methanol (1:99:400, v/v/v) as the mobile phase. The assay based on detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was finished within 2.4 min. Linearity was over the concentration range 10-5000 ng/ml with a limit of detection of 0.50 ng/ml. Intra- and inter-day precision measured as relative standard deviation were bioequivalence study of two tablet formulations of clarithromycin.

  17. A New Validated RP- HPLC Method for the Determination of Nevirapine in Human Plasma

    Directory of Open Access Journals (Sweden)

    C. H. Venkata Kumar

    2010-01-01

    Full Text Available A rapid, selective and sensitive high performance liquid chromatographic method for the estimation of nevirapine in human plasma has been developed. Chromatography was carried out on a Hypersil BDS C18 column using a mixture of ammonium acetate buffer (pH 4.0 ± 0.05 and acetonitrile (85:15 v/v as the mobile phase. The eluents were monitored for the drug by UV detection at 254 nm. Oxcarbazepine was used as an internal standard for this study. The retention times for nevirapine and oxcarbazepine were found to be 7.2 and 14.7 min respectively. The method was found to be linear in the concentration range of 50 ng/mL to 5003.7 ng/mL. The method was validated as per FDA guidelines and was found to be suitable for bioequivalence and pharmacokinetic studies.

  18. Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column.

    Science.gov (United States)

    Zhang, Doudou; Zhao, Yu; Lan, Dandan; Pang, Xiaomin; Bai, Ligai; Liu, Haiyan; Yan, Hongyuan

    2017-11-15

    In this work a polymer monolithic column was fabricated within the confines of a stainless steel column (50×4.6mm i.d.) via radical polymerization by using iron porphyrin and butyl methacrylate as co-monomers, ethylene glycol dimethacrylate as crosslinking agent, ethylene glycol, isopropyl alcohol and N, N-dimethylformamide as tri-porogens, benzoyl peroxide and N,N-dimethylaniline as initiators. The resulting monolithic column was characterized by elemental analysis, scanning electron microscopy, nitrogen adsorption BET surface area, and mercury intrusion porosimetry, respectively. Results showed that the homemade monolith occupied relatively uniform pore structure, low back pressure, and enhanced selectivity for proteins in complex bio-samples. The present work described a simple and efficient method for "fractionation separation" of human plasma proteins, and it is a promising separation method for complex bio-samples in proteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Effect of the stage of lactation in humans on carotenoid levels in milk, blood plasma and plasma lipoprotein fractions.

    Science.gov (United States)

    Schweigert, Florian J; Bathe, Katharina; Chen, Frank; Büscher, Ulrich; Dudenhausen, Joachim W

    2004-02-01

    In mammals the composition of milk changes during early lactation, with a rapid decline of fat-soluble vitamins and a continuous increase in total lipids. The mechanisms underlying this phenomenon are not well understood, but might involve selective mechanisms related to mammary uptake or secretion into the milk. Since carotenoids are specifically distributed among the lipoprotein fractions in plasma, the simultaneous determination of carotenoids in plasma, lipoprotein fractions and milk might offer an opportunity to gain insight into this phenomenon. In 21 healthy mothers carotenoids in plasma and lipoprotein fractions were investigated at day 2 and 19 and milk on day 4 and 19 after delivery. Plasma levels of alpha-tocopherol and cholesterol as well as lutein, zeaxanthin and cryptoxanthin were significantly lower later in lactation (day 19) than shortly after birth (P milk, triacylglycerol increased (P milk it was similar to the pattern found in the high density lipoprotein fraction. Based on these observations a selective mechanism might be responsible for the transfer of these components in milk involving different lipoprotein fractions at specific times of lactation.

  20. Plasma metabolomics reveal the correlation of metabolic pathways and Prakritis of humans

    Directory of Open Access Journals (Sweden)

    Amey Shirolkar

    2018-04-01

    Full Text Available Background: Ayurveda, an ancient Indian medicinal system, has categorized human body constitutions in three broad constitutional types (prakritis i.e. Vata, Pitta and Kapha. Objectives: Analysis of plasma metabolites and related pathways to classify Prakriti specific dominant marker metabolites and metabolic pathways. Materials and methods: 38 healthy male individuals were assessed for dominant Prakritis and their fasting blood samples were collected. The processed plasma samples were subjected to rapid resolution liquid chromatography–electrospray ionization–quadrupole time of flight mass spectrometry (RRLC–ESI–QTOFMS. Mass profiles were aligned and subjected to multivariate analysis. Results: Partial least square discriminant analysis (PLS-DA model showed 97.87% recognition capability. List of PLS-DA metabolites was subjected to permutative Benjamini–Hochberg false discovery rate (FDR correction and final list of 76 metabolites with p  2.0 was identified. Pathway analysis using metascape and JEPETTO plugins in Cytoscape revealed that steroidal hormone biosynthesis, amino acid, and arachidonic acid metabolism are major pathways varying with different constitution. Biological Go processes analysis showed that aromatic amino acids, sphingolipids, and pyrimidine nucleotides metabolic processes were dominant in kapha type of body constitution. Fat soluble vitamins, cellular amino acid, and androgen biosynthesis process along with branched chain amino acid and glycerolipid catabolic processes were dominant in pitta type individuals. Vata Prakriti was found to have dominant catecholamine, arachidonic acid and hydrogen peroxide metabolomics processes. Conclusion: The neurotransmission and oxidative stress in vata, BCAA catabolic, androgen, xenobiotics metabolic processes in pitta, and aromatic amino acids, sphingolipid, and pyrimidine metabolic process in kapha Prakriti were the dominant marker pathways. Keywords: Ayurveda, Prakriti, Human

  1. Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available Atmospheric pressure cold plasma (APCP might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS that kill microorganisms without substantially affecting human cells.In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC was able to inhibit or prevent damage and cell death.Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds. Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h.These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

  2. A cross-linking study on the particle species of human plasma high density lipoproteins.

    Science.gov (United States)

    Yachida, Y; Minari, O

    1983-08-01

    The present investigation was on the particle species of human plasma high density lipoprotein (HDL) characterized by the stoichiometry of their apoprotein components. HDL2-1, HDL2-2, HDL3-1, and HDL3-2 isolated from normal human plasma by sequential ultracentrifugal flotation were further subfractionated by Bio Gel A-5m gel chromatography or hydroxyapatite column chromatography, and three distinct subfractions were obtained. Subfraction 1 was obtained from all the HDL fractions and it contained mostly apolipoprotein A-I (A-I). Subfraction 2 was obtained from HDL2-2 and HDL3-1 and it contained A-I and apolipoprotein A-II (A-II) in the molar ratio of one to one, and subfraction 3 from HDL2-2 and HDL3-1 contained A-I and apolipoprotein C (C). Each subfraction was treated with bifunctional cross-linking reagents, and the intraparticle cross-linked products of apolipoproteins were examined by SDS-polyacrylamide gel electrophoresis. The results of the cross-linking studies indicated that the HDL2 fraction consisted mainly of lipoprotein particles of the (A-I)4 type and a few of the (A-I)5, (A-I)2(A-II)2, and (A-I)4(C)2 types, and that the HDL3 fraction consisted mainly of (A-I)2(A-II)2 type particles and a few (A-I)4, (A-I)3, (A-I)2, (A-I), and (A-I)3(C)2 type particles. From the results of analyses of the lipid components in the HDL of each type, it was suggested that the function of the particle species of the (A-I)n type (n = 1--5), which contained more cholesteryl ester than the (A-I)2(A-II)2 type, was concerned mainly with cholesterol metabolism.

  3. Two different spectrofluorimetric methods for simultaneous determination of gemfibrozil and rosiglitazone in human plasma.

    Science.gov (United States)

    El-Din, Mohie M K Sharaf; Attia, Khalid A M; Nassar, Mohamed W I; Kaddah, Mohamed M Y

    2010-10-15

    Two accurate, reliable, and highly sensitive spectrofluorimetric methods were developed for simultaneous determination of binary mixture gemfibrozil and rosiglitazone in human plasma without prior separation steps. The first method is based on synchronous fluorescence spectrometry using double scans. At Δλ=27nm, gemfibrozil yields detectable signal that is independent of the presence of rosiglitazone. Similarly, at Δλ=120nm the signal of rosiglitazone is not influenced by the presence of gemfibrozil. Signals at two wavelengths, 301 (Δλ=27nm) and 368nm (Δλ=120nm) vary linearly with gemfibrozil and rosiglitazone concentrations over the range 100-700ngmL(-1) (for gemfibrozil) and 20-140ngmL(-1) (for rosiglitazone), respectively. The limits of detection (LOD) were 2.3 and 2.72ngmL(-1) for gemfibrozil and rosiglitazone, respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which 258nm was selected as the excitation wavelength. Two equations are constructed based on the fact that at ( λ(EM)₂=302 nm of gemfibrozil) and (λ(EM)₂=369 nm of rosiglitazone) the fluorescence of the mixture is the sum of the individual fluorescence of gemfibrozil and rosiglitazone. The limits of detection (LOD) were 28.1 and 23.63ngmL(-1) for gemfibrozil and rosiglitazone, respectively. The proposed methods were successfully applied for the determination of the two compounds in synthetic mixtures and in human plasma with a good recovery. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Effects of argon plasma coagulation on human stomach tissue: An ex vivo study.

    Science.gov (United States)

    Gong, Eun Jeong; Ahn, Ji Yong; Jung, Hwoon-Yong; Park, Young Soo; Na, Hee Kyong; Jung, Kee Wook; Kim, Do Hoon; Lee, Jeong Hoon; Choi, Kee Don; Song, Ho June; Lee, Gin Hyug; Kim, Jin-Ho

    2017-05-01

    Argon plasma coagulation (APC) is a safe alternative treatment for gastrointestinal neoplasms and precancerous lesions. However, the extent of thermal damage after APC is difficult to predict. We investigated the effects of APC on human stomach tissue. Argon plasma coagulation was performed on 10 freshly resected human stomachs that were obtained after total gastrectomy. The effects on tissue were compared across power settings (40, 60, and 80 W), durations (5, 10, 15, 20, and 25 s), and between injection (submucosal injection of normal saline) and control (without injection) groups. Success was defined as complete mucosal necrosis without damaging the muscularis propria. Without submucosal injection, the incidence of damaging the muscularis propria increased as the power and duration increased. Tissue damage in the injection group was mostly confined to the submucosa, even when using the high-power setting. In the injection group, ablations at 40 W for 20 s, 60 W for 15 s, and 80 W for 15 or 20 s produced success rates ≥80%. In the control group, ablations at 60 W for 10 s, and 80 W for 5, or 10 s produced success rates ≥80%. The optimal energy levels to achieve complete mucosal and submucosal necrosis without damaging the muscularis propria were 800-1600 and 600-800 J in the injection and control groups, respectively. Application of APC produces good results with a low risk of perforation. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  5. Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium

    Directory of Open Access Journals (Sweden)

    Zhu W

    2015-12-01

    Full Text Available Wei Zhu,1 George Teel,1 Christopher M O’Brien,1 Taisen Zhuang,1 Michael Keidar,1 Lijie Grace Zhang1–3 1Department of Mechanical and Aerospace Engineering, 2Department of Biomedical Engineering, 3Department of Medicine, The George Washington University, Washington, DC, USA Abstract: Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium’s osseointegration involves inducing biomimetic nanotopography to enhance cell–implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications

  6. Persistent organohalogen contaminants in plasma from groups of humans with different occupations in Bangladesh.

    Science.gov (United States)

    Zamir, R; Athanasiadou, M; Nahar, N; Mamun, M I R; Mosihuzzaman, M; Bergman, A

    2009-01-01

    The present study is aimed to assess persistent organic halogenated pollutants in humans living in Bangladesh. The results are compared to other similar studies in the region and globally. Human blood plasma were collected from groups of men and women with different occupations, i.e. being students, garment industry workers, employees at the Power Development Board (PDB), all groups in Dhaka, fishermen and fishermen wife's from Dhaka and another group from Barisal district. The plasma was analysed for hexachlorobenzene (HCB), the hexachlorocyclohexane isomers, alpha-HCH, beta-HCH, gamma-HCH and delta-HCH, the DDT group of chemicals, chlordane compounds, trans-chlordane, cis-chlordane, oxychlordane, trans-nonachlor, trans-heptachlorepoxide, methoxychlor and mirex. The most abundant contaminant, in all groups studied, p,p'-DDE is dominating, with p,p'-DDT/Sigma DDT ratios indicating recent and ongoing DDT exposure. Among the other pesticides analysed beta-HCH is the most abundant indicating the use of technical HCH products instead of Lindane (gamma-HCH). While the Sigma DDT is present in the low ppm range the beta-HCH is detected in up to approx. 400 ppb, lipid basis. The beta-HCH is most abundant in the groups of students. In contrast to the pesticides analysed very low concentrations of polychlorinated biphenyls (PCB) are present in all study groups, with e.g. CB-153 in the range of 5-30 ng g(-1) fat. The concentrations of the DDT group of chemical differ significantly between fishermen and fishermen's wives living and working in the Dhaka area versus those living and working in Barisal. Also, fishermen and their wives had significantly different concentrations of DDT compared to garment industry workers.

  7. Validated assay for the simultaneous quantification of total vincristine and actinomycin-D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high-performance liquid chromatography coupled with tandem mass spectrometry

    NARCIS (Netherlands)

    Damen, Carola W. N.; Israëls, Trijn; Caron, Huib N.; Schellens, Jan H. M.; Rosing, Hilde; Beijnen, Jos H.

    2009-01-01

    A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin-D concentrations in human plasma and an assay for the determination of unbound vincristine are presented.

  8. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    Energy Technology Data Exchange (ETDEWEB)

    Yang, H.; Gan, L.; Yang, X., E-mail: luxinpei@hotmail.com, E-mail: yangxl@mail.hust.edu.cn [College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074 (China); Lu, R. [School Hospital of Huazhong University of Science and Technology, Wuhan, Hubei 430074 (China); Xian, Y.; Lu, X., E-mail: luxinpei@hotmail.com, E-mail: yangxl@mail.hust.edu.cn [State Key Laboratory of Advanced Electromagnetic Engineering and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074 (China)

    2015-12-15

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-kB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  9. A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma, using a tracer with an immobilized glycouril agent

    International Nuclear Information System (INIS)

    Rosmalen, F.M.A.; Tan, A.C.I.T.L.; Benraad, T.J.

    1987-01-01

    A highly specific and sensitive radioimmunoassay (RIA) for alpha-human atrial natriuretic peptide (hANP[1-28]) in plasma was developed. The assay used a [ 125 I]monoiodotyrosyl-hANP[1-28] tracer, prepared with an immobilized glycouril agent (Protag) and purified by high pressure liquid chromatography (HPLC), and a highly specific antiserum raised against hANP[1-28], coupled to keyhole limpet haemocyanin, in sheep. Plasma was extracted using C-18 Seppak cartridges. A good parallelism was found after dilution prior to extraction of plasma of patients with congestive heart failure (CHF) or of plasma of healthy subjects. Recovery of hANP[1-28] added to plasma was 96%. The limit of detection was 0.8 pg/tube, intra- and inter-assay variation were 9 and 12%, respectively. Mean plasma ANP values in 25 normal persons with a normal salt intake was 26.0 ± 15.5 (± SD) pg/ml. Plasma levels of 18 subjects (7 normals, 11 CHF) were measured using four different antisera after the extraction step. High correlations were found between the values obtained with these four antisera. (Auth.)

  10. Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

    Science.gov (United States)

    Yang, H.; Lu, R.; Xian, Y.; Gan, L.; Lu, X.; Yang, X.

    2015-12-01

    Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-кB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

  11. Plasma levels of 27-hydroxycholesterol in humans and mice with monogenic disturbances of high density lipoprotein metabolism

    DEFF Research Database (Denmark)

    Karuna, Ratna; Holleboom, Adriaan G; Motazacker, Mohammad M

    2011-01-01

    Secretion of 27-hydroxycholesterol (27OHC) from macrophages is considered as an alternative to HDL-mediated reverse transport of excess cholesterol. We investigated 27OHC-concentrations in plasma of humans and mice with monogenic disorders of HDL metabolism. As compared to family controls mutations...... activities of LCAT and CETP, respectively, than the formation and transfer of cholesterylesters. 27OHC plasma levels were also decreased in apoA-I-, ABCA1- or LCAT-knockout mice but increased in SR-BI-knockout mice. Transplantation of ABCA1- and/or ABCG1-deficient bone marrow into LDL receptor deficient mice...... decreased plasma levels of 27OHC. In conclusion, mutations or absence of HDL genes lead to distinct alterations in the quantity, esterification or lipoprotein distribution of 27OHC. These findings argue against the earlier suggestion that 27OHC-metabolism in plasma occurs independently of HDL....

  12. Accurate measurement of stable isotopes 46Ca and 48Ca in human feces, plasma, and urine in relation to human nutrition of calcium

    International Nuclear Information System (INIS)

    Janghorbani, M.; Sundaresan, A.; Young, V.R.

