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Sample records for human 12-h plasma

  1. Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH

    Science.gov (United States)

    Gilbert, B.; Perfetti, L.; Fauchoux, O.; Redondo, J.; Baudat, P.-A.; Andres, R.; Neumann, M.; Steen, S.; Gabel, D.; Mercanti, Delio; Ciotti, M. Teresa; Perfetti, P.; Margaritondo, G.; de Stasio, Gelsomina

    2000-07-01

    Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 μm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

  2. Tank 12H residuals sample analysis report

    Energy Technology Data Exchange (ETDEWEB)

    Oji, L. N. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Shine, E. P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Diprete, D. P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Coleman, C. J. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Hay, M. S. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2015-06-11

    The Savannah River National Laboratory (SRNL) was requested by Savannah River Remediation (SRR) to provide sample preparation and analysis of the Tank 12H final characterization samples to determine the residual tank inventory prior to grouting. Eleven Tank 12H floor and mound residual material samples and three cooling coil scrape samples were collected and delivered to SRNL between May and August of 2014.

  3. Human ovulation and plasma oxytocin

    Energy Technology Data Exchange (ETDEWEB)

    Kumaresan, Perianna; Kumaresan, Malathi; Hossini, Mahmood; Arellano, Carolina; Vasicka, Alois (State Univ. of New York Downstate Medical Center, Brooklyn (USA))

    1983-10-01

    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 ..mu..U/ml (Mean+-S.E.) on the 10th day of the cycle. OT level declined to 13+-6 ..mu..U/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.

  4. Peanut consumption increases levels of plasma very long chain fatty acids in humans.

    Science.gov (United States)

    Lam, Christina; Wong, Derek; Cederbaum, Stephen; Lim, Bennie; Qu, Yong

    2012-11-01

    Peanut consumption has been suspected of raising plasma very long chain fatty acid (VLCFA) levels in humans. The effect of peanut consumption on VLCFAs was studied in six human subjects. After 3 to 4h of peanut butter ingestion, plasma C26:0 and C26:0/C22:0 were found to be significantly elevated to levels seen in patients with peroxisomal disorders. These levels returned to normal within 12h. Peanut consumption needs to be accounted for when interpreting VLCFAs.

  5. 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.

  6. 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 Ltd., 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/1 when 0.1 ml plasma samples are analysed. Direct analysis of sodium cromoglycate concentrations in plasma samples collected up to several hours after the administration of single therapeutic doses of the compound is possible. 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/1 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.

  7. Hemagglutinin activity of human plasma fibronectin.

    Science.gov (United States)

    Vuento, M

    1979-09-01

    Purified human plasma fibronectin at concentrations of about 30 microgram/ml was found to agglutinate trypsin-treated erythrocytes from certain species. The hemagglutination reaction was inhibited by specific antibodies to fibronectin, by relatively low concentrations of polyamines and by higher concentrations of basic amino acids and nonacetylated amino sugars. The divalent cations Ca2+ and Mg2+ and the chelating agent ethylenediaminetetraacetate did not affect the reaction. None of the neutral amino acids, neutral sugars or polyanions tested was inhibitory. The results imply that plasma fibronectin is capable of interacting with cell surfaces and support the idea of a similarity between cellular and plasma fibronectins.

  8. Determination and distribution of human plasma selenoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Plecko, T.; Nordmann, S.; Ruekgauer, M.; Kruse-Jarres, J.D. [Institute for Clinical Chemistry and Laboratory Medicine, Stuttgart (Germany)

    1999-03-01

    Major portions of plasma-selenium are incorporated in the proteins glutathione peroxidase (GSH-Px), selenoprotein P (Sel P) and albumin. A chromatographic method, adapted from a procedure by Harrison et al. [6], uses heparin- and blue-sepharose to separate the three protein fractions. The determination of selenium was carried out by electrothermal atomic absorption spectroscopy (ETAAS) using the Zeeman effect. The selenium distribution of 17 healthy subjects was 68 {+-} 7% of the total plasma selenium associated to Sel P, 25 {+-} 4% associated to p-GSH-Px and 7{+-}4% associated to albumin. The recovery of selenium was 99 {+-} 4%. For precision measurements a plasma pool has been separated seven times. The selectivity of this method was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and GSH-Px activity measurements. A fast method, adapted for clinical applications, is described which allows to determine the human plasma selenium distribution in about an hour. (orig.) With 2 tabs., 8 refs.

  9. HPLC determination of tolperisone in human plasma.

    Science.gov (United States)

    Bae, Jung-Woo; Park, Young-Seo; Sohn, Uy-Dong; Myung, Chang-Sun; Ryu, Byung-Kwon; Jang, Choon-Gon; Lee, Seok-Yong

    2006-04-01

    A simple high performance liquid chromatographic (HPLC) method was developed for the determination of tolperisone in human plasma. Tolperisone and internal standard (chlorphenesin) were isolated from 1 mL of plasma using 8 mL of dichlormethane. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with 300 mL aliquot of mobile phase and a 100 mL aliquot was injected onto the C18 reverse-phased column. The mobile phase, 45% methanol containing 1% glacial acetic acid and 0.05% 1-hexanesulfonic acid was run at a flow rate of 1 mL/min. The column effluent was monitored using UV detector at 260 nm. The retention times for tolperisone and the internal standard were approximately 7.1 and 8.4 min, respectively. The standard curve was linear with minimal intra-day and inter-day variability. The quantification limit of tolperisone in human plasma was 10 ng/ mL. The proposed method has been applied to the determination of pharmacokinetic profile of tolperisone in Koreans. The Tmax of tolperisone in Koreans (0.94 +/- 0.42 h) was not significantly differ from that reported in Europeans (0.5-1 h), but the mean half-life in Koreans (1.14 +/- 0.27 h) was shorter than that in Europeans (2.56 +/- 0.2 h). The proposed HPLC method is simple, accurate, reproducible and suitable for pharmacokinetic study of tolperisone.

  10. Fundamentals of gas phase plasmas for treatment of human tissue.

    Science.gov (United States)

    Kushner, Mark J; Babaeva, Natalia Yu

    2011-01-01

    The use of gas phase plasmas for treating human tissue is at the intersection of two disciplines - plasma physics and engineering, and medicine. In this paper, a primer will be provided for the medical practitioner on the fundamentals of generating gas phase plasmas at atmospheric pressure in air for the treatment of human tissue. The mechanisms for gas phase plasmas interacting with tissue and biological fluids will also be discussed using results from computer modeling.

  11. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  12. 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.

  13. Plasma adiponectin and endogenous glucose production in humans

    DEFF Research Database (Denmark)

    Stefan, Norbert; Stumvoll, Michael; Vozarova, Barbora

    2003-01-01

    High plasma adiponectin is associated with reduced risk of type 2 diabetes, probably a consequence of its insulin-sensitizing properties. In vivo data in rodents suggest that the insulin-sensitization responsible for improvement of glycemia occurs in muscle and liver. Whereas associations of plasma...... adiponectin with muscle insulin sensitivity in humans have been examined, this has not been done for the liver....

  14. 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.

  15. Isolation of biologically-active exosomes from human plasma.

    Science.gov (United States)

    Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

    2014-09-01

    Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

  16. Rheology of human blood plasma: Viscoelastic versus Newtonian behavior

    CERN Document Server

    Brust, M; Pan, L; Garcia, M; Arratia, P E; Wagner, C; 10.1103/PhysRevLett.110.078305

    2013-01-01

    We investigate the rheological characteristics of human blood plasma in shear and elongational flows. While we can confirm a Newtonian behavior in shear flow within experimental resolution, we find a viscoelastic behavior of blood plasma in the pure extensional flow of a capillary break-up rheometer. The influence of the viscoelasticity of blood plasma on capillary blood flow is tested in a microfluidic device with a contraction-expansion geometry. Differential pressure measurements revealed that the plasma has a pronounced flow resistance compared to that of pure water. Supplementary measurements indicate that the viscoelasticity of the plasma might even lead to viscoelastic instabilities under certain conditions. Our findings show that the viscoelastic properties of plasma should not be ignored in future studies on blood flow.

  17. Human maternal plasma proteomic changes with parturition

    Directory of Open Access Journals (Sweden)

    Robert J. Phillips

    2014-12-01

    Significance: Proteomic technology is constantly advancing, and the latest techniques enable gel-free analysis of minimally preprocessed, complex biological samples, enabling simultaneous identification and quantification of many hundreds of proteins. The technique of TMT labelling and Orbitrap mass spectrometry is applicable to the analysis of serial maternal plasma samples in order to identify potential markers of the onset of labour.

  18. Metabolic Signatures of Exercise in Human Plasma

    Science.gov (United States)

    Lewis, Gregory D.; Farrell, Laurie; Wood, Malissa J.; Martinovic, Maryann; Arany, Zoltan; Rowe, Glenn C; Souza, Amanda; Cheng, Susan; McCabe, Elizabeth L.; Yang, Elaine; Shi, Xu; Deo, Rahul; Roth, Frederick P.; Asnani, Aarti; Rhee, Eugene P.; Systrom, David M.; Semigran, Marc J.; Vasan, Ramachandran S.; Carr, Steven A.; Wang, Thomas J.; Sabatine, Marc S.; Clish, Clary B.; Gerszten, Robert E.

    2010-01-01

    Exercise provides numerous salutary effects, but our understanding of how these occur is limited. To gain a clearer picture of exercise-induced metabolic responses, we have developed comprehensive plasma metabolite signatures by using mass spectrometry to measure over 200 metabolites before and after exercise. We identified plasma indicators of glycogenolysis (glucose-6-phosphate), tricarboxylic acid (TCA) cycle span 2 expansion (succinate, malate, and fumarate), and lipolysis (glycerol), as well as modulators of insulin sensitivity (niacinamide) and fatty acid oxidation (pantothenic acid). Metabolites that were highly correlated with fitness parameters were found in subjects undergoing acute exercise testing, marathon running, and in 302 subjects from a longitudinal cohort study. Exercise-induced increases in glycerol were strongly related to fitness levels in normal individuals and were attenuated in subjects with myocardial ischemia. A combination of metabolites that increased in plasma in response to exercise (glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate) upregulated the expression of nur77, a transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle. Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise. PMID:20505214

  19. EFFECT OF ACUPUNCTURE ON PLASMA GLUCOSE LEVEL IN HUMAN VOLUNTEERS

    Institute of Scientific and Technical Information of China (English)

    Amit Kumar Chakraborty; Mrigendranath Gantait; Biswapati Mukherjee

    2006-01-01

    Objective To observe the changes of plasma glucose level (PGL) in human volunteers after acupuncture. Methods Seventy-seven human volunteers were taken up from the acupuncture clinic. All of pletion of acupuncture. All cases were at four hours abstinence from food before doing acupuncture. Results Plasma glucose level varied 5 mg% or more in 62 cases (80.51%) and only those were considered for computation. PGL increased in cases who had generally plasma glucose level below 90 mg% before acupuncture;and PGL decreased in cases who had plasma glucose 90 mg% or above. In 10 control cases there was no variation of the considerable level of 5 mg% in any case. Conclusion Bi-directional variation of PGL after acupuncture indicates that acupuncture can be used to maintain optimum PGL through endogenous mechanism,suggesting that it is applicable in controlling hyperglycemia in diabetes mellitus patients.

  20. Human plasma DNP level after severe brain injury

    Institute of Scientific and Technical Information of China (English)

    GAO Yi-lu; XIN Hui-ning; FENG Yi; FAN Ji-wei

    2006-01-01

    Objective: To determine the relationship between DNP level after human severe brain injury and hyponatremia as well as isorrhea.Methods: The peripheral venous plasma as control was collected from 8 volunteers. The peripheral venous plasma from 14 severe brain injury patients were collected in the 1, 3, 7 days after injury. Radioimmunoassay was used to detect the DNP concentration. Meanwhile, daily plasma and urine electrolytes, osmotic pressure as well as 24 h liquid intake and output volume were detected.Results: The normal adult human plasma DNP level was 62. 46 pg/ml ± 27. 56 pg/ml. In the experimental group, the plasma DNP levels were higher from day 1 today 3 in 8 of the 14 patients than those in the control group (P1 =0.05, P3 =0.03). Negative fluid balance occurred in 8 patients and hyponatremia in 7 patients. The increase of plasma DNP level was significantly correlated with the development of a negative fluid balance (r=-0.69,P<0.01) and hyponatremia (x2 =4.38, P<0.05).Conclusions: The increase of plasma DNP level is accompanied by the enhancement of natriuretic and diuretic responses in severe brain-injured patients, which is associated with the development of a negative fluid balance and hyponatremia after brain injury.

  1. Effects of continuous white light and 12h white-12h blue light-cycles on the expression of clock genes in diencephalon, liver, and skeletal muscle in chicks.

    Science.gov (United States)

    Honda, Kazuhisa; Kondo, Makoto; Hiramoto, Daichi; Saneyasu, Takaoki; Kamisoyama, Hiroshi

    2017-05-01

    The core circadian clock mechanism relies on a feedback loop comprised of clock genes, such as the brain and muscle Arnt-like 1 (Bmal1), chriptochrome 1 (Cry1), and period 3 (Per3). Exposure to the light-dark cycle synchronizes the master circadian clock in the brain, and which then synchronizes circadian clocks in peripheral tissues. Birds have long been used as a model for the investigation of circadian rhythm in human neurobiology. In the present study, we examined the effects of continuous light and the combination of white and blue light on the expression of clock genes (Bmal1, Cry1, and Per3) in the central and peripheral tissues in chicks. Seventy two day-old male chicks were weighed, allocated to three groups and maintained under three light schedules: 12h white light-12h dark-cycles group (control); 24h white light group (WW group); 12h white light-12h blue light-cycles group (WB group). The mRNA levels of clock genes in the diencephalon were significantly different between the control and WW groups. On the other hand, the alteration in the mRNA levels of clock genes was similar between the control and WB groups. Similar phenomena were observed in the liver and skeletal muscle (biceps femoris). These results suggest that 12h white-12h blue light-cycles did not disrupt the circadian rhythm of clock gene expression in chicks. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Proteins involved in the Vroman effect during exposure of human blood plasma to glass and polyethylene

    NARCIS (Netherlands)

    Turbill, P.; Beugeling, T.; Poot, A.A.

    1996-01-01

    The amounts of fibrinogen adsorbed to glass from various human blood plasmas have been measured as a function of time. The plasmas were 11 single donor plasmas, pooled plasma, a single donor high molecular weight kininogen (HMWK)-deficient plasma and HMWK-deficient plasma, which had been reconstitut

  3. Statistical Analysis of Variation in the Human Plasma Proteome

    Directory of Open Access Journals (Sweden)

    Todd H. Corzett

    2010-01-01

    Full Text Available Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

  4. Determination of Glimepiride in Human Plasma by Liquid Chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ho Hyun; Lee, Hee Joo [Seoul Clinical Laboratories, Seoul (Korea, Republic of); Chang, Kyu Young [Korean Biochip Society, Seoul (Korea, Republic of); Han, Sang Beom [ChungAng University, Seoul (Korea, Republic of)

    2004-01-15

    A sensitive method for quantitation of glimepiride in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Glipizide was used as an internal standard. Glimepiride and internal standard in plasma sample was extracted using diethyl etherethyl acetate (1 : 1). A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of acetonitrile-5 mM ammonium acetate (60:40, pH 3.0). The reconstituted samples were injected into a C{sub 18} reversed-phase column. Using MS/MS in the multiple reaction monitoring (MRM) mode, glimepiride and glipizide were detected without severe interference from human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]{sup +}) at m/z 491 and a corresponding product ion at m/z 352. And the internal standard produced a protonated precursor ion ([M+H]{sup +}) at m/z 446 and a corresponding product ion at m/z 321. Detection of glimepiride in human plasma by the LC-ESI/MS/MS method was accurate and precise with a quantitation limit of 0.1 ng/mL. The validation, reproducibility, stability, and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of glimepiride in human plasma

  5. 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......(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....

  6. 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.

  7. Lipid hydroperoxides in human plasma after ethanol consumption.

    Science.gov (United States)

    Asano, Migiwa; Nushida, Hideyuki; Adachi, Junko; Nagasaki, Yasushi; Nakagawa, Kanako; Kuse, Azumi; Ueno, Yasuhiro

    2009-04-01

    Oxidative stress contributes to the pathogenesis of alcoholic liver disease. The purpose of this study is to estimate the amount of oxidative stress that is present when healthy humans consume moderate amounts of ethanol. Blood was collected from healthy volunteers before, 1 h, and 3 h after drinking 400 ml of Japanese rice wine at the rate of 100 ml per 5 min. The aldehyde dehydrogenase 2 genotype and the concentrations of blood ethanol, total lipid hydroperoxides (LOOH), and cholesterol hydroperoxides were determined. The plasma LOOH was found to have significantly increased 1h after drinking. Cholesterol hydroperoxides were not detected in plasma, either before or after drinking. There was no relationship between the LOOH and the ethanol concentration. We showed that one-shot of moderate ethanol consumption temporarily increases the plasma LOOH in healthy volunteers but excessive plasma LOOH compounds were eliminated within a short time.

  8. Coffee polyphenols protect human plasma from postprandial carbonyl modifications.

    Science.gov (United States)

    Sirota, Roman; Gorelik, Shlomit; Harris, Raviv; Kohen, Ron; Kanner, Joseph

    2013-05-01

    The antioxidant capability of coffee polyphenols to inhibit red-meat lipid peroxidation in stomach medium and absorption into blood of malondialdehyde (MDA) in humans was studied. Roasted-ground coffee polyphenols that were found to inhibit lipid peroxidation in stomach medium are 2- to 5-fold more efficient antioxidant than those found in instant coffee. Human plasma from ten volunteers analyzed after a meal of red-meat cutlets (250 g) revealed a rapid accumulation of MDA. The accumulation of MDA in human plasma modified low-density lipoprotein is known to trigger atherogenesis. Consumption of 200 mL roasted coffee by ten volunteers during a meal of red-meat cutlets, resulted after 2 and 4 h in the inhibition by 80 and 50%, respectively, of postprandial plasma MDA absorption. The results obtained in vitro simulated stomach model on MDA accumulation were predictive for the amount of MDA absorbed into circulating human plasma, in vivo. Timing the consumption of coffee during the meals may make it a very active functional food.

  9. Differences between human plasma and serum metabolite profiles.

    Directory of Open Access Journals (Sweden)

    Zhonghao Yu

    Full Text Available BACKGROUND: Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01. Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10, which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices. CONCLUSIONS/SIGNIFICANCE: Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is

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

    Science.gov (United States)

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

    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.

  11. Site Specific Modification of the Human Plasma Proteome by Methylglyoxal

    Science.gov (United States)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig; 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. PMID:26435215

  12. 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.

  13. Evaluation of measurement of human TNF in plasma by ELISA.

    Science.gov (United States)

    Engelberts, I; Möller, A; Schoen, G J; van der Linden, C J; Buurman, W A

    1991-04-01

    The performance of a sandwich-ELISA for TNF measurement in plasma and serum was studied. The ELISA was first statistically analyzed. Interassay coefficient of variance and the intraassay coefficient of variance for the concentration range between 0.5 and 5 ng/ml was less than 10%. The sensitivity of the sandwich-ELISA for TNF in culture medium was 10 pg/ml. The ELISA was shown to be specific for biologically active TNF, since a good correlation between the ELISA and the WEHI bioassay was observed when partially inactive, denatured TNF was measured. The effect of various anticoagulation systems on the reliability of human TNF measurement has been evaluated. The oxalate/NaF and EDTA systems were both appropriate, as appeared from the observed blockade of the production of TNF in the tube, either in the cell-glycolysis-blocked or in the calcium-depleted situation, respectively. An eventual decrease in the recovery of rTNF after collection of blood was prevented in the oxalate/NaF tubes. Recovery of TNF in the ELISA was diminished in the presence of plasma or serum. Techniques to enhance the efficiency of the measurement of TNF in plasma by ELISA were compared. The data indicate that in the presence of 1.1 M NaCl, the TNF masking effect of normal plasma was largely abrogated. The presence and role of inhibiting plasma components in plasma of healthy and diseased individuals are discussed.

  14. Human papillomavirus DNA in plasma of patients with cervical cancer

    Directory of Open Access Journals (Sweden)

    Voravud Narin

    2001-03-01

    Full Text Available Abstract Background Human papillomavirus (HPV is a crucial etiological factor for cervical cancer (CC development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients. Results Whereas 6 out of 50 (12% HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67. Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA. Conclusions The plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

  15. [Radioimmunoassay for human plasma 8-arginine-vasopressin (author's transl)].

    Science.gov (United States)

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

    1977-01-01

    The authors have developed a radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormon (ADH) levels as low as 0,8 pg/ml. The average plasma level of AVP after overnight water restriction was found to be 14,3 pg/ml (sd = 4,4 pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2 mg 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.

  16. PIXE analysis of human spermatozoa isolated from seminal plasma

    Science.gov (United States)

    Maeda, K.; Sasa, Y.; Kusuyama, H.; Yoshida, K.; Uda, M.

    1990-04-01

    PIXE has been applied to the multielemental and microanalysis of human spermatozoa. This is the first attempt to determine the chemical compositions of the motile spermatozoa free from contaminations of seminal plasma without loss of component elements during washing. The spermatozoa were isolated from semen by letting them swim into a kind of physiological saline, Tyrode's solution. Relative concentrations of P, K, Ca, Ti, Fe, Zn and Br in motile spermatozoa were determined by the use of the chlorine K X-ray peak intensity for evaluating the amount of Tyrode's solution contained in the sample targets. The concentrations of calcium and iron in spermatozoa were considerably higher than in seminal plasma. The concentrations of P, K, Zn and Br in spermatozoa were not so different from those in seminal plasma.

  17. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    Science.gov (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  18. An iron stable isotope comparison between human erythrocytes and plasma.

    Science.gov (United States)

    von Blanckenburg, Friedhelm; Oelze, Marcus; Schmid, Dietmar G; van Zuilen, Kirsten; Gschwind, Hans-Peter; Slade, Alan J; Stitah, Sylvie; Kaufmann, Daniel; Swart, Piet

    2014-11-01

    We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The (56)Fe/(54)Fe ratio in erythrocytes, expressed as permil difference from the "IRMM-014" iron reference standard (δ(56/54)Fe), ranges from -3.1‰ to -2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ(56/54)Fe values measured in two different laboratories range from -3.0‰ to -2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ(56/54)Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

  19. Demonstration in human plasma of a lectin activity analogous to that of bovine conglutinin

    DEFF Research Database (Denmark)

    Baatrup, G; Thiel, S; Isager, H

    1987-01-01

    Evidence of the existence in human plasma of an activity analogous to that of bovine conglutinin is presented. The human plasma component was characterized antigenically and functionally. Human plasma was shown to agglutinate complement-coated erythrocytes in the presence of Ca2+, and this conglu...

  20. Electrochemical determination of methylglyoxal as a biomarker in human plasma.

    Science.gov (United States)

    Chatterjee, Sanghamitra; Wen, Jiali; Chen, Aicheng

    2013-04-15

    A novel electrochemical approach for the quantitative analysis of methylglyoxal as a biomarker in human plasma has been developed. An electrochemical sensor employing a single walled carbon nanotube modified glassy carbon electrode for the sensitive detection of methylglyoxal is delineated for the first time using square wave voltammetry. This modified electrode exhibits potent and sustained electron-mediating behavior and a well-defined reduction peak in response to methylglyoxal was observed. Under optimized experimental conditions, a wide linear dynamic range, from 0.1 to 100 μM, and high sensitivity of 76.3 nA μM⁻¹ were achieved for the detection of methylglyoxal. The interfering effect of common coexisting metabolites in human whole blood has also been investigated. The developed assay was shown to be specific and sensitive for the analysis of plasma levels of methylglyoxal in healthy volunteer and diabetic patients.

  1. Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates

    Directory of Open Access Journals (Sweden)

    Hulya Leventerler

    2013-08-01

    Full Text Available Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml of asthenoteratospermic (40.0±25.7 and azospermic (38.0±43.6 groups were significantly lower than normozoospermic, (93.9±52.1 males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x106 sperm of teratospermic group (136.8±61.8 was significantly higher compared to the normozoospermic (87.3±26.5 males. Glucose concentration (mg/dl in asthenoteratospermic (4.0±1.4 and azospermic (15.4±6.4 groups were significantly higher than fertile (2.0±2.1 males. Also fructose concentration (mg/dl in asthenoteratospermic (706.6±143.3 and azospermic (338.1±228.2 groups were significantly high compared to the normozoospermic (184.7±124.8 group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility. [Cukurova Med J 2013; 38(4.000: 648-658

  2. Plasma proteomic alterations in non-human primates and humans after chronic alcohol self-administration.

    Science.gov (United States)

    Freeman, Willard M; Vanguilder, Heather D; Guidone, Elizabeth; Krystal, John H; Grant, Kathleen A; Vrana, Kent E

    2011-08-01

    Objective diagnostics of excessive alcohol use are valuable tools in the identification and monitoring of subjects with alcohol use disorders. A number of potential biomarkers of alcohol intake have been proposed, but none have reached widespread clinical usage, often due to limited diagnostic sensitivity and specificity. In order to identify novel potential biomarkers, we performed proteomic biomarker target discovery in plasma samples from non-human primates that chronically self-administer high levels of ethanol. Two-dimensional difference in-gel electrophoresis (2D-DIGE) was used to quantify plasma proteins from within-subject samples collected before exposure to ethanol and after 3 months of excessive ethanol self-administration. Highly abundant plasma proteins were depleted from plasma samples to increase proteomic coverage. Altered plasma levels of serum amyloid A4 (SAA4), retinol-binding protein, inter-alpha inhibitor H4, clusterin, and fibronectin, identified by 2D-DIGE analysis, were confirmed in unmanipulated, whole plasma from these animals by immunoblotting. Examination of these target plasma proteins in human subjects with excessive alcohol consumption (and control subjects) revealed increased levels of SAA4 and clusterin and decreased levels of fibronectin compared to controls. These proteins not only serve as targets for further development as biomarker candidates or components of biomarker panels, but also add to the growing understanding of dysregulated immune function and lipoprotein metabolism with chronic, excessive alcohol consumption.

  3. [HPLC-MS/MS method for determination of sodium cromoglycate concentration in human plasma and its pharmacokinetics].

    Science.gov (United States)

    Xu, Xiao-yan; Zhang, Rui; Yuan, Gui-yan; Wang, Ben-jie; Liu, Xiao-yan; Guo, Rui-chen

    2008-09-01

    The study established an HPLC-MS/MS method for determining the concentrations of sodium cromoglycate in human plasma and evaluated the pharmacokinetics of nasal drops and nasal spray. A C18 column was used to separate sodium cromoglycate in plasma with a mobile phase of a mixture of ammonium-methanol (involves 50% acetonitrile) (15:85) at a flow rate of 0.4 mL x min(-1). Electronic spray ionization (ESI) and multiple-reaction monitoring (MRM) were used for the determination of sodium cromoglycate in human plasma. The linear range of the standard curve of sodium cromoglycate was from 0.3 to 20 ng x mL(-1), and the minimum concentration of detection was 0.3 ng x mL(-1). The extraction recovery was more than 94.1%, intra-day and inter-day RSD were less than 14.3%. After a single dose of sodium cromoglycate, the main pharmacokinetic parameters of nasal spray and nasal drops were as follows, T(1/2)(1.82 +/- 0.54) h, (1.59 +/- 0.52) h; Tmax (0.47 +/- 0.12) h, (0.44 +/- 0.15) h; Cmax, (9.79 +/- 4.66) ng x mL(-1), (10.88 +/- 4.05) ng x mL(-1); AUC(0-5 h)(11.52 +/- 3.46) ng x mL(-1) x h x h, (12.63 +/- 4.23) ng x mL(-1) x h, Fr(93.6 +/- 13.8)%. The method is sensitive, rapid and accurate. It is suitable for therapeutic drug monitoring and human pharmacokinetic study of sodium cromoglycate.

  4. Red wine activates plasma membrane redox system in human erythrocytes.

    Science.gov (United States)

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  5. Effects of water immersion on plasma catecholamines in normal humans

    Science.gov (United States)

    Epstein, M.; Johnson, G.; Denunzio, A. G.

    1983-01-01

    An investigation was conducted in order to determine whether water immersion to the neck (NI) alters plasma catecholamines in normal humans. Eight normal subjects were studied during a seated control study (C) and during 4 hr of NI, and the levels of norepinephrine (NE) and epinephrine (E) as determined by radioenzymatic assay were measured hourly. Results show that despite the induction of a marked natriuresis and diuresis indicating significant central hypervolemia, NI failed to alter plasma NE or E levels compared with those of either C or the corresponding prestudy 1.5 hr. In addition, the diuresis and natriuresis was found to vary independently of NE. These results indicate that the response of the sympathetic nervous system to acute volume alteration may differ from the reported response to chronic volume expansion.

  6. Determination of actarit from human plasma for bioequivalence studies.

    Science.gov (United States)

    Loya, P; Saraf, M N

    2010-11-01

    An analytical method based on high-performance liquid chromatography with ultraviolet detection (245 nm) was developed for the determination of actarit in human plasma. Coumarin was used as an internal standard. Chromatographic separation was achieved with a C8 column using a mobile phase of methanol: 1% acetic acid (50-50, v/v) with a flow rate of 1.0 ml/min. The calibration curve was linear over the range of 0.1-4.0 μg/ml (r(2) > 0.99) and the lower limit of quantification was 0.1 μg/ml. The method was validated for sensitivity, accuracy, precision, recovery and stability. The method was used to determine the concentration-time profiles of actarit in the plasma following oral administration of 100 mg actarit tablets.

  7. A physiologically based in silico kinetic model predicting plasma cholesterol concentrations in humans

    NARCIS (Netherlands)

    Pas, van de N.C.A.; Woutersen, R.A.; Ommen, van B.; Rietjens, I.M.C.M.; Graaf, de A.A.

    2012-01-01

    Increased plasma cholesterol concentration is associated with increased risk of cardiovascular disease. This study describes the development, validation, and analysis of a physiologically based kinetic (PBK) model for the prediction of plasma cholesterol concentrations in humans. This model was dire

  8. A physiologically based in silico kinetic model predicting plasma cholesterol concentrations in humans

    NARCIS (Netherlands)

    Pas, N.C.A. van de; Woutersen, R.A.; Ommen, B. van; Rietjens, I.M.C.M.; Graaf, A.A. de

    2012-01-01

    Increased plasma cholesterol concentration is associated with increased risk of cardiovascular disease. This study describes the development, validation, and analysis of a physiologically based kinetic (PBK) model for the prediction of plasma cholesterol concentrations in humans. This model was

  9. 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.

  10. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    Directory of Open Access Journals (Sweden)

    Kentaro Kikuchi

    2003-01-01

    Full Text Available Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.

  11. Identification of calcium-binding proteins associated with the human sperm plasma membrane

    National Research Council Canada - National Science Library

    Naaby-Hansen, Soren; Diekman, Alan; Shetty, Jagathpala; Flickinger, Charles J; Westbrook, Anne; Herr, John C

    2010-01-01

    The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation...

  12. Pharmacokinetic Comparison of Soy Isoflavone Extracts in Human Plasma.

    Science.gov (United States)

    Rodríguez-Morató, Jose; Farré, Magí; Pérez-Mañá, Clara; Papaseit, Esther; Martínez-Riera, Roser; de la Torre, Rafael; Pizarro, Nieves

    2015-08-12

    The soy isoflavones daidzein and genistein produce several biological activities related to health benefits. A number of isoflavone extracts are commercially available, but there is little information concerning the specific isoflavone content of these products or differences in their bioavailability and pharmacokinetics. This study describes the development and validation of an analytical method to detect and quantify daidzein, genistein, and equol in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was applied in a crossover, randomized, bioavailability study. Twelve healthy volunteers were administered the same total isoflavones dose from two isoflavone supplement preparations (Super-Absorbable Soy Isoflavones (Life Extension, USA) and Fitoladius (Merck, Spain)). The pharmacokinetic parameters (AUC0-24/dose and Cmax/dose) of the isoflavones from the two preparations differed significantly. Such differences in bioavailability and kinetics may have relevant effects on the health benefits derived from their intake.

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

    Directory of Open Access Journals (Sweden)

    Claus Brandt

    2014-01-01

    Full Text Available 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, TNF-α, or interleukin-6 (IL-6 as well as a hyperinsulinemic euglycemic clamp were used to examine if plasma fstl3 was acutely regulated in humans. Results. Plasma fstl3 was increased in obese subjects independent of glycemic state. Moreover, plasma fstl3 was positively correlated with fat mass, 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 and associated with fat mass and low-grade inflammation. Furthermore, TNF-α increased plasma fstl3, suggesting that TNF-α is one of the inflammatory drivers of increased systemic levels of fstl3.

  14. Switch-task performance in rats is disturbed by 12 h of sleep deprivation but not by 12 h of sleep fragmentation.

    Science.gov (United States)

    Leenaars, Cathalijn H C; Joosten, Ruud N J M A; Zwart, Allard; Sandberg, Hans; Ruimschotel, Emma; Hanegraaf, Maaike A J; Dematteis, Maurice; Feenstra, Matthijs G P; van Someren, Eus J W

    2012-02-01

    Task-switching is an executive function involving the prefrontal cortex. Switching temporarily attenuates the speed and/or accuracy of performance, phenomena referred to as switch costs. In accordance with the idea that prefrontal function is particularly sensitive to sleep loss, switch-costs increase during prolonged waking in humans. It has been difficult to investigate the underlying neurobiological mechanisms because of the lack of a suitable animal model. Here, we introduce the first switch-task for rats and report the effects of sleep deprivation and inactivation of the medial prefrontal cortex. Rats were trained to repeatedly switch between 2 stimulus-response associations, indicated by the presentation of a visual or an auditory stimulus. These stimulus-response associations were offered in blocks, and performance was compared for the first and fifth trials of each block. Performance was tested after exposure to 12 h of total sleep deprivation, sleep fragmentation, and their respective movement control conditions. Finally, it was tested after pharmacological inactivation of the medial prefrontal cortex. Controlled laboratory settings. 15 male Wistar rats. Both accuracy and latency showed switch-costs at baseline. Twelve hours of total sleep deprivation, but not sleep fragmentation, impaired accuracy selectively on the switch-trials. Inactivation of the medial prefrontal cortex by local neuronal inactivation resulted in an overall decrease in accuracy. We developed and validated a switch-task that is sensitive to sleep deprivation. This introduces the possibility for in-depth investigations on the neurobiological mechanisms underlying executive impairments after sleep disturbance in a rat model.

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

    DEFF Research Database (Denmark)

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

    1989-01-01

    To clarify the origin of plasma DOPA (3,4-Dihydroxyphenylalanine), the relationship between plasma DOPA and acute or chronic changes in sympathetic activity has been studied. Plasma DOPA and noradrenaline (NA) concentrations were measured by reverse-phase high-performance liquid chromatography...... 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...

  16. 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

    2017-09-22

    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 9 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.

  17. Jejunal feeding is followed by a greater rise in plasma cholecystokinin, peptide YY, glucagon-like peptide 1, and glucagon-like peptide 2 concentrations compared with gastric feeding in vivo in humans

    DEFF Research Database (Denmark)

    Luttikhold, Joanna; van Norren, Klaske; Rijna, Herman;

    2016-01-01

    ± SD age: 21 ± 2 y) received continuous enteral nutrition that contained noncoagulating proteins for 12 h via a nasogastric tube or a nasojejunal tube placed 30-40 cm distal to the ligament of Treitz. Blood samples were collected during the 12-h postprandial period to assess the rise in plasma glucose...

  18. 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

    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......), TNF-α, or interleukin-6 (IL-6) as well as a hyperinsulinemic euglycemic clamp were used to examine if plasma fstl3 was acutely regulated in humans. RESULTS: Plasma fstl3 was increased in obese subjects independent of glycemic state. Moreover, plasma fstl3 was positively correlated with fat mass...

  19. Influence of intralipid on free propofol fraction assayed in human serum albumin solutions and human plasma

    Institute of Scientific and Technical Information of China (English)

    Rafal KALITYNSKI; Andrzej L DAWIDOWICZ; Jacek POSZYTEK

    2006-01-01

    Aim: It is generally assumed that only unbound drugs can reach the site of action by diffusing across the membranes and exerting pharmacological effects by interacting with receptors. Recent research has shown that the percentage of free drugs may depend on the total drug concentration. The aim of the paper is to verify whether the mentioned dependence reported for propofol also takes place in plasma and human serum albumin samples in the presence of intralipid-the medium used as a vehicle for propofol infusions and a parenteral nutrition agent. Methods: Artificial plasma samples and human plasma were spiked with intralipid or ethanolic solutions of propofol. The samples were then assayed for free propofol concentration using ultrafiltration and high performance liquid chromatography with fluorimetric detection. Results: The decrease of the total drug concentration results in free propofol fraction increase, irrespectively of the used type of propofol solvent and sample type. The addition of intralipid causes the lowering of the overall free drug fraction with respect to the samples spiked with ethanolic solutions of the drug. Conclusion: The presence of intralipid does not influence the phenomenon of free propofol fraction rise at low total drug concentration. Such a rise cannot be ignored in clinical conditions when the drug is applied for sedative, antiemetic or other low-dosage purposes.

  20. 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

  1. Computational lipidology: predicting lipoprotein density profiles in human blood plasma.

    Directory of Open Access Journals (Sweden)

    Katrin Hübner

    2008-05-01

    Full Text Available Monitoring cholesterol levels is strongly recommended to identify patients at risk for myocardial infarction. However, clinical markers beyond "bad" and "good" cholesterol are needed to precisely predict individual lipid disorders. Our work contributes to this aim by bringing together experiment and theory. We developed a novel computer-based model of the human plasma lipoprotein metabolism in order to simulate the blood lipid levels in high resolution. Instead of focusing on a few conventionally used predefined lipoprotein density classes (LDL, HDL, we consider the entire protein and lipid composition spectrum of individual lipoprotein complexes. Subsequently, their distribution over density (which equals the lipoprotein profile is calculated. As our main results, we (i successfully reproduced clinically measured lipoprotein profiles of healthy subjects; (ii assigned lipoproteins to narrow density classes, named high-resolution density sub-fractions (hrDS, revealing heterogeneous lipoprotein distributions within the major lipoprotein classes; and (iii present model-based predictions of changes in the lipoprotein distribution elicited by disorders in underlying molecular processes. In its present state, the model offers a platform for many future applications aimed at understanding the reasons for inter-individual variability, identifying new sub-fractions of potential clinical relevance and a patient-oriented diagnosis of the potential molecular causes for individual dyslipidemia.

  2. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    Science.gov (United States)

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma.

  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

    sources, including the HUPO PPP dataset. CONCLUSION: Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.......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...

  4. Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion

    OpenAIRE

    Edwards, Lindsay M.; Lawler, Nathan G.; Nikolic, Sonja B.; Peters, James M.; Horne, James; Wilson, Richard,; Davies, Noel W.; Sharman, James E

    2012-01-01

    Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipi...

  5. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    Science.gov (United States)

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.

  6. 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...... is the second most abundant ( approximately 34% and 30%, respectively). In contrast, CCK-58 is less abundant in human intestines ( approximately 18%) and plasma ( approximately 11%). Its predominance in feline intestines, however, was confirmed. Hence, the results show a significant species variation...

  7. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans

    DEFF Research Database (Denmark)

    Steensberg, Adam; Fischer, Christian Philip; Keller, Charlotte

    2003-01-01

    The purpose of the present study was to test the hypothesis that a transient increase in plasma IL-6 induces an anti-inflammatory environment in humans. Therefore, young healthy volunteers received a low dose of recombinant human (rh)IL-6 or saline for 3 h. Plasma IL-6 levels during rhIL-6 infusion...... number without effects on plasma epinephrine, body temperature, mean arterial pressure, or heart rate. In conclusion, this study demonstrates that physiological concentrations of IL-6 induce an anti-inflammatory rather than an inflammatory response in humans and that IL-6, independently of TNF......-induced leukocyte trafficking....