    1981-01-01

    A method based on Radiochemical Neutron Activation Analysis (RNAA) is described which allows simultaneous measurement of two stable isotopes of calcium, 46 Ca and 48 Ca, in human feces, plasma, and urine for the purpose of studying human nutrition and metabolism of calcium. It is shown that these measurements can be made with relative analytical precision of 1-5% depending on the particulars of a given experiment. The method has been applied in humans and data are given showing that kinetics of plasma appearance of 46 Ca administered orally with food can be readily investigated. This method allows investigation of a number of important nutritional and metabolic issues in all human population groups without regard to radioisotope safety considerations, and should prove especially helpful in relation to studies of calcium bioavailability from different foods in a variety of population groups for whom use of radiocalcium is not warranted. (Auth.)

  13. Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique.

    Science.gov (United States)

    Tang, Dao-quan; Li, Yin-jie; Li, Zheng; Bian, Ting-ting; Chen, Kai; Zheng, Xiao-xiao; Yu, Yan-yan; Jiang, Shui-shi

    2015-08-01

    In this work, two high-performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3-methyl-1-p-tolyl-5-pyrazolone and L-glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o-phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C18 column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate-acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)-methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Increased MMP-9 and TIMP-1 in mouse neonatal brain and plasma and in human neonatal plasma after hypoxia-ischemia: a potential marker of neonatal encephalopathy.

    Science.gov (United States)

    Bednarek, Nathalie; Svedin, Pernilla; Garnotel, Roselyne; Favrais, Géraldine; Loron, Gauthier; Schwendiman, Leslie; Hagberg, Henrik; Morville, Patrice; Mallard, Carina; Gressens, Pierre

    2012-01-01

    To implement neuroprotective strategies in newborns, sensitive and specific biomarkers are needed for identifying those who are at risk for brain damage. We evaluated the effectiveness of matrix metalloproteinases (MMPs) and their naturally occurring tissue inhibitors of metalloproteinases (TIMPs) in predicting neonatal encephalopathy (NE) damage in newborns. Plasma MMP-9 and TIMP-1 levels were upregulated as early as 1 h after the HI insult but not did not show such elevations after other types of injury (ibotenate-induced excitotoxicity, hypoxia, lipopolysaccharide-induced inflammation), and brain levels reflected this increase soon thereafter. We confirmed these results by carrying out plasma MMP-9 and TIMP-1 measurements in human newborns with NE. In these infants, protein levels of MMP-9 and TIMP-1 were found to be elevated during a short window up to 6 h after birth. This feature is particularly useful in identifying newborns in need of neuroprotection. A second peak observed 72 h after birth is possibly related to the second phase of energy failure after a HI insult. Our data, although preliminary, support the use of MMP-9 and TIMP-1 as early biomarkers for the presence and extent of perinatal brain injury in human term newborns. We first used a mouse model of neonatal HI injury to explore mechanistic aspects such as the time course of these markers after the hypoxia-ischemia event, and the correlation between the levels of these candidate markers in brain and plasma.

  15. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  16. Simultaneous determination of triple therapy for Helicobacter pylori in human plasma by reversed phase chromatography with online wavelength switching

    Science.gov (United States)

    Ahmed, Sameh; Atia, Noha N.

    2015-02-01

    The infection of gastric mucosa by Helicobacter pylori (HP) is an essential cofactor in the aetiology of gastroduodenal ulcer and gastric carcinoma. Because of the bacterial resistance, combination therapy containing omeprazole (OME), tinidazole (TNZ) and clarithromycin (CLA) is commonly used for eradication of HP. However, the simultaneous determination of the triple therapy in human plasma was not reported. A simple, reproducible, and selective HPLC method was developed for the simultaneous determination of the triple therapy mixture used for management of HP infections in human plasma. An HPLC procedure based on a liquid-liquid extraction, enrichment of the analytes and subsequent reversed-phase chromatography with UV detection was used. To enable sensitive and selective detection, the method involved the use of online wavelength switching detection, with two different detection wavelengths; 280 nm for detection of OME and TNZ and 210 nm for detection of CLA. Separations were performed on C18 analytical column with acetonitrile-10 mM phosphate buffer of pH = 3.0 at flow rate of 1.0 mL min-1. The linear ranges in human plasma were 0.05-10 μg mL-1 with correlation coefficients >0.9990. The detection limits in human plasma were 0.02-0.07 μg mL-1. Validation parameters were assessed in compliance with US-FDA guidelines. The method proved to be valuable for the therapeutic drug monitoring after oral administration of triple therapy tablets.

  17. Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard.

    Science.gov (United States)

    Nirogi, Ramakrishna V S; Kandikere, Vishwottam; Shrivastava, Wishu; Mudigonda, Koteshwara; Maurya, Santosh; Ajjala, Devender

    2007-11-01

    A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. Copyright (c) 2007 John Wiley & Sons, Ltd.

  18. Pregnancy-associated plasma protein A in human ovarian follicles and its association with intrafollicular hormone levels

    DEFF Research Database (Denmark)

    Bøtkjær, Jane Alrø; Jeppesen, Janni Vikkelsø; Wissing, Marie Louise

    2015-01-01

    To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological...... activity of PAPP-A in FF was evaluated....

  19. Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion-trap mass spectrometer

    Czech Academy of Sciences Publication Activity Database

    Žáček, Petr; Bukowski, M.; Rosenberger, T. A.; Picklo, M.

    2016-01-01

    Roč. 57, č. 12 (2016), s. 2225-2234 ISSN 0022-2275 Institutional support: RVO:61388963 Keywords : shotgun lipidomics * triple quadrupole/ion-trap * human blood plasma * phosphatidylcholines Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.810, year: 2016 http://www.jlr.org/content/57/12/2225.full

  20. Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

    DEFF Research Database (Denmark)

    Dutaud, Dominique; Aubry, Laurent; Henry, Laurent

    2002-01-01

    Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination...

  1. Modeling Zika plasma viral dynamics in non-human primates: insights into viral pathogenesis and antiviral strategies

    Energy Technology Data Exchange (ETDEWEB)

    Best, Katharine [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Guedj, Jeremie [Univ. of Paris (France). IAME; Madelain, Vincent [Univ. of Paris (France); de Lamballerie, Xavier [Aix-Marseille Univ. (France); L, So-Yonim [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Osuna, Christa E [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Whitney, James [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Perelson, Alan S. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-10-24

    The recent outbreak of Zika virus (ZIKV) has been associated with fetal abnormalities and neurological complications, prompting global concern. Here we present the first mathematical analysis of the within-host dynamics of plasma ZiKV burden in a non-human primate model, allowing for characterization of the growth and clearance of ZIKV within an individual macaque.

  2. Gadolinium-based magnetic resonance contrast agents at 7 Tesla: in vitro T1 relaxivities in human blood plasma.

    Science.gov (United States)

    Noebauer-Huhmann, Iris M; Szomolanyi, Pavol; Juras, Vladimír; Kraff, Oliver; Ladd, Mark E; Trattnig, Siegfried

    2010-09-01

    PURPOSE/INTRODUCTION: The aim of this study was to determine the T1 relaxivities (r1) of 8 gadolinium (Gd)-based MR contrast agents in human blood plasma at 7 Tesla, compared with 3 Tesla. Eight commercially available Gd-based MR contrast agents were diluted in human blood plasma to concentrations of 0, 0.25, 0.5, 1, and 2 mmol/L. In vitro measurements were performed at 37 degrees C, on a 7 Tesla and on a 3 Tesla whole-body magnetic resonance imaging scanner. For the determination of T1 relaxation times, Inversion Recovery Sequences with inversion times from 0 to 3500 ms were used. The relaxivities were calculated. The r1 relaxivities of all agents, diluted in human blood plasma at body temperature, were lower at 7 Tesla than at 3 Tesla. The values at 3 Tesla were comparable to those published earlier. Notably, in some agents, a minor negative correlation of r1 with a concentration of up to 2 mmol/L could be observed. This was most pronounced in the agents with the highest protein-binding capacity. At 7 Tesla, the in vitro r1 relaxivities of Gd-based contrast agents in human blood plasma are lower than those at 3 Tesla. This work may serve as a basis for the application of Gd-based MR contrast agents at 7 Tesla. Further studies are required to optimize the contrast agent dose in vivo.

  3. Transferrin coated nanoparticles: study of the bionano interface in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrzej S Pitek

    Full Text Available It is now well established that the surface of nanoparticles (NPs in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC and after washing (hard corona, HC in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.

  4. Oligonol supplementation modulates plasma volume and osmolality and sweating after heat load in humans.

    Science.gov (United States)

    Lee, JeongBeom; Shin, YoungOh; Murota, Hiroyuki

    2015-05-01

    Oligonol is a low-molecular-weight polyphenol that possesses antioxidant and anti-inflammatory properties. This study investigated the effects of Oligonol supplementation on sweating response, plasma volume (PV), and osmolality (Osm) after heat load in human volunteers. We conducted a placebo-controlled crossover trial. Participants took a daily dose of 200 mg Oligonol or placebo for 1 week. After a 2-week washout period, the subjects were switched to the other study arm. As a heat load, half-body immersion into hot water (42°C±0.5°C for 30 min) was performed in an automated climate chamber. Tympanic and mean body temperature (Tty, mTb) and whole-body sweat loss volume (WBSLV) were measured. Changes in PV, Osm, and serum levels of aldosterone and sodium were analyzed. Oligonol intake attenuated increases in Tty, mTb, and WBSLV after heat load compared with the placebo (Pbody temperature and excessive sweating under heat load in healthy humans, but interpretation of the results requires caution due to the potent diuretic effect of Oligonol.

  5. Development and validation of HPLC analytical method for quantitative determination of metronidazole in human plasma

    International Nuclear Information System (INIS)

    Safdar, K.A.; Shyum, S.B.; Usman, S.

    2016-01-01

    The objective of the present study was to develop a simple, rapid and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) analytical method with UV detection system for the quantitative determination of metronidazole in human plasma. The chromatographic separation was performed by using C18 RP column (250mm X 4.6mm, 5 meu m) as stationary phase and 0.01M potassium dihydrogen phosphate buffered at pH 3.0 and acetonitrile (83:17, v/v) as mobile phase at flow rate of 1.0 ml/min. The UV detection was carried out at 320nm. The method was validated as per the US FDA guideline for bioanalytical method validation and was found to be selective without interferences from mobile phase components, impurities and biological matrix. The method found to be linear over the concentration range of 0.2812 meu g/ml to 18.0 meu g/ml (r2 = 0.9987) with adequate level of accuracy and precision. The samples were found to be stable under various recommended laboratory and storage conditions. Therefore, the method can be used with adequate level of confidence and assurance for bioavailability, bioequivalence and other pharmacokinetic studies of metronidazole in human. (author)

  6. Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

    Science.gov (United States)

    Iudicone, Paola; Fioravanti, Daniela; Bonanno, Giuseppina; Miceli, Michelina; Lavorino, Claudio; Totta, Pierangela; Frati, Luigi; Nuti, Marianna; Pierelli, Luca

    2014-01-27

    Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic

  7. 3H-thymidine autoradiographic study of cell proliferation and the influence of isoproterenol and kallikrein in various cell populations of the gastrointestinal tract in animal experiments

    International Nuclear Information System (INIS)

    Albrecht, S.

    1981-01-01

    Morphological studies were carried out with the aim of studying cell proliferation in various tissues (stable and epithelial tissues) of the gastrointestinal tract. The cells were studied by 3 H thymidine autoradiography in the normal age cycle and under the influence of kallikrein or isoproterenol. Epithelial cells and smooth muscle cells of the forestomach, stomach, small intestine and colon were investigated and, in the case of the stomach, also connective-tissue cells of the mucotic stroma. Counts across the total epithelial thickness yielded similar results for the oesophagus and the forestomach. A count of 1000 cells per animal was found to yield representative information on a defined type of cell. Kallikrein was not found to have a constant mitosis-promoting effect. Isoproterenol caused the labelling index to increase or to decrease; in higher concentrations, it increased the proliferation rate in all cell types. In male animals, the labelling indices of connective-tissue cells of the gastric mucosa were significantly higher than in female animals. (orig./MG) [de

  8. Caloric Restriction and Exercise Increase Plasma ANGPTL4 Levels in Humans via Elevated Free Fatty Acids

    NARCIS (Netherlands)

    Kersten, A.H.; Lichtenstein, L.L.; Steenbergen, E.; Mudde, C.M.; Hendriks, H.F.J.; Hesselink, M.K.; Schrauwen, P.; Müller, M.R.

    2009-01-01

    Objective - Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  9. Caloric restriction and exercise increase plasma ANGPTL4 levels in humans via elevated free fatty acids.

    NARCIS (Netherlands)

    Kersten, S.; Lichtenstein, L.; Steenbergen, E.; Mudde, K.; Hendriks, H.F.; Hesselink, M.K.; Schrauwen, P.; Muller, M

    2009-01-01

    OBJECTIVE: Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  10. Caloric restriction and exercise increase plasma ANGPTL4 levels in humans via elevated free fatty acids

    NARCIS (Netherlands)

    Kersten, S.; Lichtenstein, L.; Steenbergen, E.; Mudde, K.; Hendriks, H.F.J.; Hesselink, M.K.; Schrauwen, P.; Müller, M.

    2009-01-01

    OBJECTIVE-: Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  11. Unbiased plasma metabolomics reveal the correlation of metabolic pathways and Prakritis of humans.

    Science.gov (United States)

    Shirolkar, Amey; Chakraborty, Sutapa; Mandal, Tusharkanti; Dabur, Rajesh

    2017-11-25

    Ayurveda, an ancient Indian medicinal system, has categorized human body constitutions in three broad constitutional types (prakritis) i.e. Vata, Pitta and Kapha. Analysis of plasma metabolites and related pathways to classify Prakriti specific dominant marker metabolites and metabolic pathways. 38 healthy male individuals were assessed for dominant Prakritis and their fasting blood samples were collected. The processed plasma samples were subjected to rapid resolution liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (RRLC-ESI-QTOFMS). Mass profiles were aligned and subjected to multivariate analysis. Partial least square discriminant analysis (PLS-DA) model showed 97.87% recognition capability. List of PLS-DA metabolites was subjected to permutative Benjamini-Hochberg false discovery rate (FDR) correction and final list of 76 metabolites with p  2.0 was identified. Pathway analysis using metascape and JEPETTO plugins in Cytoscape revealed that steroidal hormone biosynthesis, amino acid, and arachidonic acid metabolism are major pathways varying with different constitution. Biological Go processes analysis showed that aromatic amino acids, sphingolipids, and pyrimidine nucleotides metabolic processes were dominant in kapha type of body constitution. Fat soluble vitamins, cellular amino acid, and androgen biosynthesis process along with branched chain amino acid and glycerolipid catabolic processes were dominant in pitta type individuals. Vata Prakriti was found to have dominant catecholamine, arachidonic acid and hydrogen peroxide metabolomics processes. The neurotransmission and oxidative stress in vata, BCAA catabolic, androgen, xenobiotics metabolic processes in pitta, and aromatic amino acids, sphingolipid, and pyrimidine metabolic process in kaphaPrakriti were the dominant marker pathways. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights

  12. Development and Validation of a HPLC Method for Determination of Pefloxacin in Tablet and Human Plasma

    Directory of Open Access Journals (Sweden)

    Shahnaz Gauhar

    2009-03-01

    Full Text Available Objective(sDeveloping and validating a simple, efficient, reproducible and economic reversed phase high performance liquid chromatographic (RP-HPLC method for the quantitative determination of pefloxacin in bulk material, tablets and in human plasma. Materials and MethodsA shim-pack CLC-ODS column and a mobile phase constituting acetonitrile: 0.025 M phosphoric acid solution (13:87 v/v, pH 2.9 adjusted with KOH were used. The flow rate was 1 ml/min and the analyses performed using ultraviolet (UV detector at a wavelength of 275 nm using acetaminophen as an internal standard.ResultsThe developed method showed good resolution between pefloxacin and acetaminophen. It was selective to pefloxacin and able to resolve the drug peak from internal standard and from formulation excipients. The percentage of coefficient variation (CV of the retention times and peak areas of pefloxacin from the six consecutive injections were 0.566% and 0.989%, respectively. The results showed that the peak area responses are linear within the concentration range of 0.125 µg/ml-12 µg/ml (R2= 0.9987. The limits of detection (LOD and limits of quantitation (LOQ for pefloxacin were 0.03125 µg/ml and 0.125 µg/ml. The intra-day and inter-day variation, RSD were 0.376-0.9056 and 0.739-0.853 respectively; also, inter-day variation with relative standard deviation (RSD were 0.1465-0.821 in plasma. The accuracy results of 70%, 100%, and 130% drugs were 100.72%, 100.34%, and 100.09%, respectively.ConclusionThe method is linear, quantitative, reproducible and could be used as a more convenient, efficient and economical method for the trace analysis of drug in biological fluids, in raw material and tablets.