  8. 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

  9. A phospholipidomic analysis of all defined human plasma lipoproteins

    NARCIS (Netherlands)

    Dashti, M.; Kulik, W.; Hoek, F.; Veerman, E.C.; Peppelenbosch, M.P.; Rezaee, F.

    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

  10. 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.

  11. 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

    2015-01-01

    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.

  12. Stability of glucagon-like peptide-1 and glucagon in human plasma

    DEFF Research Database (Denmark)

    Albrechtsen, Nicolai Jacob Wewer; Bak, Monika Judyta; Hartmann, Bolette;

    2015-01-01

    To investigate the stability of glucagon-like peptide-1(GLP-1) and glucagon in plasma under short- and long-term storage conditions. Methods: Pooled human plasma (n=20), to which a dipeptidyl peptidase 4 (DPP-4) inhibitor and aprotinin were added, was spiked with synthetic GLP-1 (intact, 7-36NH2 ...

  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 h

  14. 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...

  15. Bone regenerative properties of rat, goat and human platelet-rich plasma.

    NARCIS (Netherlands)

    Plachokova, A.S.; Dolder, J. van den; Beucken, J.J.J.P. van den; Jansen, J.A.

    2009-01-01

    To explore the reported contradictory osteogenic capacity of platelet-rich plasma (PRP), the aim of the study was to examine and compare the bone regenerative effect of: PRPs of different species (rat, goat, human); human bone graft (HB) vs. HB combined with human PRP (HB+hPRP); and HB+hPRP vs. synt

  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. Plasma levels of oestriol-17 beta, oestriol and human placental lactogen during bed rest.

    Science.gov (United States)

    Chew, P C; Mok, H; Ratnam, S S

    1976-11-01

    Plasma unconjugated oestradiol-17 beta, total oestriol and human placental lactogen levels were measured in twelve healthy volunteers admitted for bed rest in the last trimester of pregnancy. No significant alteration in levels was observed.

  18. 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...

  19. Synthesis, spectral characterization and larvicidal activity of acridin-1(2H)-one analogues

    Science.gov (United States)

    Subashini, R.; Bharathi, A.; Roopan, Selvaraj Mohana; Rajakumar, G.; Abdul Rahuman, A.; Gullanki, Pavan Kumar

    Acridin-1(2H)-one analogue of 7-chloro-3,4-dihydro-9-phenyl-2-[(pyridine-2yl) methylene] acridin-1(2H)-one, 5 was prepared by using 7-chloro-3,4-dihydro-9-phenylacridin-1(2H)-one, 3 and picolinaldehyde, 4 in the presence of KOH at room temperature. These compounds were characterized by analytical and spectral analyses. The purpose of the present study was to assess the efficacy of larvicidal and repellent activity of synthesized 7-chloro-3,4-dihydro-9-phenyl-acridin-1(2H)-one analogues such as compounds 3 and 5 against the early fourth instar larvae of filariasis vector, Culex quinquefasciatus and Japanese encephalitis vector, Culex gelidus (Diptera: Culicidae). The compound exhibited high larvicidal effects at 50 mg/L against both the mosquitoes with LC50 values of 25.02 mg/L (r2 = 0.998) and 26.40 mg/L (r2 = 0.988) against C. quinquefasciatus and C. gelidus, respectively. The 7-chloro-3,4-dihydro-9-phenyl-acridin-1(2H)-one analogues that are reported for the first time to our best of knowledge can be better explored for the control of mosquito population. This is an ideal ecofriendly approach for the control of Japanese encephalitis vectors, C. quinquefasciatus and C. gelidus.

  20. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

    Inhibition of high-affinity (/sup 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/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 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 (/sup 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, Leon W.M.M.; Johnsen, Steen; Segers-Nolten, Ine 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. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

    Science.gov (United States)

    Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

    2013-11-01

    Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.

  3. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma[S

    Science.gov (United States)

    McDonald, Jeffrey G.; Smith, Daniel D.; Stiles, Ashlee R.; Russell, David W.

    2012-01-01

    We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4β-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D2 and D3. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day. PMID:22517925

  4. File list: Oth.Emb.05.AllAg.1-2h_embryos [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.1-2h_embryos dm3 TFs and others Embryo 1-2h embryos SRX197583,SRX1...97584 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.05.AllAg.1-2h_embryos.bed ...

  5. File list: ALL.Emb.50.AllAg.10-12h_embryos [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.10-12h_embryos dm3 All antigens Embryo 10-12h embryos SRX760382,SR...X760383,SRX760381,SRX760380 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/ALL.Emb.50.AllAg.10-12h_embryos.bed ...

  6. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

    Human blood plasma is a valuable specimen for the biomarker discovery process, since it is easily accessible and contains proteins that are synthesised, secreted or lost from cells and tissue. In this way, changes in plasma proteome reflect the current state of the organism. The analysis of plasma...... 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...... proteome is yet challenging due to the huge dynamic range of protein abundance. When evaluating a potential biomarker, stable basal level of the protein is needed before it can be considered a functional biomarker. However, basal level differences of plasma proteins are naturally occurring between...

  7. Tank 12H Acidic Chemical Cleaning Sample Analysis And Material Balance

    Energy Technology Data Exchange (ETDEWEB)

    Martino, C. J.; Reboul, S. H.; Wiersma, B. J.; Coleman, C. J.

    2013-11-08

    A process of Bulk Oxalic Acid (BOA) chemical cleaning was performed for Tank 12H during June and July of 2013 to remove all or a portion of the approximately 4400 gallon sludge heel. Three strikes of oxalic acid (nominally 4 wt% or 2 wt%) were used at 55°C and tank volumes of 96- to 140-thousand gallons. This report details the sample analysis of a scrape sample taken prior to BOA cleaning and dip samples taken during BOA cleaning. It also documents a rudimentary material balance for the Tank 12H cleaning results.

  8. Plasma appearance and correlation between coffee and green tea metabolites in human subjects.

    Science.gov (United States)

    Renouf, Mathieu; Guy, Philippe; Marmet, Cynthia; Longet, Karin; Fraering, Anne-Lise; Moulin, Julie; Barron, Denis; Dionisi, Fabiola; Cavin, Christophe; Steiling, Heike; Williamson, Gary

    2010-12-01

    Coffee and green tea are two of the most widely consumed hot beverages in the world. Their respective bioavailability has been studied separately, but absorption of their respective bioactive phenolics has not been compared. In a randomised cross-over design, nine healthy subjects drank instant coffee and green tea. Blood samples were collected over 12 h and at 24 h to assess return to baseline. After green tea consumption, (-)-epigallocatechin (EGC) was the major catechin, appearing rapidly in the plasma; (-)-EGC gallate (EGCg) and (-)-epicatechin (EC) were also present, but (-)-EC gallate and C were not detected. Dihydroferulic acid and dihydrocaffeic acid were the major metabolites that appeared after coffee consumption with a long time needed to reach maximum plasma concentration, suggesting metabolism and absorption in the colon. Other phenolic acid equivalents (caffeic acid (CA), ferulic acid (FA) and isoferulic acid (iFA)) were detected earlier, and they peaked at lower concentrations. Summations of the plasma area under the curves (AUC) for the measured metabolites showed 1.7-fold more coffee-derived phenolic acids than green tea-derived catechins (P = 0.0014). Furthermore, we found a significant correlation between coffee metabolites based on AUC. Inter-individual differences were observed, but individuals with a high level of CA also showed a correspondingly high level of FA. However, no such correlation was observed between the tea catechins and coffee phenolic acids. Correlation between AUC and maximum plasma concentration was also significant for CA, FA and iFA and for EGCg. This implies that the mechanisms of absorption for these two classes of compounds are different, and that a high absorber of phenolic acids is not necessarily a high absorber of catechins.

  9. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    OpenAIRE

    2003-01-01

    Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plas...

  10. Quantitation of Solifenacin in Human Plasma using a Specific and ...

    African Journals Online (AJOL)

    Methods: Solifenacin was extracted from plasma by a liquid-liquid extraction ... Solifenacin-d5 was the internal standard (IS). ... Bioassay validation followed International Bioanalytical Method Validation ... Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, ... (HPLC-MS/MS) analytical method for the.

  11. Plasma degradome affected by variable storage of human blood

    NARCIS (Netherlands)

    Kaisar, Maria; Van Dullemen, Leon F. A.; Thezenas, Marie-Laetitia; Akhtar, M. Zeeshan; Huang, Honglei; Rendel, Sandrine; Charles, Philip D.; Fischer, Roman; Ploeg, Rutger J.; Kessler, Benedikt M.

    2016-01-01

    Background: The successful application of-omics technologies in the discovery of novel biomarkers and targets of therapeutic interventions is facilitated by large collections of well curated clinical samples stored in bio banks. Mining the plasma proteome holds promise to improve our understanding o

  12. Spectrofluorimetric determination of amlodipine in human plasma without derivatization

    Directory of Open Access Journals (Sweden)

    Yucel Kadioglu

    2012-12-01

    Full Text Available A rapid and sensitive spectrofluorimetric method was developed for the determination of amlodipine (AD, a calcium channel blocker, in the plasma. The type of solvent, the wavelength range, and the range of AD concentration were selected to optimize the experimental conditions. The calibration curves were linear (r² >0.997 in the concentration range of 0.1-12.5 ppm of AD. The limit of quantitation and limit of detection values for the method for plasma samples were 0.1 ppm and 0.07 ppm, respectively. The precision calculated as the relative standard deviation was less than 3.5%, and the accuracy (relative error was better than 5.5% (n=6. The method developed in this study can be directly and easily applied for the determination of AD in the plasma without derivatization in plasma.Método espectrofluorometrico rápido e sensível é descrito para a determinação de anlodipina (AD, um bloqueador de canais de cálcio, em amostras de plasma. O tipo de solvente, a faixa de comprimento de onda e a faixa de concentração foram escolhidas a fim de otimizar as condições experimentais. As curvas de calibração foram lineares (r > 0,997 na faixa de concentração de 0,1-12,5 ppm de AD. Os valores LoQ e LoD do método para amostras de plasma foram 0,1 ppm e 0,07 ppm, respectivamente. A precisão calculada como desvio padrão relativo (RSD foi menor do que 3,5% e a precisão (erro relativo foi melhor do que 5,5% (n=6. O método desenvolvido neste estudo pode ser fácil e diretamente aplicado para a determinação de AD sem derivatização no plasma.

  13. Plasma oxytocin concentrations following MDMA or intranasal oxytocin in humans.

    Science.gov (United States)

    Kirkpatrick, Matthew G; Francis, Sunday M; Lee, Royce; de Wit, Harriet; Jacob, Suma

    2014-08-01

    MDMA (±3,4-methylenedioxymethamphetamine, 'ecstasy') is reportedly used recreationally because it increases feelings of sociability and interpersonal closeness. Prior work suggests that the pro-social effects of MDMA may be mediated by release of oxytocin. A direct examination of plasma levels of oxytocin after acute doses of oxytocin and MDMA, in the same individuals, would provide further evidence for the idea that MDMA produces its pro-social effects by increasing oxytocin. Fourteen healthy MDMA users participated in a 4-session, double-blind study in which they received oral MDMA (0.75 and 1.5mg/kg), intranasal oxytocin (20IU or 40IU), and placebo. Plasma oxytocin concentrations, as well as cardiovascular and subjective effects were assessed before and at several time points after drug administration. MDMA (1.5mg/kg only) increased plasma oxytocin levels to a mean peak of 83.7pg/ml at approximately 90-120min, compared to 18.6pg/ml after placebo. Intranasal oxytocin (40IU, but not 20IU) increased plasma oxytocin levels to 48.0pg/ml, 30-60min after nasal spray administration. MDMA dose-dependently increased heart rate, blood pressure, feelings of euphoria (e.g., 'High' and 'Like Drug'), and feelings of sociability, whereas oxytocin had no cardiovascular or subjective effects. The subjective and cardiovascular responses to MDMA were not related to plasma oxytocin levels, although the N was small for this analysis. Future studies examining the effects of oxytocin antagonists on responses to MDMA will help to determine the mechanism by which MDMA produces pro-social effects.

  14. 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

  15. 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...

  16. 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 BMI-mat

  17. Impact of 12h shift patterns in nursing: a scoping review.

    Science.gov (United States)

    Harris, Ruth; Sims, Sarah; Parr, Jenny; Davies, Nigel

    2015-02-01

    To provide a comprehensive scoping review of evidence of the impact and effectiveness of 12h shifts in the international nursing literature, supplemented by a review of evidence in other, non-nursing related industries. A search of the academic literature was undertaken in electronic databases (AMED, MEDLINE, CINAHL, PsychInfo, Scopus, HMIC, the Cochrane Library, Business Source Premier, Econ Lit, ASSIA and Social Policy and Practice). A total of 158 potentially relevant nursing research papers and reviews were published between 1973 and 2014. Two reviewers independently reviewed the articles, leaving 85 primary research studies and 10 review papers in the nursing field to be included in the scoping review. Thirty-one relevant primary research papers and reviews were also identified in the non-nursing related industries literature. Research into 12h nursing shifts fell within five broad themes: 'risks to patients', 'patient experience', 'risks to staff', 'staff experience' and 'impact on the organisation of work'. There was inconclusive evidence of the effects of 12h shift patterns in all five themes, with some studies demonstrating positive impacts and others negative or no impacts. This also mirrors the evidence in other, non-nursing related industries. The quality of research reviewed is generally weak and most studies focus on the risks, experience and work/life balance for staff, with few addressing the impact on patient outcomes and experience of care or work productivity. There is insufficient evidence to justify the widespread implementation or withdrawal of 12h shifts in nursing. It is not clearly understood where there are real benefits and where there are real and unacceptable risks to patients and staff. More research focusing on the impact of 12h nursing shifts on patient safety and experience of care and on the long term impact on staff and work organisation is required. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. 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

  19. 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.

  20. Stereoselective analysis of nebivolol isomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry: application in pharmacokinetics.

    Science.gov (United States)

    Neves, Daniel Valente; Vieira, Carolina Pinto; Coelho, Eduardo Barbosa; Marques, Maria Paula; Lanchote, Vera Lucia

    2013-12-01

    Nebivolol is available for clinical use as a racemic mixture. Isomer d-nebivolol (SRRR) is a β1 adrenergic receptor blocker and its antipode, l-nebivolol (RSSS) is responsible for endothelium-dependent NO liberation. This report describes the development and validation of a method of analysis of nebivolol isomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nebivolol isomers were extracted from 2mL aliquots of plasma spiked with tramadol as internal standard, alkalinized and added of sodium chloride and diisopropyl ether:dichloromethane (70:30, v/v). Nebivolol isomers were resolved on a Chirobiotic(®) V column using methanol:acetic acid:diethylamine (100:0.15:0.05, v/v/v) as mobile phase. Protonated ion and respective ion product were monitored in transitions 406>151 for nebivolol and 264>58 for internal standard tramadol. There was no racemization of nebivolol isomers during the procedures of sample preparation and chromatographic analysis and matrix effect was absent. Analysis of nebivolol isomers showed linearity for plasma concentrations of 25-2500pg/mL of each isomer. The quantification limit was 25pg of each isomer/mL of plasma. Variation coefficients and inaccuracy calculated in precision and accuracy determinations were lower than 15%. Nebivolol was stable in human plasma after three successive cycles of freezing and thawing, during 4h at room temperature and after processing during 12h in the auto sampler at 5°C showing deviation values lower than 15%. The method was applied in a study of the kinetic disposition of nebivolol in plasma samples collected until 48h after administration of an oral single dose of 10mg of racemic nebivolol hydrochloride to a patient with systemic arterial hypertension. The clinical study demonstrated that the nebivolol pharmacokinetics is stereoselective. Isomer l-nebivolol showed higher AUC(0-∞) (9.4ng/h/mL vs. 4.7ng/h/mL) and smaller apparent clearance (Cl/f) (531.8L/h vs

  1. Relationships of human plasma fluoride and bone fluoride to age

    Energy Technology Data Exchange (ETDEWEB)

    Parkins, F.M.; Tinanoff, N.; Moutinho, M.; Anstey, M.B.; Waziri, M.H.

    1976-04-01

    There is evidence that fluoride levels in plasma correlate with the fluoride content in bones. The authors determined whether or not fluoride in plasma and bones might correlate with age. In 41 in-patients at the University Hospital, Iowa City, 36 of whom had been residing in fluoridated communities plasma fluoride was determined in the fasting stage by the fluoride ion selective electrode. The teeth of these children were compared with those of a neighboring city where the natural fluoride content in drinking water was 0.1 ppm. For the second sampling 42 months following the beginning of the program only 10 and 11 year old children - who had been 6 and 7 years old at the beginning of the experiment - were selected. Twenty-five boys and girls in each group were compared with a similar group of children as controls. After 28 months (approximately 33 rinsing with sodium fluoride solution) the DMF index in the fluoride-treated children was 18.4% less than in the controls. In the second group among 10 and 11 year old children after 42 months with 55 rinsings the difference was 35.0%. The author acknowledged that factors other than applications of sodium fluoride may have contributed to the prevention of caries in the fluoride-treated groups.

  2. 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 < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  3. 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

  4. High-performance liquid chromatographic method for the determination of drotaverine in human plasma and urine.

    Science.gov (United States)

    Bolaji, O O; Onyeji, C O; Ogungbamila, F O; Ogunbona, F A; Ogunlana, E O

    1993-12-08

    A simple and sensitive HPLC method for the determination of drotaverine in human plasma and urine has been developed. Alkalinized plasma or urine was extracted with organic solvent and the basic components in the organic phase were back-extracted into 0.1 M HCl. An aliquot of the aqueous layer was injected onto the column and the eluent was monitored at 254 nm. Separation was performed on a C18-column with 0.02 M sodium dihydrogen phosphate-methanol (30:70, v/v) containing perchlorate ion at pH 3.2 as mobile phase. Drotaverine was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios and plasma concentrations and the intra- and inter-assay coefficients of variation were always drotaverine in humans as well as in animal models.

  5. Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

    Science.gov (United States)

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

    2009-01-16

    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-gamma (PPARgamma), 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 PPARgamma as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  6. Scavenging of hydroxyl radicals generated in human plasma following X-ray irradiation.

    Science.gov (United States)

    Hosokawa, Yoichiro; Sano, Tomoaki

    2015-11-01

    There are various antioxidant materials that scavenge free radicals in human plasma. It is possible that the radical-scavenging function causes a radiation protective effect in humans. This study estimated the hydroxyl (OH) radical-scavenging activity induced by X-ray irradiation in human plasma. The test subjects included 111 volunteers (75 males and 36 females) ranging from 22 to 35 years old (average, 24.0). OH radicals generated in irradiated human plasma were measured by electron spin resonance (ESR). The relationships between the amount of the OH radical and chemical and biological parameters [total protein, total cholesterol, triglycerides and hepatitis B surface (HBs) antibodies] were estimated in the plasma of the 111 volunteers by a multivariate analysis. The presence of HBs antibodies had the greatest influence on OH radical-scavenging activity. One volunteer who did not have the HBs antibody was given an inoculation of the hepatitis B vaccine. There was a remarkable decrease in the amount of OH radical generated from plasma after the HBs antibody was produced. The results indicate that the HBs antibody is an important factor for the scavenging of OH radicals initiated by X-ray irradiation in the human body.

  7. A model of human sleep-related growth hormone secretion in dogs: effects of 3, 6, and 12 hours of forced wakefulness on plasma growth hormone, cortisol, and sleep stages.

    Science.gov (United States)

    Takahashi, Y; Ebihara, S; Nakamura, Y; Takahashi, K

    1981-07-01

    Twenty-four canine GH (cGH) and cortisol secretion patterns associated with sleep stages were studied in 10 male adult dogs. Plasma samples were obtained at 30- or 15-min intervals via an indwelling catheter. Under baseline conditions, all dogs showed irregular polyphasic sleep, and the episodic cGH secretion had no apparent relationship with sleep or the light-dark cycle. Five dogs were subjected to regular sleep-wake cycles; 3, 6, and 12 h of forced wakefulness (FW) were repeated at 3-, 6-, and 12-h intervals (recovery sleep periods), respectively. Peak cGH secretion (mean +/- SD, 6.4 ng/ml +/- 2.4) occurred soon after recovery sleep onset in 25 of 40 total recovery periods. The incidence of sleep-onset cGH peaks and cGH secretion during the first hour of recovery sleep significantly increased with the length of the preceding FW, but were not affected by the time of day. Delta wave sleep increased during this hour, suggesting a possible correlation with the sleep-onset cGH peak. During the first 3 h of recovery after 6 and 12 h of FW, cGH secretion was significantly enhanced, but cortisol was not. Considering the characteristics of human sleep-related GH secretion, we suggest that this peak cGH secretion represents a model of human GH secretion. Possibly, a close association of cGH secretion with sleep is concealed under the baseline condition and uncovered by inducing longer sleep-wake cycles in dogs. No circadian cortisol variation was detected under the baseline or the experimental conditions.

  8. Kinetic parameters and intraindividual fluctuations of ochratoxin A plasma levels in humans

    Energy Technology Data Exchange (ETDEWEB)

    Studer-Rohr, I. [Inst. of Toxicology, Swiss Federal Inst. of Tech. and Univ. of Zurich, Schwerzenbach (Switzerland); Dept. of Food Science, Swiss Federal Inst. of Tech., Zurich (Switzerland); Schlatter, J. [Toxicology Section, Div. of Food Science, Swiss Federal Office of Public Health, Zurich (Switzerland); Dietrich, D.R. [Dept. of Environmental Toxicology, Univ. of Konstanz, Konstanz (Germany); Inst. of Toxicology, Swiss Federal Inst. of Tech. and Univ. of Zurich, Schwerzenbach (Switzerland)

    2000-11-01

    The mycotoxin ochratoxin A (OTA) is a rodent carcinogen produced by species of the ubiquitous fungal genera Aspergillus and Penicillium. OTA is found in a variety of food items and as a consequence is also found in human plasma (average concentrations found in this study: 0.1-1 ng OTA/ml plasma). To improve the scientific basis for cancer risk assessment the toxicokinetic profile of OTA was studied in one human volunteer following ingestion of 395 ng {sup 3}H-labeled OTA (3.8 {mu}Ci). A two-compartment open model consisting of a central compartment was found to best describe the in vivo data. This two-compartment model consisted of a fast elimination and distribution phase (T{sub 1/2} about 20 h) followed by a slow elimination phase (renal clearance about 0.11 ml/min.) and a calculated plasma half-life of 35.55 days. This half-life was approximately eight times longer than that determined previously in rats. In addition, the intraindividual fluctuation of OTA plasma levels was investigated in eight individuals over a period of 2 months. The concentrations determined ranged between 0.2 and 0.9 ng OTA/ml plasma. The plasma levels in some individuals remained nearly constant over time, while others varied considerably (e.g. increase of 0.4 ng/ml within 3 days, decrease of 0.3 ng/ml within 5 days) during the observation period. This intraindividual fluctuation in OTA plasma levels, which may represent differences in OTA exposure and/or metabolism, as well as the large difference in plasma half-life in humans compared to rats must be taken into consideration when the results of rat cancer study data are extrapolated to humans for risk assessment purposes. (orig.)

  9. 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

    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....

  10. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

    OpenAIRE

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in se...

  11. IL-6, but not TNF-α, increases plasma YKL-40 in human subjects

    DEFF Research Database (Denmark)

    Nielsen, Anders R; Plomgaard, Peter; Krabbe, Karen S

    2011-01-01

    Plasma levels of YKL-40 are elevated in patients with systemic infection, inflammatory disorders and cancer. Both monocytes/macrophages, neutrophils, and cancer cells have the capacity to produce YKL-40, but the regulation during the inflammatory response is unknown. To study the possible role...... of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-a in the regulation of YKL-40 plasma levels, we included healthy men, who received either recombinant human (rh)IL-6 (n=6), rhTNF-a (n=8) or vehicle (n=7) for 3h. The plasma levels of IL-6 and TNF-a reached ~ 150 and ~ 18 pg/ml, respectively, during...

  12. 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.

  13. Epicatechin inhibits human plasma lipid peroxidation caused by haloperidol in vitro.

    Science.gov (United States)

    Dietrich-Muszalska, Anna; Kontek, Bogdan; Olas, Beata; Rabe-Jabłońska, Jolanta

    2012-03-01

    Epicatechin belongs to flavonoids protecting cells against oxidative/nitrative stress. Oxidative/nitrative stress observed in schizophrenia may be caused partially by the treatment of patients with various antipsychotics. The aim of our study was to establish the effects of epicatechin and antipsychotics action (the first generation antipsychotic (FGA)--haloperidol and the second generation antipsychotic (SGA)--amisulpride) on peroxidation of plasma lipids in vitro. Lipid peroxidation in human plasma was measured by the level of thiobarbituric acid reactive species (TBARS). The properties of epicatechin were also compared with the action of a well characterized antioxidative commercial polyphenol-resveratrol (3,4',5-trihydroxystilbene) and quercetin (3,5,7,3',4'-pentahydroxyflavone). Amisulpride, contrary to haloperidol (after 1 and 24 h) does not significantly influence the increase of plasma TBARS level in comparison with control samples (P > 0.05). After incubation (1 and 24 h) of plasma with haloperidol in the presence of epicatechin we observed a significantly decreases the level of TBARS (P < 0.001, P < 0.001, respectively). In our other experiments, we found that epicatechin also decreased the amount of TBARS in human plasma treated with amisulpride. In conclusion, the presented results indicate that epicatechin-the major polyphenolic component of green tea reduced significantly human plasma lipid peroxidation caused by haloperidol. Moreover, epicatechin was found to be a more effective antioxidant, than the solution of pure resveratrol or quercetin.

  14. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator.

    Science.gov (United States)

    Gardell, S J; Hare, T R; Bergum, P W; Cuca, G C; O'Neill-Palladino, L; Zavodny, S M

    1990-12-15

    The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications.

  15. Plasma matrix metalloproteinase-9 response to downhill running in humans.

    Science.gov (United States)

    Welsh, M C; Allen, D L; Byrnes, W C

    2014-05-01

    Matrix metalloproteinase-9 is a proteolytic enzyme capable of degrading proteins of the muscle extracellular matrix. Systemic levels of MMP-9 or its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), have the potential to serve as blood markers of exercise-induced muscle damage. The purpose of this study was to determine if an eccentrically-dominated task, downhill running (DHR), produces changes in plasma MMP-9 or TIMP-1 and examine the relationship between MMP-9/TIMP-1 levels and indirect indicators of muscle damage. Subjects were sedentary (SED, n=12) or had a history of concentrically-biased training (CON, n=9). MMP-9 and TIMP-1 were measured before (Pre-Ex), immediately after (Post-Ex), and 1-, 2-, 4-, and 7-days post-DHR (-10°), and compared to discomfort ratings, creatine kinase activity and strength loss. At 1-day Post-Ex, discomfort increased (5.6 ± 7.8 to 45.5 ± 19.9 mm; 0-100 mm scale), strength decreased (-6.9 ± 1.6%) and CK increased (162.9 ± 177.2%). MMP-9 was modestly but significantly increased at Post-Ex in both CONC and SED (32.7 ± 33.6%) and at 4-days in SED (66.9 ± 88.1%), Individual responses were variable, however. There were no correlations between MMPs and discomfort ratings, plasma CK or strength. While plasma MMP-9 changes may be detectable in the systemic circulation after DHR, they are small and do not correspond to other markers of damage. © Georg Thieme Verlag KG Stuttgart · New York.

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

    DEFF Research Database (Denmark)

    Larsen, Margit Hørup; 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......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...... 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...

  17. Neuroendocrine recovery after 2-week 12-h day and night shifts

    DEFF Research Database (Denmark)

    Merkus, Suzanne L; Holte, Kari Anne; Huysmans, Maaike A

    2015-01-01

    PURPOSE: The study aimed to investigate the course and duration of neuroendocrine recovery after 2-week 12-h day and night shift working periods and to study whether there were differences in recovery between the shift groups. METHODS: Twenty-nine male offshore employees working 2-week 12-h shift....... Differences were tested using generalised estimating equations analysis. RESULTS: Compared to the reference day, night shift workers had a significantly flatter cortisol profile on the 1st day off, significantly lower cortisol concentrations at 30 min after awakening on day 4 and at awakening on day 7......, and a significantly smaller decline to evening concentration on days 4 and 11. Compared to the reference day, day shift workers only showed a significantly lower cortisol concentration at awakening on the 1st day off. Compared to day workers, night shift workers had a flatter profile on the 1st day off and a lower...

  18. Development of a microplate coagulation assay for Factor V in human plasma

    Directory of Open Access Journals (Sweden)

    Samis John A

    2011-06-01

    Full Text Available Abstract Background Factor V (FV in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. Methods The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. Results The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC, the FV 1-stage, 2-stage (With activation by thrombin, and total (2-stage activity - 1-stage activity activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP. The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Conclusions The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in

  19. Antioxidant Capacity of Plasma after Pomegranate Intake in Human Volunteers

    Directory of Open Access Journals (Sweden)

    Hajimahmoodi Mannan

    2009-05-01

    Full Text Available Dietary antioxidants including phenolic compounds are believed to be effective nutrients in the prevention of oxidative stress related disease. Pomegranate has been used for centuries in ancient cultures for its medicinal purpose and is widely acknowledged for antioxidant properties. The present study was designed to assess the effect of pomegranate fresh fruit consumption on the plasma antioxidant capacity. Thirty healthy volunteers were recruited for the study. Volunteers were randomly divided into three groups (pomegranate, vitamin E and water consumption. Blood samples were collected, after at least 12 hours overnight fast, the day before beginning supplementation period and the day after supplementation had finished. Total antioxidant capacity measurement by FRAP method and clinical laboratory test were performed for all volunteers in two selected times. The obtained data revealed that consumption of 100 grams pomegranate and vitamin E per day for ten days resulted in a significant rise (14.05%, 8.28% plasma antioxidant capacity respectively, but this difference was not significant for water group.

  20. Two independent apolipoprotein a5 Haplotypes influence human plasma triglyceride levels

    Energy Technology Data Exchange (ETDEWEB)

    Pennacchio, Len A.; Olivier, Michael; Hubacek, Jaroslav A.; Krauss, Ronald M.; Rubin, Edward M.; Cohen, Jonathan C.

    2002-09-16

    The recently identified apolipoprotein A5 gene (APOA5) has been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. We previously identified an APOA5 haplotype (designated APOA5*2) that is present in {approx}16 percent of Caucasians and is associated with increased plasma triglyceride concentrations. In this report we describe another APOA5 haplotype (APOA5*3) containing the rare allele of the single nucleotide polymorphism c.56C>G that changes serine to tryptophan at codon 19 and is independently associated with high plasma triglyceride levels in three different populations. In a sample of 264 Caucasian men and women with plasma triglyceride concentrations above the 90th percentile or below the 10th percentile, the APOA5*3 haplotype was more than three-fold more common in the group with high plasma triglyceride levels. In a second independently ascertained sample of Caucasian men and women (n 1/4 419) who were studied while consuming their self-selected diets as well as after high-carbohydrate diets and high-fat diets, the APOA5*3 haplotype was associated with increased plasma triglyceride levels on all three dietary regimens. In a third population comprising 2660 randomly selected individuals, the APOA5*3 haplotype was found in 12 percent of Caucasians, 14 percent of African-Americans and 28 percent of Hispanics and was associated with increased plasma triglyceride levels in both men and women in each ethnic group. These findings establish that the APOA5 locus contributes significantly to inter-individual variation in plasma triglyceride levels in humans. Together, the APOA5*2 and APOA5*3 haplotypes are found in 25 50 percent of African-Americans, Hispanics and Caucasians and support the contribution of common human variation to quantitative phenotypes in the general population.

  1. Observations of a 12 H wave in the mesopause region at the South Pole

    Energy Technology Data Exchange (ETDEWEB)

    Collins, R.L.; Senft, D.C.; Gardner, C.S. (Univ. of Illinois, Urbana (United States))

    1992-01-03

    In December 1989 a Na lidar was installed at the Amundsen-Scott South Pole Station and was used to measure aerosol, stratospheric temperature and mesospheric Na profiles through October 1990. The mesospheric Na data are used to characterize the gravity wave field in the mesopause region, These first lidar observations of Na layer dynamics at the South Pole show strong wave activity during the Antarctic winter. Data for 25 June and 19 August 1990 UT are presented here. The total wave induced variances in atmospheric density are respectively 29 and 35(%){sup 2}. The Na layer centroid height is very low during both observation periods. On 25 June a strong 12 h oscillation is observed in the bottomside of the Na layer which extends to altitudes as low as 74 km. The vertical displacement and temperature amplitudes associated with the 12 h oscillation are respectively 1.9 km and 19 K. The characteristics of the 12 h wave are similar to the pseudotide observed at Svalbard by Walterscheid et al.

  2. The Inhibitory Effect of Propranolol and Isoproterenol on Human Plasma Cholinesterase

    Directory of Open Access Journals (Sweden)

    Ali Awsat Mellati

    2002-10-01

    Full Text Available The effect of propranolol and isoproterenol on the hydrolysis of 4- nitrophenylbutyrate (PNPB by the purified human plasma cholinesterase was studied. During the hydrolysis of PNPB, enzyme obeyed to Michaelis-Menten model. Propranolol was found to be a competitive inhibitor, and isoproterenol yielded a complex inhibition pattern. It could be explained that the inhibitory effect of propranolol shows noncooperativity between subunits of human plasma cholinesterase upon binding of PNPB. In contrast, isoproternol inhibitory effects indicate more than one type of binding sites on this enzyme.

  3. 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...

  4. File list: Oth.Emb.05.AllAg.4-12h_embryos [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.4-12h_embryos dm3 TFs and others Embryo 4-12h embryos SRX681817,SR...X681772,SRX681796,SRX681794,SRX681770,SRX681771,SRX681795 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.05.AllAg.4-12h_embryos.bed ...

  5. May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

    Science.gov (United States)

    Olas, Beata; Kołodziejczyk, Joanna; Malinowska, Joanna

    2010-06-01

    Homocysteine (Hcys) may be implicated in different diseases, especially in cardiovascular illnesses. The most reactive form of Hcys is its cyclic thioester-homocysteine thiolactone (HTL), which is formed in plasma and represents up to 0.29% of plasma total Hcys. Recently, it has been observed that Hcys and HTL may modify plasma proteins, including albumin, hemoglobin or fibrinogen, but the role of this process is not yet well known. The aim of our study in vitro was to investigate the modifications of human plasma total proteins after incubation with the reduced form of Hcys in concentrations 10-100 micromol/l, and HTL in concentrations 1-0.1 micromol/l, which correspond to levels found in human plasma during hyperhomocysteinemia in vivo. The aim of our study was also to explain the effects of Hcys and HTL on coagulation activity of human plasma. We showed that in model system in vitro Hcys and HTL change the level of thiol, amino and carbonyl groups in plasma total proteins. Moreover, our studies reported that not only Hcys (10-100 micromol/l), but also HTL (at lower concentrations than Hcys) modulates the coagulation properties of human plasma.

  6. Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus.

    Science.gov (United States)

    Aboubakr, Hamada A; Williams, Paul; Gangal, Urvashi; Youssef, Mohammed M; El-Sohaimy, Sobhy A A; Bruggeman, Peter J; Goyal, Sagar M

    2015-06-01

    Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log10 unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O2, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O2 plasma treatment had the highest virucidal effect: more than 6.0 log10 units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log10 unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H2O2 were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.

  7. A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma.

    Science.gov (United States)

    Grant, Ryan; Ansa-Addo, Ephraim; Stratton, Dan; Antwi-Baffour, Samuel; Jorfi, Samireh; Kholia, Sharad; Krige, Lizelle; Lange, Sigrun; Inal, Jameel

    2011-08-31

    The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 μm in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 μm pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 μm) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 μM) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects.

  8. Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion.

    Science.gov (United States)

    Edwards, Lindsay M; Lawler, Nathan G; Nikolic, Sonja B; Peters, James M; Horne, James; Wilson, Richard; Davies, Noel W; Sharman, James E

    2012-09-01

    Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression.

  9. Effect of resveratrol on hemostatic properties of human fibrinogen and plasma during model of hyperhomocysteinemia.

    Science.gov (United States)

    Malinowska, Joanna; Olas, Beata

    2010-11-01

    Resveratrol (3,4', 5 - trihydroxystilben), a phenolic antioxidant synthesized in grapes and vegetables and presents in wine, has been supposed to be beneficial for the prevention of cardiovascular events. In this study the influence of resveratrol on the clot formation (using human plasma and purified fibrinogen) and the fibrin lysis during model of hyperhomocysteinemia was investigated. We induced this process using a reduced form of Hcys (at final dose of 0.1mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 0.5μM). The aim of our study in vitro was to investigate the modifications of human plasma total proteins after incubation with Hcys, HTL and resveratrol. We observed that HTL, like its precursor, Hcys stimulated polymerization of fibrinogen. Our present results also demonstrated that Hcys (0.1mM) and HLT at lower doses than Hcys (0.5μM) reduced the fibrin lysis in human plasma. Moreover, Hcys and HTL change the level of thiol and amino groups in plasma total proteins. Our results indicate that resveratrol reduced the toxicity action of Hcys and HTL on hemostatic properties of fibrinogen or plasma, suggesting its possible protector role in hyperhomocysteinemia - induced cardiovascular diseases.

  10. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress

    Science.gov (United States)

    KIM, KI CHEON; PIAO, MEI JING; HEWAGE, SUSARA RUWAN KUMARA MADDUMA; HAN, XIA; KANG, KYOUNG AH; JO, JIN OH; MOK, YOUNG SUN; SHIN, JENNIFER H.; PARK, YEUNSOO; YOO, SUK JAE; HYUN, JIN WON

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorodihydro-fluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  11. Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

    Science.gov (United States)

    Maarof, Ghyath; Bouchet-Delbos, Laurence; Gary-Gouy, Hélène; Durand-Gasselin, Ingrid; Krzysiek, Roman; Dalloul, Ali

    2010-03-04

    Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

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

    DEFF Research Database (Denmark)

    Xu, G; Hansen, Jakob; Zhao, X J

    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...... in the exercising leg. In plasma and in the exercising muscle, exercise induced an increase of most acylcarnitines followed by a rapid decline to preexercise values during recovery. In contrast, free carnitine was decreased in the exercising muscle and quickly restored thereafter. C8-, C10-, C10:1-, C12-, and C12...

  13. Identification and quantification of diadenosine polyphosphate concentrations in human plasma

    DEFF Research Database (Denmark)

    Jankowski, Joachim; Jankowski, Vera; Laufer, Udo;

    2003-01-01

    Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasm...