  13. Plasma Indoleamine 2, 3-Dioxygenase, a Biomarker for Tuberculosis in Human Immunodeficiency Virus-Infected Patients.

    Science.gov (United States)

    Adu-Gyamfi, Clement G; Snyman, Tracy; Hoffmann, Christopher J; Martinson, Neil A; Chaisson, Richard E; George, Jaya A; Suchard, Melinda S

    2017-10-15

    There is no biomarker for diagnosing active tuberculosis in patients with human immunodeficiency virus (HIV) infection. Indoleamine 2, 3-dioxygenase (IDO) is an immunoregulatory enzyme that breaks down tryptophan (Trp) to metabolites known as kynurenines (Kyns). We investigated whether IDO activity, as measured by the ratio of Kyn to Trp, could be used to diagnose or predict active tuberculosis disease in HIV-infected adults. Kyn and Trp concentrations were measured using ultraperformance liquid chromatography mass spectrometry in plasma samples from 32 HIV-infected patients in whom active tuberculosis developed and who were followed up prospectively. We compared to 70 HIV-infected control subjects from the same cohort in whom tuberculosis did not develop, matched by age, sex, and CD4 cell count, and 37 unmatched HIV-infected patients with a diagnosis of pneumonia. Clinical parameters, including body mass index, CD4 cell count, HIV load, and C-reactive protein levels were analyzed. At the time of tuberculosis diagnosis, IDO activity was significantly higher in patients with tuberculosis than in controls (P tuberculosis diagnosis, IDO activity was significantly higher in all patients who later developed tuberculosis (P tuberculosis treatment, IDO activity in patients with tuberculosis declined to levels similar to those in controls. IDO activity was 4-fold higher in patients with tuberculosis than in those with pneumonia, and could be used to distinguish them. With a receiver operating characteristic curve, IDO activity had a sensitivity of 97%, a specificity of 99%, and positive and negative predictive values of 89% and 100% for detecting active tuberculosis disease. Plasma IDO activity is suitable as a biomarker of active tuberculosis in HIV-positive patients. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  14. [Measurement of sialic acid from lipoproteins and human plasma by liquid chromatography-tandem mass spectrometry].

    Science.gov (United States)

    Guo, Shoudong; Sang, Hui; Yang, Nana; Kan, Yujie; Li, Fuyu; Li, Yu; Li, Fangyuan; Qin, Shucun

    2014-11-01

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to quantify sialic acid (N-acetylneuraminic acid, NANA) from lipoproteins and human plasma. The method was used to investigate the different contents of NANA from lipoproteins between diabetic with an average age of 51.6 years and healthy participants with an average age of 50.7 years. The NANA from lipoprotein samples was hydrolyzed by acetic acid (pH = 2) at 80 °C for 2 h and analyzed by the optimized LC-MS/MS method after high speed centrifugation and filtration. The limits of detection and quantification of NANA were 7.4 and 24.5 pg, respectively. The linear range was 2.5-80 ng/mL for NANA and the correlation coefficient (R2) was more than 0.998. The levels of NANA in the plasma of type II diabetics and healthy participants were (548.3 ± 88.9) and (415.3 ± 55.5) mg/L, respectively; and the levels of NANA from very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) of the type II diabetics and the healthy participants were (4.91 ± 0.19), (6.95 ± 0.28), (3.61 ± 0.22) μg/mg and (2.90 ± 0.27), (7.03 ± 0.04), (2.40 ± 0.09) μg/mg, respectively. The sialic acid levels of VLDL and HDL from the type II diabetics were markedly higher than those of the corresponding healthy participants with the similar ages (P lipoproteins, and is reproducible and time saving.

  15. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

    International Nuclear Information System (INIS)

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

    1988-01-01

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

  16. Effect of obesity and metabolic syndrome on plasma oxysterols and fatty acids in human.

    Science.gov (United States)

    Tremblay-Franco, Marie; Zerbinati, Chiara; Pacelli, Antonio; Palmaccio, Giuseppina; Lubrano, Carla; Ducheix, Simon; Guillou, Hervé; Iuliano, Luigi

    2015-07-01

    Obesity and the related entity metabolic syndrome are characterized by altered lipid metabolism and associated with increased morbidity risk for cardiovascular disease and cancer. Oxysterols belong to a large family of cholesterol-derived molecules known to play crucial role in many signaling pathways underlying several diseases. Little is known on the potential effect of obesity and metabolic syndrome on oxysterols in human. In this work, we questioned whether circulating oxysterols might be significantly altered in obese patients and in patients with metabolic syndrome. We also tested the potential correlation between circulating oxysterols and fatty acids. 60 obese patients and 75 patients with metabolic syndrome were enrolled in the study along with 210 age- and sex-matched healthy subjects, used as control group. Plasma oxysterols were analyzed by isotope dilution GC/MS, and plasma fatty acids profiling was assessed by gas chromatography coupled with flame ionization detection. We found considerable differences in oxysterols profiling in the two disease groups that were gender-related. Compared to controls, males showed significant differences only in 4α- and 4β-hydroxycholesterol levels in obese and metabolic syndrome patients. In contrast, females showed consistent differences in 7-oxocholesterol, 4α-hydroxycholesterol, 25-hydroxycholesterol and triol. Concerning fatty acids, we found minor differences in the levels of these variables in males of the three groups. Significant changes were observed in plasma fatty acid profile of female patients with obesity or metabolic syndrome. We found significant correlations between various oxysterols and fatty acids. In particular, 4β-hydroxycholesterol, which is reduced in obesity and metabolic syndrome, correlated with a number of saturated and mono-unsaturated fatty acids that are end-products of de novo lipogenesis. Our data provide the first evidence that obesity and metabolic syndrome are associated with

  17. Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation.

    Science.gov (United States)

    Ekaney, Michael Liembo; Otto, Gordon Philipp; Sossdorf, Maik; Sponholz, Christoph; Boehringer, Michael; Loesche, Wolfgang; Rittirsch, Daniel; Wilharm, Arne; Kurzai, Oliver; Bauer, Michael; Claus, Ralf Alexander

    2014-09-24

    Circulating histones have been identified as mediators of damage in animal models of sepsis and in patients with trauma-associated lung injury. Despite existing controversies on actual histone concentrations, clinical implications and mechanism of action in various disease conditions, histone levels in human sepsis, association with disease progression and mediated effects on endothelial and immune cells remain unreported. This study aimed to determine histone levels and its clinical implication in septic patients and to elucidate histone-mediated effects ex-vivo. Histone levels, endogenous activated protein C (APC) levels and clinical data from two independent cohorts of septic patients were obtained. Histone levels were compared with various control groups including healthy individuals, intensive care unit (ICU) patients without sepsis, ICU patients with multiple organ failure and patients with minor or multiple trauma, all without infection. Endothelial and monocytic cells were stimulated with histones. Cellular integrity and sepsis prototypical cytokines were evaluated. The mechanism of action of histones via Toll-like receptor 4 (TLR4) was evaluated using a function blocking antibody. Histone degradation in plasma was studied by immunoblotting. Histone H4 levels were significantly elevated in patients with sepsis (cohort I; n = 15 and cohort II; n = 19) versus ICU controls (n = 12), patients with multiple organ failure (n = 12) or minor trauma (n = 7), associated with need for renal replacement therapy and decrease in platelet count during disease progression, and remarkably were significantly associated with increased mortality rates in septic patients (ICU-, 28 day- and 90 day mortality rates). There was an inverse correlation between plasma histones and endogenous APC levels. Histone stimulation induced the release of sepsis prototypic cytokines and decreased cell integrity indicated by a significant increase of lactate dehydrogenase (LDH) and propidium

  18. Bioanalytical method development and validation for determination of metoprolol tartarate and hydrochlorothiazide using HPTLC in human plasma

    Directory of Open Access Journals (Sweden)

    Ambadas Ranganath Rote

    2013-12-01

    Full Text Available A simple, sensitive, rapid and economic chromatographic method has been developed for determination of metoprolol tartarate and hydrochlorothiazide in human plasma using paracetamol as an internal standard. The analytical technique used for method development was high-performance thin-layer chromatography. HPTLC Camag with precoated silica gel Plate 60F254 (20 cm×10 cm at 250 µm thicknesses (E. Merck, Darmstadt, Germany was used as the stationary phase. The mobile phase used consisted of chloroform: methanol: ammonia (9:1:0.5v/v/v. Densitometric analysis was carried out at a wavelength of 239 nm. The rf values for hydrochlorothiazide, paracetamol and metoprolol tartarate were 0.13±0.04, 0.28±0.05, 0.48±0.04, respectively. Plasma samples were extracted by protein precipitation with methanol. Concentration ranges of 200, 400, 600, 800, 1000, 1200 ng/mL and 2000, 4000, 6000, 8000, 10000, 12000 ng/mL of hydrochlorothiazide and metoprolol tartarate, respectively, were used with plasma for the calibration curves. The percent recovery of metoprolol tartarate and hydrochlorothiazide was found to be 77.30 and 77.02 %, respectively. The stability of metoprolol tartarate and hydrochlorothiazide in plasma were confirmed during three freeze-thaw cycles (-20 ºC on a bench for 24 hours and post-preparatively for 48 hours. The proposed method was validated statistically and proved suitable for determination of metoprolol tartarate and hydrochlorothiazide in human plasma.

  19. Determination of selenoprotein P in human plasma by solid phase extraction and inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    Bendahl, L.; Sidenius, U.; Gammelgaard, Bente

    2000-01-01

    measured by inductively coupled plasma mass spectrometry (ICP-MS) monitoring the Se-82 isotope. Linear response was observed in the concentration range 0.3-70.8 mu g/l selenium as selenoprotein P with a correlation coefficient of 0.9994. The precision expressed as relative standard deviation was better...... than 2% in this range. The estimated limit of detection was 2 mu g/l and the experimentally verified quantification limit was 5 mu g/l, giving a relative standard deviation less than 2%. (C) 2000 Elsevier Science B.V. All rights reserved...

  20. A simple high-performance liquid chromatography for the determination of linezolid in human plasma and saliva.

    Science.gov (United States)

    Hara, Shuuji; Uchiyama, Masanobu; Yoshinari, Masami; Matsumoto, Taichi; Jimi, Shiro; Togawa, Atsushi; Takata, Tohru; Takamatsu, Yasushi

    2015-09-01

    Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. A practical high-performance liquid chromatography method was developed for the determination of linezolid in human plasma and saliva. Linezolid and an internal standard (o-ethoxybenzamide) were extracted from plasma and saliva with ethyl acetate and analyzed on a Capcell Pak C18 MG column with UV detection at 254 nm. The calibration curve was linear through the range 0.5-50 µg/mL using a 200 μL sample volume. The intra- and interday precisions were all plasma and 5.60% for saliva. The accuracies ranged from 98.8 to 110% for both matrices. The mean recoveries of linezolid were 80.8% for plasma and 79.0% for saliva. This method was used to determine the plasma and saliva concentrations of linezolid in healthy volunteers who were orally administered a 600 mg dose of linezolid. Our liquid-liquid extraction procedure is easy and requires a small volume of plasma or saliva (200 μL). This small volume can be advantageous in clinical pharmacokinetic studies, especially if children participate. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Modulation of plasma N-acylethanolamine levels and physiological parameters by dietary fatty acid composition in humans.

    Science.gov (United States)

    Jones, Peter J H; Lin, Lin; Gillingham, Leah G; Yang, Haifeng; Omar, Jaclyn M

    2014-12-01

    N-Acylethanolamines (NAEs) are endogenous lipid-signaling molecules involved in satiety and energetics; however, how diet impacts circulating NAE concentrations and their downstream metabolic actions in humans remains unknown. Objectives were to examine effects of diets enriched with high-oleic canola oil (HOCO) or HOCO blended with flaxseed oil (FXCO), compared with a Western diet (WD), on plasma NAE levels and the association with energy expenditure and substrate oxidation. Using a randomized controlled crossover design, 36 hypercholesterolemic participants consumed three isoenergetic diets for 28 days, each containing 36% energy from fat, of which 70% was HOCO, FXCO, or WD. Ultra-performance liquid chromatography-MS/MS was used to measure plasma NAE levels and indirect calorimetry to assess energy expenditure and substrate oxidation. After 28 days, compared with WD, plasma oleoylethanolamide (OEA) and alpha-linolenoyl ethanolamide (ALEA) levels were significantly increased in response to HOCO and FXCO (P = 0.002, P < 0.001), respectively. Correlation analysis demonstrated an inverse association between plasma OEA levels and percent body fat (r = -0.21, P = 0.04), and a positive association was observed between the plasma arachidonoyl ethanolamide (AEA)/OEA ratio and android:gynoid fat (r = 0.23, P = 0.02), respectively. Results suggest that plasma NAE levels are upregulated via their dietary lipid substrates and may modulate regional and total fat mass through lipid-signaling mechanisms. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  2. Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

    International Nuclear Information System (INIS)

    Morrison, S.A.; Jesty, J.

    1984-01-01

    A comparism was made of the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and a study was made of the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H-labeled factor X to the plasma resulted, after a short lag, in burst-like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin

  3. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  4. Plasma endocannabinoid levels in lean, overweight and obese humans: relationships with intestinal permeability markers, inflammation and incretin secretion.

    Science.gov (United States)

    Little, Tanya J; Cvijanovic, Nada; DiPatrizio, Nicholas V; Argueta, Donovan A; Rayner, Christopher K; Feinle-Bisset, Christine; Young, Richard L

    2018-02-13

    Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation, however little is known of these effects in humans. This study aimed to: (i) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl-sn-glycerol (2-AG) and OEA in humans, and (ii) examine relationships with intestinal permeability, inflammation markers and incretin hormone secretion. 20 lean, 18 overweight and 19 obese participants underwent intraduodenal Intralipid® infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumour necrosis factor-α (TNF-α), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and toll-like receptor-4 (TLR4) (RT-PCR), were assessed. Fasting plasma AEA was increased in obese, compared with lean and overweight (Plean (Plean and overweight. The relationships between plasma AEA with duodenal ZO-1 and IAP, and GIP, suggest that altered endocannabinoid signalling may contribute to changes in intestinal permeability, inflammation and incretin release in human obesity.

  5. The detection of ovulation with a two-hour radioimmunoassay for human plasma luteinizing hormone using the Centria Analyzer

    International Nuclear Information System (INIS)

    Schwarz, S.

    1980-01-01

    We describe a rapid (2-h) radioimmunoassay for human plasma luteinizing hormone which utilizes the reagents from a commercially available kit. Standardization of the assay was achieved using plasma standards instead of a buffer system and the Centria radioimmunoassay centrifugal analyzer which allowed simultaneous initiation and termination of reactions in all assay tubes. The specificity, precision, and accuracy of the assay were equal to or better than the conventional 24-h assay. Since this assay is designed to detect the mid-cycle surge of luteinizing hormone, its decreased sensitivity was small price to pay for the speed with which a result could be obtained. (orig.) [de

  6. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

  7. Predictive value of four kallikrein markers for pathologically insignificant compared with aggressive prostate cancer in radical prostatectomy specimens: results from the European Randomized Study of Screening for Prostate Cancer section Rotterdam.

    Science.gov (United States)

    Carlsson, Sigrid; Maschino, Alexandra; Schröder, Fritz; Bangma, Chris; Steyerberg, Ewout W; van der Kwast, Theo; van Leenders, Geert; Vickers, Andrew; Lilja, Hans; Roobol, Monique J

    2013-11-01

    Treatment decisions can be difficult in men with low-risk prostate cancer (PCa). To evaluate the ability of a panel of four kallikrein markers in blood-total prostate-specific antigen (PSA), free PSA, intact PSA, and kallikrein-related peptidase 2-to distinguish between pathologically insignificant and aggressive disease on pathologic examination of radical prostatectomy (RP) specimens as well as to calculate the number of avoidable surgeries. The cohort comprised 392 screened men participating in rounds 1 and 2 of the Rotterdam arm of the European Randomized Study of Screening for Prostate Cancer. Patients were diagnosed with PCa because of an elevated PSA ≥3.0 ng/ml and were treated with RP between 1994 and 2004. We calculated the accuracy (area under the curve [AUC]) of statistical models to predict pathologically aggressive PCa (pT3-T4, extracapsular extension, tumor volume >0.5cm(3), or any Gleason grade ≥4) based on clinical predictors (age, stage, PSA, biopsy findings) with and without levels of four kallikrein markers in blood. A total of 261 patients (67%) had significant disease on pathologic evaluation of the RP specimen. While the clinical model had good accuracy in predicting aggressive disease, reflected in a corrected AUC of 0.81, the four kallikrein markers enhanced the base model, with an AUC of 0.84 (p limitation of the present study is that clinicians may be hesitant to make recommendations against active treatment on the basis of a statistical model. Our study provided proof of principle that predictions based on levels of four kallikrein markers in blood distinguish between pathologically insignificant and aggressive disease after RP with good accuracy. In the future, clinical use of the model could potentially reduce rates of immediate unnecessary active treatment. Copyright © 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  8. Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

    Science.gov (United States)

    Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

    2018-02-01

    1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

  9. Fabrication and physical and biological properties of fibrin gel derived from human plasma

    Science.gov (United States)

    Zhao, Haiguang; Ma, Lie; Zhou, Jie; Mao, Zhengwei; Gao, Changyou; Shen, Jiacong

    2008-03-01

    The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 °C, which is about 30 °C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of ~50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml-1) and thrombin (5 U ml-1) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

  10. Mesoporous silica particles modified with graphitic carbon: interaction with human red blood cells and plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Diego Stefani Teodoro; Franqui, Lidiane Silva; Bettini, Jefferson; Strauss, Mathias, E-mail: diego.martinez@lnnano.cnpem.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Damasceno, Joao Paulo Vita; Mazali, Italo Odone [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2016-07-01

    Full text: In this work the interaction of the mesoporous silica particles (SBA-15, ∼700 nm) modified with graphitic carbon (SBA-15/C) on human red blood cells (hemolysis) and plasma proteins (protein corona formation) is studied. XPS and CHN analysis showed that the carbon content on the SBA-15/C samples varied from 2 to 10% and was tuned by the functionalization step. The formed carbon structures where associated to graphitic nanodomains coating the pores surface as verified by Raman spectroscopy and {sup 13}C NMR. Advanced TEM/EELS analysis showed that the carbon structures are distributed along the SBA-15 mesopores. SAXS and textural analyses were used to confirm that the porous structure of the silica support is kept after the modification procedure and to calculate the number of graphitic carbon stacked layers coating the mesopores. After incubation of SBA-15 with human red blood cells (RBCs), it was observed a dose-dependent hemolytic effect, probably, due to binding of the material silanol-rich surface to the phosphatidylcholine molecules from the RBC membrane. The graphitic carbon modifications have mitigated this effect, indicating that the graphitic carbon coating protected the silanol groups of the particle surface hindering the hemolysis. Considering the protein corona formation, selective biomolecular interaction of proteins was observed for the different materials using gel electrophoresis (SDS-PAGE) analysis. Besides, graphitic carbon modification decreased the amount of proteins on the corona. Together, the in vitro hemolysis and protein corona assays are promising biological models to understand the influence of silica surface functionalization on their bionano-interactions. Finally, our work contributes to the development of fundamental research on such nanomaterials chemistry in the emerging field of nanobioscience and nanotoxicology. (author)

  11. Development and validation of RP-HPLC method for estimation of eplerenone in spiked human plasma

    Directory of Open Access Journals (Sweden)

    Paraag Gide

    2012-10-01

    Full Text Available A rapid and simple high performance liquid chromatography (HPLC method with a UV detection (241 nm was developed and validated for estimation of eplerenone from spiked human plasma. The analyte and the internal standard (valdecoxib were extracted with a mixture of dichloromethane and diethyl ether. The chromatographic separation was performed on a HiQSil C-18HS column (250 mm×4.6 mm, 5 μm with a mobile phase consisting of acetonitrile:water (50:50, v/v at flow rate of 1 mL/min. The calibration curve was linear in the range 100–3200 ng/mL and the heteroscedasticity was minimized by using weighted least squares regression with weighting factor 1/X. Keywords: Eplerenone, Liquid–liquid extraction, Weighted regression, HPLC–UV

  12. Phospholipid transfer from vesicles to high density lipoproteins, catalyzed by human plasma phospholipid transfer protein

    International Nuclear Information System (INIS)

    Sweeny, S.A.