  14. Analysis of free hydroxytyrosol in human plasma following the administration of olive oil.

    Science.gov (United States)

    Pastor, Antoni; Rodríguez-Morató, Jose; Olesti, Eulàlia; Pujadas, Mitona; Pérez-Mañá, Clara; Khymenets, Olha; Fitó, Montserrat; Covas, María-Isabel; Solá, Rosa; Motilva, María-José; Farré, Magí; de la Torre, Rafael

    2016-03-11

    Hydroxytyrosol (HT) from olive oil, a potent bioactive molecule with health benefits, has a poor bioavailability, its free form (free HT) being undetectable so far. This fact leads to the controversy whether attained HT concentrations after olive oil polyphenol ingestion are too low to explain the observed biological activities. Due to this, an analytical methodology to determine free HT in plasma is crucial for understanding HT biological activity. Plasma HT instability and low concentrations have been major limitations for its quantification in clinical studies. Here, we describe a method to detect and quantify free HT in human plasma by using liquid chromatography coupled to tandem mass spectrometry. The method encompasses different steps of sample preparation including plasma stabilization, protein precipitation, selective derivatization with benzylamine, and purification by solid-phase extraction. A high sensitivity (LOD, 0.3ng/mL), specificity and stability of HT is achieved following these procedures. The method was validated and its applicability was demonstrated by analyzing human plasma samples after olive oil intake. A pharmacokinetic comparison was performed measuring free HT plasma concentrations following the intake of 25mL of ordinary olive oil (nearly undetectable concentrations) versus an extra-virgin olive oil (Cmax=4.40ng/mL). To our knowledge, this is the first time that an analytical procedure for quantifying free HT in plasma after olive oil dietary doses has been reported. The present methodology opens the door to a better understanding of the relationship between HT plasma concentrations and its beneficial health effects. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Determination of loperamide in human plasma and saliva by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Arafat, Tawfiq; Arafat, Basil; awad, Riad; awwad, Ahmad Abu

    2014-12-01

    A simple and sensitive liquid chromatography-tandem mass spectrometric method for quantification of loperamide in human plasma and saliva was developed and validated, and then successfully applied in pharmacokinetic clinical study to investigate and correlate bioavailability of Imodium(®) 2mg quartet tablet dose in both human plasma and saliva. Loperamide with labeled internal standard was extracted from its biological matrix by methanol as protein direct precipitant in single extraction step. Adequate chromatographic separation for analytes from plasma and saliva matrices was achieved using ACE C18 (50mm×2.1mm, 5μm) column, eluted by water/methanol/formic acid (30:70:0.1%, v/v), delivered isocratically at constant flow rate of 0.75ml/min. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to European guideline, and partial validation was applied on saliva, specificity, matrix effect, recovery, sensitivity, within and between day precision and accuracy. The calibration curve was linear through the range of 20-3000pg/ml in both plasma and saliva using a 50μl sample volume. The partial validation sections outcome in saliva was so close to those in plasma. The within- and between-day precisions were all below 8.7% for plasma and below 11.4% for saliva. Accuracies ranged from 94 to 105% for both matrices. In this study, 26 healthy volunteers participated in the clinical study, and 6 of gave their saliva samples in addition to plasma at the same time schedule. The pharmacokinetic parameters of Cmax, AUC0-t and AUC0-∞, Tmax and T1/2 in both plasma and saliva were calculated and correlated.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-05-01

    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.

  17. Fibrin glue from stored human plasma. An inexpensive and efficient method for local blood bank preparation.

    Science.gov (United States)

    Spotnitz, W D; Mintz, P D; Avery, N; Bithell, T C; Kaul, S; Nolan, S P

    1987-08-01

    European surgeons have used fibrin glue extensively during thoracic, cardiovascular, and general surgical operations. Until now, however, it has been available only as a commercial preparation made from pooled human plasma, and it has not been approved by the U.S. Food and Drug Administration for use in the United States because of a high associated risk of hepatitis and acquired immune deficiency syndrome. Methods of obtaining fibrinogen, an essential component of fibrin glue, from cryoprecipitate or fresh frozen plasma have been published recently. However, the cryoprecipitate method results in relatively low concentrations of fibrinogen, which can reduce glue effectiveness. The fresh frozen plasma method is more expensive and does not meet the standards of the American Association of Blood Banks for the "closed" system required for safe handling and management of blood component products. Both the cryoprecipitate and the fresh frozen plasma methods result in waste of unstable clotting factors. These factors are necessary to replace human plasma clotting deficiencies but are not necessary for the production of fibrin glue. The authors have developed an efficient, high-concentration blood bank method for producing and maintaining a local supply of a safer and less expensive but equally effective material derived from stored human plasma. This material is produced using approved blood bank techniques for a "closed" system in blood component production, thus reducing the risks of contamination and infection, and its fibrinogen concentration is higher than that of standard cryoprecipitate. The cost of 1 unit of this fibrin glue is comparable to that for 1 unit of cryoprecipitate and less than that for 1 unit of fresh frozen plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. 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.

  19. Affinity separation of human plasma gelsolin on Affi-Gel Blue.

    Science.gov (United States)

    Yamamoto, H; Terabayashi, M; Egawa, T; Hayashi, E; Nakamura, H; Kishimoto, S

    1989-05-01

    Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

  20. 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.

  1. Human papillomavirus DNA in plasma of patients with HPV16 DNA-positive uterine cervical cancer.

    OpenAIRE

    Shimada, Takako; Yamaguchi, Naohiro; Nishida, Noriyuki; Yamasaki, Kentaro; Miura, Kiyonori; Katamine, Shigeru; Masuzaki, Hideaki

    2010-01-01

    OBJECTIVES: The squamous cell carcinoma antigen is considered the most accurate serologic tumor marker for uterine cervical carcinoma. However, serum squamous cell carcinoma antigen levels were found to correlate significantly with clinical severity of atopic dermatitis and chronic renal failure. The present study was conducted in patients with human papillomavirus 16 DNA-positive uterine cervical cancer to determine the plasma level of human papillomavirus 16 DNA and the diagnostic values of...

  2. Buspirone, fexofenadine, and omeprazole: Quantification of probe drugs and their metabolites in human plasma

    OpenAIRE

    Gor, Parul; Alnouti, Yazen; Reed, Gregory A.

    2011-01-01

    Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a —cocktail . This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The anal...

  3. A comprehensive analysis of the Streptococcus pyogenes and human plasma protein interaction network.

    Science.gov (United States)

    Sjöholm, Kristoffer; Karlsson, Christofer; Linder, Adam; Malmström, Johan

    2014-07-01

    Streptococcus pyogenes is a major human bacterial pathogen responsible for severe and invasive disease associated with high mortality rates. The bacterium interacts with several human blood plasma proteins and clarifying these interactions and their biological consequences will help to explain the progression from mild to severe infections. In this study, we used a combination of mass spectrometry (MS) based techniques to comprehensively quantify the components of the S. pyogenes-plasma protein interaction network. From an initial list of 181 interacting human plasma proteins defined using liquid chromatography (LC)-MS/MS analysis we further subdivided the interacting protein list using selected reaction monitoring (SRM) depending on the level of enrichment and protein concentration on the bacterial surface. The combination of MS methods revealed several previously characterized interactions between the S. pyogenes surface and human plasma along with many more, so far uncharacterised, possible plasma protein interactions with S. pyogenes. In follow-up experiments, the combination of MS techniques was applied to study differences in protein binding to a S. pyogenes wild type strain and an isogenic mutant lacking several important virulence factors, and a unique pair of invasive and non-invasive S. pyogenes isolates from the same patient. Comparing the plasma protein-binding properties of the wild type and the mutant and the invasive and non-invasive S. pyogenes bacteria revealed considerable differences, underlining the significance of these protein interactions. The results also demonstrate the power of the developed mass spectrometry method to investigate host-microbial relationships with a large proteomics depth and high quantitative accuracy.

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

    Directory of Open Access Journals (Sweden)

    Tae Jung Oh

    Full Text Available 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.

  5. Oxygen-dependent injury by a human plasma factor associated with minimal change disease

    NARCIS (Netherlands)

    Cheung, PK; Baller, JFW; Bakker, WW

    1998-01-01

    The mechanism by which a human plasma factor associated with proteinuria is able to cause experimental glomerular albumin leakage is unknown. This factor (called 100KF) is able to induce glomerular alterations in the rat kidney, similar to those seen in minimal change disease, including loss of glom

  6. Induction of experimental proteinuria in vivo following infusion of human plasma hemopexin

    NARCIS (Netherlands)

    Cheung, PK; Klok, PA; Baller, JFW; Bakker, WW

    2000-01-01

    Background. The human plasma constituent hemopexin (Hx), following incubation with renal tissue, is able to induce glomerular alterations in vitro that are similar to those seen in minimal change disease (MCD). Whether this acute phase reactant is also able to induce proteinuria and minimal change-l

  7. 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

    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 was to invest...

  8. 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.

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

    Science.gov (United States)

    Christensen, James C; Shiyanov, Pavel A; Estepp, Justin R; Schlager, John J

    2014-01-01

    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.

  10. Preferential association of apolipoprotein E Leiden with very low density lipoproteins of human plasma

    NARCIS (Netherlands)

    Fazio, S.; Horie, Y.; Weisgraber, K.H.; Havekes, L.M.; Rall Jr., S.C.

    1993-01-01

    Apolipoprotein (apo) E Leiden is a rare variant of human apoE characterized by defective receptor binding and associated with dominant transmission of type III hyperlipoproteinemia. In heterozygotes, apoE Leiden is present in higher concentrations in both total plasma and very low density

  11. 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

    1999-01-01

    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.

  12. 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...

  13. Characterization of a lectin in human plasma analogous to bovine conglutinin

    DEFF Research Database (Denmark)

    Thiel, S; Baatrup, G; Friis-Christiansen, P

    1987-01-01

    The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine con...

  14. 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

  15. Study on Speciation of Pr(III) in Human Blood Plasma by Computer Simulation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Speciation of Pr(III) in human blood plasma has been investigated by computer simulation. The speciation and distribution of Pr(III) has been obtained. It has been found that most of Pr(III) is bound to phosphate and to form precipitate. The results obtained are in accord with experimental observations.

  16. Non enzymatic glycosylation of alpha-1-proteinase inhibitor of human plasma.

    Directory of Open Access Journals (Sweden)

    Phadke M

    1998-04-01

    Full Text Available Human plasma contains inhibitors, which control the activity of proteolytic enzymes. Alpha-1-proteinase inhibitor and alpha-2-macroglobulin are two of them present in high concentration in human plasma, which inhibit action of trypsin among other proteinases. The trypsin inhibitory capacity (TIC of human plasma is observed to be decreased in pathological conditions like diabetes mellitus. The mechanisms of decrease in TIC was due to nonenzymatic glycosylation of alpha-1-proteinase inhibitor (A1PI. A1PI was partially purified from normal human plasma by steps involving ammonium sulphate precipitation, DEAE Sepharose CL6B chromatography, Concanavalin A Sepharose Chromatography and Sephadex G-100 Gel filtration. Purified inhibitor was glycosylated in vitro by incubating it with varying glucose concentrations, under nitrogen for different periods of time in reducing conditions. After glycosylation, the molecular weight of inhibitor increased from 52 kDa to 57 KDa because of binding with glucose molecules. The percent free amino groups in the protein decreased with increasing glucose concentration and days of incubation. The TIC of such modified inhibitor decreased significantly. Decrease in TIC was dependent on the glucose concentration and period of incubation used during in-vitro glycosylation of native inhibitor.

  17. Determination of Febuxostat in Human Plasma Using RP-LC-UV Method.

    Science.gov (United States)

    Younes, Kareem M; El-Kady, Ehab F; Elzanfaly, Eman S

    2016-07-01

    A simple and sensitive bioanalytical high-performance liquid chromatographic method with ultraviolet detection was developed and validated for the quantification of febuxostat (FEB) in human plasma. Ketoprofen was used as an internal standard. The analytes were extracted from human plasma samples by precipitation with acetonitrile. The reconstituted samples were chromatographed on C18 Bondapack 10 µm, 250 × 4.6 mm, Waters Column (Ireland) by using a mixture of acetonitrile and 0.5% aqueous phosphoric acid (pH 3) (52 : 48, v/v) as the mobile phase. The chromatographic separation was isocratically performed at room temperature at a flow rate of 1.0 mL/min with UV detection at 315 nm. The method exhibited a linear dynamic range over 0.05-5.00 µg/mL FEB in human plasma. The lower limit of quantification was 0.05 µg/mL. The results of the intra- and interday precision and accuracy studies were within the acceptable limits. The validated method was successfully applied for the determination of FEB in human plasma samples for application in bioequivalence studies.

  18. ADENOSINE-TRIPHOSPHATE DEPENDENT TAUROCHOLATE TRANSPORT IN HUMAN LIVER PLASMA-MEMBRANES

    NARCIS (Netherlands)

    WOLTERS, H; KUIPERS, F; SLOOFF, MJH; VONK, RJ

    1992-01-01

    Transport systems involved in uptake and biliary secretion of bile salts have been extensively studied in rat liver; however, little is known about these systems in the human liver. In this study, we investigated taurocholate (TC) transport in canalicular and basolateral plasma membrane vesicles iso

  19. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

    Directory of Open Access Journals (Sweden)

    Zhiyun Cao

    2013-01-01

    Full Text Available Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1 to provide increased depth in plasma proteome coverage and (2 to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS. Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

  20. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow.

    Science.gov (United States)

    Cao, Zhiyun; Yende, Sachin; Kellum, John A; Robinson, Renã A S

    2013-01-01

    Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

  1. Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma.

    Science.gov (United States)

    Uno, T; Ohkubo, T; Sugawara, K

    1997-09-26

    A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (-)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C18 bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C1 stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (-)-nicardipine was possible in a concentration range of 5-100 ng ml(-1) and the limit of detection in plasma was 2.5 ng ml(-1). The recoveries of (+)- and (-)-nicardipine added to plasma were 91.4-98.4% and 93.3-96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (-)-nicardipine in plasma from healthy volunteers.

  2. Ultra-high performance liquid chromatographic determination of levofloxacin in human plasma and prostate tissue with use of experimental design optimization procedures.

    Science.gov (United States)

    Szerkus, O; Jacyna, J; Wiczling, P; Gibas, A; Sieczkowski, M; Siluk, D; Matuszewski, M; Kaliszan, R; Markuszewski, M J

    2016-09-01

    plasma and 0.300-30μg/g for human prostate tissue samples. The intra-day and inter-day variability for levofloxacine from both plasma and prostate samples were less than 10%, with accuracies between 93 and 108% of the nominal values. The limit of detection and the limit of quantification for human plasma were 0.01μg/mL and 0.03μg/mL, respectively. For the prostate tissue, the limit of detection and the limit of quantification were 0.1μg/g and 0.3μg/g, respectively. The average recoveries of levofloxacin were in the range from 99 to 106%. Also, the method fulfills requirements of robustness what was determined and proved by Design of Experiments. The developed method was successfully applied to examine prostate tissue and plasma samples from 140 hospitalized patients enrolled into the clinical study, 12h after oral administration of LVF at a dose of 500mg. The mean (±SD) LVF concentration in prostate was 6.22±3.52μg/g and in plasma 2.54±1.14μg/mL. Due to simplicity of the method and relative small amount of sample needed for the assay, the method can be applied in clinical practice for monitoring of LVF concentrations in plasma and prostate gland.

  3. Thermal Decomposition of Anhydrous Alkali Metal Dodecaborates M2B12H12 (M = Li, Na, K

    Directory of Open Access Journals (Sweden)

    Liqing He

    2015-11-01

    Full Text Available Metal dodecaborates M2/nB12H12 are regarded as the dehydrogenation intermediates of metal borohydrides M(BH4n that are expected to be high density hydrogen storage materials. In this work, thermal decomposition processes of anhydrous alkali metal dodecaborates M2B12H12 (M = Li, Na, K synthesized by sintering of MBH4 (M = Li, Na, K and B10H14 have been systematically investigated in order to understand its role in the dehydrogenation of M(BH4n. Thermal decomposition of M2B12H12 indicates multistep pathways accompanying the formation of H-deficient monomers M2B12H12−x containing the icosahedral B12 skeletons and is followed by the formation of (M2B12Hzn polymers. The decomposition behaviors are different with the in situ formed M2B12H12 during the dehydrogenation of metal borohydrides.

  4. Simultaneous determination of niacin, niacinamide and nicotinuric acid in human plasma.

    Science.gov (United States)

    Pfuhl, P; Kärcher, U; Häring, N; Baumeister, A; Tawab, Mona Abdel; Schubert-Zsilavecz, M

    2005-01-04

    A sensitive, specific, accurate, and reproducible HPLC/MS-method for the simultaneous quantitative determination of niacin (NA) and its main metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma using chinolin-3-carboxylic acid as an internal standard was developed and validated according to international guidelines for method validation. All analytes and the internal standard were separated from acidified plasma by solid phase extraction. Afterwards the extracted samples were analyzed by HPLC/MS in the positive electrospray ionization mode (ESI) and selected ion monitoring (SIM). The total run time was 7 min between injections. The assay had a lower limit of quantification of 50.0 ng/mL for each analyte using 1 mL of plasma. The calibration curves were linear in the measured range between 50.0 and 750 ng/mL plasma. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. No indications were found for possible instabilities of niacin, niacinamide and nicotinuric acid in plasma at -20 degrees C, in the extraction solvent or after repeated thawing/freezing cycles. In stabilities were observed in whole blood and in plasma at room temperature. The recovery of the extraction method ranged from 86 to 89% for the three analytes.

  5. Comparison of apoprotein B of low density lipoproteins of human interstitial fluid and plasma.

    Science.gov (United States)

    Hong, J L; Pflug, J; Reichl, D

    1984-08-15

    Virtually all apoprotein B (apoB)-containing lipoproteins of the peripheral interstitial fluid of subjects with primary lymphoedema float in the ultracentrifugal field in the density interval 1.019-1.063 g/ml; in this respect they are similar to plasma low-density lipoproteins (LDL). 2. Virtually all apo-B-containing lipoproteins of interstitial fluid migrate in the electrophoretic field with pre-beta mobility; in this respect they are similar to plasma very-low-density lipoproteins. 3. The apoB of lipoproteins of interstitial fluid does not differ in terms of Mr from apoB-100 of human plasma [Kane, Hardman & Paulus (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2465-2469] as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. Both apoB of interstitial fluid and plasma are heterogenous in terms of their charge as determined by isoelectric focusing of their complexes with the nonionic detergent Nonidet P40. ApoB of plasma LDL focuses between pH5.9 and 6.65, and that of interstitial fluid LDL between pH 5.9 and 6.1. Thus the overall charge of apoB of interstitial fluid is more negative than that of its plasma LDL counterpart.

  6. Sphingosine 1-Phosphate Distribution in Human Plasma: Associations with Lipid Profiles

    Directory of Open Access Journals (Sweden)

    Samar M. Hammad

    2012-01-01

    Full Text Available The physiological significance of sphingosine 1-phosphate (S1P transport in blood has been debated. We have recently reported a comprehensive sphingolipid profile in human plasma and lipoprotein particles (VLDL, LDL, and HDL using HPLC-MS/MS (Hammad et al., 2010. We now determined the relative concentrations of sphingolipids including S1P in the plasma subfraction containing lipoproteins compared to those in the remaining plasma proteins. Sphingomyelin and ceramide were predominantly recovered in the lipoprotein-containing fraction. Total plasma S1P concentration was positively correlated with S1P concentration in the protein-containing fraction, but not with S1P concentration in the lipoprotein-containing fraction. The percentage of S1P transported in plasma lipoproteins was positively correlated with HDL cholesterol (HDL-C concentration; however, S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. Thus, different plasma pools of S1P may have different contributions to S1P signaling in health and disease.

  7. Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components.

    Science.gov (United States)

    Kolodziejczyk-Czepas, Joanna; Olas, Beata; Malinowska, Joanna; Wachowicz, Barbara; Moniuszko-Szajwaj, Barbara; Kowalska, Iwona; Oleszek, Wieslaw; Stochmal, Anna

    2013-02-01

    Clovers (genus: Trifolium) have been used in traditional medicine by many cultures, but the biological activity of the most of these plants still remains unknown. The aim of our in vitro study was to assess the antioxidative action of phenolic extracts from aerial parts of Trifolium scabrum and Trifolium pallidum in human blood plasma, exposed to oxidative stress. In the present study we also demonstrate, for the first time the effects of the tested extracts on coagulative properties and fibrinolytic activity of blood plasma. The protective properties of the examined extracts (0.5-50 μg/ml) against peroxynitrite-induced oxidative stress were estimated by the measurements of 3-nitrotyrosine, thiol groups and the thiobarbituric acid-reactive substances levels. The extracts considerably prevented the oxidative and nitrative damage to plasma proteins. Even the lowest doses of the Trifolium extracts (0.5 μg/ml) were able to markedly reduce 3-nitrotyrosine formation (by about 50%) and to increase the level of -SH groups (by about 30%), in comparison to the plasma exposed to ONOO(-) in the absence of the extracts. The protective action of all the used concentrations of the Trifolium extracts in the prevention of lipid peroxidation was also found. The tested extracts influenced neither the coagulative properties nor fibrinolytic activity of plasma. Moreover, the extracts were able to significantly reduce the inhibitory effect of ONOO(-) on fibrinolytic activity of plasma (assessed with the use of a chromogenic substrate for plasmin).

  8. Increased FGF21 plasma levels in humans with sepsis and SIRS.

    Science.gov (United States)

    Gariani, Karim; Drifte, Geneviève; Dunn-Siegrist, Irène; Pugin, Jérôme; Jornayvaz, François R

    2013-01-01

    Fibroblast growth factor 21 (FGF21) is a key regulator in glucose and lipid metabolism and its plasma levels have been shown to be increased not only in humans in different situations such as type 2 diabetes, obesity, and nonalcoholic fatty liver disease but also in animal models of sepsis and pancreatitis. FGF21 is considered as a pharmacological candidate in conditions associated with insulin resistance. The aim of this study was to compare FGF21 plasma levels in patients with sepsis, in patients with systemic inflammatory response syndrome (SIRS), and in healthy controls. We measured FGF21 plasma concentrations in 22 patients with established sepsis, in 11 with SIRS, and in 12 healthy volunteers. Here, we show that FGF21 levels were significantly higher in plasma obtained from patients with sepsis and SIRS in comparison with healthy controls. Also, FGF21 levels were significantly higher in patients with sepsis than in those with noninfectious SIRS. FGF21 plasma levels measured at study entry correlated positively with the APACHE II score, but not with procalcitonin levels, nor with C-reactive protein, classical markers of sepsis. Plasma concentrations of FGF21 peaked near the onset of shock and rapidly decreased with clinical improvement. Taken together, these results indicate that circulating levels of FGF21 are increased in patients presenting with sepsis and SIRS, and suggest a role for FGF21 in inflammation. Further studies are needed to explore the potential role of FGF21 in sepsis as a potential therapeutic target.

  9. Methodological factors influencing measurement and processing of plasma reelin in humans

    Directory of Open Access Journals (Sweden)

    Keller Flavio

    2003-09-01

    Full Text Available Abstract Background Reelin, intensively studied as an extracellular protein that regulates brain development, is also expressed in a variety of tissues and a circulating pool of reelin exists in adult mammals. Here we describe the methodological and biological foundation for carrying out and interpreting clinical studies of plasma reelin. Results Reelin in human plasma was sensitive to proteolysis, freeze-thawing and heating during long-term storage, sample preparation and electrophoresis. Reelin in plasma was a dimer under denaturing conditions. Boiling of samples resulted in laddering, suggesting that each of the 8 repeats expressed in reelin contains a heat-labile covalent bond susceptible to breakage. Urinary-type and tissue-type plasminogen activator converted reelin to a discrete 310 kDa fragment co-migrating with the major immunoreactive reelin fragment seen in plasma and also detected in brain. (In contrast, plasmin produced a spectrum of smaller unstable reelin fragments. We examined archival plasma of 10 pairs of age-matched male individuals differing in repeat length of a CGG repeat polymorphism of the 5'-untranslated region of the reelin gene (both alleles 11 repeats. Reelin 310 kDa band content was lower in subjects having the long repeats in all 10 pairs, by 25% on average (p Conclusions Our studies indicate the need for caution in measuring reelin in archival blood samples, and suggest that assays of plasma reelin should take into account three dimensions that might vary independently: a the total amount of reelin protein; b the relative amounts of reelin vs. its proteolytic processing products; and c the aggregation state of the native protein. Reelin-plasminogen activator interactions may affect their roles in synaptic plasticity. Our results also suggest that the human CGG repeat polymorphism affects reelin gene expression, and may affect susceptibility to human disease.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-12-01

    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 /sup 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 /sup 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 /sup 3/H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown.

  11. 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

    Science.gov (United States)

    Cayer, Marie-Pierre; Drouin, Mathieu; Proulx, Maryse; Jung, Daniel

    2010-04-15

    2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

  12. Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies.

    Science.gov (United States)

    Rigsby, Peter; Ison, Catherine; Brierley, Matthew; Ballard, Ron; Hagedorn, Hans-Jochen; Lewis, David A; Notermans, Daan W; Riis, Jørn; Robertson, Peter; Seppälä, Ilkka J T; Rijpkema, Sjoerd

    2009-08-01

    A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.

  13. New physiological function of secoiridoids: neuritogenic activity in PC12h cells.

    Science.gov (United States)

    Chiba, Kenzo; Yamazaki, Matsumi; Kikuchi, Masafumi; Kakuda, Rie; Kikuchi, Masao

    2011-01-01

    Previously, we have reported that geniposide isolated from an extract of Gardenia fructus has neuritogenic activity in PC12h cells, a subclone of rat pheochromocytoma cells. Furthermore, we have indicated that several geniposide-related iridoid compounds also had similar potent neuritogenic activity. In this study, we have examined the effects of various secoiridoid compounds [K-1, sweroside; K-2, swertiamarin; K-3, gentiopicroside; K-4, 6'-O-β-D: -glucopyranosylsweroside; K-5, 6'-O-β-D: -glucopyranosylgentiopicroside; K-6, 6'-O-β-D: -glucopyranosylswertiamarin; K-7, 5'-O-β-D: -glucopyranosylamarogentin; K-8, 5'-O-β-D: -glucopyranosylamaroswertin; H-1, n-butyl vogeloside; H-2, n-butyl epivogeloside; H-3, (7S)-secologanin butyl methyl acetal; H-4, (7R)-secologanin butyl methyl acetal; H-5, secologanin dimethyl acetal] isolated from various medicinal herbs. The secoiridoids H-1, H-2, H-3, H-4, and H-5 induced significant neurite outgrowth. Among these H-series compounds, H-2 was the most potent neuritogenic compound. Among the K-series compounds, K-1, K-2, K-3, and K-8 showed the most potent activity. These results suggest that secoiridoids have neuritogenic activity in PC12h cells and that these secoiridoid compounds are promising starting compounds for the development of neurotrophic factor-like and iridoid compounds.

  14. Evolving brain lesions in the follow-up CT scans 12 h after traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Muhammad Sohail Umerani; Asad Abbas; Saqib Kamran Bakhshi; Ujala Muhammad Qasim; Salman Sharif

    2016-01-01

    Objective: To establish the frequency of evolution in CT appearance from an initial scan to a subsequent scan within 12 h and the prognostic significance of such deterioration. Methods: All patients who presented to Department of Neurosurgery, Liaquat National Hospital and Medical College with traumatic brain injury and received their CT scan within the first 4 h of injury were included in the study. Indications for repeat CT scan were: any deterioration in neurological status after the initial scan, potentially deterio-rating lesion on initial scan with or without worsening neurology, worsening neurological status after the initial CT scan findings, or no neurological improvement after initial management in patients with normal CT scan with significant head injury. This compiled with the data of 107 patients. Results: There were 67 males and 40 females. The cause of trauma of the 70%patients was road traffic accident. In 11 patients, the lesion evolved towards resorption while 32 patients had no significant changes in the subsequent CT scan. Sixty four patients showed an increase in the size of the lesion and 65.6%of them were required surgical intervention subsequently. Conclusions: In case where the initial CT scan performed within 4 h of significant head injury was not correlated with the patient's neurology, it should be repeated within 12 h.

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

    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.

    2011-01-01

    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 (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 1H-NMR spectroscopy. SAA-free food increased peak intensity in the plasma 1H-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 1H-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. PMID:21677075

  16. "Determination of mycophenolic acid in human plasma by high-performance liquid chromatography"

    OpenAIRE

    "Mehdi Ahadi Barzoki; Mohammadreza Rouini; Kheirolla Gholami; Mahboob Lessan-Pezeshki; Saeed Rezaee

    2005-01-01

    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....

  17. 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 solv...... 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....

  18. Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Lagerwerf, A J; Vanlinthout, L E; Vree, T B

    1991-10-04

    A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 micrograms/ml of succinylcholine. In a pilot study the plasma concentration-time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.

  19. Determination of talniflumate and niflumic acid in human plasma by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Kang, Wonku; Kim, Kibum

    2009-04-01

    A simple and rapid quantification method was developed for determining both talniflumate and niflumic acid in human plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed-phase C(18) column and detected by LC/MS/MS with electrospray ionization. The assay accuracy and precision were within the FDA guidance for the analytical method validation. This method was used to measure the plasma concentrations of both compounds from healthy subjects after a single oral dose of talniflumate, 740 mg.

  20. 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

  1. Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases.

    Science.gov (United States)

    Ruggeri, Roger B; Buckbinder, Leonard; Bagley, Scott W; Carpino, Philip A; Conn, Edward L; Dowling, Matthew S; Fernando, Dilinie P; Jiao, Wenhua; Kung, Daniel W; Orr, Suvi T M; Qi, Yingmei; Rocke, Benjamin N; Smith, Aaron; Warmus, Joseph S; Zhang, Yan; Bowles, Daniel; Widlicka, Daniel W; Eng, Heather; Ryder, Tim; Sharma, Raman; Wolford, Angela; Okerberg, Carlin; Walters, Karen; Maurer, Tristan S; Zhang, Yanwei; Bonin, Paul D; Spath, Samantha N; Xing, Gang; Hepworth, David; Ahn, Kay; Kalgutkar, Amit S

    2015-11-12

    Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.

  2. Vitamin K plasma levels determination in human health.

    Science.gov (United States)

    Fusaro, Maria; Gallieni, Maurizio; Rizzo, Maria Antonietta; Stucchi, Andrea; Delanaye, Pierre; Cavalier, Etienne; Moysés, Rosa M A; Jorgetti, Vanda; Iervasi, Giorgio; Giannini, Sandro; Fabris, Fabrizio; Aghi, Andrea; Sella, Stefania; Galli, Francesco; Viola, Valentina; Plebani, Mario

    2017-05-01

    Vitamin K (phylloquinone or vitamin K1 and menaquinones or vitamin K2) plays an important role as a cofactor in the synthesis of hepatic blood coagulation proteins, but recently has also aroused an increasing interest for its action in extra-hepatic tissues, in particular in the regulation of bone and vascular metabolism. The accurate measurement of vitamin K status in humans is still a critical issue. Along with indirect assays, such as the undercarboxylated fractions of vitamin K-dependent proteins [prothrombin, osteocalcin (OC), and matrix gla protein], the direct analysis of blood levels of phylloquinone and menaquinones forms might be considered a more informative and direct method for assessing vitamin K status. Different methods for direct quantification of vitamin K serum levels are available. High-performance liquid chromatography (HPLC) methods coupled with post-column reduction procedures and fluorimetric or electrochemical detection are commonly used for food and blood analysis of phylloquinone, but they show some limitations when applied to the analysis of serum menaquinones because of interferences from triglycerides. Recent advancements include liquid chromatography tandem mass spectrometry (LCMS/MS) detection, which assures higher specificity. The optimization and standardization of these methods requires specialized laboratories. The variability of results observed in the available studies suggests the need for further investigations to obtain more accurate analytical results.

  3. Method for breast cancer diagnosis by phase spectrophotometry of human blood plasma

    Science.gov (United States)

    Mintser, Ozar P.; Oliinychenko, B. P.

    2012-01-01

    The possibility of breast cancer diagnostics by means of phase structure measurements of laser radiation transformed by human blood plasma samples. The theoretical fundamentals of polarization filtration method for direct phase shifts measurements of microscopic images are provided. The optical model of polycrystalline networks of blood plasma proteins is suggested. The results of investigating the interrelation between the values of statistical (statistical moments of the 1st-4th order), correlation (correlation area, asymmetry coefficient and autocorrelation function excess) and fractal (dispersion of logarithmic dependencies of power spectra) parameters are presented. They characterize the coordinate distributions of phase shifts between the orthogonal components of the amplitude in the points of laser images of blood plasma smears and pathological changes in the mammary gland tissue. The diagnostic criteria of breast cancer nascency are determined.

  4. 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.

  5. IL-6, but not TNF-α, increases plasma YKL-40 in human subjects

    DEFF Research Database (Denmark)

    Nielsen, Anders R; Plomgaard, Peter; Krabbe, Karen S

    2011-01-01

    Plasma levels of YKL-40 are elevated in patients with systemic infection, inflammatory disorders and cancer. Both monocytes/macrophages, neutrophils, and cancer cells have the capacity to produce YKL-40, but the regulation during the inflammatory response is unknown. To study the possible role...... of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α in the regulation of YKL-40 plasma levels, we included healthy men, who received either recombinant human (rh)IL-6 (n=6), rhTNF-α (n=8) or vehicle (n=7) for 3h. The plasma levels of IL-6 and TNF-α reached ∼ 150 and ∼ 18 pg/ml, respectively, during...

  6. Determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization.

    Science.gov (United States)

    Chen, Xiaoyan; Zhong, Dafang; Han, Ying; Xie, Zhiyong

    2003-01-01

    A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine carbocysteine in human plasma using 2-pyridylacetic acid as the internal standard (IS). The method employed derivatization with 10 M hydrochloric acid/methanol, which significantly improved the ionization efficiency of carbocysteine. After methanol-induced protein precipitation of plasma samples, carbocysteine and the IS were derivatized and subjected to LC/MS/MS analysis using atmospheric pressure chemical ionization. The method has a lower limit of quantitation of 20 ng/mL for a 0.2-mL plasma aliquot. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was less than 7%. The accuracy, determined using QC samples, was within +/- 1%. The method offered increased sensitivity, selectivity and speed of analysis over existing methods. The method was utilized to support clinical pharmacokinetic studies of carbocysteine in volunteers following oral administration. Copyright 2002 John Wiley & Sons, Ltd.

  7. [Changes in Kinetics of Chemiluminescence of Plasma as a Measure of Systemic Oxidative Stress in Humans].

    Science.gov (United States)

    Sozarukova, M M; Polimova, A M; Proskurnina, E V; Vladimirov, Yu A

    2016-01-01

    Oxidative stress is a pathogenetic factor of many diseases. The control of its level is important for early diagnosis and therapy adjustment. In this work, antioxidant status was estimated in blood plasma. In the system of 2,2'-azo-bis(2-amidinopropane)dihydrochloride-luminol a set of chemiluminescence kinetic curve parameters is proposed for oxidative stress level estimation (the latent period τ(lat) and the increasing of analytical signal ΔI(CL)). Uric acid and albumin were shown as the main components that responsible for changes in chemiluminescence kinetic curve of plasma. Serum albumin undergoes oxidative modification in dose-depend manner under the action of UV irradiation, it causes the enhancement of antioxidant properties. Changes in plasma chemiluminescence kinetics are proposed as a measure of oxidative stress in human body.

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

    Science.gov (United States)

    Fachi, Mariana Millan; Cerqueira, Letícia Bonancio; Leonart, Letícia Paula; de Francisco, Thais Martins Guimarães

    2016-01-01

    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. PMID:27930700

  9. Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Redeuil, Karine; Smarrito-Menozzi, Candice; Guy, Philippe; Rezzi, Serge; Dionisi, Fabiola; Williamson, Gary; Nagy, Kornél; Renouf, Mathieu

    2011-07-22

    This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.

  10. Human Plasma Lipidome Is Pleiotropically Associated with Cardiovascular Risk Factors and Death

    Science.gov (United States)

    Mamtani, Manju; Kent, Jack W.; Wong, Gerard; Weir, Jacquelyn M.; Barlow, Christopher K.; Diego, Vincent; Almeida, Marcio; Dyer, Thomas D.; Göring, Harald H.H.; Almasy, Laura; Mahaney, Michael C.; Comuzzie, Anthony G.; Williams-Blangero, Sarah; Meikle, Peter J.; Blangero, John; Curran, Joanne E.

    2014-01-01

    Background Cardiovascular disease (CVD) is the most common cause of death in the United States and is associated with a high economic burden. Prevention of CVD focuses on controlling or improving the lipid profile of patients at risk. The human lipidome is made up of thousands of ubiquitous lipid species. By studying biologically simple canonical lipid species, we investigated whether the lipidome is genetically redundant and whether its genetic influences can provide clinically relevant clues of CVD risk. Methods and Results We performed a genetic study of the human lipidome in 1,212 individuals from 42 extended Mexican American families. High-throughput mass spectrometry enabled rapid capture of precise lipidomic profiles, providing 319 unique species. Using variance-component based heritability analyses and bivariate trait analyses, we detected significant genetic influences on each lipid assayed. Median heritability of the plasma lipid species was 0.37. Hierarchical clustering based on complex genetic correlation patterns identified 12 genetic clusters that characterized the plasma lipidome. These genetic clusters were differentially but consistently associated with risk factors of CVD, including central obesity, obesity, type 2 diabetes, raised serum triglycerides and metabolic syndrome. Also these clusters consistently predicted occurrence of cardiovascular deaths during follow-up. Conclusions The human plasma lipidome is heritable. Shared genetic influences reduce the dimensionality of the human lipidome into clusters that are associated with risk factors of CVD. PMID:25363705

  11. Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application.

    Science.gov (United States)

    Choi, Chang-Ik; Park, Jung-In; Lee, Hye-In; Lee, Yun-Jeong; Jang, Choon-Gon; Bae, Jung-Woo; Lee, Seok-Yong

    2012-12-12

    We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC-MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C(18) column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5) - methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6min. The calibration curves were linear over a range of 0.5-300ng/mL (r>0.999). The lower limit of quantification, using 200μL human plasma, was 0.5ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC-MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.

  12. [The measurement of antioxidant activity in human plasma using cumene hydroperoxide].

    Science.gov (United States)

    Sugita, O; Nakano, M; Matsuto, T; Miida, T; Okada, M

    1998-03-01

    We describe a new method using cumene hydroperoxide to determine antioxidant activity (AO) in human plasma. We used a kit (Determiner LPO: Kyowa Medex Co., LTD. Tokyo Japan) for the determination of lipid peroxides in plasma or serum. 30 microliters 1 of sample was mixed with 70 microliters 1 of cumene hydroperoxide (50 nmol/ml) and incubated at 30 degrees C for 120 min before analysis. Samples were mixed with 1.0 ml of reagent-I (Determiner LPO) and incubated at 30 degrees C for 5 min. Then 2.0 ml of reagent-II (Determiner LPO) was added and incubated at 30 degrees C for 10 min, at which time the absorbance at 675 nm was measured. AO were calculated using the following formula: AO nmol/ml = 35 nmol/ml-(Es-Eb)/(Estd-Eb) x 35 nmol/ml (Es = sample abs., Eb = blank abs., Estd = standard abs.). Within-run precision for plasma AO was 2.3%. AO in plasma samples stored for 4 h at 4 degrees C was decreased by 1 nmol/ml. After 3 h at room temperature, AO was decreased by the same amount. Because this method measured ascorbic acid, alpha-tocopherol, glutathione peroxidase and quercetin as antioxidant compounds, we were able to measure antioxidant activity in human plasma. Our reference values were calculated from the volunteers group which consisted of 172 students and 82 soldiers. The reference intervals for plasma AO by this procedure were 15.4-20.9 nmol/ml.

  13. Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry.

    Science.gov (United States)

    Reimund, Mart; Kovrov, Oleg; Olivecrona, Gunilla; Lookene, Aivar

    2017-01-01

    LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietin-like protein (ANGPTL)3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. 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.

  15. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma.

    Science.gov (United States)

    McDonald, Jeffrey G; Smith, Daniel D; Stiles, Ashlee R; Russell, David W

    2012-07-01

    We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C₁₈ core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4β-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D₂ and D₃. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day.

  16. Microdevice for plasma separation from whole human blood using bio-physical and geometrical effects

    Science.gov (United States)

    Tripathi, Siddhartha; Kumar, Y. V. BalaVarun; Agrawal, Amit; Prabhakar, Amit; Joshi, Suhas S.

    2016-01-01

    In this research work, we present a simple and efficient passive microfluidic device for plasma separation from pure blood. The microdevice has been fabricated using conventional photolithography technique on a single layer of polydimethylsiloxane, and has been extensively tested on whole blood and enhanced (upto 62%) hematocrit levels of human blood. The microdevice employs elevated dimensions of about 100 μm; such elevated dimensions ensure clog-free operation of the microdevice and is relatively easy to fabricate. We show that our microdevice achieves almost 100% separation efficiency on undiluted blood in the flow rate range of 0.3 to 0.5 ml/min. Detailed biological characterization of the plasma obtained from the microdevice is carried out by testing: proteins by ultra-violet spectrophotometric method, hCG (human chorionic gonadotropin) hormone, and conducting random blood glucose test. Additionally, flow cytometry study has also been carried on the separated plasma. These tests attest to the high quality of plasma recovered. The microdevice developed in this work is an outcome of extensive experimental research on understanding the flow behavior and separation phenomenon of blood in microchannels. The microdevice is compact, economical and effective, and is particularly suited in continuous flow operations. PMID:27279146

  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

    2016-01-01

    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. Rheological characterization of a gel produced using human blood plasma and alginate mixtures.

    Science.gov (United States)

    Malagón-Romero, Dionisio; Hernández, Nicolás; Cardozo, Carmen; Godoy-Silva, Rubén D

    2014-06-01

    Human blood plasma is a material used to generate tissue equivalents due to presence of fibrinogen. However, gels formed using human blood plasma has weak mechanical properties. In this study, different mixtures of sodium alginate and blood plasma were performed and evaluated. By determining ζ potential can be established the stability of the plasma-alginate mixture and by dynamic rheology can determine the most suitable parameters for the gelation of the above mixtures, when calcium chloride is used as a crosslinker. Experimental results evidence an increment in ζ potential at alginate concentrations of 0.8% and 1.6% with a resulting pseudoplastic behavior of evaluated mixtures, which described the homogenization of the mixture. On the other hand, mixtures were gelled by using aspersion of calcium chloride and characterized by dynamic rheology. Solid behavior is dominant in all range of frequency sweep test between 0.1Hz and 100Hz. Finally, the ultimate tensile strength of a gel reach 6.36938±0.24320kPa, which is enough for manual handling of the gel. Between the tasks of the gel would be used for cell entrapment, for controlled release of drugs or in the manufacture of wound dressings.

  19. The Effect of Drinking on Plasma Vasopressin and Renin in Dehydrated Human Subjects

    Science.gov (United States)

    Geelen, G.; Keil, L. C.; Kravik, S. E.; Wade, C. E.; Thrasher, T. N.; Barnes, P. R.; Pyka, G.; Nesvig, C.; Greenleaf, J. E.

    1996-01-01

    Oropharyngeal mechanisms activated by drinking have been shown to induce a rapid decline in plasma vasopressin which preceeds postabsorptive changes in plasma composition in the dehydrated dog. The present study was undertaken to determine what factor(s) inhibit(s) vasopressin secretion after rehydration in water deprived human subjects. Hematocrit (Hct) and hemoglobin (Hb) were determined on the day of the experiment, together with electrolytes and osmolalities which were measured on freshly separated serum. Plasma was immediately frozen and further analyzed by radioimmunoassay for renin activity (PRA), vasopressin (AVP), and aldosterone. The data were analyzed using an analysis of variance for repeated measurements and significant differences between the dehydrated control period and various time points after the start of rehydration were determined using a multiple-range test. began and reached water replete levels 15 minutes after drinking in the absence of any detectable decline in serum sodium or osmolality, we conclude that 427 oropharyngeal factors, alone or combined with gastric distension account for the extremely rapid inhibition of AVP secretion after drinking in the water-deprived human, as has been shown to be the case in dogs. Our findings are also in agreement wiht the recent demonstration that at the onset of drinking in the dehydrated monkey, there is an abrupt fall in plasma AVP concentration associated with a considerable decrease in the firing rate of the supraoptic neurosecretory neurons.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1984-12-01

    A new sensitive direct radioimmunoassay for human plasma renin has been developed. Renin was purified from Haas' preparation utilizing a pepstatin-C/sub 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. Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC-DAD.

    Science.gov (United States)

    Locatelli, Marcello; Cifelli, Roberta; Carlucci, Giuseppe; Romagnoli, Annalisa

    2016-01-01

    A new and specific HPLC-DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5 mM, pH 5.8)/acetonitrile (both with 1% Et(3)N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500 nm and quantitative analyses were carried out at 278 nm. The LOQ of the method was 1 μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25 μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of + 25, + 4 and -20 °C in order to evaluate plasma stability profiles.

  2. Synthesis and Crystal Structure of Compound [ZnS6 (C12H8N2)

    Institute of Scientific and Technical Information of China (English)

    陈之荣; 李健晖; 李鸿钧; 苏卫平; 曹荣

    2000-01-01

    The title compound ZnS6(C12H8N2) was obtained by reaction of zinic powder with sulfur and 1,10-phenathtroline in DMF solvent, and its structure has been determined by single crystal X-ray diffraction analysis. The crystal data for the compound: triclinic, P-1, Z = 2, a = 8. 159(2), b = 9. 485(2), c = 11. 997(2)A , α =110. 00(3)°,β = 99.22(3)°, γ = 100.57(3)°, V = 832A3, Dc =1. 748g/cm3, μ(MoKa)=0. 2219 cm-1, F(000)=440. The structure was refined to R = 0. 0723 and Rw = 0. 0865 for 1427 independent observed reflections. The tilte crystal consists of a discrete molecules, in which the core of ZnS2N2 shows a distorted tetrahedral geometry.

  3. Measurement of factor v activity in human plasma using a microplate coagulation assay.

    Science.gov (United States)

    Tilley, Derek; Levit, Irina; Samis, John A

    2012-09-09

    In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

  4. Sleep quantity and quality is not compromised during planned burn shifts of less than 12 h.

    Science.gov (United States)

    Vincent, Grace E; Aisbett, Brad; Hall, Sarah J; Ferguson, Sally A

    Planned burning is a preventative strategy aimed at decreasing fuel loads to reduce the severity of future wildfire events. During planned burn operations, firefighters can work long shifts. Furthermore, remote burning locations may require firefighters to sleep away from home between shifts. The existing evidence surrounding firefighters' sleep during such operations is exclusively anecdotal. The aims of the study were to describe firefighters' sleep during planned burn operations and evaluate the impact of the key operational factors (shift start time, shift length and sleeping location) that may contribute to inadequate sleep. Thirty-three salaried firefighters were recruited from Australia's fire agencies and sleep was measured objectively using wrist actigraphy for four weeks. All variables were examined in two conditions: (1) burn days, and (2) non-burn days. Time in bed, total sleep time, sleep latency and sleep efficiency were evaluated objectively. Subjective reports of pre- and post-sleep fatigue, sleep location, sleep quality, sleep quantity, number of times woken and sleep timing were also recorded. Analyses revealed no differences in measures of sleep quantity and quality when comparing non-burn and burn days. Total sleep time was less when planned burn shifts were >12 h. However, on burn days, work shift start time as well as sleeping location did not impact firefighters' sleep quantity. Self-reported levels of pre- and post-sleep fatigue were greater on burn days compared to non-burn days. These findings indicate that sleep quantity and quality are not compromised during planned burn operations <12 h in duration.

  5. Validated HPLC-MS-MS method for determination of azithromycin in human plasma.

    Science.gov (United States)

    Barrett, B; Borek-Dohalský, V; Fejt, P; Vaingátová, S; Huclová, J; Nemec, B; Jelínek, I

    2005-09-01

    A validated, highly sensitive, and selective HPLC method with MS-MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na2EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC-MS-MS. Multiple reaction monitoring mode (MRM) was used for MS-MS detection. The calibration plot was linear in the concentration range 2.55-551.43 ng mL(-1). Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL(-1). The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).

  6. Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Mortensen, Stefan Peter; Thaning, Pia;

    2010-01-01

    One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate....... In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion...... levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular...

  7. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  8. 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.

  9. Determination of diclofenac concentrations in human plasma using a sensitive gas chromatography mass spectrometry method

    OpenAIRE

    2016-01-01

    Background A gas chromatography mass spectrometry (GCMS) method for the determination of diclofenac in human plasma has been developed and validated. Results This method utilizes hexane which is a relatively less toxic extraction solvent compared to heptane and benzene. In addition, phosphoric acid and acetone were added to the samples as deproteination agents, which increased the recovery of diclofenac. These revised processes allow clean extraction and near-quantitative recovery of analyte ...

  10. Determination of diclofenac concentrations in human plasma using a sensitive gas chromatography mass spectrometry method

    OpenAIRE

    Shah, Iltaf; Barker, James; Naughton, Declan P; Barton, Stephen J.; Ashraf, Syed Salman

    2016-01-01

    Background\\ud A gas chromatography mass spectrometry (GCMS) method for the determination of diclofenac in human plasma has been developed and validated.\\ud \\ud Results\\ud This method utilizes hexane which is a relatively less toxic extraction solvent compared to heptane and benzene. In addition, phosphoric acid and acetone were added to the samples as deproteination agents, which increased the recovery of diclofenac. These revised processes allow clean extraction and near-quantitative recover...

  11. Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure plasma jet and plasma-treated human skin cells

    Science.gov (United States)

    Winter, J.; Wende, K.; Masur, K.; Iseni, S.; Dünnbier, M.; Hammer, M. U.; Tresp, H.; Weltmann, K.-D.; Reuter, S.

    2013-07-01

    In this study, the effect of feed gas humidity on the reactive component generation of an atmospheric-pressure argon plasma jet and its effect on human skin cells are investigated. Feed gas humidity is identified as one key parameter that strongly influences stability and reproducibility of plasma medical studies. The plasma jet is investigated by absorption spectroscopy in the ultraviolet and infrared spectral region for its ozone production depending on the humidity concentration in the feed gas. By optical emission spectroscopy the dependence of present excited plasma species such as hydroxyl radicals, molecular nitrogen, argon and atomic oxygen on the feed gas humidity is investigated. As an interface layer between the plasma jet effluent and the biological cell, a buffer solution is treated and the hydrogen peroxide (H2O2) production is studied with two independent colorimetric assays as a function of humidity admixture to the feed gas. Ultimately, the effect of varying feed gas humidity on the cell viability of indirect plasma treated adherent HaCAT cells is investigated. The highest viability is found for the driest feed gas condition. Furthermore, this work shows answers for the relevance of unwanted—or intended—feed gas humidity in plasma medical experiments and their comparatively large relevance with respect to ambient humidity. The findings will lead to more reproducible experiments in the field of plasma medicine.

  12. 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.

  13. 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.)

  14. Simultaneous liquid chromatography-mass spectrometry quantification of cefixime and clavulanic acid in human plasma.

    Science.gov (United States)

    Dubala, Anil; Nagarajan, Janaki Sankarachari Krishnan; Vimal, Chandran Sathish; George, Renjith

    2015-01-01

    A simple and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) assay method has been developed and fully validated for the simultaneous quantification of cefixime (CX) and clavulanic acid (CA) in human plasma. Analytes and internal standard were extracted from human plasma by a solid phase extraction technique using a Sam prep (3 mL, 100 mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol : acetonitrile : 2 mM ammonium acetate (pH 3.5) (25 : 25 : 50, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes were carried out using single quadrupole LC-APCI-MS through selected ion monitoring at m/z 452 and 198, respectively, for CX and CA. The assay was linear over the concentration range of 0.05-10.0 and 0.1-10.0 μg/mL, respectively, for CX and CA. The mean plasma extraction recoveries of the CX and CA were found to be 95.20-96.27% and 94.67-95.58%, respectively. The method was successfully applied for the determination of pharmacokinetics of CX and CA after oral administration of single dosage CX/CA (200/125 mg) pill to the humans (n = 12).

  15. Determination of trace amounts of morphine in human plasma by anodic adsorptive stripping differential pulse voltammetry

    Institute of Scientific and Technical Information of China (English)

    Ali Niazi; Ateesa Yazdanipour

    2008-01-01

    New adsorptive anodic differential pulse stripping voltammetry method for the direct determination of morphine at trace levels in human plasma of addicts is proposed.The procedure involves an adsorptive accumulation of morphine on a HMDE,followed by oxidation of adsorbed morphine by voltammetry scan using differential pulse modulation.The optimum conditions for the analysis of morphine are pH 10.5,Eacc of - 100 mV (vs.Ag/AgCl),and tacc of 120 s.The peak current is proportional to the concentration of morphine,and a linear calibration graph is obtained at 0.01-3.10 μg mL-1.A relative standard deviation of 1.06% (n=5) was obtained,and the limit of detection was 3 ng mL- 1.The capability of the method for the analysis of real samples was evaluated by the determination of morphine in spiked human plasma and addicts human plasma with satisfactory results.

  16. 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.

  17. Capsular polysaccharide of Group B Streptococcus mediates biofilm formation in the presence of human plasma.

    Science.gov (United States)

    Xia, Fan Di; Mallet, Adeline; Caliot, Elise; Gao, Cherry; Trieu-Cuot, Patrick; Dramsi, Shaynoor

    2015-01-01

    Group B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process.

  18. Plasma hyperosmolality improves tolerance to combined heat stress and central hypovolemia in humans.

    Science.gov (United States)

    Gagnon, Daniel; Romero, Steven A; Ngo, Hai; Poh, Paula Y S; Crandall, Craig G

    2017-03-01

    Heat stress profoundly impairs tolerance to central hypovolemia in humans via a number of mechanisms including heat-induced hypovolemia. However, heat stress also elevates plasma osmolality; the effects of which on tolerance to central hypovolemia remain unknown. This study examined the effect of plasma hyperosmolality on tolerance to central hypovolemia in heat-stressed humans. With the use of a counterbalanced and crossover design, 12 subjects (1 female) received intravenous infusion of either 0.9% iso-osmotic (ISO) or 3.0% hyperosmotic (HYPER) saline. Subjects were subsequently heated until core temperature increased ~1.4°C, after which all subjects underwent progressive lower-body negative pressure (LBNP) to presyncope. Plasma hyperosmolality improved LBNP tolerance (ISO: 288 ± 193 vs. 382 ± 145 mmHg × min, P = 0.04). However, no differences in mean arterial pressure (P = 0.10), heart rate (P = 0.09), or muscle sympathetic nerve activity (P = 0.60, n = 6) were observed between conditions. When individual data were assessed, LBNP tolerance improved ≥25% in eight subjects but remained unchanged in the remaining four subjects. In subjects who exhibited improved LBNP tolerance, plasma hyperosmolality resulted in elevated mean arterial pressure (ISO: 62 ± 10 vs. 72 ± 9 mmHg, P < 0.01) and a greater increase in heart rate (ISO: +12 ± 24 vs. HYPER: +23 ± 17 beats/min, P = 0.05) before presyncope. No differences in these variables were observed between conditions in subjects that did not improve LBNP tolerance (all P ≥ 0.55). These results suggest that plasma hyperosmolality improves tolerance to central hypovolemia during heat stress in most, but not all, individuals. Copyright © 2017 the American Physiological Society.

  19. Saliva vs. plasma bioequivalence of metformin in humans: validation of class II drugs of the salivary excretion classification system.

    Science.gov (United States)

    Idkaidek, N; Arafat, T

    2014-11-01

    To study saliva and plasma bioequivalence of metformin in humans, and to investigate the robustness of using saliva instead of plasma as surrogate for bioequivalence of class II drugs according to the salivary excretion classification system (SECS).Plasma and saliva samples were collected for 12 h after 500 mg oral dosing of metformin to 16 healthy humans. Plasma and saliva pharmacokinetic parameters, 90% confidence intervals and intra-subject variability values were calculated using Kinetica V5. Descriptive statistics and dimensional analysis were calculated by Excel. SimCYP program V13 was used for estimation of effective intestinal permeability.Metformin was subjected to salivary excretion since it falls into class II (Low permeability/High fraction unbound to plasma proteins), with correlation coefficients of 0.95-0.99 between plasma and saliva concentrations. Saliva/plasma concentration ratios were 0.29-0.39. The 90% confidence limits of all parameters failed in both saliva and plasma. Intra-subject variability values in saliva were higher than plasma leading to need for higher number of subjects to be used in saliva.Saliva instead of plasma can be used as surrogate for bioequivalence of class II drugs according to SECS when adequate sample size is used. Future work is planned to demonstrate SECS robustness in drugs that fall into class III.

  20. Proteomic characterization of freeze-dried human plasma: providing treatment of bleeding disorders without the need for a cold chain.

    Science.gov (United States)

    Steil, Leif; Thiele, Thomas; Hammer, Elke; Bux, Jürgen; Kalus, Monika; Völker, Uwe; Greinacher, Andreas

    2008-11-01

    Transfusion of human plasma is a basic treatment for severe coagulopathies, especially in major bleeding. The required logistics to provide plasma is challenging because of the need to maintain a cold chain. This disadvantage could be overcome by lyophilized plasma. However, it is unknown to what extent lyophilization alters plasma proteins. Quantitative proteomic technologies were applied to monitor protein changes during production of lyophilized, solvent/detergent (S/D)-treated plasma. The impact of S/D treatment and lyophilization on the plasma proteome was evaluated by differential in-gel electrophoresis (2D-DIGE), and proteins were characterized by mass spectrometry. Clotting factor activities were determined in lyophilized S/D-treated plasma after 24 months of storage at room temperature. By 2D-DIGE, 600 individual protein spots were compared. Lyophilization did not change any of the 600 spots, whereas pathogen inactivation caused significant changes of 38 spots including alpha1-antitrypsin, alpha1-antichymotrypsin, and alpha2-antiplasmin. Clotting factor activities remained stable over 24 months of storage. Lyophilization of human plasma neither alters its protein composition nor impairs its clotting capacity. It does not require cost-intensive logistics for storage and transport and can be quickly reconstituted. It is suggested that lyophilized, pathogen-inactivated plasma is an attractive option to provide the most important basic treatment for severe coagulopathies in areas without cold chain and to provide plasma with reduced time delay in emergency situations.

  1. The effect of the plasma needle on the human keratinocytes related to the wound healing process

    Science.gov (United States)

    Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

    2016-01-01

    In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

  2. Effects of repetitive platelet-rich plasma application on human tenocyte proliferation.

    Science.gov (United States)

    Mazzocca, Augustus D; O'Malley, Michael; Beitzel, Knut; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Bradley, James P; Romeo, Anthony; Arciero, Robert A

    2015-01-01

    Current clinical application of platelet-rich plasma is showing a trend toward multiple treatments. The goal of this study was to show the benefit of interval platelet-rich plasma application in the healing and recovery of human tenocytes using an in vitro cell model. Eight volunteers (6 men and 2 women) were included in this study (mean±SD age, 31.6±10.9 years). Venous blood was collected from new blood draws at 3 different times. Two blood products were prepared on each day of treatment: platelet-rich plasma derived from a single-spin process (PRPSS) and platelet-rich plasma derived from a double-spin process (PRPDS). The study had 2 limbs: 2-day and 4-day intervals. Cell proliferation, measured as disintegrations per minute, was then examined via a radioactive thymidine assay. In the 2-day-interval group, the difference in disintegrations per minute between days 0 and 2 in the PRPSS group reached statistical significance (P =.006). In the PRPDS group, statistical difference was seen between days 0 and 4 (P=.001) and between days 2 and 4 (P=.030). In the 4-day-interval group, the difference in disintegrations per minute between days 4 and 8 in the PRPSS group reached statistical significance, showing a decrease in cell proliferation (P =.013). In the PRPDS group, a statistical difference was seen between days 0 and 8 (P=.021), also showing a decrease in cell proliferation. The greatest effect of platelet-rich plasma, which has a positive effect on tenocyte proliferation and recovery, is seen on initial application. Its effect is diminished with repetitive application, and this finding leads to questioning of the efficacy of interval platelet-rich plasma dosing.

  3. Apolipoprotein modulation of streptococcal serum opacity factor activity against human plasma high-density lipoproteins.

    Science.gov (United States)

    Rosales, Corina; Gillard, Baiba K; Courtney, Harry S; Blanco-Vaca, Francisco; Pownall, Henry J

    2009-08-25

    Human plasma HDL are the target of streptococcal serum opacity factor (SOF), a virulence factor that clouds human plasma. Recombinant (r) SOF transfers cholesteryl esters (CE) from approximately 400,000 HDL particles to a CE-rich microemulsion (CERM), forms a cholesterol-poor HDL-like particle (neo HDL), and releases lipid-free (LF) apo A-I. Whereas the rSOF reaction requires labile apo A-I, the modulation effects of other apos are not known. We compared the products and rates of the rSOF reaction against human HDL and HDL from mice overexpressing apos A-I and A-II. Kinetic studies showed that the reactivity of various HDL species is apo-specific. LpA-I reacts faster than LpA-I/A-II. Adding apos A-I and A-II inhibited the SOF reaction, an effect that was more profound for apo A-II. The rate of SOF-mediated CERM formation was slower against HDL from mice expressing human apos A-I and A-II than against WT mice HDL and slowest against HDL from apo A-II overexpressing mice. The lower reactivity of SOF against HDL containing human apos is due to the higher hydropathy of human apo A-I, particularly its C-terminus relative to mouse apo A-I, and the higher lipophilicity of human apo A-II. The SOF-catalyzed reaction is the first to target HDL rather than its transporters and receptors in a way that enhances reverse cholesterol transport (RCT). Thus, effects of apos on the SOF reaction are highly relevant. Our studies show that the "humanized" apo A-I-expressing mouse is a good animal model for studies of rSOF effects on RCT in vivo.

  4. Plasma mannose-binding lectin is stimulated by PPARα in humans.

    Science.gov (United States)

    Rakhshandehroo, Maryam; Stienstra, Rinke; de Wit, Nicole J; Bragt, Marjolijn C E; Haluzik, Martin; Mensink, Ronald P; Müller, Michael; Kersten, Sander

    2012-03-01

    The peroxisome proliferator activated receptor-α (PPARα) is a major transcriptional regulator of lipid metabolism in liver and represents the molecular target for hypolipidemic fibrate drugs. Effects of PPARα on lipid metabolism are partially mediated by circulating proteins such as FGF21 and ANGPTL4. The present study was undertaken to screen for and identify circulating proteins produced by human liver that are under the control of PPARα. Toward that aim, primary human hepatocytes were treated with the synthetic PPARα agonist Wy-14643 and whole genome expression data selected for secreted proteins. Expression of FGF21, ANGPTL4, and mannose-binding lectin (MBL), a soluble mediator of innate immunity and primary component of the lectin branch of the complement system, was markedly upregulated by Wy-14643 in primary human hepatocytes. Mice express two MBL isomers, Mbl1 and Mbl2. Mbl1 mRNA was weakly induced by Wy-14643 in primary mouse hepatocytes and remained unaltered by Wy-14643 in mouse liver. Mbl2 mRNA was unchanged by Wy-14643 in primary mouse hepatocytes and was strongly reduced by Wy-14643 in mouse liver. Remarkably, plasma Mbl1 levels were increased by chronic PPARα activation in lean and obese mice. Importantly, in two independent clinical trials, treatment with the PPARα agonist fenofibrate at 200 mg/day for 6 wk and 3 mo increased plasma MBL levels by 73 (P = 0.0016) and 86% (P = 0.017), respectively. It is concluded that hepatocyte gene expression and plasma levels of MBL are stimulated by PPARα and fenofibrate in humans, linking PPARα to regulation of innate immunity and complement activation in humans and suggesting a possible role of MBL in lipid metabolism.

  5. Susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1.

    Science.gov (United States)

    Da Silva, Aleksandro S; 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

    2011-12-01

    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.

  6. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS

    Science.gov (United States)

    Cai, Xiaoming; Li, Ruibin

    2016-11-01

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc.

  7. Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines

    Science.gov (United States)

    Dezest, Marlène; Chavatte, Laurent; Bourdens, Marion; Quinton, Damien; Camus, Mylène; Garrigues, Luc; Descargues, Pascal; Arbault, Stéphane; Burlet-Schiltz, Odile; Casteilla, Louis; Clément, Franck; Planat, Valérie; Bulteau, Anne-Laure

    2017-01-01

    Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N2) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.

  8. 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.

  9. Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines

    Science.gov (United States)

    Dezest, Marlène; Chavatte, Laurent; Bourdens, Marion; Quinton, Damien; Camus, Mylène; Garrigues, Luc; Descargues, Pascal; Arbault, Stéphane; Burlet-Schiltz, Odile; Casteilla, Louis; Clément, Franck; Planat, Valérie; Bulteau, Anne-Laure

    2017-01-01

    Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N2) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events. PMID:28120925

  10. Surface modification by allylamine plasma polymerization promotes osteogenic differentiation of human adipose-derived stem cells.

    Science.gov (United States)

    Liu, Xujie; Feng, Qingling; Bachhuka, Akash; Vasilev, Krasimir

    2014-06-25

    Tuning the material properties in order to control the cellular behavior is an important issue in tissue engineering. It is now well-established that the surface chemistry can affect cell adhesion, proliferation, and differentiation. In this study, plasma polymerization, which is an appealing method for surface modification, was employed to generate surfaces with different chemical compositions. Allylamine (AAm), acrylic acid (AAc), 1,7-octadiene (OD), and ethanol (ET) were used as precursors for plasma polymerization in order to generate thin films rich in amine (-NH2), carboxyl (-COOH), methyl (-CH3), and hydroxyl (-OH) functional groups, respectively. The surface chemistry was characterized by X-ray photoelectron spectroscopy (XPS), the wettability was determined by measuring the water contact angles (WCA) and the surface topography was imaged by atomic force microscopy (AFM). The effects of surface chemical compositions on the behavior of human adipose-derive stem cells (hASCs) were evaluated in vitro: Cell Count Kit-8 (CCK-8) analysis for cell proliferation, F-actin staining for cell morphology, alkaline phosphatase (ALP) activity analysis, and Alizarin Red S staining for osteogenic differentiation. The results show that AAm-based plasma-polymerized coatings can promote the attachment, spreading, and, in turn, proliferation of hASCs, as well as promote the osteogenic differentiation of hASCs, suggesting that plasma polymerization is an appealing method for the surface modification of scaffolds used in bone tissue engineering.

  11. In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma.

    Science.gov (United States)

    Natsume, Midori; Osakabe, Naomi; Yasuda, Akiko; Baba, Seigo; Tokunaga, Takashi; Kondo, Kazuo; Osawa, Toshihiko; Terao, Junji

    2004-12-01

    Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34,840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.

  12. Metabolism of Oxycodone in Human Hepatocytes from Different Age Groups and Prediction of Hepatic Plasma Clearance

    Science.gov (United States)

    Korjamo, Timo; Tolonen, Ari; Ranta, Veli-Pekka; Turpeinen, Miia; Kokki, Hannu

    2012-01-01

    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 h, 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. The data suggests that there are no children-specific metabolites of oxycodone. Moreover, CYP3A activity seems to be the major determinant in metabolic clearance of oxycodone regardless of age group or individual variability in hepatocyte batches. PMID:22291644

  13. Enhanced Biological Behavior of In Vitro Human Gingival Fibroblasts on Cold Plasma-Treated Zirconia.

    Directory of Open Access Journals (Sweden)

    Miao Zheng

    Full Text Available To evaluate whether atmospheric-pressure dielectric-barrier-discharge plasma treatment of zirconia enhances its biocompatibility with human gingival fibroblasts.The zirconia disks were divided into four groups and treated using helium atmospheric-pressure dielectric-barrier-discharge plasmas for 30, 60 or 90 s or left untreated. The surface morphology, wettability and chemical elements were analyzed. Fibroblasts density, morphology, morphometry and attachment-related genes expression were measured at different time points from 3 to 72 h.After plasma treatment, the surface morphology and roughness remained the same, while the contact angle decreased from 78.31° to 43.71°, and the surface C/O ratio decreased from 3.17 to 0.89. The surficial areas and perimeters of HGFs were increased two-fold in the treated groups at 3 h. Fibroblasts density increased on treated disks at all time points, especially the ones treated for 60 s. Attachment-related genes in the groups treated for 30 and 60 s were significantly higher at 3 and 24 h.The helium atmospheric-pressure dielectric-barrier-discharge plasma treatment enhances the biological behavior of fibroblasts on zirconia by increasing the expression of attachment-related genes within 24 h and promoting the cell density during longer culture times. Wettability of zirconia, an important physicochemical property, has a vital influence on the cell behaviors.

  14. Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

    Science.gov (United States)

    Larsson, Anders; Hansson, Lars-Olof

    2004-01-01

    A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.

  15. Heart rate variability, overnight urinary norepinephrine, and plasma cholesterol in apparently healthy human adults.

    Science.gov (United States)

    Thayer, Julian F; Fischer, Joachim E

    2013-01-20

    The aim of this study was to assess the relationship between autonomic nervous system activity as indexed by measures of heart rate variability and overnight urinary norepinephrine, and plasma cholesterol levels in a large sample of working adults. The study population comprised 611 apparently healthy employees of an airplane manufacturing plant in Southern Germany. Heart rate variability was calculated as beat-to-beat intervals over the course of one 24-hour weekday measured with an ambulatory ECG recorder. Overnight urine collection and blood samples were also obtained. We found an inverse association between indices of vagally-mediated heart rate variability and plasma levels of total cholesterol, low density lipoprotein (LDL), and the ratio of LDL to high density lipoprotein (HDL) that remained significant in multivariate models after controlling for relevant covariates including norepinephrine. Urinary norepinephrine was not significantly related to any measure of cholesterol in multivariate models. We report here for the first time, in a large sample of healthy human adults, evidence supporting the hypothesis of a clinically relevant inverse relationship between measures of plasma cholesterol and vagally-mediated heart rate variability after controlling for sympathetic nervous system activity. This suggests an important role for the vagal control of plasma cholesterol levels in cardiovascular disease. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Effect of plasma on composition of human enamel and cementum pellicle

    Energy Technology Data Exchange (ETDEWEB)

    Kajisa, L.; Prakobphol, A. (Division of Oral Biology, Department of Stomatology, School of Dentistry, University of California at San Francisco (USA)); Schioedt, M. (Department of Oral Medicine and Oral Surgery, University Hospital, Copenhagen (Denmark)); Fisher, S.J. (Department of Anatomy, School of Medicine, University of California at San Francisco (USA))

    1990-01-01

    Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed {sup 125}I-iodination and by mild periodate oxidation followed by NaB{sup 3}H{sub 4} reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no {sup 125}I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In Addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major {sup 125}I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa {sup 3}H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva. (author).

  17. 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 chromosom

  18. RNASET2 in human spermatozoa and seminal plasma: a novel relevant indicator for asthenozoospermia.

    Science.gov (United States)

    Liu, Y; Chen, G; Lu, L; Sun, H; Guo, Q; Xue, K; Fan, Y; Ding, Z

    2013-01-01

    Adequate sperm motility is requisite for human fertilization, whereas the underlying causes or mechanisms of impaired sperm motility, asthenozoospermia, still remain largely unknown. RNASET2 (Ribonuclease T2) may be one of the effectors modulating human sperm motility. We determined if there is a correlation between RNASET2 expression levels in human semen from asthenozoospermia and fertile individuals. Thus, RNASET2 expression levels in spermatozoa and seminal plasma of healthy and asthenozoospermia individuals were evaluated using Western blot, laser scanning confocal microscope analysis, ELISA and flow cytometry. The results revealed that RNASET2 expression was identified in both human spermatozoa and seminal plasma. In spermatozoa from fertile individuals, it was localized to the acrosome, neck and the middle piece of tail regions. However, in spermatozoa from asthenozoospermia individuals (n = 67), RNASET2 staining was especially more frequent and evident in the neck and middle piece than that in fertile individuals (n = 59, p asthenozoospermia than in fertile individuals (p asthenozoospermia individuals may contribute to sperm motility impairment.

  19. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a in Plasma.

    Directory of Open Access Journals (Sweden)

    Masayuki Ozawa

    Full Text Available High lipoprotein(a [Lp(a] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a [apo(a], the unique component of Lp(a, is found only in primates and humans, the study of human Lp(a has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT techniques, we produced transgenic miniature pigs expressing human apo(a in the plasma. First, we placed the hemagglutinin (HA-tagged cDNA of human apo(a under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a; one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a. Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL of Lp(a in plasma, and the transgenic apo(a gene was transmitted to the offspring. Thus, we generated a human apo(a-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

  20. 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.

  1. Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans

    DEFF Research Database (Denmark)

    Stefan, Norbert; Vozarova, Barbora; Funahashi, Tohru;

    2002-01-01

    -induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin......Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin...... concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity...

  2. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application

    Directory of Open Access Journals (Sweden)

    Jung-Hwan Lee

    2016-01-01

    Full Text Available Nonthermal atmospheric pressure plasma jets (APPJ have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS. Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells. In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it.

  3. Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma.

    Science.gov (United States)

    Kirk, James T; Brault, Norman D; Baehr-Jones, Tom; Hochberg, Michael; Jiang, Shaoyi; Ratner, Daniel M

    2013-04-15

    A widely acknowledged goal in personalized medicine is to radically reduce the costs of highly parallelized, small fluid volume, point-of-care and home-based diagnostics. Recently, there has been a surge of interest in using complementary metal-oxide-semiconductor (CMOS)-compatible silicon photonic circuits for biosensing, with the promise of producing chip-scale integrated devices containing thousands of orthogonal sensors, at minimal cost on a per-chip basis. A central challenge in biosensor translation is to engineer devices that are both sensitive and specific to a target analyte within unprocessed biological fluids. Despite advances in the sensitivity of silicon photonic biosensors, poor biological specificity at the sensor surface remains a significant factor limiting assay performance in complex media (i.e. whole blood, plasma, serum) due to the non-specific adsorption of proteins and other biomolecules. Here, we chemically modify the surface of silicon microring resonator biosensors for the label-free detection of an analyte in undiluted human plasma. This work highlights the first application of a non-fouling zwitterionic surface coating to enable silicon photonic-based label-free detection of a protein analyte at clinically relevant sensitivities in undiluted human plasma.

  4. METHOD DEVELOPMENT AND VALIDATION OF FENOFIBRATE BY HPLC USING HUMAN PLASMA

    Directory of Open Access Journals (Sweden)

    M.T. Zzaman

    2009-01-01

    Full Text Available The objectives of the study were to develop a rapid, simple, and economical HPLC method for the estimation of Fenofibrate in plasma and to validate the developed bioanalytical method. This research paper describes an specific procedure for determination of Fenofibrate in human plasma. The method employs reverse phase liquid chromatography with UV detection. The mobile phase used was simple and column friendly and was sufficiently sensitive to quantify Fenofibrate in amounts as low as 0.095 µg/ml. This limit of quantitation (LOQ was determined as the lowest concentration with a coefficient of variation lower than 20% and the total accuracy of the method ranged from 101.99 to 107.41%. The LOQ of this method is better than those of other reported methods. The calibration curve plotted concentration versus area and was linear from 0.095 µg/ml to 19.924 µg/ml, having r2 greater than 0.98 during the course of validation. The above method is valid for the analysis of drug in human plasma. The method with slight modification could be used for the drug analysis in various dosage forms.

  5. Determination of Candesartan in Human Plasma with Liquid Chromatography - Tandem Mass Spectrometry.

    Science.gov (United States)

    Forjan, Vanja; Cvitkovič Maričič, Lea; Prosen, Helena; Brodnjak Vončina, Darinka

    2016-01-01

    A sensitive, specific and rapid liquid chromatography - tandem mass spectrometry method was developed and validated for the determination of candesartan in human plasma. Analyte was separated from endogenous components present in plasma by solid phase extraction. Chromatographic separation was performed on Gemini C18 analytical column using mobile phase acetonitrile - 5 mM ammonium formate pH 2 (90:10, v/v) at flow rate of 0.3 mL/min. For detection, tandem mass spectrometry in SRM mode with positive electrospray ionization was used. The mass transitions m/z 441.1 > 263.1 and 445.1 > 267.1 were used to determine candesartan by using candesartan-d4 as an internal standard. After development, the method was validated according to the requirements of EMA regulatory guidelines in the concentration range 1 - 400 ng/ml in human plasma. Limit of quantification (LLOQ) was 1 ng/ml. The developed and validated method proved to be very fast and reproducible and was therefore successfully implemented in pharmacokinetic and bioequivalence studies with large number of study samples.

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

    Science.gov (United States)

    Olas, Beata; Kontek, Bogdan; Malinowska, Paulina; Żuchowski, Jerzy; Stochmal, Anna

    2016-01-01

    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.

  7. 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.

  8. Effects of a Nonthermal Atmospheric Pressure Plasma Jet on Human Gingival Fibroblasts for Biomedical Application.

    Science.gov (United States)

    Lee, Jung-Hwan; Kim, Kyoung-Nam

    2016-01-01

    Nonthermal atmospheric pressure plasma jets (APPJ) have been developed and applied in biomedical research as a cancer treatment or bacterial sterilization. However, the drawback of APPJ on normal oral cells during plasma treatment and underlying cell death mechanisms have not been studied and clearly explained, although there is known to be an influence from reactive oxygen species (ROS). Hence, this study investigates whether and how a nonthermal atmospheric pressure air plasma jet kills human normal gingival cells using immortalized human gingival fibroblasts (hTERT-hNOF cells). In this study, a set of physicochemical or biological methods were used to illuminate the killing mechanisms. It was found that ROS were induced intracellularly without a breakdown of the cell wall and apoptosis was involved in cell death when an air APPJ treatment was performed on the cells directly without media; the air treatment only supported a detachment of the cells without increase of ROS. It was also revealed that a correlation between intracellular ROS concentration and cells viability existed. These results indicated that the direct air APPJ treatment possibly raises safety issue to normal tissue and thereby APPJ application in biomedical field needs more in vitro and in vivo study to optimize it.

  9. Synthesis, structural and spectroscopic studies of 2-oxoacenaphthylen-1(2H)-ylidene nicotinohydrazide.

    Science.gov (United States)

    Henriques, M S C; Del Amparo, R; Pérez-Álvarez, D; Nogueira, B A; Rodríguez-Argüelles, M C; Paixão, J A

    2017-02-05

    The synthesis of a new hydrazone, 2-oxoacenaphthylen-1(2H)-ylidene nicotinohydrazide, and its structural and spectroscopic characterization is reported. The obtained powder was recrystallized from DMSO and ethanol that afforded small crystals used for single-crystal X-ray diffraction studies. The compound was found to crystallize in two polymorphs, depending on the crystallization conditions. One of the polymorphs (form I) crystallizes in the centrosymmetric P21/c monoclinic space group, the other (form II) crystallizes in the non-centrosymmetric, but achiral, orthorhombic space group P212121. Conformation of the molecules is similar in both polymorphs, but the network of weak intermolecular interactions determining the crystal packing is different. In form II an additional C-H⋯O bond connects molecules related by the screw-axis running parallel to the a-axis. Crystals of both polymorphs were also screened by FT-IR and Raman microscopy; a detailed analysis of the spectra and comparison with those of the isolated molecule calculated by ab-initio HF/MP2 and DFT/B3LYP methods using a correlation consistent cc-pVDZ basis set is presented. In addition, UV-vis and NMR studies were performed in solution.

  10. Synthesis, structural and spectroscopic studies of 2-oxoacenaphthylen-1(2H)-ylidene nicotinohydrazide

    Science.gov (United States)

    Henriques, M. S. C.; Del Amparo, R.; Pérez-Álvarez, D.; Nogueira, B. A.; Rodríguez-Argüelles, M. C.; Paixão, J. A.