    1985-01-01

    Human plasma phospholipid transfer protein (PLTP) catalyzes the mass transfer of phosphatidylcholine (PC). Partial purification of PLTP yielded proteins with apparent M/sub r/ = 59,000 and 40,000 by SDS-PAGE. PLTP activity was measured by transfer of [ 14 C]L-α-dipalmitoyl PC from egg-PC vesicles to HDL. Activity was enhanced at low pH (4.5) upon addition of β-mercaptoethanol while Ca +2 and Na + had no effect. E/sub act/ for facilitated PC transfer was 18.2 +/- 2 kcal/mol. The donor specificity of PLTP was examined using vesicles containing egg-PC plus cholesterol or sphingomyelin. The fluidity of the donor membrane (measured by fluorescence polarization of diphenylhexatriene) correlated strongly with a decrease in PLTP activity. Phosphatidic acid did not affect activity. Increase in vesicle size reduced activity. The acceptor specificity of PLTP was examined using chemically modified HDL. PLTP activity increased up to 1.7-fold with an initial increase in negative charge and then decreased upon extensive modification. A mechanism is proposed where PLTP binds to vesicls and enhances the diffusion of PC into the medium where it is adsorbed by HDL

  13. Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

    Science.gov (United States)

    Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

    2017-03-01

    To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  14. LC-MS/MS determination of tranexamic acid in human plasma after phospholipid clean-up.

    Science.gov (United States)

    Fabresse, Nicolas; Fall, Fanta; Etting, Isabelle; Devillier, Philippe; Alvarez, Jean-Claude; Grassin-Delyle, Stanislas

    2017-07-15

    Tranexamic acid is a widely used antifibrinolytic drug but its pharmacology and pharmacokinetics remains poorly understood. Owing to the recent knowledge on phospholipid-induced matrix effects during human plasma analysis, our aim was to develop a liquid chromatography-mass spectrometry method for the quantitation of tranexamic acid after efficient sample clean-up. Sample preparation consisted in phospholipid removal and protein precipitation. Hydrophilic interaction liquid chromatography was used and the detection was achieved with multiple reaction monitoring. The method was validated according to the European Medicine Agency guideline in the range 1.0-1000.0μg/mL. The performance of the method was excellent with a precision in the range 1.2-3.0%, an accuracy between 88.4 and 96.6% and a coefficient of variation of the internal standard-normalized matrix factor below 6.7%. This method is suitable for the quantification of tranexamic acid in the wide range of concentrations observed during clinical studies, with all the advantages related to phospholipid removal. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

    Directory of Open Access Journals (Sweden)

    Irena Trbojević-Akmačić

    2016-06-01

    Full Text Available Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License.

  16. Inactivation of animal and human prions by hydrogen peroxide gas plasma sterilization.

    Science.gov (United States)

    Rogez-Kreuz, C; Yousfi, R; Soufflet, C; Quadrio, I; Yan, Z-X; Huyot, V; Aubenque, C; Destrez, P; Roth, K; Roberts, C; Favero, M; Clayette, P

    2009-08-01

    Prions cause various transmissible spongiform encephalopathies. They are highly resistant to the chemical and physical decontamination and sterilization procedures routinely used in healthcare facilities. The decontamination procedures recommended for the inactivation of prions are often incompatible with the materials used in medical devices. In this study, we evaluated the use of low-temperature hydrogen peroxide gas plasma sterilization systems and other instrument-processing procedures for inactivating human and animal prions. We provide new data concerning the efficacy of hydrogen peroxide against prions from in vitro or in vivo tests, focusing on the following: the efficiency of hydrogen peroxide sterilization and possible interactions with enzymatic or alkaline detergents, differences in the efficiency of this treatment against different prion strains, and the influence of contaminating lipids. We found that gaseous hydrogen peroxide decreased the infectivity of prions and/or the level of the protease-resistant form of the prion protein on different surface materials. However, the efficiency of this treatment depended strongly on the concentration of hydrogen peroxide and the delivery system used in medical devices, because these effects were more pronounced for the new generation of Sterrad technology. The Sterrad NX sterilizer is 100% efficient (0% transmission and no protease-resistant form of the prion protein signal detected on the surface of the material for the mouse-adapted bovine spongiform encephalopathy 6PB1 strain and a variant Creutzfeldt-Jakob disease strain). Thus, gaseous or vaporized hydrogen peroxide efficiently inactivates prions on the surfaces of medical devices.

  17. Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma

    International Nuclear Information System (INIS)

    Miida, T.; Fielding, C.J.; Fielding, P.E.

    1990-01-01

    The transfer of [ 3 H]cholesterol in low-density lipoprotein (LDL) to different high-density lipoprotein (HDL) species in native human plasma was determined by using nondenaturing two-dimensional electrophoresis. Transfer from LDL had a t 1/2 at 37 degree C of 51 ± 8 min and an activation energy of 18.0 kCal mol -1 . There was unexpected specificity among HDL species as acceptors of LDL-derived labeled cholesterol. The largest fraction of the major α-migrating class (HDL 2b ) was the major initial acceptor of LDL-derived cholesterol. Kinetic analysis indicated a rapid secondary transfer from HDL 2b to smaller αHDL (particularly HDL 3 ) driven enzymatically by the lecithin-cholesterol acyltransferase reaction. Rates of transfer among αHDL were most rapid from the largest αHDL fraction (HDL 2b ), suggesting possible protein-mediated facilitation. Simultaneous measurements of the transport of LDL-derived and cell-derived isotopic cholesterol indicated that the former preferably utilized the αHDL pathyway, with little label in pre-βHDL. The same experiments confirmed earlier data that cell-derived cholesterol is preferentially channeled through pre-βHDL. The authors suggest that the functional heterogeneity of HDL demonstrated here includes the ability to independently process cell- and LDL-derived free cholesterol

  18. Phosphorylation of chloroform soluble compounds in plasma membranes of human epidermoid carcinoma A431 cells

    International Nuclear Information System (INIS)

    Brautigan, D.L.; Randazzo, P.; Shriner, C.; Fain, J.N.

    1985-01-01

    This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. The authors found a novel chloroform-soluble product radiolabeled with [gamma- 32 P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32 P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid

  19. Ionospheric disturbances generated by different natural processes and by human activity in Earth plasma environment

    Directory of Open Access Journals (Sweden)

    J. Blecki

    2004-06-01

    Full Text Available The magnetosphere-ionosphere-thermosphere subsystem is strongly coupled via the electric field, particle precipitation, heat flows and small scale interaction. Satellites in situ measurements and ground based complex diagnostics can provide comprehensive coverage of both time and geomagnetic place effects. Human activity also can perturb Earth s environment, but few are connected with controlled experiments in the ionosphere and are transient. Most of them are related to industrial activity and have increased in recent years. The most important power sources are broadcasting transmitters, power stations, power lines and heavy industry. At ionospheric altitude some disturbances and physical processes are related to seismic activity, thunderstorm activity and some global changes in the Earth environment such as ozone holes. Various natural and artificial indicators can affect satellite telecommunication quality. The aim of this presentation is to report progress in understanding the physical processes in the ionosphere described above and to assess the application of these considerations to the study of plasma effects on Earth-space and satellite-to-satellite communication.

  20. Evaluation of effects of platelet-rich plasma on human facial skin.

    Science.gov (United States)

    Yuksel, Esra Pancar; Sahin, Gokhan; Aydin, Fatma; Senturk, Nilgun; Turanli, Ahmet Yasar

    2014-10-01

    Platelet-rich plasma (PRP) has been used for rapid healing and tissue regeneration in many fields of medicine. This study was conducted to evaluate the effects of PRP application procedure on human facial skin. PRP was applied thrice at 2-week intervals on the face of ten healthy volunteers. It was applied to individual's forehead, malar area, and jaw by a dermaroller, and injected using a 27-gauge injector into the wrinkles of crow's feet. Participants were asked to grade on a scale from 0 to 5 for general appearance, skin firmness-sagging, wrinkle state and pigmentation disorder of their own face before each PRP procedure and 3 months after the last PRP procedure. While volunteers were evaluating their own face, they were also assessed by three different dermatologists at the same time by the same five-point scale. There was statistically significant difference regarding the general appearance, skin firmness-sagging and wrinkle state according to the grading scale of the patients before and after three PRP applications. Whereas there was only statistically significant difference for the skin firmness-sagging according to the assessment of the dermatologists. PRP application could be considered as an effective procedure for facial skin rejuvenation.

  1. Analytical method by high resolution liquid chromatography for the determination of carbamazepine in human plasma

    International Nuclear Information System (INIS)

    Jimenez Aleman, Narda M; Calero Carbonell, Jorge E; Padron Yaquis, Alejandro S; Izquierdo Lozano, Julio C

    2007-01-01

    One of the requirements to develop the studies of bioavailability and bioequivalence is to have analytic methodologies validated for the work with samples in biological fluids. A method was developed by high resolution liquid chromatography for the determination of carbamazepine in human plasma. A mixture of hydrogen phosphate of sodium: acetonitrile (65:35) adjusted to pH= 3.3 with phosphoric acid, flow of 1.2 mL/min and ultraviolet detection at 210 nm, was used as mobile phase. Propylparabene was used as an internal standard. According to the established regulations for the validation of the methods in biological fluids, the following parameters were studied: stability of the samples, lineality, specificity, precision, accuracy and limit of detection and quantification. The method proved to be specific and sensitive with a detection and quantification limit of 0.9 and 1.0 ng, respectively. The method was lineal, precise and exact in the range of concentrations of 1. 07 at 12.67 μg/mL. The mean recovery was not statistically different from 100.0 %. The analito in the proposed biological matrix remained in the studied period. The methodology described in this work is applied in our case to the study that evaluates the bioavailability and bioequivalence of a Cuban formulation of carbamazepine in healthy volunteers. (Author)

  2. Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma.

    Science.gov (United States)

    Bagheri, Akbar; Hosseini, Hadi

    2012-12-01

    The electrochemical behavior of raloxifene (RLX) on the surface of a glassy carbon electrode (GCE) has been studied by cyclic voltammetry (CV). The CV studies were performed in various supporting electrolytes, wide range of potential scan rates, and pHs. The results showed an adsorption-controlled and quasi-reversible process for the electrochemical reaction of RLX, and a probable redox mechanism was suggested. Under the optimum conditions, differential pulse voltammetry (DPV) was applied for quantitative determination of the RLX in pharmaceutical formulations. The DPV measurements showed that the anodic peak current of the RLX was linear to its concentration in the range of 0.2-50.0μM with a detection limit of 0.0750μM, relative standard deviation (RSD %) below 3.0%, and a good sensitivity. The proposed method was successfully applied for determination of the RLX in pharmaceutical and human plasma samples with a good selectivity and suitable recovery. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study

    International Nuclear Information System (INIS)

    Fenske, D.B.; Chana, R.S.; Parmar, Y.I.; Treleaven, W.D.; Cushley, R.J.

    1990-01-01

    The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31 P NMR. Lateral diffusion coefficients, D T , obtained from the viscosity dependence of the 31 P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL 2 , HDL 3 ), and egg PC/TO microemulsions at 25 degree C, for VLDL at 40 degree C, and for LDL at 45 degree C. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, Δσ, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence for the 31 P NMR line widths. These results suggest differences in the orientation and/or ordering of the head-group in the HDLs. The dynamic behavior of the phosphate moiety in LDL and HDL 3 has been obtained from the temperature dependence of the 31 P spin-lattice relaxation rates. Values of the correlation time for phosphate group reorientation and the activation energy for the motion are nearly identical in LDL and HDL 3 and are similar to values obtained for phospholipid bilayers. This argues against long-lived protein-lipid interactions being the source of either the slow diffusion in LDL or the altered head-group orientation in the HDLs

  4. Effects of itopride hydrochloride on plasma gut-regulatory peptide and stress-related hormone levels in healthy human subjects.

    Science.gov (United States)

    Katagiri, Fumihiko; Shiga, Toru; Inoue, Shin; Sato, Yuhki; Itoh, Hiroki; Takeyama, Masaharu

    2006-01-01

    Itopride hydrochloride (itopride), a gastrokinetic drug, has recently been evaluated for its clinical usefulness in functional dyspepsia. We investigated effects of itopride on human plasma gastrin-, somatostatin-, motilin-, and cholecystokinin (CCK)-like immunoreactive substances (IS); adrenocorticotropic hormone (ACTH)-immunoreactive substances (IS), and cortisol under stress conditions in healthy subjects. A single administration of itopride caused significant increases in plasma somatostatin- and motilin-IS levels compared to placebo. Itopride significantly decreased plasma CCK-IS, and suppressed the ACTH-IS level compared to placebo. We hypothesize that itopride may have an accelerating gastric emptying effect, and a modulatory effect on the hypothalamo-pituitary-adrenal axis and autonomic nervous functions. These effects might be beneficial in stress-related diseases, suggesting that itopride has clinicopharmacological activities.

  5. Routine ultramicro-measurement of human growth hormone in plasma by solid-phase radioimmunoassay and its clinical application

    International Nuclear Information System (INIS)

    Ogawa, Norio; Takahara, Jiro; Ofuji, Tadashi

    1975-01-01

    The development and application of the method for the ultramicromeasurement of human growth hormone (HGH) in plasma based on the solid-phase radioimmunoassay (RIA) by using antibody-coated disposable microtiter trays was reported. There was no statistical significance between the basal HGH level obtained from this method and that from the double-antibody RIA in normal subjects. On the other hand, the mean basal plasma HGH level after an overnight fasting in 21 hypopituitary patients was 484 pg/ml (+-282; 1 SD) by this method, and this was significantly lower (P<0.001) than 1376 pg/ml (+-498; 1 SD) by the double-antibody RIA. These data show that determination of basal plasma HGH levels by this method is of much value in the diagnosis of hypopituitarism. (JPN)

  6. Interrelation between human fertility and seminal plasma lipids, prostaglandins and zinc

    International Nuclear Information System (INIS)

    Hafiez, A.A.; Zaki, K.; Abbas, E.Z.; Halawa, F.A.; Abdel-Azis, A.

    1986-01-01

    In adult fertile men (32), men with oligospermia (43) and men with azoospermia (31) seminal plasma lipids, prostaglandins (PG) and Zn were determined. The PGs were determined by radioimmunoassay. In oligospermia the seminal plasma levels of PGE phospholipids, triglycerides and Zn were significantly increased, while the PGF/sub 2α/ level was unchanged. In azoospermia the seminal plasma total lipids, phospholipids and cholesterol were significantly decreased, PGE revealed an insignificant decrease only

  7. Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma.

    Science.gov (United States)

    Staempfli, Henry R; Constable, Peter D

    2003-08-01

    The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.

  8. Proteomics reveals the effects of sustained weight loss on the human plasma proteome

    DEFF Research Database (Denmark)

    Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

    2016-01-01

    Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed...... by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly...

  9. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    Science.gov (United States)

    Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

    2008-10-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

  10. iTRAQ based investigation of plasma proteins in HIV infected and HIV/HBV coinfected patients - C9 and KLK are related to HIV/HBV coinfection.