    2017-02-01

    The synthesis of a new hydrazone, 2-oxoacenaphthylen-1(2H)-ylidene nicotinohydrazide, and its structural and spectroscopic characterization is reported. The obtained powder was recrystallized from DMSO and ethanol that afforded small crystals used for single-crystal X-ray diffraction studies. The compound was found to crystallize in two polymorphs, depending on the crystallization conditions. One of the polymorphs (form I) crystallizes in the centrosymmetric P21/c monoclinic space group, the other (form II) crystallizes in the non-centrosymmetric, but achiral, orthorhombic space group P212121. Conformation of the molecules is similar in both polymorphs, but the network of weak intermolecular interactions determining the crystal packing is different. In form II an additional C-H⋯O bond connects molecules related by the screw-axis running parallel to the a-axis. Crystals of both polymorphs were also screened by FT-IR and Raman microscopy; a detailed analysis of the spectra and comparison with those of the isolated molecule calculated by ab-initio HF/MP2 and DFT/B3LYP methods using a correlation consistent cc-pVDZ basis set is presented. In addition, UV-vis and NMR studies were performed in solution.

  11. MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma

    Science.gov (United States)

    Wiesner, Philipp; Tafelmeier, Maria; Chittka, Dominik; Choi, Soo-Ho; Zhang, Li; Byun, Young Sup; Almazan, Felicidad; Yang, Xiaohong; Iqbal, Navaid; Chowdhury, Punam; Maisel, Alan; Witztum, Joseph L.; Handel, Tracy M.; Tsimikas, Sotirios; Miller, Yury I.

    2013-01-01

    Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis. PMID:23667177

  12. Determination of barnidipine in human serum and dog plasma by HPLC with electrochemical detection.

    Science.gov (United States)

    Takamura, K; Abdel-Wadood, H M; Kusu, F; Rafaat, I H; Saleh, G A; el-Rabbat, N A; Otagiri, M

    1995-10-01

    Barnidipine is a 1,4-dihydropyridine calcium antagonist. HPLC was conducted on a polybutadiene coated alumina column using an alkaline mobile phase and an electrochemical detector to determine the content of this drug in serum and plasma. A good linear relationship between barnidipine concentration and peak height was found in 5-500 ng/ml with a correlation coefficient of 0.998. The detection limit was 1 ng/ml. The within-day and day-to-day variations were examined for control human serum. Relative standard deviation of within-day assay for serum spiked with 10 ng/ml barnidipine.HCl was 6.9% and the recovery was 104%. A pharmacokinetic study was made in which the time course of barnidipine in dog plasma was followed.

  13. 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.

  14. Improved high-performance liquid chromatographic analysis of terazosin in human plasma.

    Science.gov (United States)

    Cheah, P Y; Yuen, K H; Liong, M L

    2000-08-18

    A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25-100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25-100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.

  15. 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.

  16. LC-MS/MS quantitation of antimalarial drug piperaquine and metabolites in human plasma.

    Science.gov (United States)

    Aziz, Mohd Yusmaidie; Hoffmann, Kurt-Jürgen; Ashton, Michael

    2017-09-15

    This study aimed to develop a sensitive, quantitative assay for the antimalarial piperaquine (PQ) and its metabolites M1 and M2 in human plasma. Analytes were gradiently separated on a C18 column and detected with a Sciex API 4000 MS/MS with an ESI source operated in the positive ion mode with deuterated PQ as internal standard. The response was linear in the range 3.9-2508nM with a runtime of 7.0min per sample. The method was applied to clinical samples from healthy volunteers. This LC-MS/MS method for the simultaneous quantitation of PQ and two of its metabolites in plasma may prove helpful for assessment of metabolite safety issues in vivo. Copyright © 2017. Published by Elsevier B.V.

  17. LC-MS-MS Method for Determination of Metolazone in Human Plasma

    Directory of Open Access Journals (Sweden)

    Shikha M. N. Roy

    2008-01-01

    Full Text Available A rapid, sensitive and specific method for quantification of metolazone in human plasma using metaxalone as internal standard is described. Sample preparation involved a simple liquid-liquid extraction procedure. The extract was analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC–MS–MS. Chromatography was performed isocratically on a 5 μm C18 analytical column (50 mm × 4.6 mm i.d. with buffer–acetonitrile 20:80 (v/v as mobile phase. The response to metolazone was a linear function of concentration over the range 1.00 to 2000.00 ng mL-1. The lower limit of quantification in plasma was 1.0 ng mL-1. The method was successfully applied in a bioequivalence study of a metolazone formulation after administration as a single oral dose.

  18. RP-HPLC Method Development and Its Validation for Quantitative Determination of Rimonabant in Human Plasma

    Directory of Open Access Journals (Sweden)

    Shravan Bankey

    2012-01-01

    Full Text Available A simple, accurate, and precise HPLC method was developed and validated for determination of rimonabant in human plasma. Following liquid-liquid extraction, chromatographic separation was accomplished using C18 column with mobile phase consisting of acetonitrile : water (90 : 10, v/v, drug was detected at 260 nm using UVdetector. The LOD and LOQ were 3.0 and 10.0 μg/L, respectively. The method is linear in the interval 50.0–1000.0 μg/L. The average extraction recovery of drug from plasma was found to be 92.2%. The percent CV of the method was found to be less than 10.8%, and accuracy was found between 94.5 and 106.7%. The assay may be applied to a pharmacokinetic and bioequivalence study of rimonabant.

  19. Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

    Science.gov (United States)

    Kagel, J R; Rossi, D T; Nordblom, G D; Dudeck, R C; Barksdale, C M; Kuo, B S; Wright, D S

    1995-09-01

    A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

  20. Plasma clearance of human low-density lipoprotein in human apolipoprotein B transgenic mice is related to particle diameter.

    Science.gov (United States)

    Berneis, Kaspar; Shames, David M; Blanche, Patricia J; La Belle, Michael; Rizzo, Manfredi; Krauss, Ronald M

    2004-04-01

    To test for intrinsic differences in metabolic properties of low-density lipoprotein (LDL) as a function of particle size, we examined the kinetic behavior of 6 human LDL fractions ranging in size from 251 to 265 A injected intravenously into human apolipoprotein (apo) B transgenic mice. A multicompartmental model was formulated and fitted to the data by standard nonlinear regression using the Simulation, Analysis and Modeling (SAAM II) program. Smaller sized LDL particles (251 to 257 A) demonstrated a significantly slower fractional catabolic rate (FCR) (0.050 +/- 0.045 h(-1)) compared with particles of larger size (262 to 265 A) (0.134 +/- -0.015 h(-1), P particles are cleared more slowly from plasma than larger LDL and are exchanged more slowly with the extravascular space. This might be due to compositional or structural features of smaller LDL that lead to retarded clearance.

  1. In vitro human chondrocyte culture on plasma-treated poly(glycerol sebacate) scaffolds.

    Science.gov (United States)

    Theerathanagorn, Tharinee; Klangjorhor, Jeerawan; Sakulsombat, Morakot; Pothacharoen, Peraphan; Pruksakorn, Dumnoensun; Kongtawelert, Prachya; Janvikul, Wanida

    2015-01-01

    Porous poly(glycerol sebacate) (PGS) scaffolds were prepared using a salt leaching technique and subsequently surface modified by a low oxygen plasma treatment prior to the use in the in vitro culture of human chondrocytes. Condensation polymerization of glycerol and sebacic acid used at various mole ratios, i.e. 1:1, 1:1.25, and 1:1.5, was initially conducted to prepare PGS prepolymers. Porous elastomeric PGS scaffolds were directly fabricated from the mixtures of each prepolymer and 90% (w/w) NaCl particles and then subjected to the plasma treatment to enhance the surface hydrophilicity of the materials. The properties of both untreated and plasma-treated PGS scaffolds were comparatively evaluated, in terms of surface morphology, surface chemical composition, porosity, and storage modulus using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, micro-computed tomography, and dynamic mechanical analysis, respectively. The responses of chondrocytes cultured on individual PGS scaffolds were assessed, in terms of cell proliferation and ECM production. The results revealed that average pore sizes and porosity of the scaffolds were increased with an increasing sebacic acid concentration used. The storage moduli of the scaffolds were raised after the plasma treatment, possibly due to the further crosslinking of PGS upon treatment. Moreover, the scaffold prepared with a higher sebacic acid content demonstrated a greater capability of promoting cell infiltration, proliferation, and ECM production, especially when it was plasma-treated; the greatest HA, sGAG, uronic acid, and collagen contents were detected in matrix of this scaffold. The H & E and safranin O staining results also strongly supported this finding. The storage modulus of the scaffold was intensified after incubation with the chondrocytes for 21 days, indicating the accretion and retention of matrix ECM on the cell-cultured scaffold.

  2. 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.

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

    Science.gov (United States)

    Roldán-Pijuán, Mercedes; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2015-04-01

    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 wells in a 96-well donor plate and diluted with HCl to protonate the acidic drugs. The acidic drugs were extracted as protonated species from the individual plasma samples, through corresponding artificial liquid membranes each comprising 2 μL of dihexyl ether, and into corresponding acceptor solutions each comprising 50 μL of 25 mM ammonia solution (pH 10). The liquid membranes and the acceptor solutions were located in a 96-well filter plate, which was sandwiched with the 96-well donor plate during extraction. Parallel extraction of several samples was performed for 15 to 60 min, followed by high-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 extraction was accomplished from plasma. Linearity was obtained for all six drugs in the range 0.025-10 μg/mL, with r (2) values ranging between 0.998 and 1.000. Precision data were in the range 3-22% RSD, and accuracy data were within 72-130% with spiked plasma samples. Based on the current experiences, PALME showed substantial potential for future high-throughput bioanalysis of non-polar acidic drugs.

  4. 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.

  5. Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tao; Qian, Weijun; Gritsenko, Marina A.; Camp, David G.; Monroe, Matthew E.; Moore, Ronald J.; Smith, Richard D.

    2005-12-01

    The enormous complexity, wide dynamic range of relative protein abundance of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenges the capabilities of existing analytical methodologies. We describe here the comprehensive analysis of human plasma N-glycoproteins using the combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin, enzymatically digested, and the bound, N-linked glycopeptides were released using peptide-N-glycosidase F. Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A total of 2140 different N-glycopeptides were confidently identified using stringent criteria, covering 371 non-redundant N-glycoproteins with the majority of them being extracellular or membrane proteins. The strategy significantly improved the detection, enabling the identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein ({approx}200 pg/mL), cathepsin L ({approx}1 ng/mL), and transforming growth factor beta 1 ({approx}2 ng/mL). A total of 712 N-glycosylation sites were identified and the confidence of these site identifications was further validated by accurate mass measurements using high resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR). This study provides the basis for future high-throughput measurements using the accurate mass and time tag approach.

  6. Buspirone, fexofenadine, and omeprazole: quantification of probe drugs and their metabolites in human plasma.

    Science.gov (United States)

    Gor, Parul; Alnouti, Yazen; Reed, Gregory A

    2011-07-15

    Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a "cocktail". This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5'-hydroxyomeprazole; buspirone and its metabolite 1-[2-pyrimidyl]-piperazine (1PP); and fexofenadine. These analytes and the internal standard lansoprazole were extracted from plasma using protein precipitation with acetonitrile. Gradient reverse-phase chromatography was performed with 7.5mM ammonium bicarbonate and acetonitrile, and the analytes were quantified in positive ion electrospray mode with multiple reaction monitoring. The method was validated to quantify the concentration ranges of 1.0-1000ng/ml for omeprazole, omeprazole sulfone, 5'-hydroxyomeprazole, and fexofenadine; 0.1-100ng/ml for buspirone, and 1.0-100ng/ml for 1PP. These linear ranges span the plasma concentrations for all of the analytes from probe drug studies. The intra-day precision was between 2.1 and 16.1%, and the accuracy ranged from 86 to 115% for all analytes. Inter-day precision and accuracy ranged from 0.3 to 14% and from 90 to 110%, respectively. The lower limits of quantification were 0.1ng/ml for buspirone and 1ng/ml for all other analytes. This method provides a fast, sensitive, and selective analytical tool for quantification of the six analytes in plasma necessary to support the use of this probe drug cocktail in clinical studies.

  7. Toxic metals signature in the human seminal plasma of Pakistani population and their potential role in male infertility.

    Science.gov (United States)

    Zafar, Ambreen; Eqani, Syed Ali Musstjab Akber Shah; Bostan, Nazish; Cincinelli, Alessandra; Tahir, Faheem; Shah, Syed Tahir Abbas; Hussain, Alamdar; Alamdar, Ambreen; Huang, Qingyu; Peng, Siyuan; Shen, Heqing

    2015-06-01

    Aims of this study were to provide firsthand data on the incidence of trace metals in human seminal plasma and find possible correlations between levels of toxic metals and semen quality of Pakistani population. Human semen samples were collected from male partners of couples undergoing infertility assessment at the National Institute of Health Islamabad (Pakistan). We investigated seventy-five seminal plasma samples, which were further categorized into three groups (normozoospermia, oligozoospermia and azoospermia) according to WHO guidelines. The concentration of 17 different toxic metals in human seminal plasma was determined simultaneously by using Inductively coupled plasma mass spectrometry (ICP-MS). Out of 17 trace metals, Cd and Ni showed significant difference (p metals were lower and/or comparable to that found in populations of other countries. The results show the first evidence of the effect of toxic metals on semen quality and male infertility in Pakistan.

  8. Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

    DEFF Research Database (Denmark)

    Udby, Lene; Sørensen, Ole E; Pass, Jesper

    2004-01-01

    Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin......-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein...... (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein...

  9. Is a deuterated internal standard appropriate for the reliable determination of olmesartan in human plasma?

    Science.gov (United States)

    Piórkowska, Edyta; Musijowski, Jacek; Buś-Kwaśnik, Katarzyna; Rudzki, Piotr J

    2017-01-01

    A right choice of the internal standard is one of the most challenging tasks during bioanalytical method development. Surprisingly, among the HPLC-MS methods for the determination of a cardiovascular drug olmesartan in plasma only structural analogues or similar compounds were used as internal standards. We have tried to answer the question whether the stable isotope labelled (deuterated) internal standard, as recommended by regulatory agencies, can be used for the reliable determination of olmesartan in human plasma. An HPLC-MS method using this standard in a simplified liquid-liquid extraction procedure led to accurate and precise results in the linearity range of 5-2500ng/mL. The method is well suited for pharmacokinetic studies following a single 40mg oral dose of olmesartan medoxomil in humans. The method was fully validated according to international guidelines and successfully applied in a bioequivalence study in humans. The use of deuterated olmesartan as the internal standard afforded a reliable tool for regulatory bioanalysis that can indirectly contribute to therapy efficacy and improve the safety of patients treated with generic medicines. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Directory of Open Access Journals (Sweden)

    Elena Matteucci

    2007-01-01

    Full Text Available Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  11. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Science.gov (United States)

    Matteucci, Elena; Giampietro, Ottavio

    2007-09-17

    Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na(+)/H(+) exchange and HC(3) (-)/Cl(-) anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs) are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia) and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  12. 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.

  13. Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.

    Directory of Open Access Journals (Sweden)

    Sharon J Pitteri

    Full Text Available The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

  14. An investigation of four chemically peculiar stars with photometric periods below 12 h

    Science.gov (United States)

    Hümmerich, Stefan; Bernhard, Klaus; Paunzen, Ernst; Hambsch, Franz-Josef; Bohlsen, Terry; Powles, Jonathan

    2017-04-01

    We present an investigation of three chemically peculiar (CP) stars and one CP star candidate that exhibit photometric periods below 12 h. New spectroscopic observations have been acquired that confirm the peculiar nature of all objects. HD 77013 and HD 81076 are classical CP1 (Am) stars, HD 67983 is a marginal CP1 (Am:) star and HD 98000 is a CP2 (Ap) star. We have procured observations from the ASAS-3 and SuperWASP archives and obtained additional photometry in order to verify the results from the sky survey data. We have derived astrophysical parameters and investigated the positions of our target stars in the MBol versus log Teff diagram, from which information on evolutionary status has been derived. We present period analyses and discuss each object in detail. From the available data, we propose pulsational variability as the underlying mechanism for the variability in HD 67983, HD 77013 and HD 81076, which offer the opportunity to study the interaction of atomic diffusion and pulsation. HD 67983 and HD 77013 exhibit multiperiodic variability in the γ Doradus frequency realm; HD 81076 is a δ Scuti star. The CP2 star HD 98000 exhibits monoperiodic variability with a frequency of f ≈ 2.148 cycles d-1 (P ≈ 0.466 d), which we interpret as the rotational period. If this assumption is correct, HD 98000 is the α2 Canum Venaticorum variable with the shortest period hitherto observed and thus a very interesting object that might help to investigate the influence of rotational mixing on chemical peculiarities.

  15. 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

  16. Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one based inhibitors of the dual-specificity phosphatase Cdc25B.

    Science.gov (United States)

    George Rosenker, Kara M; Paquette, William D; Johnston, Paul A; Sharlow, Elizabeth R; Vogt, Andreas; Bakan, Ahmet; Lazo, John S; Wipf, Peter

    2015-06-15

    The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes, particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus, Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the 3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling methodology to synthesize a new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a Ki of 1.9μM. Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint bypass. Molecular docking studies support binding near the catalytic site. Copyright © 2015. Published by Elsevier Ltd.

  17. Determination of rhoifolin and daidzin in human plasma by high-performance liquid chromatography.

    Science.gov (United States)

    Ishii, K; Urano, S; Furuta, T; Kasuya, Y

    1994-05-13

    A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C18 cartridge. The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol-0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.

  18. The Serum Amyloid p Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels

    DEFF Research Database (Denmark)

    Poulsen, Ebbe Toftgaard; Pedersen, Kata Wolff; Marzeda, Anna Maria

    2017-01-01

    containing the physiological Ca2+ 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 integration partners not previously reported. The SAP...... involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to binding to the generic structure of amyloid deposits and possibly to protect these against proteolysis. In this study, we have characterized the SAP interactome in human plasma...

  19. Influence of ritodrine on plasma steroids and human placental lactogen levels in third-trimester pregnancies.

    Science.gov (United States)

    Schreyer, P; Zer, Y; Ariely, S; Herman, A; Caspi, E

    1989-01-01

    The effects of ritodrine, administered by intravenous, intramuscular or per os routes, upon plasma levels of unconjugated estriol, estradiol, progesterone and human placental lactogen (HPL) were investigated in 24 third-trimester pregnancies. Ritodrine administered at a constant rate of 200 micrograms/min for 3 h, injected intramuscularly 8 x 10 mg/day for 5 days or given orally in doses of 6 x 20 mg/day for 10 days resulted in a significant decrease in the steroid hormone concentration and a significant elevation of HPL levels.

  20. 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...

  1. A Fast Response Highly Selective Probe for the Detection of Glutathione in Human Blood Plasma

    Directory of Open Access Journals (Sweden)

    Robert M. Strongin

    2012-05-01

    Full Text Available A fluorescent probe for glutathione (GSH detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys and homocysteine (Hcy. The proposed method is precise with a relative standard deviation (R.S.D lower than 6% (n = 3 and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.

  2. Saliva versus plasma bioequivalence of rusovastatin in humans: validation of class III drugs of the salivary excretion classification system.

    Science.gov (United States)

    Idkaidek, Nasir; Arafat, Tawfiq

    2015-03-01

    Bioequivalence of rusovastatin in healthy human volunteers was done using saliva and plasma matrices in order to investigate the robustness of using saliva instead of plasma as a surrogate for bioequivalence of class III drugs according to the salivary excretion classification system (SECS). Saliva and plasma samples were collected for 72 h after oral administration of rusovastatin 40 mg to 12 healthy humans. Saliva and plasma pharmacokinetic parameters were calculated by non-compartmental analysis. Analysis of variance, 90 % confidence intervals, and intra-subject and inter-subject variability values of pharmacokinetic parameters were calculated using Kinetica program V5. Human effective intestinal permeability was also calculated by SimCYP program V13. Rusovastatin falls into class III (high permeability/low fraction unbound to plasma proteins) and hence was subjected to salivary excretion. A correlation coefficient of 0.99 between saliva and plasma concentrations, and a saliva/plasma concentration ratio of 0.175 were observed. The 90 % confidence limits of area under the curve (AUClast) and maximum concentration (C max) showed similar trends in both saliva and plasma. On the other hand, inter- and intra-subject variability values in saliva were higher than in plasma, leading to the need for a slightly higher number of subjects to be used in saliva studies. Non-invasive saliva sampling instead of the invasive plasma sampling method can be used as a surrogate for bioequivalence of SECS class III drugs when an adequate sample size is used.

  3. 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.

  4. Determination of moxonidine in human plasma by liquid chromatography-electrospray ionisation-mass spectrometry.

    Science.gov (United States)

    Zhao, Luhua; Ding, Li; Wei, Xin

    2006-01-23

    A sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of moxonidine in human plasma. After the addition of clonidine-HCl, the internal standard (IS) and sodium hydrogen carbonate, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on a Lichrospher ODS (5 microm, 250 mm x 4.6 mm) column using an elution system of 10 mmol/L ammonium acetate buffer-methanol (20:80 v/v) as the mobile phase. Analytes were determined using electrospary ionization in a single quadrupole mass spectrometer. LC-ESI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z: 242.2 for moxonidine and m/z: 230.1 for the IS. The method has shown to be sensitive and specific by testing six different blank plasma batches. Linearity was established for the range of concentrations 0.01976-9.88 ng/mL with a coefficient of correlation (r) of 0.9999. The lower limit of quantification (LOQ) was identifiable and reproducible at 0.01976 ng/mL. The method has been successfully applied to study the pharmacokinetics of moxonidine in healthy male Chinese volunteers.

  5. Zinc and iron concentration and SOD activity in human semen and seminal plasma.

    Science.gov (United States)

    Marzec-Wróblewska, Urszula; Kamiński, Piotr; Lakota, Paweł; Szymański, Marek; Wasilow, Karolina; Ludwikowski, Grzegorz; Kuligowska-Prusińska, Magdalena; Odrowąż-Sypniewska, Grażyna; Stuczyński, Tomasz; Michałkiewicz, Jacek

    2011-10-01

    The aim of the present study was to measure zinc (Zn) and iron (Fe) concentration in human semen and superoxide dismutase (SOD) activity in seminal plasma and correlate the results with sperm quality. Semen samples were obtained from men (N = 168) undergoing routine infertility evaluation. The study design included two groups based on the ejaculate parameters. Group I (n = 39) consisted of males with normal ejaculate (normozoospermia), and group II (n = 129) consisted of males with pathological spermiogram. Seminal Zn and Fe were measured in 162 samples (group I, n = 38; group II, n = 124) and SOD activity in 149 samples (group I, n = 37; group II, n = 112). Correlations were found between SOD activity and Fe and Zn concentration, and between Fe and Zn concentration. SOD activity was negatively associated with volume of semen and positively associated with rapid progressive motility, nonprogressive motility, and concentration. Negative correlation was stated between Fe concentration and normal morphology. Mean SOD activity in seminal plasma of semen from men of group I was higher than in seminal plasma of semen from men of group II. Fe concentration was higher in teratozoospermic males than in males with normal morphology of spermatozoa in group II. Our results suggest that Fe may influence spermatozoa morphology.

  6. Growth of Yersinia pseudotuberculosis in human plasma: impacts on virulence and metabolic gene expression

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2008-12-01

    Full Text Available Abstract Background In man, infection by the Gram-negative enteropathogen Yersinia pseudotuberculosis is usually limited to the terminal ileum. However, in immunocompromised patients, the microorganism may disseminate from the digestive tract and thus cause a systemic infection with septicemia. Results To gain insight into the metabolic pathways and virulence factors expressed by the bacterium at the blood stage of pseudotuberculosis, we compared the overall gene transcription patterns (the transcriptome of bacterial cells cultured in either human plasma or Luria-Bertani medium. The most marked plasma-triggered metabolic consequence in Y. pseudotuberculosis was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow" in Escherichia coli. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to E. coli acetate may be further metabolized in Y. pseudotuberculosis. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively; the yadA adhesin gene and most of the transcriptional units of the pYV-encoded type III secretion apparatus were found to be upregulated, whereas transcription of the pH6 antigen locus was strongly repressed. Conclusion Our results suggest that plasma growth of Y. pseudotuberculosis is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence.

  7. from Spiked Human Plasma Using RP-HPLC with UV Detection

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    Monita Gide

    2014-01-01

    Full Text Available A rapid, simple, selective, and specific reverse phase high performance liquid chromatography (RP-HPLC method with UV detection (315 nm was developed and validated for estimation of febuxostat from spiked human plasma. The analyte and internal standard (diclofenac were extracted using LLE with diethyl ether. The chromatographic separation was performed on Shodex C-18-4E (5 μm; 250×4.6 mm with a mobile phase comprised of methanol : acetate buffer pH 4, 20 mM (90 : 10 v/v, at a flow rate of 1 mL/min. Febuxostat was well resolved from plasma constituents and internal standard. The calibration curve was linear in the range of 250–8000 ng/mL. The heteroscedasticity was minimized by using weighted least square regression with weighing factor of 1/x. The intraday and interday %RSD was less than 15. Results of recovery studies prove the extraction efficiency. Stability data indicated that febuxostat was stable in plasma after three freeze thaw cycles and upon storage at −20°C for 30 days.

  8. Human plasma concentrations of cytochrome P450 probes extrapolated from pharmacokinetics in cynomolgus monkeys using physiologically based pharmacokinetic modeling.

    Science.gov (United States)

    Shida, Satomi; Utoh, Masahiro; Murayama, Norie; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-01-01

    1. Cynomolgus monkeys are widely used in preclinical studies as non-human primate species. Pharmacokinetics of human cytochrome P450 probes determined in cynomolgus monkeys after single oral or intravenous administrations were extrapolated to give human plasma concentrations. 2. Plasma concentrations of slowly eliminated caffeine and R-/S-warfarin and rapidly eliminated omeprazole and midazolam previously observed in cynomolgus monkeys were scaled to human oral biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Results of the simplified human PBPK models were consistent with reported experimental PK data in humans or with values simulated by a fully constructed population-based simulator (Simcyp). 3. Oral administrations of metoprolol and dextromethorphan (human P450 2D probes) in monkeys reportedly yielded plasma concentrations similar to their quantitative detection limits. Consequently, ratios of in vitro hepatic intrinsic clearances of metoprolol and dextromethorphan determined in monkeys and humans were used with simplified PBPK models to extrapolate intravenous PK in monkeys to oral PK in humans. 4. These results suggest that cynomolgus monkeys, despite their rapid clearance of some human P450 substrates, could be a suitable model for humans, especially when used in conjunction with simple PBPK models.

  9. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  10. Determination of platinum surface contamination in veterinary and human oncology centres using inductively coupled plasma mass spectrometry

    NARCIS (Netherlands)

    Janssens, T.; Brouwers, E. E M; de Vos, J. P.; de Vries, N.; Schellens, J. H M; Beijnen, J. H.

    2015-01-01

    The objective of this study was to determine the surface contamination with platinum-containing antineoplastic drugs in veterinary and human oncology centres. Inductively coupled plasma mass spectrometry was used to measure platinum levels in surface samples. In veterinary and human oncology

  11. 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.

  12. The comparison of DNA damage induced by micro DBD plasma and low energy electron for curing human diseases

    Science.gov (United States)

    Park, Yeunsoo

    2015-09-01

    It is well known that low energy electrons (LEE, especially below 10 eV) can generate DNA damage via indirect action named dissociative electron attachment (DEA). We can now explain some parts of the exact mechanism on DNA damage by LEE collision with direct ionization effect when cancer patients get the radiotherapy. It is kind of remarkable information in the field of radiation therapy. However, it is practically very difficult to directly apply this finding to human disease cure due to difficulty of LEE therapy actualization and request of further clinical studies. Recently, there is a novel challenge in plasma application, that is, how we can apply plasma technology to diagnosis and treatment of many serious diseases like cancer. Cold atmospheric pressure plasma (CAPP) is a very good source to apply to plasma medicine and bio-applications because of low temperature, low cost, and easy handling. Some scientists have already reported good results related to clinical plasma application. The purposes of this study are to further find out exact mechanisms of DNA damage by LEE at the molecular level, to verify new DNA damage like structural alteration on DNA subunits and to compare DNA damage by LEE and plasma source. We will keep expanding our study to DNA damage by plasma source to develop plasma-based new medical and biological applications. We will show some recent results, DNA damage by LEE and non-thermal plasma.

  13. Stability of glucagon-like peptide 1 and glucagon in human plasma

    Science.gov (United States)

    Wewer Albrechtsen, Nicolai J; Bak, Monika J; Hartmann, Bolette; Christensen, Louise Wulff; Kuhre, Rune E; Deacon, Carolyn F; Holst, Jens J

    2015-01-01

    To investigate the stability of glucagon-like peptide 1 (GLP-1) and glucagon in plasma under short- and long-term storage conditions. Pooled human plasma (n=20), to which a dipeptidyl peptidase 4 (DPP4) inhibitor and aprotinin were added, was spiked with synthetic GLP-1 (intact, 7–36NH2 as well as the primary metabolite, GLP-1 9–36NH2) or glucagon. Peptide recoveries were measured in samples kept for 1 and 3 h at room temperature or on ice, treated with various enzyme inhibitors, after up to three thawing–refreezing cycles, and after storage at −20 and −80 °C for up to 1 year. Recoveries were unaffected by freezing cycles or if plasma was stored on ice for up to 3 h, but were impaired when samples stood at RT for more than 1 h. Recovery of intact GLP-1 increased by addition of a DPP4 inhibitor (no ice), but was not further improved by neutral endopeptidase 24.11 inhibitor or an inhibitor cocktail. GLP-1, but not glucagon, was stable for at least 1 year. Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared with immediate analysis, regardless of storage time. Plasma handling procedures can significantly influence results of subsequent hormone analysis. Our data support addition of DPP4 inhibitor for GLP-1 measurement as well as cooling on ice of both GLP-1 and glucagon. Freeze–thaw cycles did not significantly affect stability of GLP-1 or glucagon. Long-term storage may affect glucagon levels regardless of storage temperature and results should be interpreted with caution. PMID:25596009

  14. Individual variation in macronutrient regulation measured by proton magnetic resonance spectroscopy of human plasma.

    Science.gov (United States)

    Park, Youngja; Kim, Seoung Bum; Wang, Bing; Blanco, Roberto A; Le, Ngoc-Anh; Wu, Shaoxiong; Accardi, Carolyn J; Alexander, R Wayne; Ziegler, Thomas R; Jones, Dean P

    2009-07-01

    Proton nuclear magnetic resonance ((1)H-NMR) spectroscopy of plasma provides a global metabolic profiling method that shows promise for clinical diagnostics. However, cross-sectional studies are complicated by a lack of understanding of intraindividual variation, and this limits experimental design and interpretation of data. The present study determined the diurnal variation detected by (1)H NMR spectroscopy of human plasma. Data reduction methods revealed three time-of-day metabolic patterns, which were associated with morning, afternoon, and night. Major discriminatory regions for these time-of-day patterns included the various kinds of lipid signals (-CH(2)- and -CH(2)OCOR), and the region between 3 and 4 ppm heavily overlapped with amino acids that had alpha-CH and alpha-CH(2). The phasing and duration of time-of-day patterns were variable among individuals, apparently because of individual difference in food processing/digestion and absorption and clearance of macronutrient energy sources (fat, protein, carbohydrate). The times of day that were most consistent among individuals, and therefore most useful for cross-sectional studies, were fasting morning (0830-0930), postprandial afternoon (1430-1630), and nighttime samples (0430-0530). Importantly, the integrated picture of metabolism provided by (1)H-NMR spectroscopy of plasma suggests that this approach is suitable to study complex regulatory processes, including eating patterns/eating disorders, upper gastrointestinal functions (gastric emptying, pancreatic, biliary functions), and absorption/clearance of macronutrients. Hence, (1)H-NMR spectroscopy of plasma could provide a global metabolic tolerance test to assess complex processes involved in disease, including eating disorders and the range of physiological processes causing dysregulation of energy homeostasis.

  15. Streptococcal Serum Opacity Factor Increases Hepatocyte Uptake of Human Plasma High Density Lipoprotein-Cholesterol1

    Science.gov (United States)

    Gillard, Baiba K.; Rosales, Corina; Pillai, Biju K.; Lin, Hu Yu; Courtney, Harry S.; Pownall, Henry J.

    2010-01-01

    Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM), that contains the cholesterol esters (CE) of up to ~400,000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. [3H]CE uptake by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was respectively 2.4 and 4.5 times faster than from control HDL. CERM-[3H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[3H]CE uptake by both receptors. RAP and heparin inhibit CERM-[3H]CE but not HDL-[3H]CE uptake thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase hepatic disposal of plasma cholesterol in a way that is therapeutically useful. PMID:20879789

  16. Simulation of Human Plasma Concentrations of Thalidomide and Primary 5-Hydroxylated Metabolites Explored with Pharmacokinetic Data in Humanized TK-NOG Mice.

    Science.gov (United States)

    Nishiyama, Sayako; Suemizu, Hiroshi; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2015-11-16

    Plasma concentrations of thalidomide and primary 5-hydroxylated metabolites including 5,6-dihydroxythalidomide and glutathione (GSH) conjugate(s) were investigated in chimeric mice with highly "humanized" liver cells harboring cytochrome P450 3A5*1. Following oral administration of thalidomide (100 mg/kg), plasma concentrations of GSH conjugate(s) of 5-hydroxythalidomide were higher in humanized mice than in controls. Simulation of human plasma concentrations of thalidomide were achieved with a simplified physiologically based pharmacokinetic model in accordance with reported thalidomide concentrations. The results indicate that the pharmacokinetics in humans of GSH conjugate and/or catechol primary 5-hydroxylated thalidomide contributing in vivo activation can be estimated for the first time.

  17. Cloud point extraction for analysis of antiretrovirals in human plasma by UFLC-ESI-MS/MS

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    Gabriel A. Hunzicker

    2015-12-01

    Full Text Available An analytical methodology based on cloud point extraction (CPE coupled to Ultra-Fast Liquid Chromatography and electrospray tandem mass spectrometry (UFLC-MS/MS was developed for analysis of Abacavir (ABC, Efavirenz (EFV, Lamivudine (3 TC and Nelfinavir (NFV in human plasma. It is the first time that CPE was used for extraction of antiretrovirals (ARV from plasma. The effects of relevant physic-chemical variables on analytical response of each ARV, including pH, surfactant concentration, equilibration time and temperature, were study and optimized; as well as its coupling to UFLC-ESI-MS/MS. Under optimized conditions, the resulting methodology was as follows: a 500 μL aliquot of human plasma was diluted with 2 mL deionized water in a 10 mL centrifuge tube. A 500 μL aliquot Triton X-114 5% w/v was added and homogenized using a vortex stirrer. The resulting cloudy solution was kept at 65 °C for 20 min for promoting the condensation of surfactant micelles. Then it was centrifuged at 3000 × g for 5 min for separation of the surfactant-rich phase. After discarding the aqueous supernatant, 400 μL ACN were added to the remaining surfactant rich phase and centrifuged in order to precipitate proteins and separate them. A 150 μL aliquot of the supernatant was transferred to 2 mL vial and further diluted with 400 μL deionized water. A 30 μL aliquot of the so-prepared solution was injected and analyzed into the UFLC-MS/MS. The method detection limits for ABC, EFV, 3 TC and NFV under optimized conditions were 31, 77, 57 and 21 ng mL−1, respectively. The RSD% for the studied analytes were <15%, except at the LOQ, which were <19%. Recovery values ranged from 81 to 107%. The proposed methodology was successfully applied for the analysis of ABC, EFV, 3 TC and NFV in human plasma within the concentration range of 43–6816, 125–4992, 81–3248 and 49–7904 ng mL−1, respectively. Under optimized working conditions the proposed

  18. Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass.

    Science.gov (United States)

    Liu, Xiao-yan; Qu, Ting-ting; Wang, Ben-jie; Wei, Chun-min; Yuan, Gui-yan; Zhang, Rui; Guo, Rui-chen

    2008-09-01

    A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-12-01

    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.

  20. Purification and characterization of an insulin-like growth factor II variant from human plasma.

    Science.gov (United States)

    Hampton, B; Burgess, W H; Marshak, D R; Cullen, K J; Perdue, J F

    1989-11-15

    An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.

  1. Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin.

    Science.gov (United States)

    Sagmeister, Peter; Gibson, Matthew A; McDade, Kyle H; Gailer, Jürgen

    2016-08-01

    Although low-level chronic exposure of humans to cadmium (Cd(2+)) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.

  2. Determination of triapine, a ribonucleotide reductase inhibitor, in human plasma by liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Feng, Ye; Kunos, Charles A; Xu, Yan

    2015-09-01

    Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation-mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC-MS/MS method for the determination of triapine in human plasma. In this method, 2-[(3-fluoro-2-pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP18 column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mM, pH 8.50; v/v); column eluate was monitored by positive turbo-ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple-reaction-monitoring mode. The method developed had a linear calibration range of 0.250-50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS-normalized recovery and the IS-normalized matrix factor of triapine were 101-104% and 0.89-1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC-MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial.

  3. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    Science.gov (United States)

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

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

    Science.gov (United States)

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

    1984-09-01

    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. [14C]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 [14C]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.

  5. Enantiomeric resolution of ibuprofen and flurbiprofen in human plasma by SPE-chiral HPLC methods.

    Science.gov (United States)

    Ali, Imran; Hussain, Iqbal; Saleem, Kishwar; Aboul-Enein, Hassan Y

    2012-07-01

    Chiral analysis of profens in human plasma is an important area of research due to different pharmaceutical activities of their enantiomers. The solid phase extraction of ibuprofen and flurbiprofen from human plasma was carried out on C18 cartridges by using phosphate buffer (50 mM, pH 6.0) followed by elution with methanol. Chiral-HPLC was performed on AmyCoat RP (150 mm x 46 mm, 3 μm particle size) column by using different combinations of water-acetonitrile-trifluoro acetic acid at 1.5 mLmin-1 flow rate. The detection was achieved at 236 and 254 nm for ibuprofen and flurbiprofen, respectively with 27±1°C as working temperature. The chromatographic parameters i.e. retention (k), separation (α) and resolution (Rs) factors ranged from 4.54-14.42, 1.10-1.30 and 1.01-1.49, respectively. The binding differences of enantiomers of ibuprofen and flurbiprofen were 4.4 and 5.2, respectively. These values suggest that S-(+)- enantiomer of flurbiprofen is more active than ibuprofen due to low enantiomeric difference of the later drug. The developed SPE-Chiral HPLC methods were validated, which are selective, efficient and reproducible.

  6. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  7. 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.

  8. Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Vivek Upadhyay; Vikas Trivedi; Gaurang Shah; Manish Yadav; Pranav S. Shrivastav

    2014-01-01

    A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50 mL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100mm*2.1mm,1.7mm) column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000 ng/mL. The mean extraction recovery for the analyte and IS was 495%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.

  9. Purification of human seminal plasma no. 7 antigen by immunoaffinity chromatography on bound monoclonal antibody.

    Science.gov (United States)

    Isojima, S; Koyama, K; Fujiwara, N

    1982-01-01

    Human seminal plasma (HSP) No. 7 antigen was purified by immunoaffinity chromatography on bound 1C4 monoclonal antibody (Moab) (Shigeta et al., 1980b). The pooled HSP protein was applied to a CNBr-activated Sepharose 4B column of bound 1C4 Moab gamma globulin and the antibody bound fraction (fr) eluted was further purified by rechromatography in the same way. The purified antigen in the antibody bound fr obtained by rechromatography gave a single band on SDS-PAGE in a position corresponding to a molecular weight of 15,000 daltons. This preparation was 196.2 times more effective than the original HSP protein in neutralizing the sperm immobilizing activity of 1C4 Moab. The purified HSP No. 7 antigen contained iron, but was different from lactoferrin and transferrin. It did not show any enzymatic activities, such as those of acid phosphatase, LDH or trypsin inhibitor, and shared antigenicity with human milk protein. It was present in seminal plasma as a molecule with a higher molecular weight but seemed to be cleaved to a monomer of 15,000 daltons during purification procedures. This antigen is present on spermatozoa as sperm-coating antigen and the corresponding antibody can immobilize spermatozoa with complement. Images Fig. 3 PMID:7127911

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

    Energy Technology Data Exchange (ETDEWEB)

    Jin Gu [Department of Chemistry, University of Science and Technology of China, HeFei, 230026 (China)], E-mail: Gjin@ustc.edu.cn; Yao Qizhi; Zhang Shanzi [Department of Chemistry, University of Science and Technology of China, HeFei, 230026 (China); Zhang Lei [Department of Chemistry, University of Science and Technology of China, HeFei, 230026 (China); AnHui Entry Exit Inspection and Quarantine Bureau, HeFei, 230001 (China)

    2008-12-01

    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.

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

    Science.gov (United States)

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

    2009-01-01

    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 P4322 and contained three β-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 Å resolution. PMID:19407392

  12. Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Vivek Upadhyay

    2014-06-01

    Full Text Available A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS from 50 µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100 mm×2.1 mm, 1.7 µm column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 (75:25, v/v as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000 ng/mL. The mean extraction recovery for the analyte and IS was >95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.

  13. Development and Validation of Acyclovir HPLC External Standard Method in Human Plasma: Application to Pharmacokinetic Studies

    Directory of Open Access Journals (Sweden)

    Selvadurai Muralidharan

    2014-01-01

    Full Text Available A simple, rapid, and selective RP-HPLC method was developed for the estimation of acyclovir in human plasma. The method involves a simple protein precipitation technique. Chromatographic separation was carried out on a reverse phase C18 column using mixture of 5 mM ammonium acetate (pH 4.0 and acetonitrile (40 : 60, v/v at a flow rate of 1.0 mL/min with UV detection at 290 nm. The retention time of acyclovir was 4.12 minutes. The method was validated and found to be linear in the range of 25.0–150.0 ng/mL. Validation studies were achieved by using the fundamental parameters, including accuracy, precision, selectivity, sensitivity, linearity and range, stability studies, limit of detection (LOD, and limit of quantitation (LOQ. It shows recovery at 91.0% which is more precise and accurate compared to the other method. These results indicated that the bioanalytical method was linear, precise, and accurate. The new bioanalytical method was successfully applied to a pharmacokinetic linearity study in human plasma.

  14. 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.

  15. Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans.

    Science.gov (United States)

    Stefan, Norbert; Vozarova, Barbora; Funahashi, Tohru; Matsuzawa, Yuji; Weyer, Christian; Lindsay, Robert S; Youngren, Jack F; Havel, Peter J; Pratley, Richard E; Bogardus, Clifton; Tataranni, P Antonio

    2002-06-01

    Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity (insulin-stimulated glucose disposal, hyperinsulinemic clamp), and glucose tolerance (75-g oral glucose tolerance test) were measured in 55 Pima Indians (47 men and 8 women, aged 31 +/- 8 years, body fat 29 +/- 8% [mean +/- SD]; 50 with normal glucose tolerance, 3 with impaired glucose tolerance, and 2 with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation. Group 2 (38 subjects) had follow-up measurements of insulin-stimulated glucose disposal. Cross-sectionally, plasma adiponectin concentration was positively associated with insulin-stimulated glucose disposal (r = 0.58, P < 0.0001) and negatively associated with percent body fat (r = -0.62, P < 0.0001) in the whole group. In group 1 plasma adiponectin was negatively associated with the basal (r = -0.65, P = 0.003) and positively associated with the fold increase in IR

  16. Rapid and Sensitive Method for Quantitative Determination of Lopinavir and Ritonavir in Human Plasma by Liquid Chromatography- Tandem Mass Specrtometry

    Directory of Open Access Journals (Sweden)

    G. A. Temghare

    2009-01-01

    Full Text Available A rapid and sensitive liquid chromatography-mass spectrometric (LC-MS-MS method for the simultaneous determination of lopinavir and ritonavir in human plasma using abacavir as internal standard has been developed and validated. Sample preparation of plasma involved solid phase extraction. Detection was performed using an Applied Biosystems Sciex API 2000 Mass spectrometer. The assay of lopinavir and ritonavir was linear over the range of 50 ng mL-1 to 20000 ng mL -1 and 20 ng mL -1 to 3000 ng mL-1 respectively with a precision of <15% and accuracy in the range of 85-115%. The limit of quantification in plasma for lopinavir and ritonavir was 50 ng mL -1 and 20 ng mL -1 respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma

  17. 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.

  18. Saliva versus Plasma Relative Bioavailability of Tolterodine in Humans: Validation of Class III Drugs of the Salivary Excretion Classification System.

    Science.gov (United States)

    Idkaidek, N; Najib, N; Salem, I I; Najib, O

    2016-06-01

    Relative bioavailability study of tolterodine in healthy human volunteers was done using saliva and plasma matrices in order to investigate the robustness of using saliva instead of plasma as a surrogate for bioavailability and bioequivalence of class III drugs according to the salivary excretion classification system (SECS). Saliva and plasma samples were collected up to 16 h after 2 mg oral dose. Saliva and plasma pharmacokinetic parameters were calculated by non compartmental analysis using Kinetica program V5. Human effective intestinal permeability was optimized by SimCYP program V13. Tolterodine falls into class III (High permeability/Low fraction unbound to plasma proteins) and hence was subjected to salivary excretion. A high pearsons correlation coefficient of 0.97 between mean saliva and plasma concentrations, and saliva/plasma concentrations ratio of 0.33 were observed. In addition, correlation coefficients and saliva/plasma ratios of area under curve and maximum concentration were 0.98, 0.95 and 0.42, 0.34 respectively. On the other hand, time to reach maximum concentration was higher in saliva by 2.37 fold. In addition, inter subject variability values in saliva were slightly higher than plasma leading to need for slightly higher number of subjects to be used in saliva studies (55 vs. 48 subjects). Non-invasive saliva sampling instead of invasive plasma sampling method can be used as a surrogate for bioavailability and bioequivalence of SECS class I drugs when adequate sample size is used. © Georg Thieme Verlag KG Stuttgart · New York.

  19. Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

    OpenAIRE

    Hsueh, W A; Carlson, E J; Dzau, V J

    1983-01-01

    An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 4 degrees C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we f...

  20. Increased plasma 8-iso-prostaglandin F2α concentration in severe human traumatic brain injury.

    Science.gov (United States)

    Yu, Guo-Feng; Jie, Yuan-Qing; Wu, An; Huang, Qiang; Dai, Wei-Min; Fan, Xiao-Feng

    2013-06-05

    8-Iso-Prostaglandin F2α (8-iso-PGF2α) is considered as a gold standard for measuring oxidative stress in vivo. The present study was undertaken to investigate plasma 8-iso-PGF2α concentrations in severe human traumatic brain injury (TBI) and to analyze its correlation with disease outcome. One hundred six healthy subjects and 106 severe TBI patients were recruited. The correlations of plasma 8-iso-PGF2α concentration with 1-year mortality and unfavorable outcome (Glasgow Outcome Scale score of 1-3) were analyzed. Thirty-one patients (29.2%) died and 48 patients (45.3%) had an unfavorable outcome at 1 year after TBI. Patients had significantly higher plasma 8-iso-PGF2α levels compared to healthy controls (572.1±157.5 pg/ml vs. 84.3±18.9 pg/ml, Piso-PGF2α level as an independent predictor for 1-year unfavorable outcome [odds ratio (OR) 1.401, 95% confidence interval (CI) 1.107-2.371, P=0.005] and mortality (OR 1.609, 95% CI 1.113-3.142, P=0.003). A receiver operating characteristic curve analysis showed plasma 8-iso-PGF2α level predicted 1-year unfavorable outcome [area under curve (AUC), 0.871; 95% CI, 0.792-0.928] and mortality (AUC, 0.881; 95% CI, 0.804-0.936) as statistically significantly. The prognostic value of 8-iso-PGF2α was similar to that of Glasgow Coma Scale score for 1-year clinical outcomes (both P>0.05). However, 8-iso-PGF2α did not improve the prognostic value of Glasgow Coma Scale score for 1-year clinical outcomes (both P>0.05). Plasma 8-iso-PGF2α level is highly associated with 1-year clinical outcomes of TBI. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Comparison of extraction and quantification methods of perfluorinated compounds in human plasma, serum, and whole blood

    Energy Technology Data Exchange (ETDEWEB)

    Reagen, William K. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States)], E-mail: wkreagen@mmm.com; Ellefson, Mark E. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States); Kannan, Kurunthachalam [Wadsworth Center, New York State Department of Health and Department of Environmental Health Sciences (United States); State University of New York at Albany, NY 12201-0509 (United States); Giesy, John P. [Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK (Canada); Department of Biology and Chemistry, Center for Coastal Pollution and Conservation, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Zoology Department, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI (United States); School of Environment, Nanjing University, Nanjing (China)

    2008-11-03

    Perfluorinated compounds are ubiquitous in the environment and have been reported to occur in human blood. Accurate risk assessments require accurate measurements of exposures, but identification and quantification of PFCs in biological matrices can be affected by both ion suppression and enhancement in liquid chromatography-tandem mass spectrometry techniques (LC/MS-MS). A study was conducted to quantify potential biases in LC/MS-MS quantification methods. Using isotopically labeled perfluorooctanoic acid ([{sup 13}C{sub 2}]-PFOA), perfluorononanoic acid ([{sup 13}C{sub 2}]-PFNA), and ammonium perfluorooctanesulfonate ([{sup 18}O{sub 2}]-PFOS) spiked tissues, ion-pairing extraction, solid-phase extraction, and protein precipitation sample preparation techniques were compared. Analytical accuracy was assessed using both solvent calibration and matrix-matched calibration for quantification. Data accuracy and precision of 100 {+-} 15% was demonstrated in both human sera and plasma for all three sample preparation techniques when matrix-matched calibration was used in quantification. In contrast, quantification of ion-pairing extraction data using solvent calibration in combination with a surrogate internal standard resulted in significant analytical biases for all target analytes. The accuracy of results, based on solvent calibration was highly variable and dependent on the serum and plasma matrices, the specific target analyte [{sup 13}C{sub 2}]-PFOA, [{sup 13}C{sub 2}]-PFNA, or [{sup 18}O{sub 2}]-PFOS, the target analyte concentration, the LC/MS-MS instrumentation used in data generation, and the specific surrogate internal standard used in quantification. These results suggest that concentrations of PFCs reported for human blood using surrogate internal standards in combination with external solvent calibration can be inaccurate unless biases are accounted for in data quantification.

  2. 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.

  3. Human papillomavirus (HPV detected in restored plasma DNA from women diagnosed with pre-invasive lesions and invasive cervical cancer

    Directory of Open Access Journals (Sweden)

    Yazmín Rocío Arias

    2010-06-01

    Full Text Available Objective: To improve the sensitivity of Human Papillomavirus (HPV detection in plasma from high-grade cervical neoplasia patients (CIN III and cervical cancer (CC evaluating any likely correlation with disease stage. Method: We subjected plasma DNA isolates from 112 patients (CIN and ICC to a pre-PCR restoration treatment to improve detection sensitivity. HPV-specific sequences were detected by conventional PCR both in cervical scrapes and plasma DNA obtained from each patient. For every single DNA sample, both non-restored and restored isolates were PCR analyzed. Results: We detected HPV in plasma DNA isolates with significantly higher efficiency on restored plasma-DNA as compared to each non-restored equivalent, still maintaining close correlation with the clinical stage of the cases. By analyzing plasma-DNA isolates we could classify as HPV positive >50.0% of the cases that were previously known to be positive from the cervical scrape based assay. Interestingly, 100% of the cases in which subtype HPV18 was detected in cervical scrapes were also positive in plasma DNA. Conclusions: Restoration of plasma DNA from cervical cancer patients allows a more sensitive PCR-based HPV detection, maintaining the correlation to disease stage traditionally observed.

  4. Human papillomavirus (HPV detected in restored plasma DNA from women diagnosed with pre-invasive lesions and invasive cervical cancer

    Directory of Open Access Journals (Sweden)

    Edward Fabián Carrillo

    2010-06-01

    Full Text Available Objective: To improve the sensitivity of Human Papillomavirus (HPV detection in plasma from high-grade cervical neoplasia patients (CIN III and cervical cancer (CC evaluating any likely correlation with disease stage.Method: We subjected plasma DNA isolates from 112 patients (CIN and ICC to a pre-PCR restoration treatment to improve detection sensitivity. HPV-specific sequences were detected by conventional PCR both in cervical scrapes and plasma DNA obtained from each patient. For every single DNA sample, both non-restored and restored isolates were PCR analyzed.Results: We detected HPV in plasma DNA isolates with significantly higher efficiency on restored plasma-DNA as compared to each non-restored equivalent, still maintaining close correlation with the clinical stage of the cases. By analyzing plasma-DNA isolates we could classify as HPV positive >50.0% of the cases that were previously known to be positive from the cervical scrape based assay. Interestingly, 100% of the cases in which subtype HPV18 was detected in cervical scrapes were also positive in plasma DNA.Conclusions: Restoration of plasma DNA from cervical cancer patients allows a more sensitive PCR-based HPV detection, maintaining the correlation to disease stage traditionally observed.

  5. Characterization of isomeric VX nerve agent adducts on albumin in human plasma using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Saeidian, Hamid; Mirkhani, Valioallah; Mousavi Faraz, Sajjad; Taghi Naseri, Mohammad; Babri, Mehran

    2015-01-01

    This study includes the characterization of isomeric VX organophosphorus adducts on albumin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). VX or its structural isomers were spiked into a vial containing plasma in order to obtain phosphorylated albumin. After pronase and trypsin digestion, the resulting solutions were analyzed to confirm adduct formation with the amino acid tyrosine on the albumin in human plasma. The LC-MS/MS experiments show that VX and its isomers can be attached to tyrosine on the albumin in human blood. Mass spectrometric studies revealed some interesting fragmentation pathways during the ionization process, such as ethylene, formic acid and ammonia elimination and an intermolecular electrophilic aromatic substitution reaction. The proposed mechanisms for the formation of the fragments were confirmed through the analysis of fragments of deuterated adducts.

  6. Determination of Ephedrine Alkaloids in Human Urine and Plasma by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study

    Science.gov (United States)

    Trujillo, William A.; Sorenson, Wendy R.

    2008-01-01

    A collaborative study was conducted to evaluate the accuracy and precision of a method for ephedrine-type alkaloids (i.e., norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine) in human urine and plasma. The amount of ephedrine-type alkaloids present was determined using liquid chromatography (LC) with tandem mass selective detection. The test samples were diluted to reflect a concentration of 5.00–100 ng/mL for each alkaloid. An internal standard was added and the alkaloids were separated using a 5 μm phenyl LC column with an ammonium acetate, glacial acetic acid, acetonitrile, and water mobile phase. Eight blind duplicates of human urine and eight blind duplicates of human plasma were analyzed by 10 collaborators. In addition to negative controls, test portions of urine and plasma were fortified at 3 different levels with each of the 6 ephedrine-type alkaloids at approximately 1, 2, and 5 μg/mL for urine and 100, 200, and 500 ng/mL for plasma. On the basis of the accuracy and precision results for this collaborative study, it is recommended that this method be adopted Official First Action for the determination of 6 different ephedrine-type alkaloids in human urine and plasma. PMID:14509420

  7. Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents.

    Science.gov (United States)

    Carr, A C; Hawkins, C L; Thomas, S R; Stocker, R; Frei, B

    2001-03-01

    Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.

  8. 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.

  9. Maldi-tof fingerprinting of seminal plasma lipids in the study of human male infertility.

    Science.gov (United States)

    Camargo, Mariana; Intasqui, Paula; de Lima, Camila Bruna; Montani, Daniela Antunes; Nichi, Marcílio; Pilau, Eduardo Jorge; Gozzo, Fabio Cesar; Lo Turco, Edson Guimarães; Bertolla, Ricardo Pimenta

    2014-09-01

    This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. For this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub-study 1.1), high and low sperm nuclear DNA fragmentation (sub-study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub-study 1.1 and 89 for sub-study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix-Assisted Laser Desorption Ionization Quadrupole-Time-of-Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters(®) MassLynx, and MetaboAnalyst online software was used for statistical analyses. For sub-studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS-DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5%. PLS-DA generated a list of 19, 24 and 23 VIP ions for sub-studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.

  10. Piperine Decreases Binding of Drugs to Human Plasma and Increases Uptake by Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Dubey, Raghvendra K; Leeners, Brigitte; Imthurn, Bruno; Merki-Feld, Gabriele Susanne; Rosselli, Marinella

    2017-09-26

    We previously reported that piperine, an active alkaloidal principal of black and long peppers, enhances drug bioavailability by inhibiting drug metabolism. Another mechanism influencing drug availability/uptake is its free fraction. Since piperine is highly lipophilic, we hypothesize that it could also interact with drugs through binding displacement and influence their bioavailability. Accordingly, using equilibrium dialysis, we investigated whether piperine alters the binding of model drug ligands, that is flunitrazepam, diazepam, warfarin, salicylic acid, propranolol, lidocaine, and disopyramide to human plasma (n = 4). Since alterations in binding influence drug disposition, we also studied the effects of piperine on the uptake of plasma bound (3) H-propranolol and (14) C-warfarin by cultured bovine brain microvascular endothelial cells (BMECs). Piperine (1-1000 μM) increased the free fraction (fu) of both albumin and alpha-acid glycoprotein bound drugs in a concentration-dependent manner (p < 0.01). Moreover, piperine (10 μM) increased the uptake of (3) H-propranolol and (14) C-warfarin by BMECs (p < 0.01). In conclusion, our findings provide the first evidence that piperine displaces plasma bound drugs from both albumin and alpha-acid glycoprotein and facilitates drug uptake across biological membranes (e.g. BMEC). Moreover, it is feasible that piperine may similarly facilitate the transport of drugs into tissues, in vivo, and alter both pharmacokinetics and pharmacodynamics of administered drugs. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  11. 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%.

  12. Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

    Science.gov (United States)

    Song, Yanting; Takatsuki, Katsuya; Sekiguchi, Tetsushi; Funatsu, Takashi; Shoji, Shuichi; Tsunoda, Makoto

    2016-07-01

    This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.

  13. 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...

  14. Aqueous two phase system based on ionic liquid for isolation of quinine from human plasma sample.

    Science.gov (United States)

    Flieger, J; Czajkowska-Żelazko, A

    2015-01-01

    Aqueous two phase system was applied for selective extraction of quinine from human plasma. Bi-phase was constructed from ionic liquid: butyl-methyl-imidazolium chloride after addition kosmotropic salts K₃PO₄ or KH₂PO₄. Quinine was determined in plasma samples after drinking of tonic containing quinine. Determination was performed by HPLC on 5-μm Zorbax SB-CN column and eluent containing 40% acetonitrile (v/v), 20 mM phosphate buffer at pH 3 and 40 mM NaPF₆ using external standard method. The spectrophotometric detection was set λ=214 nm. Selective fluorescence detection was performed at excitation of 325 nm and emission of 375 nm. Proposed strategy provides suitable sample purification and gives extraction yields in the range of 89-106%. The determination coefficient (R(2)) has a value ≥0.997 in the range of 50-800 ng/ml quinine concentration. The limit of quantification was set at 27.9 ng/ml and the detection limit was found to be 8.4 ng/ml under fluorescence detection.

  15. "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.

  16. [Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268].

    Science.gov (United States)

    Zhalimov, V K; Sklifas, A N; Kukushkin, N I

    2012-01-01

    It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.

  17. Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection.

    Science.gov (United States)

    Kontrimaviciūte, Violeta; Larroque, Michel; Briedis, Vitalis; Margout, Delphine; Bressolle, Françoise

    2005-08-05

    A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.

  18. The interaction of the carbon nanoparticles with human cell plasma membrane

    Science.gov (United States)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

  19. On-chip purification and detection of hepatitis C virus RNA from human plasma.

    Science.gov (United States)

    Vaghi, V; Potrich, C; Pasquardini, L; Lunelli, L; Vanzetti, L; Ebranati, E; Lai, A; Zehender, G; Mombello, D; Cocuzza, M; Pirri, C F; Pederzolli, C

    2016-01-01

    Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. A simple sample preparation method for measuring amoxicillin in human plasma by hollow fiber centrifugal ultrafiltration.

    Science.gov (United States)

    Dong, Wei-Chong; Hou, Zi-Li; Jiang, Xin-Hui; Jiang, Ye

    2013-02-01

    A simple sample preparation method has been developed for the determination of amoxicillin in human plasma by hollow fiber centrifugal ultrafiltration (HF-CF-UF). A 400-μL plasma sample was placed directly into the HF-CF-UF device, which consisited of a slim glass tube and a U-shaped hollow fiber. After centrifugation at 1.25 × 10(3) g for 10 min, the filtrate was withdrawn from the hollow fiber and 20 µL was directly injected into the high-performance liquid chromatography (HPLC) for analysis. The calibration curve was linear over the range of 0.1-20 µg/mL (r = 0.9996) and the limit of detection was as low as 0.025 µg/mL. The average recovery and absolute recovery were 99.9% and 84.5%, respectively. Both the intra-day and inter-day precisions (relative standard deviation) were less than 3.1% for three concentrations (0.25, 2.5 and 10 µg/mL). The sample preparation process was simplified. Only after a single centrifugal ultrafiltration can the filtrate be injected directly into HPLC. The present method is simple, sensitive and accurate. It could be effective for the analysis of biological samples with high protein contents, especially for the biopharmaceutical analysis of drugs that use traditional isolation techniques for sample preparation such as the protein precipitation method.

  3. Sensitive quantification of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Hofmann, Ute; Schwab, Matthias; Treiber, Gerd; Klotz, Ulrich

    2006-02-02

    A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC-electrospray mass spectrometry. Following liquid-liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH(+) ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.

  4. Determination of triamterene in human plasma and urine after its cloud point extraction

    Directory of Open Access Journals (Sweden)

    Ahad Bavili Tabrizi

    2014-01-01

    Full Text Available A new analytical approach was developed involving cloud point extraction (CPE and spectrofluorimetric determination of triamterene (TM in biological fluids. A urine or plasma sample was prepared and adjusted to pH 7, then TM was quickly extracted using CPE, using 0.05% (w/v of Triton X-114 as the extractant. The main factors that affected the extraction efficiency (the pH of the sample, the Triton X-114 concentration, the addition of salt, the extraction time and temperature, and the centrifugation time and speed were studied and optimized. The method gave calibration curves for TM with good linearities and correlation coefficients (r higher than 0.99. The method showed good precision and accuracy, with intra- and inter-assay precisions of less than 8.50% at all concentrations. Standard addition recovery tests were carried out, and the recoveries ranged from 94.7% to 114%. The limits of detection and quantification were 3.90 and 11.7 µg L-1, respectively, for urine and 5.80 and 18.0 µg L-1, respectively, for plasma. The newly developed, environmentally friendly method was successfully used to extract and determine TM in human urine samples.

  5. Validated microbiological and HPLC methods for the determination of moxifloxacin in pharmaceutical preparations and human plasma

    Directory of Open Access Journals (Sweden)

    Ahmed A. Abdelaziz

    2012-12-01

    Full Text Available The article presents a comparison between microbiological and high performance liquid chromatographic (HPLC assays for quantification of moxifloxacin in tablets, ophthalmic solutions and human plasma. The microbiological method employed a cylinder-plate agar diffusion assay using a strain of Esherichia coli ATCC 25922 as the test organism and phosphate buffer (pH8 as the diluent. The calibration curves were linear (R²> 0.98 over a concentration range of 0.125 to 16 µgml-1. The within day and between days precisions were 0.999 over the range of 0.125 to 16 µg ml-1. The within day and between days precisions were < 4.07% and < 5.09% respectively. Recovery values were between 97.7 and 107.6%. Similar potencies were obtained after the analysis of moxifloxacin tablets and ophthalmic solutions by both methods. Also pharmacokinetic parameters were calculated after the analysis of plasma samples of six male healthhy volunteers by both validated methods.

  6. Differential expression of SLAMS and other modulatory molecules by human plasma cells during normal maturation.

    Science.gov (United States)

    Rodríguez-Bayona, Beatriz; Ramos-Amaya, Ana; Brieva, José A

    2011-01-30

    Plasma cells (PCs) are specialized in antibody (Ab) production and they are, therefore, responsible for maintaining humoral immune responses. The human PC compartment is heterogeneous. PCs from inductive secondary lymphoid organs and from peripheral blood (PB) show less capability for prolonged survival and Ab production than bone marrow (BM) PCs, a pool consisting of fully mature cells. The HLDA9 workshop has allowed the use of labeled-monoclonal Abs (moAbs) recognizing a variety of recently identified lymphocyte modulatory surface receptors. In this study, flow cytometry analysis has been used to define the presence of these receptors on human PCs obtained from human tonsil (as an example of inductive organ), from PB and from BM. It was found that human PCs commonly expressed SLAMF1 (CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF6 (CD352) and SLAMF7 (CD319), but not SLAMF4 (CD244). In addition, PCs distinctively showed a low level of SLAMF5 (CD84) and a very high level of SLAMF7 expression in comparison with earlier stages of B cell maturation. All PC subsets exhibited a similar pattern of expression of SLAMF receptors suggesting a stage-dependent role for these proteins. In addition, most circulating PCs clearly expressed TNFRSF14 (CD270), BTLA (CD272), B7-1 (CD80) and B7-2 (CD86), and a substantial fraction of them were also positive for TNFRSF18 (CD357), FCRL1 (CD307a) and LAIR-1 (CD305). In contrast, tonsil and BM PCs only exhibited partial expression of TNFRSF14 and B7-2, a pattern of molecular expression similar to that detected on germinal center (GC) B cells. Present results indicate that human PCs exhibit a common pattern of SLAMF proteins, but differ in the rest of the receptors examined; this difference might be associated with their distinctive homing and functional requirements. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Direct quantification of brown algae-derived fucoidans in human plasma by a fluorescent probe assay

    CERN Document Server

    Warttinger, Ulrich; Harenberg, Job; Krämer, Roland

    2016-01-01

    Fucoidan is a generic term for a class of fucose rich, structurally diverse sulfated polysaccharides that are found in brown algae and other marine organisms. Depending on the species from which the fucoidan is extracted, a wide variety of biological activities including antitumor, antiinflammatory, immune-modulating, antiviral, antibacterial and pro- and anticoagulant activities has been described. Fucoidans have the advantage of low toxicity and oral bioavailibiity and are viable drug candidates, preclinical and pilot clinical trials show promising results. The availability of robust assays, in particular for analysing the blood levels of fucoidan, is a fundamental requirement for pharmacokinetic analysis in drug development projects. This contribution describes the application of a commercially availbale, protein-free fluorescent probe assay (Heparin Red) for the direct quantification of several fucoidans (from Fucus vesiculosus, Macrocystis pyrifera, and Undaria pinnatifida) in human plasma. By only minor...

  8. 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.

  9. Tb(Ⅲ) Speciation in Human Blood Plasma by Computer Simulation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A multi-phase model was developed and Tb(Ⅲ) speciation in human blood plasma was studied. At a concentration below 3.744×10-4mol/L (or at the concentration), Tb(Ⅲ) is mostly bound to phosphate to form precipitate of TbPO4. As the concentration of Tb(Ⅲ) increases,phosphate is exceeded and another kind of precipitate of Tb2(CO3)3 appears. Among soluble Tb(Ⅲ) species, Tb(Ⅲ) mainly distribute in [Tb (Tf)] at low concentration and in [Tb (HSA)], [Tb2(Tf], [Tb (IgG)], [Tb (Lactate)]2+, [Tb (CitArgH)] and free Tb(Ⅲ) at high concentration.

  10. Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for barnidipine in human plasma.

    Science.gov (United States)

    Pawula, M; Watson, D; Teramura, T; Watanabe, T; Higuchi, S; Cheng, K N

    1998-11-20

    A sensitive and specific LC-MS-MS assay has been developed and validated for barnidipine (1-benzyl-3-pyrrolidinyl)methyl-2,6-dimethyl-4(m-nitrophenyl)-1,4-dihydr opyridine-3,5-dicarboxylate). The assay involves a simple and rapid solid-phase extraction procedure. Sample analysis was on a Spherisorb S3ODS2 100 mmX2 mm I.D. column, with a Finnigan TSQ 7000 mass spectrometer, using an electrospray interface and selective reaction monitoring (SRM). The intra- and inter-day precision and accuracy, determined as the coefficient of variation and relative error, respectively, were 11.8% or less. The limit of quantitation was 0.03 ng/ml, and the calibration was linear between 0.03 and 3.0 ng/ml. The method has been used successfully for the measurement of over two thousand human plasma samples from pharmacokinetic clinical trials.

  11. Feasibility analysis of a Plasma Focus neutron source for BNCT treatment of transplanted human liver

    Science.gov (United States)

    Benzi, V.; Mezzetti, F.; Rocchi, F.; Sumini, M.

    2004-01-01

    Boron Neutron Capture Therapy preliminary treatments on transplanted human liver have been recently conducted at Pavia University. The need of high fluences of thermal neutrons imposed the use of the available thermal channel of a TRIGA reactor properly modified for this application. We analyse the possibility of using the Plasma Focus (PF) machine as a pulsed neutron source for this medical application instead of a nuclear reactor. Thermalization of the fast (2.45 MeV for D-D reactions) neutrons produced by the PF is gained with a paraffin or polyethylene moderator which contains both the neutron source and the irradiation chamber. The design parameters of a PF optimized for such an application are discussed, as well as other considerations on the advantages that this machine can bring to this kind of cancer therapy.

  12. Computer Simulation for Effect of Tb3+ on Ca2+Speciation in Human Plasma

    Institute of Scientific and Technical Information of China (English)

    卢兴; 王悦; 张海元; 王进平; 牛春吉; 倪嘉缵

    2002-01-01

    Effect of Tb3+ on Ca2+ speciation in human plasma was studied by means of the computer program of MINTEQA2. When Tb3+ ions are not added into the system, Ca2+ ions mostly distribute in free Ca2+ (74.7%) and the surplus distributes in Ca2+ complexes, such as [CaHCO3]+(7.9%), [Ca(Lac)]+(6.4%), CaHPO4 (1.3%), [CaHistidinateThreoninateH3]3+(2.4%), [CaCitrateHistidinateH2] (2.3%) and CaCO3(1.1%). Tb3+ can compete with Ca2+ for inorganic as well as biological ligands. An increase of concentration of Tb3+ in the system results in an increase of content of free Ca2+ and a decrease of contents of Ca2+ complexes.

  13. A NEW VALIDATED RP- HPLC METHOD FOR DETERMINATION OF PENCICLOVIR IN HUMAN PLASMA

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    VENKATA KUMAR, ANANTAHAKUMAR, SESHAGIRI RAO

    2013-09-01

    Full Text Available A rapid, selective and sensitive high performance liquid chromatographic method for the estimation of Penciclovir in human plasma has been developed. Chromatography was carried out on a Hypersil BDS C18 column using a mixture of 20mM Potassium dihydrogen phosphate buffer (pH 3.5 + 0.05 and methanol (95:5 v/v as the mobile phase. The eluents were monitored for the drug by UV detection at 254 nm. Acyclovir was used as an internal standard for this study. The retention times for Penciclovir and Acyclovir were found to be 6.2 and 9.2 min respectively. The method was found to be linear in the concentration range of 50.1 ng/mL to 7000.9 ng/mL. The method was validated as per FDA guidelines and was found to be suitable for bioequivalence and pharmacokinetic studies.

  14. Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

    Energy Technology Data Exchange (ETDEWEB)

    Carter, M.G.; Shukla, S.D. (Univ. of Missouri School of Medicine, Columbia (USA))

    1987-05-01

    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 {sup 32}P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 {times} 10{sup {minus}7} M PAF at 37{degree}C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for {sup 32}P-phosphoinositides. The percent stimulation of {sup 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.

  15. Development and validation for estimation of eplerenone of RP-HPLC method in spiked human plasma

    Institute of Scientific and Technical Information of China (English)

    Paraag Gide; Sandeep Sonawane; Abhishek Chitnis

    2012-01-01

    A rapid and simple high performance liquid chromatography (HPLC) mcthod wiih 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 um) 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 I/X.

  16. Analysis of nabumetone in human plasma by HPLC. Application to single dose pharmacokinetic studies.

    Science.gov (United States)

    Kobylińska, Kamila; Barlińska, Małgorzata; Kobylińska, Maria

    2003-06-01

    A simple and sensitive high performance liquid chromatography method for the determination of nabumetone in human plasma is described. The procedure involves liquid-liquid extraction with ethyl acetate and reversed-phase chromatography with fluorimetric detection (excitation 230 nm, emission 356 nm). The chromatographic conditions and the extraction procedure gave a clean chromatogram for the compound. The limit of quantitation was established as 0.313 ng/ml and the calibration curve was linear up to 20 ng/ml. The within-day and between-day relative standard deviations were less than 10% and the accuracy of the assay was in the range of 99-104%. The suitability of the method is shown for pharmacokinetic studies.

  17. Caffeine intake increases plasma ketones: an acute metabolic study in humans.

    Science.gov (United States)

    Vandenberghe, Camille; St-Pierre, Valérie; Courchesne-Loyer, Alexandre; Hennebelle, Marie; Castellano, Christian-Alexandre; Cunnane, Stephen C

    2017-04-01

    Brain glucose uptake declines during aging and is significantly impaired in Alzheimer's disease. Ketones are the main alternative brain fuel to glucose so they represent a potential approach to compensate for the brain glucose reduction. Caffeine is of interest as a potential ketogenic agent owing to its actions on lipolysis and lipid oxidation but whether it is ketogenic in humans is unknown. This study aimed to evaluate the acute ketogenic effect of 2 doses of caffeine (2.5; 5.0 mg/kg) in 10 healthy adults. Caffeine given at breakfast significantly stimulated ketone production in a dose-dependent manner (+88%; +116%) and also raised plasma free fatty acids. Whether caffeine has long-term ketogenic effects or could enhance the ketogenic effect of medium chain triglycerides remains to be determined.

  18. Long-term performance and stability of molecular shotgun lipidomic analysis of human plasma samples.

    Science.gov (United States)

    Heiskanen, Laura A; Suoniemi, Matti; Ta, Hung Xuan; Tarasov, Kirill; Ekroos, Kim

    2013-09-17

    The stability of the lipid concentration levels in shotgun lipidomics analysis was tracked over a period of 3.5 years. Concentration levels in several lipid classes, such as phospholipids, were determined in human plasma lipid extracts. Impact of the following factors on the analysis was investigated: sample amount, internal standard amount, and sample dilution factor. Moreover, the reproducibility of lipid profiles obtained in both polarity modes was evaluated. Total number of samples analyzed was approximately 6800 and 7300 samples in negative and positive ion modes, respectively, out of which 610 and 639 instrument control samples were used in stability calculations. The assessed shotgun lipidomics approach showed to be remarkably robust and reproducible, requiring no batch corrections. Coefficients of variation (CVs) of lipid mean concentration measured with optimized analytical parameters were typically less than 15%. The high reproducibility indicated that no lipid degradation occurred during the monitored time period.

  19. DETERMINATION OF FLUCONAZOLE IN HUMAN PLASMA BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.

    Science.gov (United States)

    Safaei, Zahra; Alipour, Eskandar; Shafaati, Alireza; Zarghp, Afshin

    2015-01-01

    A rapid, simple and reproducible high performance liquid chromatographic method was developed and validated for determination of fluconazole in human plasma. The separation was performed on MZ C8 column (125 x 4 mm, 5 µm) using acetonitrile - potassium dihydrogen phosphate buffer (15 : 85, v/v), pH 3.0, as the mobile phase at a flow rate of 1.5 mL/min. The wavelength was set at 261 nm. The assay enables the measurement of fluconazole for therapeutic drug monitoring with a minimum quantification limit of 20 ng/mL. The method involves simple, protein precipitation procedure and analytical recovery was complete. The calibration curve was linear over the concentration range 0.1-4 µg/mL. The coefficients of variation for inter-day and intra-day assay were found to be less than 10%.

  20. Effects of pirenzepine on Dai-kenchu-to-induced elevation of the plasma neuropeptide levels in humans.

    Science.gov (United States)

    Sato, Yuhki; Inoue, Shin; Katagiri, Fumihiko; Itoh, Hiroki; Takeyama, Masaharu

    2006-01-01

    Dai-kenchu-to has been used for the treatment of abdominal obstructions, including bowel obstructions and a feeling of coldness in the abdomen. We reported that Dai-kenchu-to increases plasma neuropeptide [motilin, vasoactive intestinal polypeptide (VIP), serotonin, calcitonin gene-related peptide (CGRP), and substance P]-like immunoreactive substances (IS) levels and that its pharmacologic effects on the gastrointestine are due to changes in gastrointestinal mucosa-regulatory peptide levels. We examined the effects of the selective M(1) muscarinic receptor antagonist pirenzepine on the elevation of Dai-kenchu-to-induced plasma neuropeptide (gastrin, motilin, somatostatin, VIP, CGRP, substance P)-IS levels in human volunteers and the area under the plasma neuropeptide concentration-time curve from 0 to 240 min (AUC(0-->240 min)), which were calculated from the plasma neuropeptide concentration-time curves from each volunteers. Oral pretreatment with pirenzepine reduced the Dai-kenchu-to-induced elevation of plasma motilin and VIP-IS levels and AUC(0-->240 min). Combined treatment with Dai-kenchu-to and pirenzepine increased plasma somatostatin-IS levels and decreased plasma gastrin-IS levels and had no effects on plasma CGRP- and substance P-IS levels and AUC(0-->240 min) compared with administration of Dai-kenchu-to alone. Dai-kenchu-to appeared to induce the release of motilin and VIP into plasma mainly through the activation of M(1) muscarinic receptors, and pirenzepine may affect the pharmacologic action of Dai-kenchu-to by elevation of plasma motilin and VIP levels.

  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. 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.

  3. Determination of lamivudine in human plasma by HPLC and its use in bioequivalence studies.

    Science.gov (United States)

    Kano, Eunice Kazue; dos Reis Serra, Cristina Helena; Koono, Eunice Emiko Mori; Andrade, Simone Schramm; Porta, Valentina

    2005-06-13

    A simple, accurate, precise and sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed to quantificate lamivudine (3-TC) in human plasma samples from bioequivalence studies. 3-TC and stavudine (internal standard, I.S.) were extracted from 0.5 ml of human plasma by acetonitrile protein precipitation. The method was validated over a concentration range of 0.05-3.00 microg/ml and used in a bioequivalence trial between two lamivudine formulations, to assess its usefulness in this kind of study. FURP-lamivudine (Fundação para o Remédio Popular, Brazil, as test formulation) and Epivir (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 150 mg oral dose to 24 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determined by calculating 90% confidence intervals (90% CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, using logarithmic transformed data. The 90% confidence intervals for the ratio of Cmax (0.86-1.06), AUC0-t (0.96-1.04) and AUC0-inf (0.97-1.05) values for the test and reference products are within the 0.80-1.25 interval proposed by FDA and EMEA. It was concluded that the two 3-TC formulations are bioequivalent in their rate and extent of absorption, and thus, may be used interchangeably.