    Science.gov (United States)

    Sun, Tao; Liu, Li; Wu, Ao; Zhang, Yujiao; Jia, Xiaofang; Yin, Lin; Lu, Hongzhou; Zhang, Lijun

    2017-10-01

    Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) share similar routes of transmission, and rapid progression of hepatic and immunodeficiency diseases has been observed in coinfected individuals. Our main objective was to investigate the molecular mechanism of HIV/HBV coinfections. We selected HIV infected and HIV/HBV coinfected patients with and without Highly Active Antiretroviral Therapy (HAART). Low abundance proteins enriched using a multiple affinity removal system (MARS) were labeled with isobaric tags for relative and absolute quantitation (iTRAQ) kits and analyzed using liquid chromatography-mass spectrometry (LC-MS). The differential proteins were analyzed by Gene Ontology (GO) database. A total of 41 differential proteins were found in HIV/HBV coinfected patients as compared to HIV mono-infected patients with or without HAART treatment, including 7 common HBV-regulated proteins. The proteins involved in complement and coagulation pathways were significantly enriched, including plasma kallikrein (KLK) and complement component C9 (C9). C9 and KLK were verified to be down-regulated in HIV/HBV coinfected patients through ELISA analysis. The present iTRAQ based proteomic analyses identified 7 proteins that are related to HIV/HBV coinfection. HBV might influence hepatic and immune functions by deregulating complement and coagulation pathways. C9 and KLK could potentially be used as targets for the treatment of HIV/HBV coinfections. Copyright © 2017. Published by Elsevier Ltd.

  11. Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

    Science.gov (United States)

    Kratzer, Alexander; Kees, Frieder; Dorn, Christoph

    2016-12-15

    Ultrafiltration is a rapid and convenient method to determine the free concentrations of drugs in plasma. Several ultrafiltration devices based on Eppendorf cups are commercially available, but are not validated for such use by the manufacturer. Plasma pH, temperature and relative centrifugal force as well as membrane type can influence the results. In the present work, we developed an ultrafiltration method in order to determine the free concentrations of linezolid or fluconazole, both neutral and moderately lipophilic antiinfective drugs for parenteral as well as oral administration, in plasma of patients. Whereas both substances behaved relatively insensitive in human plasma regarding variations in pH (7.0-8.5), temperature (5-37°C) or relative centrifugal force (1000-10.000xg), losses of linezolid were observed with the Nanosep Omega device due to adsorption onto the polyethersulfone membrane (unbound fraction 75% at 100mg/L and 45% at 0.1mg/L, respectively). No losses were observed with Vivacon which is equipped with a membrane of regenerated cellulose. With fluconazole no differences between Nanosep and Vivacon were observed. Applying standard conditions (pH 7.4/37°C/1000xg/20min), the mean unbound fraction of linezolid in pooled plasma from healthy volunteers was 81.5±2.8% using Vivacon, that of fluconazole was 87.9±3.5% using Nanosep or 89.4±3.3% using Vivacon. The unbound fraction of linezolid was 85.4±3.7% in plasma samples from surgical patients and 92.1±6.2% in ICU patients, respectively. The unbound fraction of fluconazole was 93.9±3.3% in plasma samples from ICU patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Effect of the long-term regular intake of virgin olive oil on the phenolic metabolites in human fasting plasma.

    Science.gov (United States)

    Valls, Rosa-Maria; Soler, Aranzazu; Girona, Josefa; Heras, Mercedes; Romero, Maria-Paz; Covas, Maria-Isabel; Solà, Rosa; Masana, Lluis; Motilva, Maria-Jose

    2010-09-21

    The effect of repeated consumption of virgin olive oil on endogenous phenolic metabolites of fasting plasma is unknown. For this reason, we hypothesized that regular long-term virgin olive oil intake could have an indirect protection effect on the endogenous phenols. Thus, the aim of the study was to determine the phenolic profile of human plasma in a fasting state of long-term regular virgin olive oil consumers, using the fasting plasma of non-consumers as a natural control. Forty participants living in the area of Reus (Catalonia, Spain) were selected, 20 life-long regular consumers of virgin olive oil and a natural control of 20 non-consumers, the latter being Rumanians who dislike the taste of olive oil. The diet was obtained from 3-day food records. The results showed similar phenolic composition of fasting plasmas of the two volunteer groups. Of special interest is that more of the compounds quantified showed higher concentration in fasting plasma from habitual virgin olive oil consumers. The compounds were semi-quantified using caffeic acid as the calibration standard. The quantification of fasting consumer's plasma showed higher concentration of a hydroxyflavanone type compound (2.90+/-0.04 microM vs 1.5+/-0.04 microM) and a catecholamine derivative (0.70+/-0.03 microM vs 0.56+/-0.03 microM) than the plasma of non-consumers (P<0.05). The results suggest an indirect protective mechanism of long-term regular virgin olive oil consumption related to the protection of the endogenous antioxidant system. Copyright 2010 Elsevier B.V. All rights reserved.

  13. Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.

    Science.gov (United States)

    Graham, Mark J; Lee, Richard G; Bell, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M

    2013-05-24

    Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

  14. A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle.

    Science.gov (United States)

    Hussey, Sophie E; Lum, Helen; Alvarez, Andrea; Cipriani, Yolanda; Garduño-Garcia, Jesús; Anaya, Luis; Dube, John; Musi, Nicolas

    2014-03-01

    Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals. Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp. Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of inhibitor of kappa Bα (p = 0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation. A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. ClinicalTrials.gov NCT01740817.

  15. Sensitive quantification of apomorphine in human plasma using a LC-ESI-MS-MS method.

    Science.gov (United States)

    Abe, Emuri; Alvarez, Jean-Claude

    2006-06-01

    An analytical method based on liquid chromatography coupled with ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the identification and quantification of apomorphine in human plasma. Apomorphine was isolated from 0.5 mL of plasma using a liquid-liquid extraction with diethyl ether and boldine as internal standard, with satisfactory extraction recoveries. Analytes were separated on a 5-microm C18 Highpurity (Thermohypersil) column (150 mm x 2.1 mm I.D.) maintained at 30 degrees C, coupled to a precolumn (C18, 5-microm, 10 mm x 2.0 mm I.D., Thermo). The elution was achieved isocratically with a mobile phase of 2 mM NH4COOH buffer pH 3.8/acetonitrile (50/50, vol/vol) at a flow rate of 200 microL per minute. Data were collected either in full-scan MS mode at m/z 150 to 500 or in full-scan tandem mass spectrometry mode, selecting the [M+H]ion at m/z 268.0 for apomorphine and m/z 328.0 for boldine. The most intense daughter ion of apomorphine (m/z 237.1) and boldine (m/z 297.0) were used for quantification. Retention times were 2.03 and 2.11 minutes for boldine and apomorphine, respectively. Calibration curves were linear in the 0.025 to 20 ng/mL range. The limits of detection and quantification were 0.010 ng/mL and 0.025 ng/mL, respectively. Accuracy and precision of the assay were measured by analyzing 54 quality control samples for 3 days. At concentrations of 0.075, 1.5, and 15 ng/mL, intraday precisions were less than 10.1%, 5.3%, and 3.8%, and interday precisions were less than 4.8%, 6.6%, and 6.5%, respectively. Accuracies were in the 99.5 to 104.2% range. An example of a patient who was given 6 mg of apomorphine subcutaneously is shown, with concentrations of 14.1 ng/mL after 30 minutes and 0.20 ng/mL after 6 hours. The method described enables the unambiguous identification and quantification of apomorphine with very good sensitivity using only 0.5 mL of sample, and is very convenient for therapeutic drug

  16. The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age.

    Science.gov (United States)

    Trabado, Séverine; Al-Salameh, Abdallah; Croixmarie, Vincent; Masson, Perrine; Corruble, Emmanuelle; Fève, Bruno; Colle, Romain; Ripoll, Laurent; Walther, Bernard; Boursier-Neyret, Claire; Werner, Erwan; Becquemont, Laurent; Chanson, Philippe

    2017-01-01

    Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the "normal" metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the "normal" metabolome and its main sources of variation.

  17. In vitro effect of sodium nitrite on platelet aggregation in human platelet rich plasma--preliminary report.

    Science.gov (United States)

    Kadan, M; Doğanci, S; Yildirim, V; Özgür, G; Erol, G; Karabacak, K; Avcu, F

    2015-10-01

    The role of nitrates and nitric oxide on platelet functions has obtained an increasing attention with respect to their potential effects on cardiovascular disorders. In this study we aimed to analyze the effect of sodium nitrite on platelet functions in human platelets. This in vitro study was designed to show the effect of sodium nitrite on platelet functions in seven healthy volunteers. Blood samples were centrifuged to prepare platelet rich plasma and platelet poor plasma. Platelet rich plasma was diluted with the platelet poor plasma to have a final count of 300,000 ± 25,000 platelets. Platelet rich plasma was incubated with six different increasing doses (from 10 μM to 5 mM) of sodium nitrite for 1 hour at 37°C. Then stimulating agents including collagen (3 μg ml-1), adenosine diphosphate (10 μM), and epinephrine (10 μM) were added to the cuvette. Changes in light transmission were observed for 10 minutes. In addition spontaneous aggregation were performed in control group with all aggregating agents separately. Effect of sodium nitrite on agonist-induced platelet aggregation depends on the concentration of sodium nitrite. Compared with control group, agonist-induced platelet aggregations were significantly suppressed by sodium nitrite at the concentration of 5, 1.0 and 0.5 mM. Our results suggested that sodium nitrite has inhibitory effects in vitro on platelet aggregation in a dose-dependent manner.

  18. Beta-lipotropin is the major component of the plasma opioid response to surgical stress in humans

    Energy Technology Data Exchange (ETDEWEB)

    Porro, C.A.; Facchinetti, F.; Bertellini, E.; Petraglia, F.; Stacca, R.; Barbieri, G.C.; Genazzani, A.R.

    1987-12-07

    There is growing experimental evidence that beta-endorphin immunoreactivity is raised by surgical stress in patients undergoing general anesthesia. As the assay methods employed to date did not allow to fully discriminate between beta-endorphin and its immediate precursor, beta-lipotropin, the authors have investigated in the present study plasma levels of these two peptides by separating them by chromatography on plasma extracts prior to radioimmunoassay. Beta-lipotropin, but not beta-endorphin, plasma levels were found to be significantly elevated during surgery in the general anesthesia group, while no change was found in either peptide concentration in the spinal one. Cortisol plasma levels also increased significantly 90 minutes after the beginning of surgery. Although the sampling time they adopted may have prevented them from detecting an early peak of beta-endorphin during the first 30 minutes of surgery, the major component of the pituitary opioid response to surgical stress appears to be related to beta-lipotropin. This is in agreement with results of experimental work on various kinds of stress in animals and humans and seems to rule out a role for plasma beta-endorphin in post-operative analgesia. 38 references, 1 figure, 1 table.

  19. Exercise-induced shear stress is associated with changes in plasma von Willebrand factor in older humans.

    Science.gov (United States)

    Gonzales, Joaquin U; Thistlethwaite, John R; Thompson, Benjamin C; Scheuermann, Barry W

    2009-07-01

    Shear stress is the frictional force of blood against the endothelium, a stimulus for endothelial activation and the release of von Willebrand factor (vWF). This study tested the hypothesis that the increase in shear stress associated with exercise correlates with plasma vWF. Young (n = 14, 25.7 +/- 5.4 years) and older (n = 13, 65.6 +/- 10.7 years) individuals participated in 30 min of dynamic handgrip exercise at a moderate intensity. Brachial artery diameter and blood flow were measured using ultrasound Doppler and blood samples were collected before, immediately after, and following 30 min of recovery from exercise with plasma levels of vWF. Plasma levels of vWF increased (P exercise. The change in plasma vWF was linearly correlated with the increase in shear stress during exercise in older individuals (post-exercise: r = 0.78, 30 min recovery: r = 0.77, P < 0.01), but no association was found in the young individuals. These changes in plasma levels of vWF in humans suggest that aging influences endothelial activation and hemostasis.

  20. Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Krämer, Lisa; Jäger, Christian; Trezzi, Jean-Pierre; Jacobs, Doris M; Hiller, Karsten

    2018-02-14

    Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13 C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13 C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13 C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13 C-labeled bread and quantified 13 C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated.

  1. The effects of eating marine- or vegetable-fed farmed trout on the human plasma proteome profiles of healthy men

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Lametsch, René; Bügel, Susanne

    2015-01-01

    Most human intervention studies have examined the effects on a subset of risk factors, some of which may require long-term exposure. The plasma proteome may reflect the underlying changes in protein expression and activation, and this could be used to identify early risk markers. The aim of the p......Most human intervention studies have examined the effects on a subset of risk factors, some of which may require long-term exposure. The plasma proteome may reflect the underlying changes in protein expression and activation, and this could be used to identify early risk markers. The aim...... of the present study was to evaluate the impact of regular fish intake on the plasma proteome. We recruited thirty healthy men aged 40 to 70 years, who were randomly allocated to a daily meal of chicken or trout raised on vegetable or marine feeds. Blood samples were collected before and after 8 weeks...... of intervention, and after the removal of the twelve most abundant proteins, plasma proteins were separated by two-dimensional gel electrophoresis. Protein spots 4·3 visualised by silver staining were matched by two-dimensional imaging software. Within-subject changes in spots were compared...

  2. Quantitative determination of 21-hydroxy-deflazacort in human plasma using gradient semi-microbore liquid chromatography.

    Science.gov (United States)

    Reynolds, D L; Burmaster, S D; Eichmeier, L S

    1994-01-01

    A sensitive and selective liquid chromatographic procedure to quantitate the deflazacort metabolite 21-hydroxy-deflazacort (DF-21OH) in human plasma was developed and validated. DF-21OH and fludrocortisone acetate (internal standard, IS) were isolated from human plasma (2 mL) by solid-phase extraction onto C-18 cartridges. Potential interferences were selectively removed and analytes were eluted with ethyl acetate. Following evaporation, the residue was reconstituted for HPLC analysis. Separation was achieved by gradient elution using a 5 microns YMC Basic column (2.0 x 100 mm) with mobile phases consisting of 20% methanol and 50% acetonitrile in 50 mM phosphate buffer (pH 3) at a temperature of 50 degrees C. Flow rate was maintained at 0.3 mL/min., and analytes were quantified spectrophotometrically at 246 nm. The assay was validated over the range 1.0 to 500 ng/mL DF-21OH. Calibration curves were prepared using a weighted (1/concentration) nonlinear quadratic regression algorithm. Peak-height ratios were proportional to the amount of DF-21OH added to plasma. Assay precision (%RSD) ranged from 4.2 to 11%, with a corresponding assay accuracy (% relative error) of +/- 2.8%. Absolute recovery of DF-21OH from plasma was 78-86% over the concentration range. The minimum quantitation limit was 1.0 ng/mL.

  3. Improvement in the performance of external quality assessment in Korean HIV clinical laboratories using unrecalcified human plasma.

    Science.gov (United States)

    Wang, Jin-Sook; Kee, Mee-Kyung; Choi, Byeong-Sun; Kim, Chan-Wha; Kim, Hyon-Suk; Kim, Sung Soon

    2012-01-01

    The external quality assessment schemes (EQAS) organizer provides a suitable program to monitor and improve the quality of human immunodeficiency virus (HIV) testing laboratories with EQAS panels prepared under various conditions. The aim of the current study was to investigate the effects of human plasma samples on the EQAS results of HIV obtained from hospital-based clinical laboratories. From 2007 to 2009, HIV EQAS panels consisted of four to six samples that consisted of undiluted positive and negative samples and were provided to laboratories twice per year. Up until the first half EQAS in 2008, EQAS panel materials were obtained by converting acid citrate dextrose treated plasma to serum via chemical treatment with CaCl2. Beginning with the second EQAS in 2008, all materials were prepared without the defibrination process. Approximately 300 HIV clinical laboratories participated in this program. The overall performance of clinical laboratories was shown to be improved when using unrecalcified plasma panels compared with recalcified panels. Significant differences were observed in EIA analyses of plasma for both positive (plaboratories.

  4. Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry

    Science.gov (United States)

    Krämer, Lisa; Jäger, Christian; Jacobs, Doris M.; Hiller, Karsten

    2018-01-01

    Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS). The limit of quantification was increased by optimizing (1) the metabolite extraction from plasma, (2) the GC-MS measurement, and (3) most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine). Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated. PMID:29443915

  5. Quantification of Stable Isotope Traces Close to Natural Enrichment in Human Plasma Metabolites Using Gas Chromatography-Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Lisa Krämer

    2018-02-01

    Full Text Available Currently, changes in metabolic fluxes following consumption of stable isotope-enriched foods are usually limited to the analysis of postprandial kinetics of glucose. Kinetic information on a larger diversity of metabolites is often lacking, mainly due to the marginal percentage of fully isotopically enriched plant material in the administered food product, and hence, an even weaker 13C enrichment in downstream plasma metabolites. Therefore, we developed an analytical workflow to determine weak 13C enrichments of diverse plasma metabolites with conventional gas chromatography-mass spectrometry (GC-MS. The limit of quantification was increased by optimizing (1 the metabolite extraction from plasma, (2 the GC-MS measurement, and (3 most importantly, the computational data processing. We applied our workflow to study the catabolic dynamics of 13C-enriched wheat bread in three human subjects. For that purpose, we collected time-resolved human plasma samples at 16 timepoints after the consumption of 13C-labeled bread and quantified 13C enrichment of 12 metabolites (glucose, lactate, alanine, glycine, serine, citrate, glutamate, glutamine, valine, isoleucine, tyrosine, and threonine. Based on isotopomer specific analysis, we were able to distinguish catabolic profiles of starch and protein hydrolysis. More generally, our study highlights that conventional GC-MS equipment is sufficient to detect isotope traces below 1% if an appropriate data processing is integrated.