  4. 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

  5. Separation and Determination of Methylnaltrexone in Human Plasma Samples After Oral Administration by HPLC Coupled with Electrochemical Detection

    Institute of Scientific and Technical Information of China (English)

    WANG An-bao; Joseph F. Foss; YUAN Chun-su; Joachim Osinski

    2005-01-01

    A high performance liquid chromatography(HPLC) method coupled with electrochemical detection and solid phase extraction is described for the separation and determination of methylnaltrexone(MNTX), a quaternary opioid antagonist, in human clinical plasma samples after oral administration. Linearity of the standard curve for MNTX was found in the range of 4.0_150 ng/mL and was statistically conformed. The correlation coefficient(r2) and calibration equation obtained from linear regression analysis are 0.9999 and Y=54.27X-0.22, where Y and X represent the peak area and concentration of MNTX, respectively. The detection limit of MNTX under the present experimental conditions is 2.0 ng/mL by estimating at a ratio of 3 of signal to noise. The mean recovery of MNTX in human plasma is higher than 97%. The analytical method was applied to the pharmacokinetic determination of MNTX after single dose oral administration. These data demonstrate that the change of MNTX plasma concentration versus time is obvious. MNTX level of plasma reaches to a plateau between 45 to 120 minutes and then falls slowly. The content of MNTX in plasma sample maintains at an obviously detectable level after twelve hours of oral administration. The pharmacokinetic parameters for a single dose of 19.2 mg/kg in plasma are cmax=206.42(±16.53) ng/mL and tmax=60 min.

  6. Validation of a simple gas chromatographic-mass spectrometric method for the determination of gamma-butyrolactone in human plasma.

    Science.gov (United States)

    Fukui, Yousuke; Matsusima, Eiji; Muramoto, Kouichi; Nagai, Nobutaka; Ohama, Koso; Yamashita, Kazumasa

    2003-02-25

    A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.

  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. Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

    Science.gov (United States)

    Yoon, Jeongheon; Wang, Hongsheng; Kim, Yong Chan; Yoshimoto, Momoko; Abbasi, Sadia; Morse Iii, Herbert C

    2016-09-01

    Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

  9. In vivo triglyceride synthesis in subcutaneous adipose tissue of humans correlates with plasma HDL parameters

    Science.gov (United States)

    Tuvdendorj, Demidmaa; Munoz, Alejandro O.; Ruiz-Barros, Viviana; Schwarz, Jean-Marc; Montalto, Giuseppe; Chandalia, Manisha; Sowers, Lawrence C.; Rizzo, Manfredi; Murphy, Elizabeth J; Abate, Nicola

    2016-01-01

    Backgrounds and aims Low concentrations of plasma HDL-C are associated with the development of atherosclerotic cardiovascular diseases and type 2 diabetes. Here we aimed to explore the relationship between the in vivo fractional synthesis of triglycerides (fTG) in subcutaneous (s.q.) abdominal adipose tissue (AT), HDL-C concentrations and HDL particle size composition in non-diabetic humans. Methods The fTG in s.q. abdominal AT was measured in 16 non-diabetic volunteers (7 women, 9 men; Age: 49±20 years; BMI: 31±5 kg/m; Fasting Plasma Glucose: 90±10 mg/dl) after 2H2O labeling. HDL-C concentration and subclasses, large (L-HDL), intermediate (I-HDL) and small (S-HDL) were measured. Results Linear regression analyses demonstrated significant associations of fTG with plasma concentration of HDL-C (r=0.625,p=0.009) and percent contribution of L-HDL (r=0.798,pHDL (r=−0.765,pHDL (r=−0.629, p=0.009). When analyses were performed by gender, the associations remained significant in women (HDL-C: r=0.822,p=0.023; L-HDL: r=0.892,p=0.007; I-HDL: r=−0.927,p=0.003) but not men. Conclusions Our study demonstrated an in vivo association between subcutaneous abdominal adipose tissue lipid dynamics and HDL parameters in humans, but this was true for women not men. Positive association with L-HDL and negative with I-HDL suggest that subcutaneous abdominal adipose tissue lipid dynamics may play an important role in production of mature functional HDL particles. Further studies evaluating the mechanism responsible for these associations and the observed gender differences are important and warranted to identify potential novel targets of intervention to increase the production of atheroprotective subclasses of HDL-Cs and thus decreasing the risks of development of atherosclerotic conditions. PMID:27323227

  10. 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.

  11. 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.

  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 (Polive oil consumption related to the protection of the endogenous antioxidant system.

  13. 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.

  14. The effect of different alcoholic beverages on blood alcohol levels, plasma insulin and plasma glucose in humans.

    Science.gov (United States)

    Nogueira, L C; Couri, S; Trugo, N F; Lollo, P C B

    2014-09-01

    In the present work we studied the effects of four alcoholic beverages on blood alcohol levels, plasma insulin concentrations and plasma glucose concentrations in men and women. The volunteers were healthy non-smokers and they were divided according to sex into two groups of ten individuals. The alcoholic beverages used in the study were beer, red wine, whisky and "cachaça". In men, ingestion of the distilled drinks promoted a spike in blood alcohol levels more quickly than ingestion of the fermented drinks. In women, beer promoted the lowest blood alcohol levels over the 6h of the experiment. Whisky promoted highest blood alcohol levels in both sexes. The ingestion of wine promoted a significant difference in relation to the blood alcohol concentration (BAC) as a function of gender. The ingestion of cachaça by women produced BAC levels significantly smaller than those obtained for wine.

  15. Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

    Science.gov (United States)

    Miller, L; Weissmüller, S; Ringler, E; Crauwels, P; van Zandbergen, G; Seitz, R; Waibler, Z

    2015-08-01

    Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

  16. Room temperature synthesis and solid-state structure of Ni 2P 2O 6·12H 2O

    Science.gov (United States)

    Haag, J. M.; LeBret, G. C.; Cleary, D. A.; Twamley, B.

    2005-04-01

    The synthesis and crystal structure of Ni 2P 2O 6·12H 2O are reported. The compound was synthesized from aqueous solutions of nickel chloride and sodium hypodiphosphate at room temperature. The space group is orthorhombic, Pnnm. Unit cell dimensions are a=11.2418(5) Å, b=18.5245(8) Å, and c=7.3188(3) Å, Z=4. The product is unstable with respect to dehydration above room temperature.

  17. High performance liquid chromatography for the determination of glucosamine sulfate in human plasma after derivatization with 9-fluorenylmethyl chloroformate.

    Science.gov (United States)

    Huang, Tao-min; Deng, Chun-Hui; Chen, Nian-zhu; Liu, Zhen; Duan, Geng-Li

    2006-10-01

    In this study, we developed a simple, rapid, sensitive, and reliable method for the determination of glucosamine sulfate in human plasma, which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD. For the first time, FMOC-Cl was introduced into derivatization of glucosamine sulfate in human plasma. The amino groups of glucosamine sulfate and vertilmicin sulfate (the internal standard) were trapped with FMOC-Cl to form glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts, which can be very suitable for HPLC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150 x 4 mm id, 5 microm) with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1 mL/min. The retention times of glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts were 8.9 and 21.2 min, respectively. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 15 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-10 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 5.2-8.1% and 6.1- 8.5%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 90%. The validated method was successfully applied to the determination of glucosamine sulfate in human plasma samples.

  18. Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study.

    Science.gov (United States)

    Główka, Franciszek K; Karaźniewicz-Łada, Marta

    2007-06-01

    A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.

  19. Thermal decomposition of Pr[(C5H8NS2)3(C12H8N2)

    Institute of Scientific and Technical Information of China (English)

    GUO; Pengjiang; JIAO; Baojuan; CHEN; Sanping; HU; Rongzu

    2005-01-01

    The thermal behavior of the complex Pr[(C5H8NS2)3(C12H8N2)] in a dry nitrogen flow was examined by TG-DTG analysis. The TG-DTG investigations indicated that Pr[(C5H8NS2)3-(C12H8N2)] was decomposed into Pr2S3 and deposited carbon in one step where Pr2S3 predominated in the final products. The results of non-isothermal kinetic calculations showed that the decomposition stage was the random nucleation and subsequent growth mechanism (n =nential constant In[A/s] was 7.8697. The empirical kinetics model equation was proposed as f(α) =3/2(1-α)[-ln(1-α)]1/3. The X-ray powder diffraction patterns of the thermal decomposition products at 800℃ under N2 atmosphere show that the product can be indexed to the cubic Pr2S3 phase. The transmission electron microscopy (TEM) of the final product reveals the particle appearance of a diameter within 40 nm. The experimental results show that the praseodymium sulfide nanocrystal can be prepared from thermal decomposition of Pr[(C5H8NS2)3(C12H8N2)].

  20. Modulatory Effect of Human Plasma on the Internal Nanostructure and Size Characteristics of Liquid-Crystalline Nanocarriers

    DEFF Research Database (Denmark)

    Mat Azmi, Intan Diana Binti; Wu, Linping; Wibroe, Peter Popp

    2015-01-01

    and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Through an integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA) we have studied the time-dependent effect of human plasma (and...

  1. 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.

  2. Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion trap mass spectrometer

    Science.gov (United States)

    Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PC. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PC. This approach is comprised of two mass spectr...

  3. 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.

  4. 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.

  5. A single method for detecting 11 organophosphate pesticides in human plasma and breastmilk using GC-FPD.

    Science.gov (United States)

    Naksen, Warangkana; Prapamontol, Tippawan; Mangklabruks, Ampica; Chantara, Somporn; Thavornyutikarn, Prasak; Robson, Mark G; Ryan, P Barry; Barr, Dana Boyd; Panuwet, Parinya

    2016-07-01

    Organophosphate (OP) pesticides are widely used for crop protection in many countries including Thailand. Aside from causing environmental contamination, they affect human health especially by over-stimulating of the neurotransmission system. OP pesticides, as with other non-persistent pesticides, degrade quickly in the environment as well as are metabolized quite rapidly in humans. Assessing human exposures to these compounds requires analytical methods that are sensitive, robust, and most importantly, suitable for specific laboratory settings. The aim of this study was to develop and validate an analytical method for measuring 11 OP pesticide residues in human plasma and breast milk. Analytes in both plasma and breast milk samples were extracted with acetone and methylene chloride, cleaned-up using aminopropyl solid phase extraction cartridges, and analyzed by gas chromatography with flame photometric detection. The optimized method exhibited good linearity, with the coefficients of determination of 0.996-0.999 and pesticides measured, seven were detected in plasma samples and five were detected in breast milk samples. Mass spectrometry was used to confirm results. Overall, this method is rapid and reliable. It offers the laboratories with limited access to mass spectrometry a capacity to investigate levels OP pesticides in plasma and breastmilk in those occupationally exposed for health risk assessment. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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 ...

  7. Iron(III)-chelating resins. X. Iron detoxification of human plasma with iron(III)-chelating resins

    NARCIS (Netherlands)

    Feng, M.; Feng, M.H.; van der Does, L.; Bantjes, A.; Bantjes, A.

    1994-01-01

    Iron detoxification of human blood plasma was studied with resins containing desferrioxamine B (DFO) or 3-hydroxy-2-methyl-4(1H)-pyridinone (HMP) as iron(III)-chelating groups. The behaviour of four resins was investigated: DFO-Sepharose, HMP-Sepharose and crosslinked copolymers of

  8. Hemolytic plate assay for quantification of active human complement component C3 using methylamine-treated plasma as complement source

    DEFF Research Database (Denmark)

    Ploug, M; Jessen, T E; Welinder, K. G.

    1985-01-01

    A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbi...

  9. Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier.

    Science.gov (United States)

    Noguchi, Saki; Nishimura, Tomohiro; Fujibayashi, Ayasa; Maruyama, Tetsuo; Tomi, Masatoshi; Nakashima, Emi

    2015-09-01

    Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [(3) H]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a Km of 20 μM, and was increased in the absence of chloride. [(3) H]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. 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.

  11. Complex formation between human prostate-specific antigen and protease inhibitors in mouse plasma.

    Science.gov (United States)

    Hekim, Can; Riipi, Tero; Zhu, Lei; Laakkonen, Pirjo; Stenman, Ulf-Håkan; Koistinen, Hannu

    2010-04-01

    When secreted from the prostate, most of prostate-specific antigen (PSA) is free and enzymatically active. Upon reaching circulation, active PSA is inactivated by complex formation with protease inhibitors. To justify the use of mouse models for evaluation of the function of PSA and for studies on therapeutic modalities based on modulation of PSA activity, it is important to know whether PSA complexation is similar in mouse and man. To characterize the circulating forms of PSA in mouse, we used subcutaneous LNCaP and 22RV1 human prostate cancer cell xenograft tumor models. We also added PSA directly to mouse serum. Free and total PSA were measured by immunoassay, and PSA complexes were extracted by immunopurification followed by SDS-PAGE, in-gel trypsin digestion and identification of signature peptides by mass spectrometry. In mice bearing xenograft tumors, 68% of the immunoreactive PSA occurred in complex, and when added to mouse serum, over 70% of PSA forms complexes that comprises alpha(2)-macroglobulin and members of the alpha(1)-antitrypsin (AAT) family. In mouse plasma, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than alpha(1)-antichymotrypsin, which is the main complex forming serpin in man. The complex formation of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the complexation of PSA. (c) 2009 Wiley-Liss, Inc.

  12. Establishing human heart chromium, cobalt and vanadium concentrations by inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Day, Patrick L; Eckdahl, Steven J; Maleszewski, Joseph J; Wright, Thomas C; Murray, David L

    2017-05-01

    Chromium, cobalt, and vanadium are used in metallic joint prosthesis. Case studies have associated elevated heart tissue cobalt concentrations with myocardial injury. To document the long term heart metal ion concentrations, a validated inductively coupled plasma mass spectroscopy (ICP-MS) method was needed. The method utilized a closed-vessel microwave digestion system to digest the samples. An ICP-MS method utilizing Universal Cell Technology was used to determine our target analyte concentrations. Accuracy was verified using reference materials. Precision, sensitivity, recovery and linearity studies were performed. This method was used to establish a reference range for a non-implant containing cohort of 80 autopsy human heart tissues RESULTS: This method demonstrated an analytic measurement range of 0.5-100ng/mL for each element. Accuracy was within ±10% of target value for each element. Within-run precision for each element was below 20% CV. The chromium, vanadium and cobalt concentrations (mean±SD) were 0.1523±0.2157μg/g, 0.0094±0.0211μg/g and 0.1039±0.1305μg/g respectively in 80 non-implant containing human heart tissue samples. This method provides acceptable recovery of the chromium, cobalt and vanadium in heart tissue; allowing assessment of the effects of metallic joint prosthesis on myocardial health. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium.

    Science.gov (United States)

    Zhu, Wei; Teel, George; O'Brien, Christopher M; Zhuang, Taisen; Keidar, Michael; Zhang, Lijie Grace

    2015-01-01

    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 bio-mimetic 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.

  14. 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

  15. The influence of insulin on the raised plasma fibronectin concentration in human obesity

    DEFF Research Database (Denmark)

    Dejgård, A; Andersen, T; Gluud, C

    1986-01-01

    Plasma concentrations of fibronectin, free insulin, C-peptide and plasma glucose were determined in 40 morbidly obese subjects and in 51 normal weight controls, matched for sex and age. All plasma concentrations were significantly elevated (p less than 0.01) among the obese subjects. A significant...

  16. Toxicokinetics of colchicine in humans: analysis of tissue, plasma and urine data in ten cases.

    Science.gov (United States)

    Rochdi, M; Sabouraud, A; Baud, F J; Bismuth, C; Scherrmann, J M

    1992-11-01

    1. A specific and sensitive radioimmunoassay was used to study the toxicokinetics of colchicine in seven cases of acute human poisoning. Post-mortem tissue concentrations of colchicine were measured in three further cases. Depending on the time of patient admission, two disposition processes could be observed. The first, in three patients, admitted early, showed a bi-exponential plasma colchicine decrease, with distribution half-lives of 30, 45 and 90 min. The second, in four patients, admitted late, showed a mono-exponential decrease. Plasma terminal half-lives ranged from 10.6 to 31.7 h for both groups. 2. Pharmacokinetic analysis of urine colchicine data was performed for two patients. The fraction of unchanged colchicine excreted in urine was about 30%, renal clearance was about 13 l h-1 and three-fold less than total body clearance (39 l h-1). The apparent volume of distribution was 21 l kg-1. 3. Post-mortem tissue analysis showed an ubiquitous colchicine distribution. Colchicine accumulated at high concentrations in the bone marrow (more than 600 ng g-1), testicle (400 ng g-1), spleen (250 ng g-1), kidney (200 ng g-1), lung (200 ng g-1) and heart (95 ng g-1); it was also found in the brain (125 ng g-1). 4. This toxicokinetic study shows that after massive ingestion, the disposition parameters and kinetics of colchicine are not markedly modified from those occurring in healthy volunteers. The absorption process was not delayed and the distribution and elimination half-lives were in the range known to occur with therapeutic doses.

  17. 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.

  18. A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties.

    Science.gov (United States)

    Wu, Lin; Wu, Jinhui; Zhang, Jian; Zhou, Yuanyuan; Ren, Guoyan; Hu, Yiqiao

    2008-05-01

    A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.

  19. Characterization of thermoplastic microfiltration chip for the separation of blood plasma from human blood.

    Science.gov (United States)

    Chen, Pin-Chuan; Chen, Chih-Chun; Young, Kung-Chia

    2016-09-01

    In this study, we developed a fully thermoplastic microfiltration chip for the separation of blood plasma from human blood. Spiral microchannels were manufactured on a PMMA substrate using a micromilling machine, and a commercial polycarbonate membrane was bonded between two thermoplastic substrates. To achieve an excellent bonding between the commercial membrane and the thermoplastic substrates, we used a two-step injection and curing procedure of UV adhesive into a ring-shaped structure around the microchannel to efficiently prevent leakage during blood filtration. We performed multiple filtration experiments using human blood to compare the influence of three factors on separation efficiency: hematocrit level (40%, 23.2%, and 10.9%), membrane pore size (5 μm, 2 μm, and 1 μm), and flow rate (0.02 ml/min, 0.06 ml/min, 0.1 ml/min). To prevent hemolysis, the pressure within the microchannel was kept below 0.5 bars throughout all filtration experiments. The experimental results clearly demonstrated the following: (1) The proposed microfiltration chip is able to separate white blood cells and red blood cells from whole human blood with a separation efficiency that exceeds 95%; (2) no leakage occurred during any of the experiments, thereby demonstrating the effectiveness of bonding a commercial membrane with a thermoplastic substrate using UV adhesive in a ring-shaped structure; (3) separation efficiency can be increased by using a membrane with smaller pore size, by using diluted blood with lower hematocrit, or by injecting blood into the microfiltration chip at a lower flow rate.

  20. 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

  1. 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.

  2. 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.......5% in plasma and 1.6% to 6.2% in urine. Between-day reproducibility ranged from 2.9% to 5.3% in plasma and 0.6% to 1.8% in urine. The inaccuracy expressed as bias ranged from -3.1% to 1.9% in plasma and from -7.2% to 0.7% in urine. The lower limit of quantification for metformin in plasma was 5 ng....../mL and in urine was 40 ng/mL. The method was therefore considered to be precise, accurate, reproducible, and sensitive enough to be appropriate for pharmacokinetic studies of metformin. The applicability of the method for human pharmacokinetic studies was demonstrated by dosing a healthy male volunteer with 500...

  3. Interaction between Pirenzepine and Ninjinto, a Traditional Japanese Herbal Medicine, on the Plasma Gut-Regulated Peptide Levels in Humans.

    Science.gov (United States)

    Sato, Yuhki; Hiroki, Itoh; Suzuki, Yosuke; Tatsuta, Ryosuke; Takeyama, Masaharu

    2013-01-01

    The Japanese herbal medicine (Kampo) Ninjinto has been used for the treatment of gastroenteritis, esogastritis, gastric atony, gastrectasis, vomiting, and anorexia. The pharmacological effects of Ninjinto on the gastrointestine are due to changes in the levels of gut-regulated peptide, such as motilin, somatostatin, calcitonin gene-related peptide (CGRP), substance P, and vasoactive intestinal polypeptide (VIP). The release of these peptides is controlled by acetylcholine (ACh) from the preganglionic fibers of the parasympathetic nerve. Thus, we examined the effects of the selective M1 muscarinic receptor antagonist pirenzepine on the elevation of Ninjinto-induced plasma the area under the plasma gut-regulated peptide concentration-time curve from 0 to 240 min (AUC0→240 min) in humans. Oral pretreatment with pirenzepine significantly reduced the Ninjinto-induced elevation of plasma motilin and substance P release (AUC0→240 min). Combined treatment with Ninjinto and pirenzepine significantly increased the release of plasma somatostatin (AUC0→240 min) compared with administration of Ninjinto alone or placebo. Ninjinto appeared to induce the release of substance P and motilin into plasma mainly through the activation of M1 muscarinic receptors, and pirenzepine may affect the pharmacologic action of Ninjinto by the elevation of plasma substance P, motilin, and somatostatin.

  4. Interaction between Pirenzepine and Ninjinto, a Traditional Japanese Herbal Medicine, on the Plasma Gut-Regulated Peptide Levels in Humans

    Directory of Open Access Journals (Sweden)

    Yuhki Sato

    2013-01-01

    Full Text Available The Japanese herbal medicine (Kampo Ninjinto has been used for the treatment of gastroenteritis, esogastritis, gastric atony, gastrectasis, vomiting, and anorexia. The pharmacological effects of Ninjinto on the gastrointestine are due to changes in the levels of gut-regulated peptide, such as motilin, somatostatin, calcitonin gene-related peptide (CGRP, substance P, and vasoactive intestinal polypeptide (VIP. The release of these peptides is controlled by acetylcholine (ACh from the preganglionic fibers of the parasympathetic nerve. Thus, we examined the effects of the selective M1 muscarinic receptor antagonist pirenzepine on the elevation of Ninjinto-induced plasma the area under the plasma gut-regulated peptide concentration-time curve from 0 to 240 min ( in humans. Oral pretreatment with pirenzepine significantly reduced the Ninjinto-induced elevation of plasma motilin and substance P release (. Combined treatment with Ninjinto and pirenzepine significantly increased the release of plasma somatostatin ( compared with administration of Ninjinto alone or placebo. Ninjinto appeared to induce the release of substance P and motilin into plasma mainly through the activation of M1 muscarinic receptors, and pirenzepine may affect the pharmacologic action of Ninjinto by the elevation of plasma substance P, motilin, and somatostatin.

  5. Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

    Science.gov (United States)

    Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

    2017-04-01

    Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated.

  6. Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE-ICP-MS.

    Science.gov (United States)

    Nguyen, Tam T T N; Østergaard, Jesper; Stürup, Stefan; Gammelgaard, Bente

    2013-02-01

    A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 μg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

  7. Evaluation of non-thermal plasma-induced anticancer effects on human colon cancer cells

    Science.gov (United States)

    Choi, Jae-Sun; Kim, Jeongho; Hong, Young-Jun; Bae, Woom-Yee; Choi, Eun Ha; Jeong, Joo-Won; Park, Hun-Kuk

    2017-01-01

    Non-thermal atmospheric-pressure plasma has been introduced in various applications such as sterilization, wound healing, blood coagulation, and other biomedical applications. The most attractive application of non-thermal atmospheric-pressure plasma is in cancer treatment, where the plasma is used to produce reactive oxygen species (ROS) to facilitate cell apoptosis. We investigate the effects of different durations of exposure to dielectric-barrier discharge (DBD) plasma on colon cancer cells using measurement of cell viability and ROS levels, western blot, immunocytochemistry, and Raman spectroscopy. Our results suggest that different kinds of plasma-treated cells can be differentiated from control cells using the Raman data. PMID:28663896

  8. A fast and sensitive method for the determination of nitrite in human plasma by capillary electrophoresis with fluorescence detection.

    Science.gov (United States)

    Wang, Xu; Adams, Erwin; Van Schepdael, Ann

    2012-08-15

    Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE-UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (-30 kV) were used to shorten the analysis time. The good separation was achieved with 20 mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4 min. The deproteinized plasma sample was injected hydrodynamically for 5s at -50 mbar into a 60 cm × 75 μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240-400 nm), and the emitted light was measured at 418 nm by the use of a cutoff filter. A good linearity (R(2)=0.9975) was obtained in the range from 2 to 500 nM. The detection limit of nitrite was 0.6 nM in original plasma samples, which is 750 times lower than our previous CE-UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.

  9. Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.

    Directory of Open Access Journals (Sweden)

    Rohini Narayanaswamy

    Full Text Available Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S-, 25-, 27-, 7α-hydroxycholesterol (HC and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential

  10. Formation of mono(ethylhexyl)phthalate from di(ethylhexyl)phthalate in human plasma stored in PVC bags and its presence in fractionated plasma proteins.

    Science.gov (United States)

    Vessman, J; Rietz, G

    1978-01-01

    The formation of mono(ethylhexyl)phthalate (MEHP) from di(ethylhexyl)phthalate in human plasma stored in bags of polyvinylchloride has been studied. Substantial amounts were formed and in ten bags from 4 to 56microgram/ml were found. After 2 weeks at room temperature the concentration of MEHP had increased to values between 27 and 79 microgram/ml. However, MEHP was also disappearing as shown in a recovery experiment. Of the fractionated proteins albumin contained MEHP in amounts from less than 3 to 290 microgram/g.

  11. 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

  12. The Influence of Probiotic Lactobacillus casei in Combination with Prebiotic Inulin on the Antioxidant Capacity of Human Plasma.

    Science.gov (United States)

    Kleniewska, Paulina; Hoffmann, Arkadiusz; Pniewska, Ewa; Pawliczak, Rafał

    2016-01-01

    The aim of the present study was to assess whether probiotic bacteria Lactobacillus casei (4 × 10(8) CFU) influences the antioxidant properties of human plasma when combined with prebiotic Inulin (400 mg). Experiments were carried out on healthy volunteers (n = 32). Volunteers were divided according to sex (16 male and 16 female) and randomly assigned to synbiotic and control groups. Blood samples were collected before synbiotic supplementation and after 7 weeks, at the end of the study. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) activity, and the ferric reducing ability of plasma (FRAP) in human plasma were examined. The administration of synbiotics containing L. casei plus Inulin resulted in a significant increase in FRAP values (p = 0.00008) and CAT activity (p = 0.02) and an insignificant increase in SOD and GPx activity compared to controls. Synbiotics containing L. casei (4 × 10(8) CFU) with prebiotic Inulin (400 mg) may have a positive influence on human plasma antioxidant capacity and the activity of selected antioxidant enzymes.

  13. Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study.

    Science.gov (United States)

    Zhang, Dujuan; Teng, Yanni; Chen, Keguang; Liu, Sha; Wei, Chunmin; Wang, Benjie; Yuan, Guiyan; Zhang, Rui; Liu, Xiaoyan; Guo, Ruichen

    2012-10-01

    A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C(18) reversed-phase column, eluted with mobile phase of acetonitrile-ammonium acetate (5 m m; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers.

  14. 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......, GLUT4 was more abundantly expressed in slow as compared to fast fibres at the same fibre diameter (p diabetic and obese was reduced...... compared to control subjects at the same diameter (p diabetic subjects expressed a fibre-volume-dependent GLUT4 expression (p diabetic p = 0.06). Our results show that increasing...

  15. Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans

    DEFF Research Database (Denmark)

    Orskov, C; Rabenhøj, L; Wettergren, A

    1994-01-01

    plasma were 7 +/- 1 and 6 +/- 1 pM, respectively (n = 6). In response to a breakfast meal, the concentration of amidated GLP-I rose significantly amounting to 41 +/- 5 pM 90 min after the meal ingestion, whereas the concentration of glycine-extended GLP-I only rose slightly to a maximum of 10 +/- 1 p......M. Thus, both amidated and glycine-extended GLP-I molecules are produced in the small intestine and in the pancreas in humans. Both amidated and glycine-extended GLP-I are measurable in fasting plasma.(ABSTRACT TRUNCATED AT 250 WORDS)...

  16. Rapid quantification of the metabolite of valacyclovir hydrochloride in human plasma by liquid chromatography-tandem mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Objective To establish a rapid,sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of acyclovir (the metabolite of valacyclovir hydrochloride) in human plasma. Methods After addition of ganciclovir as internal standard (IS),plasma samples were prepared by one-step protein precipitation using acetonitrile as precipitant,followed by an isocratic elution with 0.1% formic acid solution-methanol (95∶5,v/v) on an Agilent ZORBAX SB-C18 (150mm×2.1mm i.d.,3....

  17. [Determination of 10 sedative-hypnotics in human plasma using pulse splitless injection technique and gas chromatography-mass spectrometry].

    Science.gov (United States)

    Chang, Qing; Ma, Hongying; Wang, Fangjie; Ou, Honglian; Zou, Ming

    2011-11-01

    A simple, precise and sensitive gas chromatography-mass spectrometry (GC-MS) method coupled with pulse splitless injection technique was developed for the determination of 10 sedative-hypnotics (barbital, amobarbital, phenobarbital, oxazepam, diazepam, nitrazepam, clonazepam, estazolam, alprazolam, triazolam) in human plasma. The drugs spiked in plasma were extracted with ethyl acetate after alkalization with 0.1 mol/L NaOH solution. The organic solvent was evaporated under nitrogen stream, and the residues were redissolved by ethyl acetate. The separation was performed on an HP-5MS column (30 m x 250 microm x 0.25 microm). The analytes were determined and identified using selected ion monitoring (SIM) mode and scan mode, respectively. The internal standard method was used for the determination. The target analytes were well separated from each other on their SIM chromatograms and also on the total ion current (TIC) chromatograms. The blank extract from human plasma gave no peaks that interfered with all the analytes on the chromatogram. The calibration curves for 10 sedative-hypnotics showed excellent linearity. The correlation coefficients of all the drugs were higher than 0.9954. The recoveries of the drugs spiked in human plasma ranged from 92.28% to 111.7%, and the relative standard deviations (RSDs) of intra-day and inter-day determinations were from 4.09% to 14.26%. The detection limits ranged from 2 to 20 microg/L. The method is simple, reliable, rapid and sensitive for the determination and the quantification of 10 sedative-hypnotics in human plasma and seems to be useful in the practice of clinical toxicological cases.

  18. Biological effects of bacterial pigment undecylprodigiosin on human blood cells treated with atmospheric gas plasma in vitro.

    Science.gov (United States)

    Lazović, Saša; Leskovac, Andreja; Petrović, Sandra; Senerovic, Lidija; Krivokapić, Nevena; Mitrović, Tatjana; Božović, Nikola; Vasić, Vesna; Nikodinovic-Runic, Jasmina

    2017-01-01

    It is known that some bacterial species are more resilient to different kinds of irradiation due to the naturally developed protective mechanisms and compounds such as pigments. On the other hand, reasoned tissue engineering using plasma remains a critical task and requires very precise control of plasma parameters in order to mitigate its potential detrimental effects. Here we isolated a natural protective agent, microbially produced undecylprodigiosin ((5'Z)-4'-methoxy-5'-[(5-undecyl-1H-pyrrol-2-yl)methylene]-1H,5'H-2,2'-bipyrrole), and investigated its effects on human blood cells independently and in combination with plasma. Two approaches were applied; the first, undecylprodigiosin (UP pigment) was added to the blood cultures, which then were exposed to plasma (pre-treatment); and the second- the blood cultures were exposed to plasma and then treated with pigment (post-treatment). The interactions of plasma and UP pigment with blood cells were investigated by conducting a series of biological tests providing the information regarding their genotoxicity, cytotoxicity and redox modulating activities. The exposure of cells to plasma induced oxidative stress as well as certain genotoxic and cytotoxic effects seen as elevated micronuclei incidence, decreased cell proliferation and enhanced apoptosis. In blood cultures treated with UP pigment alone, we found that both cytotoxic and protective effects could be induced depending on the concentration used. The highest UP pigment concentration increased lipid peroxidation and the incidence of micronuclei by more than 70% with maximal suppression of cell proliferation. On the contrary, we found that the lowest UP pigment concentration displayed protective effects. In combined treatments with plasma and UP pigment, we found that UP pigment could provide spatial shielding to plasma exposure. In the pre-treatment approach, the incidence of micronuclei was reduced by 35.52% compared to control while malondialdehyde level

  19. Induction of apoptosis in human myeloid leukemia cells by remote exposure of resistive barrier cold plasma.

    Science.gov (United States)

    Thiyagarajan, Magesh; Anderson, Heather; Gonzales, Xavier F

    2014-03-01

    Cold atmospheric plasma (CAP), an ambient temperature ionized gas, is gaining extensive interest as a promising addition to anti-tumor therapy primarily due to the ability to generate and control delivery of electrons, ions, excited molecules, UV photons, and reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) to a specific site. The heterogeneous composition of CAP offers the opportunity to mediate several signaling pathways that regulate tumor cells. Consequently, the array of CAP generated products has limited the identification of the mechanisms of action on tumor cells. The aim of this work is to assess the cell death response of human myeloid leukemia cells by remote exposure to CAP generated RNS by utilizing a novel resistive barrier discharge system that primarily produces RNS. The effect of variable treatments of CAP generated RNS was tested in THP-1 cell (human monocytic leukemia cell line), a model for hematological malignancy. The number of viable cells was evaluated with erythrosine-B staining, while apoptosis and necrosis was assessed by endonuclease cleavage observed by agarose gel electrophoresis and detection of cells with the exclusionary dye propidium iodide and fluorescently labeled annexin-V by flow cytometry and fluorescent microscopy. Our observations indicate that treatment dosage levels of 45 s of exposure to CAP emitted RNS-induced apoptotic cell death and for higher dosage conditions of ≥50 s of exposure to CAP induced necrosis. Overall the results suggest that CAP emitted RNS play a significant role in the anti-tumor potential of CAP.

  20. 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 degrees C, which is about 30 degrees C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of approximately 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.

  1. Lack of species-specific difference in pulmonary function when using mouse versus human plasma in a mouse model of hemorrhagic shock.

    Science.gov (United States)

    Peng, Zhanglong; Pati, Shibani; Fontaine, Magali J; Hall, Kelly; Herrera, Anthony V; Kozar, Rosemary A

    2016-11-01

    Clinical studies have demonstrated that the early and empiric use of plasma improves survival after hemorrhagic shock. We have demonstrated in rodent models of hemorrhagic shock that resuscitation with plasma is protective to the lungs compared with lactated Ringer's solution. As our long-term objective is to determine the molecular mechanisms that modulate plasma's protective effects in injured bleeding patients, we have used human plasma in a mouse model of hemorrhagic shock. The goal of the current experiments is to determine if there are significant adverse effects on lung injury when using human versus mouse plasma in an established murine model of hemorrhagic shock and laparotomy. Mice underwent laparotomy and 90 minutes of hemorrhagic shock to a mean arterial pressure (MAP) of 35 ± 5 mm Hg followed by resuscitation at 1× shed blood using either mouse fresh frozen plasma (FFP), human FFP, or human lyophilized plasma. Mean arterial pressure was recorded during shock and for the first 30 minutes of resuscitation. After 3 hours, animals were killed, and lungs collected for analysis. There was a significant increase in early MAP when mouse FFP was used to resuscitate animals compared with human FFP or human lyophilized plasma. However, despite these differences, analysis of the mouse lungs revealed no significant differences in pulmonary histopathology, lung permeability, or lung edema between all three plasma groups. Analysis of neutrophil infiltration in the lungs revealed that mouse FFP decreased neutrophil influx as measured by neutrophil staining; however, myeloperoxidase immunostaining revealed no significant differences in between groups. The study of human plasma in a mouse model of hemorrhagic shock is feasible but does reveal some differences compared with mouse plasma-based resuscitation in physiologic measures such as MAP postresuscitation. Measures of end organ function such as lung injury appear to be comparable in this acute model of hemorrhagic

  2. Human dermis separation via ultra-short pulsed laser plasma-mediated ablation

    Energy Technology Data Exchange (ETDEWEB)

    Huang Huan; Guo Zhixiong, E-mail: guo@jove.rutgers.ed [Department of Mechanical and Aerospace Engineering, Rutgers, State University of New Jersey, Piscataway, NJ 08854 (United States)

    2009-08-21

    In vitro layer separation of human dermal tissues via laser plasma-mediated ablation was successfully conducted for the first time. The ablation of wet dermis was generated using a 900 fs ultra-short pulsed laser with the wavelength centred at 1552 nm. The ablation and separation results were imaged and measured by scanning electron microscopy. The dermis ablation threshold was determined as 9.65 {+-} 1.21 J cm{sup -2} and the incubation factor was found as 0.46 {+-} 0.03. Histological examinations were performed to find proper laser parameters for dermis ablation and separation with minimal thermal damage. No thermal damage was found in the single line ablation results when the pulse overlap rate was not over 5 pulses {mu}m{sup -1}. Even in the multi-line ablation, thermal damage was insignificant and the lateral damage zone was generally within 5 {mu}m in the results with 100 continuously repeated line scans. The separation of a whole piece of wet dermal tissue into two thin layers was presented. Several separation tests with different layer thicknesses from 200 to 600 {mu}m were completed. The unevenness of the separated layers was generally under 10%. The cohesion and morphology of the separated tissue layers were not altered.

  3. Simultaneous Quantification of Ciprofloxacin, Quinine and 3-hyrdoxyquinine in Human Plasma using a HPLC Method

    Directory of Open Access Journals (Sweden)

    Adebanjo J. Adegbola, Julius O. Soyinka , Babatunde A. Adeagbo

    2016-02-01

    Full Text Available Malaria has been shown to strongly predispose patients in areas of malaria endemicity to bacteremia with severe outcomes, thus justifying the use of antibiotics in combination with antimalarial therapy in patients with severe malaria. This study describes a High-Performance Liquid Chromatographic (HPLC method for simultaneous determinations of Ciprofloxacin (CPN, Quinine (QN, and its major metabolite, 3-Hydroxyquinine (3-HQN, in human plasma. Following a simple precipitation with acetonitrile, chromatographic separation was achieved on a reversed-phase Agilent Zorbax (CN column (5 µm, 150 X 4.6 mm i.d using a mobile phase consisting of acetonitrile: potassium dihydrogen phosphate (pH = 2.8; 0.02 M (42:58, v/v. Retention times for CPN, 3-HQN, IS and QN were 2.7, 3.3, 3.6 and 4.9 minutes respectively. The limits of detection and validated lower limits of quantitation were 30 and 70 ng/ml for both QN and 3-HQN while the corresponding values were 50 and 100 ng/ml for CPN, respectively. The new HPLC method here developed, when compared with previous methods for the analysis of either or both drugs is simple, rapid, selective, reproducible and costeffective. It is also suitable for conducting a simultaneous therapeutic monitoring of quinine and ciprofloxacin in patients when concomittantly administered as demonstrated in five healthy volunteers.

  4. Highly sensitive spectrofluorimetric method for determination of certain aminoglycosides in pharmaceutical formulations and human plasma.

    Science.gov (United States)

    Omar, Mahmoud A; Nagy, Dalia M; Hammad, Mohamed A; Aly, Alshymaa A

    2013-06-01

    A simple, reliable, highly sensitive and selective spectrofluorimetric method has been developed for determination of certain aminoglycosides namely amikacin sulfate, tobramycin, neomycin sulfate, gentamicin sulfate, kanamycin sulfate and streptomycin sulfate. The method is based on the formation of a charge transfer complexes between these drugs and safranin in buffer solution of pH 8. The formed complexes were quantitatively extracted with chloroform under the optimized experimental conditions. These complexes showed an excitation maxima at 519-524 nm and emission maxima at 545-570 nm. The calibration plots were constructed over the range of 4-60 pg mL(-1) for amikacin, 4-50 pg mL(-1) for gentamicin, neomycin and kanamycin, 4-40 pg mL(-1) for streptomycin and 5-50 pg mL(-1) for tobramycin. The proposed method was successfully applied to the analysis of the cited drugs in dosage forms. The proposed method was validated according to ICH and USP guidelines with respect to specificity, linearity, accuracy, precision and robustness. The high sensitivity of the proposed method allowed determination of amikacin and gentamicin in spiked and real human plasma.