  6. Biological variability dominates and influences analytical variance in HPLC-ECD studies of the human plasma metabolome

    Directory of Open Access Journals (Sweden)

    Willett Walter C

    2007-11-01

    Full Text Available Abstract Background Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify ad libitum fed and caloric-restricted rats. These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease. Methods Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection. Results We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30. At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals (N = 10 at 0, 24, and 48 hours, suggesting the influence of some biological factors on parameters normally considered as analytical. Conclusion Overall analytical precision (mean median CV, ~9% and total between-person variation (median CV, ~50–70% appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk.

  7. Liquid chromatography tandem mass spectrometry method for simultaneous determination of metoprolol tartrate and ramipril in human plasma.

    Science.gov (United States)

    Gowda, K Veeran; Mandal, Uttam; Senthamil Selvan, P; Sam Solomon, W D; Ghosh, Animesh; Sarkar, Amlan Kanti; Agarwal, Sangita; Nageswar Rao, T; Pal, Tapan Kumar

    2007-10-15

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol tartrate (MT) and ramipril, in human plasma. Both the drugs were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v). The chromatographic separation was performed on a reversed-phase C8 column with a mobile phase of 10 mM ammonium formate-methanol (3:97, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 5-500 ng/ml for metoprolol and ramipril in human plasma. The precursor to product ion transitions of m/z 268.0-103.10 and m/z 417.20-117.20 were used to measure metoprolol and ramipril, respectively.

  8. Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

    Directory of Open Access Journals (Sweden)

    Srinivasa Rao Polagani

    2013-02-01

    Full Text Available A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC–MS/MS assay method has been developed and fully validated for the simultaneous quantification of atorvastatin, metformin and glimepiride in human plasma. Carbamazepine was used as internal standard (IS. The analytes were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile. The reconstituted samples were chromatographed on a Alltima HP C18 column by using a 60:40 (v/v mixture of acetonitrile and 10 mM ammonium acetate (pH 3.0 as the mobile phase at a flow rate of 1.1 mL/min. The calibration curves obtained were linear (r2≥0.99 over the concentration range of 0.50–150.03 ng/mL for atorvastatin, 12.14–1207.50 ng/mL for metformin and 4.98–494.29 ng/mL for glimepiride. The API-4000 LC–MS/MS in multiple reaction monitoring (MRM mode was used for detection. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. All the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers. Keywords: Atorvastatin, Metformin, Glimepiride, LC–MS/MS, Human plasma, Pharmacokinetics

  9. Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma

    Directory of Open Access Journals (Sweden)

    Sander Bekeschus

    2016-01-01

    Full Text Available In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α, differentiation (retinoic acid signaling and interferon inducible factors, and cell growth (Yin Yang 1. Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1 and of the neutrophil attractant chemokine interleukin-8 (IL-8. Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

  10. Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma.

    Science.gov (United States)

    Bekeschus, Sander; Schmidt, Anke; Bethge, Lydia; Masur, Kai; von Woedtke, Thomas; Hasse, Sybille; Wende, Kristian

    2016-01-01

    In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

  11. BDNF Val66Met homozygosity does not influence plasma BDNF levels in healthy human subjects

    NARCIS (Netherlands)

    Luykx, J.J.; Boks, M.P.M.; Breetvelt, E.J.; Aukes, M.F.; Strengman, E.; da Pozzo, E.; Dell'osso, L.; Marazziti, D.; van Leeuwen, A.; Vreeker, A.; Abramovic, L.; Martini, C.; Numans, M.E.; Kahn, R. S.; Ophoff, R. A.

    2013-01-01

    A putative pathway by which the BDNF Val66Met polymorphism (rs6265) leads to aberrant phenotypes is its influence on plasma BDNF. Research into the impact of rs6265 on plasma BDNF has given rise to conflicting results. Moreover, most such studies have compared Met-carriers with Val-homozygous

  12. Comparative assessment of saliva and plasma for drug bioavailability and bioequivalence studies in humans

    Directory of Open Access Journals (Sweden)

    Nasir M. Idkaidek

    2017-07-01

    Conclusion: Our results suggest that there is a potential in BA/BE studies for saliva to be considered as a surrogate for plasma concentration, which goes along with drug regulations. The use of saliva instead of plasma in such studies makes them non-invasive, easy and with a lower clinical burden.

  13. The issue of HPLC determination of endogenous lipoic acid in human plasma.

    Science.gov (United States)

    Sechovcová, Soňa; Královcová, Pavla; Kanďár, Roman; Ventura, Karel

    2018-05-01

    Lipoic acid (LA) is used extensively as a therapeutic agent for the treatment of various diseases. Many methods have been reported for the determination of LA plasma levels and its metabolites after its supplementation, but available information concerning endogenous plasma levels is still scarce. Studies which directly focused on determining the endogenous plasma levels provided highly controversial results, endogenous plasma levels of LA: 2.4 and 4.9 nmol/L respectively. However, the levels of free LA in the plasma of nonsupplemented voluntary blood donors were not detectable in all cases. The presented results of our study show that endogenous concentrations of LA are <1.85 nmol/L. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Effects of ethanol on plasma protein shedding in the human stomach

    International Nuclear Information System (INIS)

    Brassinne, A.

    1979-01-01

    Plasma protein shedding in the stomach was measured in 23 normal individuals before and after intragastric administration of a 30% solution of ethyl alcohol. Two different methods were used to assess plasma protein shedding. The first technique utilizes [ 131 I] albumin and requires neutralization of the gastric juice. It was used in 12 subjects and failed to demonstrate any increase of plasma protein shedding under the influence of ethanol. The second technique which utilizes [ 51 Cr] chloride was used in 11 subjects. It demonstrated a significant increase of the gastric clearance of plasma protein which reached 2.5 times the control values. The [ 51 Cr] chloride technique does not require prior neutralization of gastric acidity. It is concluded that, in normal man, ethanol administration increases plasma protein shedding in the stomach when it is given in the presence of an acid gastric juice. The effect is not observed when the gastric acidity is neutralized

  15. Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

    Directory of Open Access Journals (Sweden)

    Niko Hildebrandt

    2011-10-01

    Full Text Available Förster resonance energy transfer (FRET from luminescent terbium complexes (LTC as donors to semiconductor quantum dots (QDs as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.

  16. Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography.

    Science.gov (United States)

    Davies, Benjamin J; Herbert, Megan K; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

    2006-11-07

    The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.

  17. Comparison of the quantification of caffeine in human plasma by gas chromatography and ELISA

    Directory of Open Access Journals (Sweden)

    A.B. Carregaro

    2001-06-01

    Full Text Available In the present study we evaluated the precision of the ELISA method to quantify caffeine in human plasma and compared the results with those obtained by gas chromatography. A total of 58 samples were analyzed by gas chromatography using a nitrogen-phosphorus detector and routine techniques. For the ELISA test, the samples were diluted to obtain a concentration corresponding to 50% of the absorbance of the standard curve. To determine whether the proximity between the I50 of the standard curve and that of the sample would bring about a more precise result, the samples were divided into three blocks according to the criterion of difference, in modulus, of the I50 of the standard curve and of the I50 of the sample. The samples were classified into three groups. The first was composed of 20 samples with I50 up to 1.5 ng/ml, the second consisted of 21 samples with I50 ranging from 1.51 to 3 ng/ml, and the third of 17 samples with I50 ranging from 3.01 to 13 ng/ml. The determination coefficient (R² = 0.999 showed that the data obtained by gas chromatography represented a reliable basis. The results obtained by ELISA were also reliable, with an estimated Pearson correlation coefficient of 0.82 between the two methods. This coefficient for the different groups (0.88, 0.79 and 0.49 for groups 1, 2 and 3, respectively showed greater reliability for the test with dilutions closer to I50.

  18. A coverslip-based technique for evaluating Staphylococcus aureus biofilm formation on human plasma

    Directory of Open Access Journals (Sweden)

    Jennifer N Walker

    2012-03-01

    Full Text Available The ability of the opportunistic pathogen, Staphylococcus aureus, to form biofilms is increasingly being viewed as an important contributor to chronic infections. In vitro methods for analyzing S. aureus biofilm formation have focused on bacterial attachment and accumulation on abiotic surfaces, such as in microtiter plate and flow cell assays. Microtiter plates provide a rapid measure of relative biomass levels, while flow cells have limited experimental throughput but are superior for confocal microscopy biofilm visualization. Although these assays have proven effective at identifying mechanisms involved in cell attachment and biofilm accumulation, the significance of these assays in vivo remains unclear. Studies have shown that when medical devices are implanted they are coated with host factors, such as matrix proteins, that facilitate S. aureus attachment and biofilm formation. To address the challenge of integrating existing biofilm assay features with a biotic surface, we have established an in vitro biofilm technique utilizing UV-sterilized coverslips coated with human plasma. The substratum more closely resembles the in vivo state and provides a platform for S. aureus to establish a robust biofilm. Importantly, these coverslips are amenable to confocal microscopy imaging to provide a visual reference of the biofilm growth stage, effectively merging the benefits of the microtiter and flow cell assays. We confirmed the approach using clinical S. aureus isolates and mutants with known biofilm phenotypes. Altogether, this new biofilm assay can be used to assess the function of S. aureus virulence factors associated with biofilm formation and for monitoring the efficacy of biofilm treatment modalities.

  19. Spectrofluorimetric determination of sertraline in dosage forms and human plasma through derivatization with 9-fluorenylmethyl chloroformate

    Directory of Open Access Journals (Sweden)

    Belal Fathalla

    2011-10-01

    Full Text Available Abstract Background Sertraline is primarily used to treat major depression in adult outpatients as well as obsessive-compulsive, panic and social anxiety disorders in both adults and children. A survey of the literature reveals that most of the reported methods are either insufficiently sensitive or tedious and require highly sophisticated and dedicated instrumentation. The proposed method is considered to be specific for determination of SER in presence of its metabolite (deaminated form. Results A sensitive, simple and specific spectrofluorimetric method was developed for the determination of sertraline (SER in pharmaceutical formulations and biological fluids. The method is based on its reaction with 9-fluorenylmethyl chloroformate (FMOC-Cl in borate buffer of pH 8.0 to yield a highly fluorescent derivative peaking at 315 nm after excitation at 265 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence concentration plot was rectilinear over the range of 0.05-1.0 μg mL-1 with a lower detection limit of 5.34 × 10-3 μg mL-1 and limit of quantitation of 0.016 μg mL-1. Conclusions The proposed method was successfully applied to the analysis of commercial tablets and the results obtained were in good agreement with those obtained using the reference method. Furthermore, the method was applied for the determination of SER in spiked and real human plasma. The mean % recovery (n = 3 was 94.33 ± 1.53 and 92.00 ± 2.65, respectively. A proposal of the reaction pathway was postulated.

  20. Exercise-induced increase in glucose transport, GLUT-4, and VAMP-2 in plasma membrane from human muscle

    DEFF Research Database (Denmark)

    Kristiansen, S; Hargreaves, Mark; Richter, Erik

    1996-01-01

    contractions may induce trafficking of GLUT-4-containing vesicles via a mechanism similar to neurotransmitter release. Our results demonstrate for the first time exercise-induced translocation of GLUT-4 and VAMP-2 to the plasma membrane of human muscle and increased sarcolemmal glucose transport.......A major effect of muscle contractions is an increase in sarcolemmal glucose transport. We have used a recently developed technique to produce sarcolemmal giant vesicles from human muscle biopsy samples obtained before and after exercise. Six men exercised for 10 min at 50% maximal O2 uptake (Vo2max...

  1. Perfluorinated compounds in human serum and seminal plasma from an urban and rural population in Sri Lanka

    Energy Technology Data Exchange (ETDEWEB)

    Guruge, Keerthi; Miyazaki, Shigeru; Yamanaka, Noriko [National Institute of Animal Health, Tsukuba (Japan); Taniyasu, Sacgu; Yamashita, Nobuyoshi [National Institute of Advance Industrial and Technology, Tsukuba (Japan); Wijeratna, S.; Seneviratne, H. [Colombo Univ. (Sri Lanka)

    2004-09-15

    Fluorinated organic compounds (FOCs) have been used for variety of industrial applications such as surfactants, adhesives, insecticides, and their global production increase since 1970s. These compounds repel both water and oil. The high-energy carbon-fluorine covalent bonds in FOCs are strong enough to have high persistency in the environment. These compounds emerged as priory environmental pollutants since they are found in various biota throughout the world. Human contamination of some FOCs was reported mostly in developed countries such as USA, Japan and from Europe. In the present study, we report 10 FOCs in human serum including seminal plasma for the first time, collected from volunteers from Sri Lanka.

  2. [Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

    Science.gov (United States)

    Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

    2017-03-01

    Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

  3. Effects of platelet rich plasma (PRP) on human gingival fibroblast, osteoblast and periodontal ligament cell behaviour.

    Science.gov (United States)

    Kobayashi, Eizaburo; Fujioka-Kobayashi, Masako; Sculean, Anton; Chappuis, Vivianne; Buser, Daniel; Schaller, Benoit; Dőri, Ferenc; Miron, Richard J

    2017-06-02

    The use of platelet rich plasma (PRP, GLO) has been used as an adjunct to various regenerative dental procedures. The aim of the present study was to characterize the influence of PRP on human gingival fibroblasts, periodontal ligament (PDL) cells and osteoblast cell behavior in vitro. Human gingival fibroblasts, PDL cells and osteoblasts were cultured with conditioned media from PRP and investigated for cell migration, proliferation and collagen1 (COL1) immunostaining. Furthermore, gingival fibroblasts were tested for genes encoding TGF-β, PDGF and COL1a whereas PDL cells and osteoblasts were additionally tested for alkaline phosphatase (ALP) activity, alizarin red staining and mRNA levels of osteoblast differentiation markers including Runx2, COL1a2, ALP and osteocalcin (OCN). It was first found that PRP significantly increased cell migration of all cells up to 4 fold. Furthermore, PRP increased cell proliferation at 3 and 5 days of gingival fibroblasts, and at 3 days for PDL cells, whereas no effect was observed on osteoblasts. Gingival fibroblasts cultured with PRP increased TGF-β, PDGF-B and COL1 mRNA levels at 7 days and further increased over 3-fold COL1 staining at 14 days. PDL cells cultured with PRP increased Runx2 mRNA levels but significantly down-regulated OCN mRNA levels at 3 days. No differences in COL1 staining or ALP staining were observed in PDL cells. Furthermore, PRP decreased mineralization of PDL cells at 14 days post seeding as assessed by alizarin red staining. In osteoblasts, PRP increased COL1 staining at 14 days, increased COL1 and ALP at 3 days, as well as increased ALP staining at 14 days. No significant differences were observed for alizarin red staining of osteoblasts following culture with PRP. The results demonstrate that PRP promoted gingival fibroblast migration, proliferation and mRNA expression of pro-wound healing molecules. While PRP induced PDL cells and osteoblast migration and proliferation, it tended to have

  4. Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application.

    Science.gov (United States)

    Chen, Feng; Hu, Zhe-Yi; Parker, Robert B; Laizure, S Casey

    2017-06-01

    Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

    Science.gov (United States)

    Hasegawa, Chika; Kumazawa, Takeshi; Uchigasaki, Seisaku; Lee, Xiao-Pen; Sato, Keizo; Terada, Masaru; Kurosaki, Kunihiko

    2011-10-01

    Dextromethorphan was extracted from human plasma samples (100 μL) using MonoTip C(18) tips, which are packed with C(18)-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 μL of distilled water and 50 μL of 1 mol/L glycine-sodium hydroxide buffer (pH 10). The mixture was extracted to the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug. © Springer-Verlag 2011

  6. Determination of Sphingosine-1-Phosphate in Human Plasma Using Liquid Chromatography Coupled with Q-Tof Mass Spectrometry

    Science.gov (United States)

    Egom, Emmanuel E.; Fitzgerald, Ross; Canning, Rebecca; Pharithi, Rebabonye B.; Murphy, Colin; Maher, Vincent

    2017-01-01

    Evidence suggests that high-density lipoprotein (HDL) components distinct from cholesterol, such as sphingosine-1-phosphate (S1P), may account for the anti-atherothrombotic effects attributed to this lipoprotein. The current method for the determination of plasma levels of S1P as well as levels associated with HDL particles is still cumbersome an assay method to be worldwide practical. Recently, a simplified protocol based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific quantification of plasma levels of S1P with good accuracy has been reported. This work utilized a triple quadrupole (QqQ)-based LC-MS/MS system. Here we adapt that method for the determination of plasma levels of S1P using a quadrupole time of flight (Q-Tof) based LC-MS system. Calibration curves were linear in the range of 0.05 to 2 µM. The lower limit of quantification (LOQ) was 0.05 µM. The concentration of S1P in human plasma was determined to be 1 ± 0.09 µM (n = 6). The average accuracy over the stated range of the method was found to be 100 ± 5.9% with precision at the LOQ better than 10% when predicting the calibration standards. The concentration of plasma S1P in the prepared samples was stable for 24 h at room temperature. We have demonstrated the quantification of plasma S1P using Q-Tof based LC-MS with very good sensitivity, accuracy, and precision that can used for future studies in this field. PMID:28820460

  7. A Population Pharmacokinetic Model for Disposition in Plasma, Saliva and Urine of Scopolamine after Intranasal Administration to Healthy Human Subjects

    Science.gov (United States)

    Wu, L.; Tam, V. H.; Chow, D. S. L.; Putcha, L.

    2014-01-01

    An intranasal gel formulation of scopolamine (INSCOP) was developed for the treatment of Space Motion Sickness. The bioavailability and pharmacokinetics (PK) were evaluated under the Food and Drug Administration guidelines for clinical trials with an Investigative New Drug (IND) protocol. The aim of this project was to develop a PK model that can predict the relationship between plasma, saliva and urinary scopolamine concentrations using data collected from the IND clinical trials with INSCOP. Methods: Twelve healthy human subjects were administered three dose levels (0.1, 0.2 and 0.4 mg) of INSCOP. Serial blood, saliva and urine samples were collected between 5 min and 24 h after dosing and scopolamine concentrations were measured by using a validated LC-MS-MS assay. Pharmacokinetic Compartmental models, using actual dosing and sampling times, were built using Phoenix (version 1.2). Model selection was based on the likelihood ratio test on the difference of criteria (-2LL) and comparison of the quality of fit plots. Results: The best structural model for INSCOP (minimal -2LL= 502.8) was established. It consisted of one compartment each for plasma, saliva and urine, respectively, which were connected with linear transport processes except the nonlinear PK process from plasma to saliva compartment. The best-fit estimates of PK parameters from individual PK compartmental analysis and Population PK model analysis were shown in Tables 1 and 2, respectively. Conclusion: A population PK model that could predict population and individual PK of scopolamine in plasma, saliva and urine after dosing was developed and validated. Incorporating a non-linear transfer from plasma to saliva compartments resulted in a significantly improved model fitting. The model could be used to predict scopolamine plasma concentrations from salivary and urinary drug levels, allowing non-invasive therapeutic monitoring of scopolamine in space and other remote environments.

  8. Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays.

    Science.gov (United States)

    Fernando, M Rohan; Jiang, Chao; Krzyzanowski, Gary D; Ryan, Wayne L

    2018-04-12

    Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS

    DEFF Research Database (Denmark)

    Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte

    2016-01-01

    The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were...

  10. A Modified LC/MS/MS Method with Enhanced Sensitivity for the Determination of Scopolamine in Human Plasma

    Science.gov (United States)

    Wang, Zuwei; Vaksman, Zalman; Putcha, Lakshmi

    2008-01-01

    Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC

  11. Immobilization of Platelet-Rich Plasma onto COOH Plasma-Coated PCL Nanofibers Boost Viability and Proliferation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Anastasiya Solovieva

    2017-12-01

    Full Text Available The scaffolds made of polycaprolactone (PCL are actively employed in different areas of biology and medicine, especially in tissue engineering. However, the usage of unmodified PCL is significantly restricted by the hydrophobicity of its surface, due to the fact that its inert surface hinders the adhesion of cells and the cell interactions on PCL surface. In this work, the surface of PCL nanofibers is modified by Ar/CO2/C2H4 plasma depositing active COOH groups in the amount of 0.57 at % that were later used for the immobilization of platelet-rich plasma (PRP. The modification of PCL nanofibers significantly enhances the viability and proliferation (by hundred times of human mesenchymal stem cells, and decreases apoptotic cell death to a normal level. According to X-ray photoelectron spectroscopy (XPS, after immobilization of PRP, up to 10.7 at % of nitrogen was incorporated into the nanofibers surface confirming the grafting of proteins. Active proliferation and sustaining the cell viability on nanofibers with immobilized PRP led to an average number of cells of 258 ± 12.9 and 364 ± 34.5 for nanofibers with ionic and covalent bonding of PRP, respectively. Hence, our new method for the modification of PCL nanofibers with PRP opens new possibilities for its application in tissue engineering.

  12. Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice

    NARCIS (Netherlands)

    M.J. van Haperen (Rien); A. van Tol (Arie); P. Vermeulen; M. Jauhiainen; T. van Gent (Teus); P.M. van den Berg (Paul); S. Ehnholm (Sonja); A.W.M. van der Kamp (Arthur); M.P.G. de Crom (Rini); F.G. Grosveld (Frank)

    2000-01-01

    textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress

  13. PLASMA AND LUNG MACROPHAGE CAROTENOID RESPONSIVENESS TO SUPPLEMENTATION AND OZONE EXPOSURE IN HUMANS

    Science.gov (United States)

    OBJECTIVE:: To examine the effect of ozone exposure and vegetable juice supplementation on plasma and lung macrophage concentrations of carotenoids. DESIGN:: A randomized trial. SETTING:: Subjects were exposed to ambient air prior to antioxidant supplementation and to ozone after...

  14. Comparison of Three Sample Preparation Procedures for the Quantification of L-Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma Using HPLC-FLD

    DEFF Research Database (Denmark)

    Schou-Pedersen, Anne Marie Voigt; Lykkesfeldt, Jens

    2018-01-01

    Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were co...... sample preparation of human plasma samples before HPLC-FLD in providing important information regarding elevated ADMA concentrations.......Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were...... compared regarding the quantification of L-arginine and ADMA in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). The samples were analysed by using high-performance liquid...

  15. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  16. Comparison between human cord blood serum and platelet-rich plasma supplementation for Human Wharton's Jelly Stem Cells and dermal fibroblasts culture

    Directory of Open Access Journals (Sweden)

    Hashemi SS

    2016-08-01

    Full Text Available We carried out a side-by-side comparison of the effects of Human cord blood serum (HcbS versus embryonic PRP on Human Wharton's Jelly Stem Cells(hWMSCand dermal fibroblasts proliferation. Human umbilical cord blood was collected to prepare activated serum (HCS and platelet-rich plasma (CPRP.Wharton's Jelly Stem Cells and dermal fibroblasts were cultured in complete medium with10% CPRP, 10%HCSor 10% fetal bovine serumand control (serum-free media.The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion and Cell proliferation. We showed that proliferation of fibroblasts and mesenchymal stem cells in the presence of cord blood serum and platelet-rich plasma significantly more than the control group (p≤0/05. As an alternative to FBS, cord blood serum has been proved as an effective component in cell tissue culture applications and embraced a vast future in clinical applications of regenerative medicine. However, there is still a need to explore the potential of HCS and its safe applications in humanized cell therapy or tissue engineering.

  17. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid.

    Science.gov (United States)

    Hyung, Seok-Won; Piehowski, Paul D; Moore, Ronald J; Orton, Daniel J; Schepmoes, Athena A; Clauss, Therese R; Chu, Rosalie K; Fillmore, Thomas L; Brewer, Heather; Liu, Tao; Zhao, Rui; Smith, Richard D

    2014-11-01

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

  18. Gonadotropin-releasing hormone radioimmunoassay and its measurement in normal human plasma, secondary amenorrhea, and postmenopausal syndrome

    International Nuclear Information System (INIS)

    Rosenblum, N.G.; Schlaff, S.

    1976-01-01

    A sensitive and specific double antibody radioimmunoassay for gonadotropin-releasing hormone (GnRH) has been developed for measurement in ethanol extracts of human plasma. Iodinated hormone was prepared with the use of the chloramine-T method, and antibodies were developed in rabbits over a six-month period with a GnRH synthetic copolymer immunogen. A Scatchard plot revealed at least three species of antibody. The assay can measure conservatively at the 5 pg. per milliliter level and shows no cross-reactivity with other available hypothalamic and pituitary hormones. The releasing hormone was quantitatively recovered from human plasma with immunologic identity to native hormone. Unextracted plasma could not be used because of nonspecific displacement. The measurement of GnRH in individuals receiving 100 μg of intravenous bolus infusions of the synthetic decapeptide show extremely elevated values with two half-lives: one of two to four minutes and another of 35 to 40 minutes. In our experiments, we have found measurable GnRH in patients with secondary amenorrhea and at the midcycle in normal women. In the normal cycling woman during the follicular and luteal phases, GnRH was undetectable. In postmenopausal women with extreme hypoestrogenism and markedly elevated luteinizing hormone values, GnRH was also undetectable. No bursts of GnRH could be detected in normal men when sampled every ten minutes over a two-hour period and every two hours throughout the day

  19. A simple and selective liquid chromatography- tandem mass spectrometry method for determination of ε-aminocaproic acid in human plasma

    Directory of Open Access Journals (Sweden)

    Ganesh S. Moorthy

    2015-07-01

    Full Text Available Understanding the clinical pharmacology of the antifibrinolytic drug epsilon-aminocaproic acid (EACA is critical for rational drug administration in children. The aim of this study is to develop a reliable assay for the determination of EACA in human plasma. We describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS assay for EACA in human plasma. Sample preparation involved plasma dilution (1:2040, followed by reversed-phase chromatographic separation and selective detection using tandem mass spectrometry. EACA had a linear range of 1 - 250 μg/mL. The intraday precision based on the standard deviation of replicates of quality control samples ranged from 4.7 to 10.4% and the accuracy ranged from 92-106%. The interday precision ranged from 4.6 to 9.8% and the accuracy ranged from 95-103%. Stability studies showed that EACA was stable during the conditions for sample preparation and storage. The described method is robust and successfully employed for clinical studies of EACA in children

  20. A Rapid, Simple, and Validated RP-HPLC Method for Quantitative Analysis of Levofloxacin in Human Plasma

    Directory of Open Access Journals (Sweden)

    Dion Notario

    2017-04-01

    Full Text Available To conduct a bioequivalence study for a copy product of levofloxacin (LEV, a simple and validated analytical method was needed, but the previous developed methods were still too complicated. For this reason, a simple and rapid high performance liquid chromatography method was developed and validated for LEV quantification in human plasma. Chromatographic separation was performed under isocratic elution on a Luna Phenomenex® C18 (150 × 4.6 mm, 5 µm column. The mobile phase was comprised of acetonitrile, methanol, and phosphate buffer 25 mM that adjusted at pH 3.0 (13:7:80 v/v/v and pumped at a flow rate of 1.5 mL/min. Detection was performed under UV detector at wavelength of 280 nm. Samples were prepared by adding acetonitrile and followed by centrifugation to precipitate plasma protein. Then followed successively by evaporation and reconstitution step. The optimized method meets the requirements of validation parameters which included linearity (r = 0.995, sensitivity (LLOQ and LOD was 1.77 and 0.57 µg/mL respectively, accuracy (%error above LLOQ ≤ 12% and LLOQ ≤ 20%, precision (RSD ≤ 9%, and robustness in the ranges of 1.77-28.83 µg/mL. Therefore, the method can be used as a routine analysis of LEV in human plasma as well as in bioequivalence study of LEV.

  1. Determination of bifendate in human plasma by HPLC-MS and bioequivalence on bifendate pills in healthy volunteers.

    Science.gov (United States)

    Zhou, Lingjie; Gu, Lihua; Wang, Yijian; Liang, Jianying

    2006-03-03

    A sensitive and specific method for the determination of bifendate in human plasma was developed, based on high-performance liquid chromatography (HPLC)-mass spectrometry (MS). The samples were extracted from plasma with diethyl ether, followed by separation and evaporation after addition of internal standard diazepam. The residue was reconstituted in methanol and injected into the HPLC-MS. Chromatography was performed on an Inertsil ODS column with a mobile phase consisting of methanol-distilled water (70/30, v/v) at a flow rate of 0.3 mL/min. Quantitative analysis was achieved by MS detection, using a mass spectrometer equipped with an electrospray ionization interface (ESI) and operated in selected ion monitoring (SIM) and positive-ionization mode using target ions at m/z 419 for bifendate and m/z 285 for internal standard, respectively. The linearity was confirmed in the concentration range of 2-200 ng/mL in human plasma and the precision of this assay was not more than 6.79% over the entire concentration range. The method was sensitive and repeatable enough to be used in pharmacokinetic and bioavailability studies.

  2. Direct-injection HPLC method of measuring micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column.

    Science.gov (United States)

    Uranishi, Hiroaki; Nakamura, Mitsuhiro; Nakamura, Hiroki; Ikeda, Yukari; Otsuka, Mayuko; Kato, Zenichiro; Tsuchiya, Teruo

    2011-04-15

    A direct-injection HPLC-based method has been developed for determining amounts of micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column. The method is easy to perform and requires only 10 μL of a filtered plasma sample. The chromatographic separations were carried out with a gradient mode. The fluorescence detection wavelengths of excitation and emission were set at 273 nm and 464 nm, respectively. Retention times for micafungin and IS were 22.4 and 23.7 min, respectively. Micafungin and FR195743 (IS) peaks were completely separated with little tailing, and no interference was observed. The calibration curve of micafungin showed good linearity in the range of 0.5-20.0 μg/mL (r(2)=1.00). The intra-day accuracy ranged from -4.5 to 5.3%. The inter-day accuracy ranged from -9.8 to 1.5%. The precisions were less than 10%. This method is useful for the determination of micafungin in human plasma. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Cyanide binding to human plasma heme–hemopexin: A comparative study

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Leboffe, Loris; Polticelli, Fabio

    2012-01-01

    Highlights: ► Cyanide binding to ferric HHPX–heme–Fe. ► Cyanide binding to ferrous HHPX–heme–Fe. ► Dithionite-mediated reduction of ferric HHPX–heme–Fe–cyanide. ► Cyanide binding to HHPX–heme–Fe is limited by ligand deprotonation. ► Cyanide dissociation from HHPX–heme–Fe–cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme–Fe–hemopexin (HPX–heme–Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX–heme–Fe (HHPX–heme–Fe(III) and HHPX–heme–Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX–heme–Fe(III)–cyanide complex, at pH 7.4 and 20.0 °C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX–heme–Fe(III) and HHPX–heme–Fe(II) are K = (4.1 ± 0.4) × 10 −6 M, k on = (6.9 ± 0.5) × 10 1 M −1 s −1 , and k off = 2.8 × 10 −4 s −1 ; and H = (6 ± 1) × 10 −1 M, h on = 1.2 × 10 −1 M −1 s −1 , and h off = (7.1 ± 0.8) × 10 −2 s −1 , respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX–heme–Fe(III)–cyanide complex is l = 8.9 ± 0.8 M −1/2 s −1 . HHPX–heme–Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  4. Cyanide binding to human plasma heme-hemopexin: A comparative study

    Energy Technology Data Exchange (ETDEWEB)

    Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma (Italy); Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Leboffe, Loris [Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Polticelli, Fabio [Dipartimento di Biologia, Universita Roma Tre, Roma (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

  5. Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine

    Science.gov (United States)

    Herrera, Daniel; Vásquez, Camilo; Corthésy, Blaise; Franco, Manuel A; Angel, Juana

    2013-01-01

    Rotavirus (RV)–specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines. PMID:23839157

  6. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    DEFF Research Database (Denmark)

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before......-regulated by increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion......, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could...

  7. Differing Effects of Younger and Older Human Plasma on C2C12 Myocytes in Vitro

    Directory of Open Access Journals (Sweden)

    Ifigeneia Kalampouka

    2018-02-01

    Full Text Available Ageing is associated with a general reduction of physiological function and a reduction of muscle mass and strength. Endocrine factors such as myostatin, activin A, growth and differentiation factor 11 (GDF-11 and their inhibitory peptides influence muscle mass in health and disease. We hypothesised that myocytes cultured in plasma from older and younger individuals would show an ageing effect, with reduced proliferation and differentiation in older environments. C2C12 myoblasts were grown as standard and stimulated with media conditioned with 5% plasma from healthy male participants that were either younger (n = 6, 18–35 years of age or older (n = 6, >57 years of age. Concentration of plasma myostatin (total and free, follistatin-like binding protein (FLRG, GDF-11 and activin A were quantified by ELISA. Both FLRG and activin A were elevated in older individuals (109.6 and 35.1% increase, respectively, whilst myostatin (free and total and GDF-11 were not. Results indicated that plasma activin A and FLRG were increased in older vs. younger participants, GDF11 and myostatin did not differ. Myoblasts in vitro showed no difference in proliferation rate between ages, however scratch closure was greater in younger vs. older plasma stimulated myoblasts (78.2 vs. 87.2% of baseline scratch diameter, respectively. Myotube diameters were larger in cells stimulated with younger plasma than with older at 24 and 48 h, but not at 2 h. A significant negative correlation was noted between in vivo plasma FLRG concentration and in vitro myotube diameter 48 h following plasma stimulation (r2 = 0.392, p = 0.030. Here we show that myoblasts and myotubes cultured in media conditioned with plasma from younger or older individuals show an ageing effect, and further this effect moderately correlates with circulating FLRG concentration in vivo. The effect of ageing on muscle function may not be innate to the tissue, but involve a general cellular environment change

  8. Determination of suvorexant in human plasma using 96-well liquid-liquid extraction and HPLC with tandem mass spectrometric detection.

    Science.gov (United States)

    Breidinger, S A; Simpson, R C; Mangin, E; Woolf, E J

    2015-10-01

    A method, using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra(®)), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1-1000ng/mL. Stable isotope labeled (13)C(2)H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction, in the 96-well format, of a 100μL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50×2.1mm, 3μm) column with a mobile phase consisting of 30/70 (v/v %) 10mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451→186) and (13)C(2)H3-suvorexant (m/z 455→190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at -20°C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at -20°C. The method described has been used to support clinical studies during Phase I through III of clinical development. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals

    International Nuclear Information System (INIS)

    Ehresman, David J.; Froehlich, John W.; Olsen, Geary W.; Chang, Shu-Ching; Butenhoff, John L.

    2007-01-01

    Interest in human exposure to perfluorinated acids, including perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) has led to their measurement in whole blood, plasma and serum. Comparison of measurements in these different blood-based matrices, however, has not been rigorously investigated to allow for across-matrix comparisons. This research evaluated concentrations of PFBS, PFHS, PFOS, and PFOA in whole blood collected in heparin (lithium) and ethylenediamine tetraacetic acid (EDTA), plasma samples collected in heparin and EDTA, and serum (from whole blood allowed to clot). Blood samples were collected from 18 voluntary participants employed at 3M Company. Solid phase extraction methods were used for all analytical sample preparations, and analyses were completed using high-pressure liquid chromatography/tandem mass spectrometry methods. Serum concentrations ranged from: limit of quantitation (LOQ, 5 ng/mL) to 25 ng/mL for PFBS; LOQ (5 ng/mL) to 75 ng/mL for PFHS; LOQ (5 ng/mL) to 880 ng/mL for PFOS; and LOQ (5 or 10 ng/mL) to 7320 ng/mL for PFOA. Values less than the LOQ were not included in the statistical analyses of the mean of the ratios of individual values for the matrices. PFBS was not quantifiable in most samples. Serum to plasma ratios for PFHS, PFOS, and PFOA were 1:1 and this ratio was independent of the level of concentrations measured. Serum or plasma to whole blood ratios, regardless of the anticoagulant used, approximated 2:1. The difference between plasma and serum and whole blood corresponded to volume displacement by red blood cells, suggesting that the fluorochemicals are not found intracellularly or attached to the red blood cells

  10. Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

    Science.gov (United States)

    Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

    2015-01-01

    Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. PMID:25589789

  11. Spectrofluorimetric determination of aliskiren in tablets and spiked human plasma through derivatization with dansyl chloride.

    Science.gov (United States)

    Aydoğmuş, Zeynep; Sarı, Ferhat; Ulu, Sevgi Tatar

    2012-03-01

    A simple and sensitive method has been developed and validated for the determination of aliskiren (ALS) in its dosage forms and spiked plasma. The method was based on the reaction of the drug with dansyl chloride in the presence of bicarbonate solution of pH 10.5 to give a highly fluorescent derivative which was measured at 501 nm with excitition at 378 nm in dichloromethane. Different experimental parameters affecting the development of the method and stability were carefully studied and optimized. The calibration curves were linear over the concentration ranges of 100-700 and 50-150 ng/mL for standard solution and plasma, respectively. The limits of detection were 27.52 ng/mL in standard solution, 4.91 ng/mL in plasma. The developed method was successfully applied to the analysis the drug in the commercial tablets and spiked plasma samples. The mean recovery of ALS from tablets and plasma was 100.10 and 97.81%, respectively. A proposal of the reaction pathway was presented.

  12. Low plasma adiponectin concentrations do not predict weight gain in humans

    DEFF Research Database (Denmark)

    Vozarova, Barbora; Stefan, Norbert; Lindsay, Robert S

    2002-01-01

    Low concentrations of plasma adiponectin, the most abundant adipose-specific protein, are observed in obese individuals and predict the development of type 2 diabetes. Administration of adiponectin to rodents prevented diet-induced weight gain, suggesting a potential etiologic role of hypoadipone......Low concentrations of plasma adiponectin, the most abundant adipose-specific protein, are observed in obese individuals and predict the development of type 2 diabetes. Administration of adiponectin to rodents prevented diet-induced weight gain, suggesting a potential etiologic role...... of hypoadiponectinemia in the development of obesity. Our aim was to prospectively examine whether low plasma adiponectin concentrations predict future weight gain in Pima Indians, explaining the predictive effect of adiponectin on the development of type 2 diabetes. We measured plasma adiponectin concentrations in 219...... nondiabetic Pima Indians (112 M/107 F, age 31 +/- 9 years, body weight 96 +/- 20 kg [mean +/- SD]) in whom body weight and height were measured and BMI calculated at baseline and follow-up. Cross-sectionally, plasma adiponectin concentrations were negatively associated with body weight (r = -0.28, P = 0...

  13. Proteomic-Biostatistic Integrated Approach for Finding the Underlying Molecular Determinants of Hypertension in Human Plasma.

    Science.gov (United States)

    Gajjala, Prathibha R; Jankowski, Vera; Heinze, Georg; Bilo, Grzegorz; Zanchetti, Alberto; Noels, Heidi; Liehn, Elisa; Perco, Paul; Schulz, Anna; Delles, Christian; Kork, Felix; Biessen, Erik; Narkiewicz, Krzysztof; Kawecka-Jaszcz, Kalina; Floege, Juergen; Soranna, Davide; Zidek, Walter; Jankowski, Joachim

    2017-08-01

    Despite advancements in lowering blood pressure, the best approach to lower it remains controversial because of the lack of information on the molecular basis of hypertension. We, therefore, performed plasma proteomics of plasma from patients with hypertension to identify molecular determinants detectable in these subjects but not in controls and vice versa. Plasma samples from hypertensive subjects (cases; n=118) and controls (n=85) from the InGenious HyperCare cohort were used for this study and performed mass spectrometric analysis. Using biostatistical methods, plasma peptides specific for hypertension were identified, and a model was developed using least absolute shrinkage and selection operator logistic regression. The underlying peptides were identified and sequenced off-line using matrix-assisted laser desorption ionization orbitrap mass spectrometry. By comparison of the molecular composition of the plasma samples, 27 molecular determinants were identified differently expressed in cases from controls. Seventy percent of the molecular determinants selected were found to occur less likely in hypertensive patients. In cross-validation, the overall R 2 was 0.434, and the area under the curve was 0.891 with 95% confidence interval 0.8482 to 0.9349, P hypertensive patients were found to be -2.007±0.3568 and 3.383±0.2643, respectively, P hypertensives and normotensives. The identified molecular determinants may be the starting point for further studies to clarify the molecular causes of hypertension. © 2017 American Heart Association, Inc.

  14. A study of the effect on human mesenchymal stem cells of an atmospheric pressure plasma source driven by different voltage waveforms

    Science.gov (United States)

    Laurita, R.; Alviano, F.; Marchionni, C.; Abruzzo, P. M.; Bolotta, A.; Bonsi, L.; Colombo, V.; Gherardi, M.; Liguori, A.; Ricci, F.; Rossi, M.; Stancampiano, A.; Tazzari, P. L.; Marini, M.

    2016-09-01

    The effect of an atmospheric pressure non-equilibrium plasma on human mesenchymal stem cells was investigated. A dielectric barrier discharge non-equilibrium plasma source driven by two different high-voltage pulsed generators was used and cell survival, senescence, proliferation, and differentiation were evaluated. Cells deprived of the culture medium and treated with nanosecond pulsed plasma showed a higher mortality rate, while higher survival and retention of proliferation were observed in cells treated with microsecond pulsed plasma in the presence of the culture medium. While a few treated cells showed the hallmarks of senescence, unexpected delayed apoptosis ensued in cells exposed to plasma-treated medium. The plasma treatment did not change the expression of OCT4, a marker of mesenchymal stem cell differentiation.

  15. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub

    2011-02-01

    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  16. Sensitive method for precise measurement of endogenous angiotensins I, II and III in human plasma

    International Nuclear Information System (INIS)

    Kawamura, M.; Yoshida, K.; Akabane, S.

    1987-01-01

    We measured endogenous angiotensins (ANGs) I, IIandIII using a system of extraction by Sep-Pak column followed by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). An excellent separation of ANGs was obtained by HPLC. The recovery of ANGs I, IIandIII was 80-84%, when these authentic peptides were added to 6 ml of plasma. The coefficient of variation of the ANGs was 0.04-0.09 for intra-assay and 0.08-0.13 for inter-assay, thereby indicating a good reproducibility. Plasma ANGs I, IIandIII measured by this method in 5 normal volunteers were 51,4.5 and 1.2 pg/ml. In the presence of captopril, ANGs IIandIII decreased by 84% and 77%, respectively, while ANG I increased 5.1 times. This method is therefore useful to assess the precise levels of plasma ANGs

  17. Effect of mental stress on plasma homovanillic acid in healthy human subjects.

    Science.gov (United States)

    Sumiyoshi, T; Yotsutsuji, T; Kurachi, M; Itoh, H; Kurokawa, K; Saitoh, O

    1998-07-01

    Plasma levels of homovanillic acid (pHVA) have been suggested to provide a measure of dopaminergic activity in the central nervous system. The present study investigated the effect of mental stress by the Kraepelin test, a test of continuous arithmetic addition of single-digit figures for 30 min, on pHVA levels in 13 male psychiatrically normal healthy volunteers. Following an overnight fast and restricted physical activity, plasma samples were collected immediately before and after the administration of the Kraepelin test. Plasma HVA levels following the administration of the Kraepelin test were significantly lower than the pretest pHVA levels. The percent change in pHVA levels by the Kraepelin test positively correlated with pretest pHVA levels. The observed reduction in pHVA levels by mental stress in normal subjects may reflect some aspects of a dopamine-dependent restitutive system in the brain.

  18. Interleukin-7 Plasma Levels in Human Differentiate Anorexia Nervosa, Constitutional Thinness and Healthy Obesity.

    Science.gov (United States)

    Germain, Natacha; Viltart, Odile; Loyens, Anne; Bruchet, Céline; Nadin, Katia; Wolowczuk, Isabelle; Estour, Bruno; Galusca, Bogdan

    2016-01-01

    Interleukin-7 (IL-7) is a cytokine involved in energy homeostasis as demonstrated in rodents. Anorexia nervosa is characterized by restrained eating behavior despite adaptive orexigenic regulation profile including high ghrelin plasma levels. Constitutional thinness is a physiological condition of resistance to weight gain with physiological anorexigenic profile including high Peptide YY plasma level. Healthy obesity can be considered as a physiological state of resistance to weight loss with opposite appetite regulating profile to constitutional thinness including low Peptide YY plasma level. No studies in IL-7 are yet available in those populations. Therefore we evaluated circadian plasma levels of IL-7 in anorexia nervosa compared to constitutional thinness, healthy obese and control females. 10 restrictive-type anorexia nervosa women, 5 bingeing/purging anorexia nervosa woman, 5 recovered restrictive anorexia nervosa women, 4 bulimic females, 10 constitutional thinness women, 7 healthy obese females, and 10 normal weight women controls were enrolled in this cross-sectional study, performed in endocrinology unit and academic laboratory. Twelve-point circadian profiles of plasma IL-7 levels were measured in each subject. 24h mean IL-7 plasma levels (pg/ml, mean±SEM) were decreased in restrictive-type anorexia nervosa (123.4±14.4, panorexia nervosa (24.2±5.6, panorexia nervosa (64.2±16.1, p = 0.01) and healthy obese patients (51±3.2, panorexia nervosa, confirming its difference with constitutional thinness. Healthy obesity, with low IL-7, is once again in mirror image of constitutional thinness with normal high IL-7.

  19. Development and validation of an HPLC method for simultaneous determination of trimethoprim and sulfamethoxazole in human plasma.

    Science.gov (United States)

    Sayar, Esin; Sahin, Selma; Cevheroglu, Semsettin; Hincal, A Atilla

    2010-09-01

    The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is used in the treatment of many common infections such as urinary, respiratory and gastrointestinal tract infections. The aim of this study was to determine TMP and SMX simultaneously in human plasma samples by high performance liquid chromatography (HPLC) using antipyrine as the internal standard. Separation of the compounds was achieved on a reverse-phase C8 column packed with 5 microm dimethyl octadecylsilyl bonded amorphous silica (4.6 mm x 250 mm) column using a mobile phase consisted of potassium hydrogen phosphate, acetonitrile, methanol and water adjusted to pH 6.2. The mobile phase was delivered at a flow rate of 1 mL min- and the effluent was monitored using Max plot technique at 25 derees C. Retention times were 5 min for TMP, 7 min for antipyrine and 9 min for SMX. Quantitation limits were 10 ng mL(-1) for TMP and 50 ng mL(-1) for SMX. Our findings indicated that the developed HPLC method was precise, accurate, specific and sensitive for simultaneous determination of TMP and SMX. Proposed HPLC method was successfully applied for the analysis of TMP and SMX in human plasma after oral administration of a co-trimoxazole tablet to human volunteers.

  20. Rapid, simple and highly sensitive LC-ESI-MS/MS method for the quantification of tamsulosin in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Wishu, S; Varma, D P

    2005-12-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of tamsulosin (I), a highly selective alpha1-adrenoceptor antagonist used for the treatment of patients with symptomatic benign prostatic hyperplasia. The analyte and internal standard, mosapride (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Waters symmetry C18 column with a mobile phase of 0.03% formic acid-acetonitrile (30:70, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 409.1 solidus in circle 228.1 and m/z 422.3 solidus in circle 198.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1-50.0 ng/mL for tamsulosin in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. (c) 2005 John Wiley & Sons, Ltd.

  1. Validation and application of a high-performance liquid chromatography--tandem mass spectrometry assay for mosapride in human plasma.

    Science.gov (United States)

    Ramakrishna, N V S; Vishwottam, K N; Manoj, S; Koteshwara, M; Chidambara, J; Varma, D P

    2005-09-01

    A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of mosapride (I), a novel and potent gastroprokinetic agent that enhances the upper gastrointestinal motility by stimulating 5-HT(4) receptor. The analyte and internal standard, tamsulosin (II), were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase Waters symmetry C(18) column with a mobile phase of 0.03% formic acid-acetonitrile (10:90, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 422.3 -->198.3 and m/z 409.1 -->228.1 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-100.0 ng/mL for mosapride in human plasma. The lower limit of quantitation was 500 pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.0 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. Copyright (c) 2005 John Wiley & Sons, Ltd.

  2. Mistimed food intake and sleep alters 24-hour time-of-day patterns of the human plasma proteome.

    Science.gov (United States)

    Depner, Christopher M; Melanson, Edward L; McHill, Andrew W; Wright, Kenneth P

    2018-06-05

    Proteomics holds great promise for understanding human physiology, developing health biomarkers, and precision medicine. However, how much the plasma proteome varies with time of day and is regulated by the master circadian suprachiasmatic nucleus brain clock, assessed here by the melatonin rhythm, is largely unknown. Here, we assessed 24-h time-of-day patterns of human plasma proteins in six healthy men during daytime food intake and nighttime sleep in phase with the endogenous circadian clock (i.e., circadian alignment) versus daytime sleep and nighttime food intake out of phase with the endogenous circadian clock (i.e., circadian misalignment induced by simulated nightshift work). We identified 24-h time-of-day patterns in 573 of 1,129 proteins analyzed, with 30 proteins showing strong regulation by the circadian cycle. Relative to circadian alignment, the average abundance and/or 24-h time-of-day patterns of 127 proteins were altered during circadian misalignment. Altered proteins were associated with biological pathways involved in immune function, metabolism, and cancer. Of the 30 circadian-regulated proteins, the majority peaked between 1400 hours and 2100 hours, and these 30 proteins were associated with basic pathways involved in extracellular matrix organization, tyrosine kinase signaling, and signaling by receptor tyrosine-protein kinase erbB-2. Furthermore, circadian misalignment altered multiple proteins known to regulate glucose homeostasis and/or energy metabolism, with implications for altered metabolic physiology. Our findings demonstrate the circadian clock, the behavioral wake-sleep/food intake-fasting cycle, and interactions between these processes regulate 24-h time-of-day patterns of human plasma proteins and help identify mechanisms of circadian misalignment that may contribute to metabolic dysregulation.

  3. RP-HPLC Determination of Three Anti-Hyperlipidemic Drugs in Spiked Human Plasma and in Dosage Forms

    Directory of Open Access Journals (Sweden)

    Ola. M. Abdallah

    2011-01-01

    Full Text Available Sensitive, simple and accurate high performance liquid chromatographic (HPLC methods for the determination of atorvastatin (AT, fluvastatin (FL and pravastatin (PV have been developed. The proposed methods involve the use of a 150 mmx4.6 mm Zorbax Extend-C18 column (5 μm particle size and different chromatographic conditions for the separation of the three statins. Linearity range was 5-40, 5-30 and 10-60 μg mL-1 for AT, FL and PV respectively. The developed methods proved to be successful in the determination of all studied drugs in spiked human plasma samples.

  4. Non-Equilibrium Plasma Applications for Water Purification Supporting Human Spaceflight and Terrestrial Point-of-Use

    Science.gov (United States)

    Blankson, Isaiah M.; Foster, John E.; Adamovsky, Grigory

    2016-01-01

    2016 NASA Glenn Technology Day Panel Presentation on May 24, 2016. The panel description is: Environmental Impact: NASA Glenn Water Capabilities Both global water scarcity and water treatment concerns are two of the most predominant environmental issues of our time. Glenn researchers share insights on a snow sensing technique, hyper spectral imaging of Lake Erie algal blooms, and a discussion on non-equilibrium plasma applications for water purification supporting