  5. Deoxycytidine in human plasma: potential for protecting leukemic cells during chemotherapy.

    Science.gov (United States)

    Cohen, J D; Strock, D J; Teik, J E; Katz, T B; Marcel, P D

    1997-06-24

    Degradation of DNA produces deoxycytidine. Metabolism of deoxycytidine to dCTP inhibits phosphorylation of cytosine arabinoside (araC), fludarabine (FaraA) and 2-chlorodeoxyadenosine (CdA) by deoxycytidine kinase. This study measured plasma deoxycytidine in healthy adults and two leukemia patients and then determined how clinically relevant deoxycytidine levels would affect drug toxicity in human leukemia and lymphoma cells. Deoxycytidine was well below 0.05 microM in ten healthy persons. In the leukemia patients it was <0.05 and 0.44 microM before chemotherapy, rising to 10.3 and 5.5 microM during treatment. A broad range of clinically relevant deoxycytidine levels were high enough to profoundly decrease araC, FaraA and CdA toxicity in MOLT3, CA46 and HL60 leukemia/lymphoma cells and to change dCTP, DNA synthesis and drug incorporation into DNA in a manner consistent with prior mechanistic studies. Varying deoxycytidine levels could be an important factor influencing leukemia therapy.

  6. Liquid chromatography/tandem mass spectrometry assay for the quantification of troxerutin in human plasma.

    Science.gov (United States)

    Liu, Fei; Xu, Yu; Rui, Lei; Gao, Shu; Dong, Haijun; Guo, Qingxiang

    2006-01-01

    A simple, rapid, sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify troxerutin in human plasma. The analyte and rutin, used as the internal standard, were analyzed on a Phenomenex Synergi Fusion RP column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Acetonitrile/water (20:80 v/v) was used as the isocratic mobile phase, with 0.1% formic acid in water. A simple sample preparation method of protein precipitation with perchloric acid was employed. The assay was linear over the concentration range 31.25-4000 pg/mL. Correlation coefficients generated by linear regression with a 1/x(2) weighting factor ranged from 0.9991 to 0.9996. The intra- and inter-day precision over the entire concentration range were less than 12.28%. The method was successfully applied to a pharmacokinetic study after oral administration of a 300 mg troxerutin drop pill to 18 healthy volunteers.

  7. DEVELOPMENT AND VALIDATION OF A BIOANALYTICAL METHOD FOR DETERMINATION OF QUETIAPINE FROM HUMAN PLASMA

    Directory of Open Access Journals (Sweden)

    Khanvilkar Vineeta V.

    2013-02-01

    Full Text Available A simple high-performance liquid chromatographic (HPLC method for the analysis of the antipsychotic drug quetiapine fumarate in human plasma has been developed. Zolpidem tartrate was employed as the internal standard (IS. Biological samples were pretreated by liquid-liquid extraction (LLE technique using tert-butyl methyl ether (TBME. Separation was performed on a HiQSil C18HS (250x4.6 mm, 5µm column. The mobile phase used was Acetonitrile-ammonium acetate buffer (pH 3.5, 10 mM (40:60 v/v pumped at a flow rate of 1 mL/min. Samples were injected by means of an autosampler via a variable loop and detected using UV detector at a wavelength of 254 nm. A good linearity was found in the concentration range of 100-2000 ng/mL. Within and between batch precision and accuracy of the proposed method were evaluated by percent relative standard deviation (% R.S.D. and percent relative error (% RE respectively; both being within the acceptable limits. The method was also validated for recovery, carry-over and stability. Freeze and thaw stability, short term stability and long term stability were evaluated for the developed bioanalytical method. The described method can be applied for quantitation of quetiapine in real clinical samples.

  8. Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study.

    Science.gov (United States)

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

    2013-02-01

    Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

  9. Robust aptamer sol-gel solid phase microextraction of very polar adenosine from human plasma.

    Science.gov (United States)

    Mu, Li; Hu, Xiangang; Wen, Jianping; Zhou, Qixing

    2013-03-01

    Conventional solid phase microextraction (SPME) has a limited capacity to extract very polar analytes, such as adenosine. To solve this problem, aptamer conjugating sol-gel methodology was coupled with an SPME fiber. According to the authors' knowledge, this is the first reported use of aptamer SPME. The fiber of aptamer sol-gel SPME with a mesoporous structure has high porosity, large surface area, and small water contact angle. Rather than employing direct entrapment, covalent immobilization was the dominant method of aptamer loading in sol-gel. Aptamer sol-gel fiber captured a specified analyte from among the analog molecules, thereby, exhibiting an excellent selective property. Compared with commercial SPME fibers, this aptamer fiber was suitable for extracting adenosine, presenting an extraction efficiency higher than 20-fold. The values of repeatability and reproducibility expressed by relative standard deviation were low (9.4%). Interestingly, the sol-gel network enhanced the resistance of aptamer SPME to both nuclease and nonspecific proteins. Furthermore, the aptamer sol-gel fiber was applied in human plasma with LOQ 1.5 μg/L, which is an acceptable level. This fiber also demonstrates durability and regeneration over 20-cycles without significant loss of efficiency. Given the various targets (from metal ions to biomacromolecules and cells) of aptamers, this methodology will extend the multi-domain applications of SPME.

  10. 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.

  11. Determination of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Kanazawa, Hideko; Okada, Akiko; Matsushima, Yoshikazu; Yokota, Hiromitsu; Okubo, Shigeo; Mashige, Fumiko; Nakahara, Kazuhiko

    2002-03-08

    Omeprazole is a benzimidazole compound that acts as a proton-pump inhibitor. Because the metabolism of omeprazole is mainly catalyzed by cytochrome P-450 (CYP) 3A4 and CYP2C19. the genetic polymorphism of CYP2C19 could be of clinical concern in the treatment of acid-related diseases with omeprazole. Therefore, a reliable method for omeprazole phenotyping is desirable in clinical situations. This study has demonstrated the determination of omeprazole and its metabolites in human plasma by liquid chromatography-three-dimensional quadrupole mass spectrometry with a sonic spray ionization interface. The analytical column was YMC-Pack Pro C18(50x2.0 mm I.D.) using acetonitrile-50 mM ammonium acetate (pH 7.25) (1:4) at a flow-rate of 0.2 ml/min. The drift voltage was 30 V. The sampling aperture was heated at 110 degrees C and Shield temperature was 230 degrees C. In the mass spectrum, the molecular ions of omeprazole, hydroxyomeprazole and omeprazole sulfone were clearly observed as base peaks. This method is sufficiently sensitive and accurate for pharmacokinetic studies of omeprazol.

  12. Molecular recognition based iron removal from human plasma with imprinted membranes.

    Science.gov (United States)

    Yavuz, H; Andaç, M; Uzun, L; Say, R; Denizli, A

    2006-09-01

    The aim of this study is to prepare ion-imprinted poly(2-hydroxyethyl methacrylate) (HEMA) based membranes which can be used for the selective removal of Fe3+ ions from Fe3+-overdosed human plasma. N-methacryloyl-(L)-glutamic acid (MAGA) was chosen as the ion-complexing monomer. In the first step, Fe3+ was complexed with MAGA and then, the Fe3+-imprinted poly(HEMA-MAGA) membranes were prepared by UV-initiated photo-polymerization of HEMA and MAGA-Fe3+ complex in the presence of an initiator (benzoyl peroxide). After that, the template (i.e., Fe3+ ions) was removed by using 0.1 M EDTA solution at room temperature. The specific surface area of the Fe3+-imprinted poly(HEMA-MAGA) membranes was found to be 49.2 m2/g and the swelling ratio was 92%. According to the elemental analysis results, the polymeric membranes contained 145.7 micromol MAGA/g polymer. The maximum adsorption capacity was 164.2 micromol Fe3+/g membrane. The relative selectivity coefficients of ion-imprinted membranes for Fe3+/Zn2+ and Fe3+/Cr3+ were 12.6 and 62.5 times greater than the non-imprinted matrix, respectively. The Fe3+-imprinted poly(HEMA-MAGA) membranes could be used many times without decreasing their Fe3+ adsorption capacities significantly.

  13. Novel LC- ESI-MS/MS method for desvenlafaxine estimation human plasma: application to pharmacokinetic study.

    Science.gov (United States)

    Kancharla, Pushpa Kumari; Kondru, Venu Gopal Raju; Dannana, Gowri Sankar

    2016-02-01

    A simple, sensitive and specific liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the quantification of desvenlafaxine in human plasma using desvenlafaxine d6 as an internal standard (IS). Chromatographic separation was performed using a Thermo-BDS hypersil C8 column (50 × 4.6 mm, 3 µm) with an isocratic mobile phase composed of 5 mM ammonium acetate buffer: methanol (20:80, v/v), at a flow rate of 0.80 mL/min. Desvenlafaxine and desvenlafaxine d6 were detected with proton adducts at m/z 264.2/58.1 and 270.2/ 64.1 in multiple reaction monitoring positive mode, respectively. Liquid-liquid extraction was used to extract the drug and the IS. The method was linear over the concentration range 1.001-400.352 ng/mL with a correlation coefficient of ≥0.9994. This method demonstrated intra and inter-day precision within 0.7-5.5 and 1.9-6.8%, and accuracy within 95.3-107.4 and 93.4-99.5%. Desvenlafaxine was found to be stable throughout the freeze-thaw cycles, bench-top and long-term matrix stability studies. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of desvenlafaxine in pharmaceutical dosage forms. Copyright © 2015 John Wiley & Sons, Ltd.

  14. Self-assembly of human plasma fibrinogens on binary organosilane monolayers with micro domains

    Energy Technology Data Exchange (ETDEWEB)

    Lee, S.H. [Faculty of Engineering, Shinshu Universuty, 4-17-1 Wakasato, Nagano 380 8553 (Japan); Saito, N., E-mail: hiro@eco-t.esi.nagoya-u.ac.jp [Department of Materials, Physics and Energy Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464 8603 (Japan); Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464 8603 (Japan); Takai, O. [EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa, Nagoya 464 8603 (Japan)

    2009-06-30

    The adsorption behavior and self-assembly of human plasma fibrinogen (HPF) on binary methyl- and amino-terminated self-assembled monolayers (SAMs) were investigated by atomic force microscopy (AFM). The binary SAMs were fabricated through self-assembly mechanism of organosilane molecules. The height of domains is the domain height is 0.8 {+-} 0.2 nm from the AFM topographic image. It corresponds to the domain height is 0.8 {+-} 0.2 nm from the AFM topographic image. It corresponds to the difference between the length of the alkyl chain of octadecyltrichlorosilane (OTS) and that of n-(6-aminohexyl)aminopropyltrimethoxysilane (AHAPS). The fibrinogen solution used ultrapure water as the solvent and its pH was adjusted at 3 and 10. From the AFM results at pH 3, HPF only formed network structures on the OTS domains of the binary SAM at early immersion times, and then the network structures expanded and connected between OTS domains through the AHAPS surface at long immersion times. In this case, a few HPFs are discretely adsorbed on the AHAPS surface. However, HPF is uniformly adsorbed on the binary SAM under the other conditions of pH.

  15. Analysis of Pregnancy-Associated Plasma Protein A Production in Human Adult Cardiac Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Piera D’Elia

    2013-01-01

    Full Text Available IGF-binding proteins (IGFBPs and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

  16. Application of liquid chromatography method with electrochemical detection for bioequivalence study of trimetazidine in human plasma.

    Science.gov (United States)

    Grabowski, Tomasz; Swierczewska, Anna; Borucka, Beata; Sawicka, Renata; Sasinowska-Motyl, Małgorzata; Gumułka, Stanisław Witold

    2012-01-01

    A method to estimate trimetazidine (CAS: 13171-25-0) levels in human plasma by means of HPLC with electrochemical detection was developed. Trimethoprim (CAS: 26807-65-8) was used as an internal standard. This method of analysis was fully validated according to the guidelines of the United States Food and Drug Administration, European Medicines Agency and Organization for Economic Co-operation and Development and Good Laboratory Practice rules. The accuracy and precision of the developed method were found to be satisfactory and stability studies showed acceptable variation (below 15%) of trimetazidine concentrations when samples were stored frozen at -75 degrees C for 54 days. The developed method was successfully used for a comparative 2 x 2 period, crossover bioequivalence study of two extended-release preparations of trimetazidine performed on 24 healthy volunteers at the steady state after multiple dosing of 35 mg twice daily for 4 days and a single 35 mg dose on the 5th day and after a single dose of 35 mg under fasting or postprandial conditions.

  17. Increased plasma soluble human leukocyte antigen-G in persistent wheezy infants.

    Science.gov (United States)

    Tahan, Fulya; Eke Gungor, Hatice; Akar, Himmet Haluk; Saraymen, Berkay

    2017-05-01

    Human leukocyte antigen (HLA)-G is a non-classical major histocompatibility complex class I antigen characterized by limited polymorphism in its coding region, unique tissue expression pattern in physiologic conditions and immunomodulatory properties. Recently, the level of soluble (s)HLA-G was found to be higher in atopic asthma and allergic rhinitis, but this remains to be clarified in wheezy infants. The aim of the present study was therefore to investigate sHLA-G in wheezy infants. The subjects consisted of infants with persistent wheezing and positive modified asthma predictive index (mAPI; n = 30; persistent group) and those with transient wheezing and negative mAPI (n = 17; transient group). sHLA-G was measured in plasma using enzyme-linked immunosorbent assay. Total immunoglobulin E (IgE) and eosinophil count were measured, and skin testing was performed with a battery of 13 antigens with appropriate positive and negative controls. sHLA-G was significantly higher in the persistent wheezing (positive mAPI) group compared with the transient wheezing (negative mAPI) group (P = 0.008). There was no significant difference in peripheral blood eosinophil count and total IgE between the groups. The increased sHLA-G in infants with persistent wheeze suggests that sHLA-G may be able to be used to distinguish persistent from transient wheeze. Further comprehensive studies are needed on this topic. © 2016 Japan Pediatric Society.

  18. Simultaneous determination of clobazam and its major metabolite in human plasma by a rapid HPLC method.

    Science.gov (United States)

    Rouini, Mohammadreza; Ardakani, Yalda H; Hakemi, Lida; Mokhberi, Maryam; Badri, Gheise

    2005-09-05

    A rapid and specific HPLC method has been developed and validated for simultaneous determination of clobazam, the anticonvulsant agent, and its major metabolite in human plasma. The sample preparation was a liquid-liquid extraction with tuloene yielding almost near 100% recoveries of two compounds. Chromatographic separation was achieved with a Chromolith Performance RP-18e 100 mm x 4.6mm column, using a mixture of a phosphate buffer (pH 3.5; 10mM)-acetonitrile (70:30, v/v), in isocratic mode at 2 ml/min at a detection wave-length of 228 nm. The calibration curves were linear (r(2)>0.998) in the concentration range of 5-450 ng ml(-1). The lower limit of quantification was 5 ng ml(-1) for two compounds studied. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 0.89-9.1% and 2.1-10.1% R.S.D., respectively. The developed procedure was applied to assess the pharmacokinetics of clobazam and its major metabolite following administration of a single 10mg oral dose of clobazam to healthy volunteers.

  19. Quantitation of clobazam in human plasma using high-performance liquid chromatography.

    Science.gov (United States)

    Knapp, J; Bokník, P; Gumbinger, H G; Linck, B; Lüss, H; Müller, F U; Schmitz, W; Vahlensieck, U; Neumann, J

    1999-05-01

    A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.

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

    Science.gov (United States)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    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 are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive 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, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  1. Chemiluminescence Determination of Local Anaesthetic Mepivacaine in Human Plasma and Pharmaceuticals.

    Science.gov (United States)

    Mokhtari, Ali

    2016-12-01

    In this study, a new method has been developed for the simple determination of mepivacaine (carbocaine). The method is based on the enhancement effect of mepivacaine in the chemiluminescence (CL) reaction of tris(1,10 phenanthroline) ruthenium(II) with cerium(IV). A mechanism for the CL reaction has been proposed on the basis of UV-Vis, fluorescent and CL spectra. Under optimum conditions, the CL intensity was proportional to the concentration of the drug in solution over the range 0.45-226.25 μg mL-1 (R2 = 0.9996). The LOD was 0.34 μg mL-1 (S/N = 3). LOD was about 10 times lower than the therapeutic concentration of mepivacaine. The percent of relative standard deviation for determination of 11 replicates at level of 9.05 μg mL-1 and 90.50 μg mL-1 of mepivacaine were 1.8 and 3.7%, respectively. The proposed method was applied successfully for the determination of mepivacaine in human plasma and injectable solutions.

  2. 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.

  3. 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

    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...... between the treatment groups. Differentially affected spots were identified by matrix-assisted laser desorption ionisation-time of flight/time of flight MS and the human Swiss-Prot database. We found 23/681 abundant plasma protein spots, which were up- or down-regulated by the dietary treatment (P...spots differed from those in subjects given the chicken meal, but only three of these were common, and only one spot differed between the two trout groups. In both groups, the affected plasma proteins were involved in biological...

  4. Stability of individual carotenoids, retinol and tocopherols in human plasma during exposure to light and after extraction.

    Science.gov (United States)

    Su, Q; Rowley, K G; O'Dea, K

    1999-06-11

    We have modified gradient HPLC procedures for simultaneous quantification of retinol, gamma-tocopherol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, trans-lycopene, cis-lycopene, alpha-carotene and beta-carotene in 200-microl aliquots of human plasma. The photosensitivity of these analytes in plasma exposed to fluorescent lighting for up to 72 h was investigated and most were stable under these conditions. The stability of these analytes held in darkness at -20 degrees C, 4 degrees C or room temperature for up to 48 h after extraction from plasma was also investigated. Variability in measurement of most analytes was greater at room temperature than at 4 degrees C or -20 degrees C. There were statistically significant variations in the measured concentrations of some analytes in samples kept cold. However, the magnitude of these variations was small and of little biological significance, particularly over the first 24 h.

  5. Effect of major and minor surgery on plasma levels of arginine, citrulline, nitric oxide metabolites, and ornithine in humans.

    Science.gov (United States)

    Hol, Jaap W; van Lier, Felix; Valk, Madelous; Klimek, Markus; Stolker, Robert J; Fekkes, Durk

    2013-12-01

    To determine the effect of surgical invasiveness on plasma levels of arginine, citrulline, ornithine, and nitric oxide (NO) in humans. Surgical trauma may have a profound effect on the metabolism of NO. However, human studies reported both increased and decreased NO levels after hemorrhagic shock. Arginine, citrulline, and ornithine are key amino acids involved in NO metabolism, but studies evaluating these amino acids together with NO and during 2 types of surgery are lacking. This study tests the hypothesis that major surgery has a more profound effect on plasma levels of arginine, citrulline, NO, and ornithine than minor surgery. Fifteen patients undergoing minor surgery (vulvectomy) and 13 patients undergoing major surgery (laparotomy) were prospectively followed up for 4 days. Plasma was collected for evaluation of levels of arginine, citrulline, NO, and ornithine. Throughout the experiment, arginine levels did not significantly differ between experimental groups. Perioperative plasma citrulline levels were significantly lower in the laparotomy group than in the vulvectomy group, whereas both groups showed a decrease in citrulline levels at the end of the operation and 24 hours postoperatively. Roughly the same pattern was seen for plasma NO and ornithine levels. However, ornithine levels in the laparotomy group showed a more drastic decrease at the end of the operation and 24 hours postoperatively than citrulline and NO levels. The level of surgical invasiveness has the most profound effect on plasma levels of ornithine. In addition, heavier surgical trauma is paired with lower postoperative levels of citrulline and NO metabolites than lighter surgery. It is suggested that surgical trauma stimulates the laparotomy group to consume significantly more ornithine, possibly for use in wound healing.

  6. The effects of eating marine- or vegetable-fed farmed trout on the human plasma proteome profiles of healthy men.

    Science.gov (United States)

    Rentsch, Maria L; Lametsch, René; Bügel, Susanne; Jessen, Flemming; Lauritzen, Lotte

    2015-02-28

    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 between the treatment groups. Differentially affected spots were identified by matrix-assisted laser desorption ionisation-time of flight/time of flight MS and the human Swiss-Prot database. We found 23/681 abundant plasma protein spots, which were up- or down-regulated by the dietary treatment (P spots differed from those in subjects given the chicken meal, but only three of these were common, and only one spot differed between the two trout groups. In both groups, the affected plasma proteins were involved in biological processes such as regulation of vitamin A and haem transport, blood fibrinolysis and oxidative defence. Thus, regular fish intake affects the plasma proteome, and the changes may indicate novel mechanisms of effect.

  7. 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 ...... evaluates and monitors intervention in metabolic diseases....... in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly...

  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...... evaluates and monitors intervention in metabolic diseases....

  9. 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.

  10. Electron spectroscopic analysis of the human lipid skin barrier: cold atmospheric plasma-induced changes in lipid composition.

    Science.gov (United States)

    Marschewski, Marcel; Hirschberg, Joanna; Omairi, Tarek; Höfft, Oliver; Viöl, Wolfgang; Emmert, Steffen; Maus-Friedrichs, Wolfgang

    2012-12-01

    The lipids of the stratum corneum comprise the most important components of the skin barrier. In patients with ichthyoses or atopic dermatitis, the composition of the skin barrier lipids is disturbed resulting in dry, scaly, itching erythematous skin. Using the latest X-Ray Photoelectron Spectroscopy (XPS) technology, we investigated the physiological skin lipid composition of human skin and the effects of cold atmospheric plasma treatment on the lipid composition. Skin lipids were stripped off forearms of six healthy volunteers using the cyanoacrylate glue technique, plasma treated or not and then subjected to detailed XPS analysis. We found that the human lipid skin barrier consisted of 84.4% carbon (+1.3 SEM%), 10.8% oxygen (+1.0 SEM%) and 4.8% nitrogen (+0.3 SEM%). The composition of physiological skin lipids was not different in males and females. Plasma treatment resulted in significant changes in skin barrier lipid stoichiometry. The total carbon amount was reduced to 76.7%, and the oxygen amount increased to 16.5%. There was also a slight increase in nitrogen to 6.8%. These changes could be attributed to reduced C-C bonds and increased C-O, C=O, C-N and N-C-O bonds. The moderate increase in nitrogen was caused by an increase in C-N and N-C-O bonds. Our results show for the first time that plasma treatment leads to considerable changes in the human skin lipid barrier. Our proof of principle investigations established the technical means to analyse, if plasma-induced skin lipid barrier changes may be beneficial in the treatment of ichthyotic or eczematous skin.

  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.

  12. Universal pooled plasma (Uniplas(®)) does not induce complement-mediated hemolysis of human red blood cells in vitro.

    Science.gov (United States)

    Heger, Andrea; Brandstätter, Hubert; Prager, Bettina; Brainovic, Janja; Cortes, Rhoda; Römisch, Jürgen

    2015-02-01

    Pooling of plasma of different blood groups before large scale manufacturing of Uniplas(®) results in the formation of low levels of soluble immune complexes (CIC). The aim of this study was to investigate the level and removal of CIC during Uniplas(®) manufacturing. In addition, an in vitro hemolysis assay should be developed and investigate if Uniplas(®) does induce complement-mediated hemolysis of human red blood cells (RBC). In-process samples from Uniplas(®) (universal plasma) and Octaplas(LG)(®) (blood group specific plasma) routine manufacturing batches were tested on CIC using commercially available ELISA test kits. In addition, CIC was produced by admixing heat-aggregated immunoglobulins or monoclonal anti-A/anti-B antibodies to plasma and removal of CIC was followed in studies of the Uniplas(®) manufacturing process under down-scale conditions. The extent of RBC lysis was investigated in plasma samples using the in-house hemolysis assay. Levels of CIC in Uniplas(®) are within the normal ranges for plasma and comparable to that found in Octaplas(LG)(®). Down-scale experiments showed that both IgG/IgM-CIC levels are significantly removed on average by 40-50% during Uniplas(®) manufacturing. Uniplas(®) does not induce hemolysis of RBCs in vitro. Hemolysis occurs only after spiking with high titers of anti-A/anti-B antibodies and depends on the antibody specificity (i.e. titer) in the plasma sample. The results of this study confirm the safety of Uniplas(®) regarding transfusion to patients of all ABO blood groups. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. 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 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.

  14. 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.

  15. A New Surface Plasmon Resonance-Based Immunoassay for Rapid, Reproducible and Sensitive Quantification of Pentraxin-3 in Human Plasma

    Directory of Open Access Journals (Sweden)

    Mara Canovi

    2014-06-01

    Full Text Available A new immunoassay based on surface plasmon resonance (SPR for the rapid, reproducible and sensitive determination of pentraxin-3 (PTX3 levels in human plasma has been developed and characterized. The method involves a 3-min flow of plasma over a sensor chip pre-coated with a monoclonal anti-PTX3 antibody (MNB4, followed by a 3-min flow of a polyclonal anti-PTX3 antibody (pAb, required for specific recognition of captured PTX3. The SPR signal generated with this secondary antibody linearly correlates with the plasma PTX3 concentration, in the range of 5–1500 ng/mL, with a lowest limit of detection of 5 ng/mL. The PTX3 concentrations determined with the SPR-based immunoassay in the plasma of 21 patients with sepsis, ranging 15–1600 ng/mL, were superimposable to those found in a classic ELISA immunoassay. Since the PTX3 concentration in the plasma of healthy subjects is <2 ng/mL, but markedly rises in certain medical conditions, the method is useful to quantify pathological levels of this important biomarker, with important diagnostic applications. In comparison with the classic ELISA, the SPR-based approach is much faster (30 min versus 4–5 h and could be exploited for the development of new cost-effective SPR devices for point-of-care diagnosis.

  16. High-performance liquid chromatographic method for sensitive determination of the alkylating agent CB1954 in human plasma.

    Science.gov (United States)

    Anderson, D; Ferry, D R; Knox, R J; Andrews, S J; Downes, A J; Kerr, D J; Seymour, L W

    1999-08-20

    A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme-prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85 +/- 0.015 min, and for CB1954 of 10.72 +/- 0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m2), and plasma clearance of the drug showed biphasic kinetics with alpha half-life 14.6 min, and beta half-life 170.5 min.

  17. A new surface plasmon resonance-based immunoassay for rapid, reproducible and sensitive quantification of pentraxin-3 in human plasma.

    Science.gov (United States)

    Canovi, Mara; Lucchetti, Jacopo; Stravalaci, Matteo; Valentino, Sonia; Bottazzi, Barbara; Salmona, Mario; Bastone, Antonio; Gobbi, Marco

    2014-06-19

    A new immunoassay based on surface plasmon resonance (SPR) for the rapid, reproducible and sensitive determination of pentraxin-3 (PTX3) levels in human plasma has been developed and characterized. The method involves a 3-min flow of plasma over a sensor chip pre-coated with a monoclonal anti-PTX3 antibody (MNB4), followed by a 3-min flow of a polyclonal anti-PTX3 antibody (pAb), required for specific recognition of captured PTX3. The SPR signal generated with this secondary antibody linearly correlates with the plasma PTX3 concentration, in the range of 5-1500 ng/mL, with a lowest limit of detection of 5 ng/mL. The PTX3 concentrations determined with the SPR-based immunoassay in the plasma of 21 patients with sepsis, ranging 15-1600 ng/mL, were superimposable to those found in a classic ELISA immunoassay. Since the PTX3 concentration in the plasma of healthy subjects is <2 ng/mL, but markedly rises in certain medical conditions, the method is useful to quantify pathological levels of this important biomarker, with important diagnostic applications. In comparison with the classic ELISA, the SPR-based approach is much faster (30 min versus 4-5 h) and could be exploited for the development of new cost-effective SPR devices for point-of-care diagnosis.

  18. Comparative evaluation of the cytomegalovirus DNA load in polymorphonuclear leukocytes and plasma of human immunodeficiency virus-infected subjects.

    Science.gov (United States)

    Boivin, G; Handfield, J; Toma, E; Murray, G; Lalonde, R; Bergeron, M G

    1998-02-01

    The cytomegalovirus (CMV) DNA load was determined in polymorphonuclear leukocytes (PMNL) and plasma samples from 106 human immunodeficiency virus-infected subjects at risk of developing CMV disease (group 1) and from 27 AIDS patients with documented CMV disease (group 2). For both groups, the number of CMV copies in PMNL was significantly higher than in plasma when results were derived from an equivalent blood volume (P < .001, PMNL vs. plasma). Additionally, group 2 (symptomatic) patients had a greater viral DNA load than group 1 (asymptomatic) subjects (P < .001 for both PMNL and plasma). The sensitivity, specificity, and positive and negative predictive values of qualitative polymerase chain reaction using PMNL (PCR-PMNL) for the presence of CMV disease were 100%, 58%, 38%, and 100%, respectively, compared with 70%, 93%, 74%, and 92% for qualitative PCR-plasma and 93%, 92%, 76%, and 98% for quantitative PCR-PMNL using a cutoff of 16,000 copies/mL. Thus, the best strategy for diagnosing CMV disease in these individuals relies on quantitative assessment of the viral DNA load in PMNL.

  19. Improvement of antibacterial activity of some sulfa drugs through linkage to certain phthalazin-1(2H)-one scaffolds.

    Science.gov (United States)

    Ibrahim, Hany S; Eldehna, Wagdy M; Abdel-Aziz, Hatem A; Elaasser, Mahmoud M; Abdel-Aziz, Marwa M

    2014-10-06

    RAB1 5 is a lead antibacterial agent in which trimethoprim is linked to phthalazine moiety. Similarly, our strategy in this research depends on the interconnection between some sulfa drugs and certain phthalazin-1(2H)-one scaffolds in an attempt to enhance their antibacterial activity. This approach was achieved through the combination of 4-substituted phthalazin-1(2H)-ones 9a, b or 14a, b with sulfanilamide 1a, sulfathiazole 1b or sulfadiazine 1c through amide linkers 6a, b to produce the target compounds 10a-d and 15a-e, respectively. The antibacterial activity of the newly synthesized compounds showed that all tested compounds have antibacterial activity higher than that of their reference sulfa drugs 1a-c. Compound 10c represented the highest antibacterial activity against Gram-positive bacteria Streptococcus pneumonia and Staphylococcus aureus with MIC = 0.39 μmol/mL. Moreover, compound 10d displayed excellent antibacterial activity against Gram-negative bacteria Escherichia coli and Salmonella typhimurium with MIC = 0.39 and 0.78 μmol/mL, respectively.

  20. Identification of expressed genes in regenerating rat liver in 0-4-8-12 h short interval successive partial hepatectomy

    Institute of Scientific and Technical Information of China (English)

    Cun-Shuan Xu; Salman Rahman; Jing-Bo Zhang; Jin-Yun Yuan; Wen-Qiang Li; Hong-Peng Han; Ke-Jin Yang; Cui-Fang Chang; Li-Feng Zhao; Yu-Chang Li; Hui-Yong Zhang

    2005-01-01

    AIM: To identify the genes differentially expressed in the regenerating rat liver of 0-4-8-12 h short interval successive partial hepatectomy (SISPH) and to analyze their expression profiles.METHODS: Five hundred and fifty-one elements screened from subtractive cDNA libraries were made into a cDNA microarray (cDNA chip). Extensive gene expression analysis following 0-4-8-12 h SISPH was conducted by microarray.RESULTS: One hundred and eighty-three elements were selected, which were either up- or down-regulated more than 2-fold at one or more time points after SISPH. Cluster analysis and generalization analysis showed that there were five distinct temporal patterns of gene expression.Eighty-six genes were unreported, associated with liver regeneration (LR).CONCLUSION: Microarray analysis shows that the down regulated genes are much more than the up-regulated ones in SISPH; the numbers of genes expressed consistently are fewer than that expressed immediately; the genes expressed in high abundance are much fewer than that increased 2-5-fold. The comparison of SISPH with partial hepatectomy (PH) shows that the expression trends of most genes in SISPH and in PH are similar,but the expression of 43 genes is specifically altered in SISPH.

  1. Weather resistance of CaSO4 ṡ 1/2H2O-based sand-fixation material

    Science.gov (United States)

    Liu, Xin; Tie, Shengnian

    2017-07-01

    Searching for an economical and effective sand-fixing material and technology is of great importance in Northwest China. This paper described the use of a semihydrated gypsum-(CaSO4 ṡ 1/2H2O-)based composite as a sand-fixing material. Its morphology and composition were characterized by SEM, and its water resistance, freezing-thawing resistance and wind erosion resistance were tested in the field. The results indicated that semihydrated gypsum-(CaSO4 ṡ 1/2H2O-)based sand-fixing composite has good water resistance and water-holding capacity. Its strength is maintained at 1.42 MPa after 50 freezing and thawing cycles, and its wind erosion increases with increasing wind speed and slope. Its compressive strength starts to decrease after nine months of field tests with no change in appearance, but it still satisfies the requirements of fixation technology. This sand-fixing material should have wide application owing to its good weather resistance.

  2. Corticotropin releasing hormone- and adreno-corticotropin-like immunoreactivity in human placenta, peripheral and uterine vein plasma.

    Science.gov (United States)

    Schulte, H M; Healy, D L

    1987-01-01

    The presence of corticotropin releasing hormone (CRH)-like immunoreactivity (IR) in human placenta and maternal peripheral blood has been reported by many investigators. However, its physiological role has not yet been defined. We investigated plasma and placental tissue from women at different times of pregnancy and performed peripheral and uterine vein sampling during caesarean section before and after removal of the placenta. Beside IR-CRH, IR-GRF and -GnRH as well as -ACTH and cortisol were measured. The highest content of CRH was found in placental extracts from end term (40 weeks) pregnancies and lower levels at an earlier stage (10 weeks). Plasma CRH from peripheral blood could be detected in some samples and was higher as pregnancy advanced. Thirty minutes after removal of the placenta CRH levels dropped in peripheral plasma and could not be detected in uterine vein samples. IR-ACTH plasma levels were within the range of normals, cortisol was elevated. Gel- and HPLC-chromatographie revealed that placental extracts coeluted with synthetic human CRH. The material from endterm placenta showed full bioactivity in the rat pituitary bio-assay. IR-GRF could only be detected in 10 weeks placental tissue and no IR-GnRH was measured. We conclude that CRH from the placenta is biologically active, however, cannot stimulate the maternal pituitary-adrenal-axis.

  3. Interaction between Fibrinogen and Insulin-Like Growth Factor-Binding Protein-1 in Human Plasma under Physiological Conditions.

    Science.gov (United States)

    Gligorijević, N; Nedić, O

    2016-02-01

    Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.

  4. Les 2x12h : une solution au conflit de temporalités du travail posté ? The 12-hour shifts system: A way to settle conflicting temporalities in shift work

    Directory of Open Access Journals (Sweden)

    Béatrice Barthe

    2009-11-01

    Full Text Available Les horaires de travail en 2x12h semblent faciliter l’articulation des temporalités mise à mal par le travail posté, articulation avec les dimensions temporelles du fonctionnement humain : les rythmicités biologiques, l’organisation de la vie socio-familiale et la fatigue notamment. Dans un contexte social et économique favorable à la diffusion des 2x12h, cet article propose une synthèse des effets de ces horaires de travail posté sur la santé, sur la vie personnelle des opérateurs concernés et sur le travail. Même si, comparés aux autres systèmes d’horaires, les 2x12h semblent avoir des conséquences moindres sur la santé et la vie personnelle, cette synthèse montre l’importance d’autres facteurs (caractéristiques des personnes, temps d’exposition aux horaires, propriétés du système d’horaires, par exemple sur ces effets et la nécessité d’intégrer l’analyse du contenu du travail et des exigences du travail dans l’analyse de ces effets. Avant de penser le 2x12h comme une solution au conflit de temporalités du travail posté, la réflexion d’aménagement des horaires de travail se doit d’intégrer une analyse systémique et détaillée du travail, prenant en compte le contenu du travail, les exigences des tâches et les stratégies d’accord des temporalités mises en place par les opérateurs concernés au niveau de leur activité de travail.Twelve-hour shifts appear to facilitate the flow of temporalities involved in shift work schedules and the different temporal dimensions of human functioning: particularly biological rhythms, private life, and fatigue. In a social and economic context that favors the increase of 12-h shifts, this paper sums up the effects of 12-h shifts on health, personal life, and work. 12-h shifts seem to have less impact on health and personal life, when compared with other shift-work systems. This synthesis shows the importance of other factors (characteristics of the

  5. Simultaneous determination of nicotine, cotinine, and nicotine N-oxide in human plasma, semen, and sperm by LC-Orbitrap MS.

    Science.gov (United States)

    Abu-Awwad, Ahmad; Arafat, Tawfiq; Schmitz, Oliver J

    2016-09-01

    Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N'-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.

  6. Molecular distribution of gold(I) in human blood plasma after treatment with the antiarthritic disodium aurothiomalate.

    Science.gov (United States)

    Rayner, M H; Grootveld, M; Sadler, P J

    1989-01-01

    On-line separation and detection of both high- and low-molecular-weight gold species in human blood plasma treated with disodium aurothiomalate were achieved by a technique incorporating a combination of fast protein liquid chromatography with continuous monitoring of absorbance at 254 nm and eluant gold concentration by flame atomic absorption spectroscopy. The fate of gold (40 microM disodium aurothiomalate) in human blood plasma was monitored over a period of 24 h. Results obtained indicated that more than 50% of the gold is associated with a peak identified as albumin after a 5 min incubation period at 37 degrees C, and by 30 min more than 90% is found to be associated with albumin. This novel analytical combination enables the rapid, selective and sensitive determination of gold(I) and its biotransformation products (both protein--and non-protein-bound) in biological fluids.

  7. Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

    Directory of Open Access Journals (Sweden)

    Sérgio Luiz Dalmora

    2010-01-01

    Full Text Available A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 ºC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 μg/mL (r²=0.9999 and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference of phenobarbital 100 mg after single oral dose administration to healthy human volunteers.

  8. 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

    Stable peptidomimetics mimicking natural antimicrobial peptides (AMPs) have emerged as a promising class of potential novel antibiotics. In the present study, we aimed at determining whether the antibacterial activity of two α-peptide/β-peptoid peptidomimetics against a range of bacterial pathogens...... 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...... treatments might be lower than traditionally deduced from MICs determined in laboratory media. Thus, antibiotics previously considered too toxic could be developed into usable last-resort drugs, due to ensuing lowered risk of side effects. In contrast, the activity of the compounds was significantly...

  9. Evidence for local expansion of IgA plasma cell precursors in human ileum

    NARCIS (Netherlands)

    Yuvaraj, S.; Dijkstra, G.; Burgerhof, J.G.M.; Dammers, P.M.; Stoel, M.; Visser, Annie; Kroese, F.G.M.; Bos, N.A.

    2009-01-01

    IgA plays a crucial role in establishment and maintenance of mucosal homeostasis between host cells and commensal bacteria. To this end, numerous IgA plasma cells are located in the intestinal lamina propria. Whether the (immediate) precursor cells for these plasma cells can expand locally is not

  10. 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.

  11. 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, v

  12. Physical activity affects plasma coenzyme Q10 levels differently in young and old humans.

    Science.gov (United States)

    Del Pozo-Cruz, Jesús; Rodríguez-Bies, Elisabet; Ballesteros-Simarro, Manuel; Navas-Enamorado, Ignacio; Tung, Bui Thanh; Navas, Plácido; López-Lluch, Guillermo

    2014-04-01

    Coenzyme Q (Q) is a key lipidic compound for cell bioenergetics and membrane antioxidant activities. It has been shown that also has a central role in the prevention of oxidation of plasma lipoproteins. Q has been associated with the prevention of cholesterol oxidation and several aging-related diseases. However, to date no clear data on the levels of plasma Q during aging are available. We have measured the levels of plasmatic Q10 and cholesterol in young and old individuals showing different degrees of physical activity. Our results indicate that plasma Q10 levels in old people are higher that the levels found in young people. Our analysis also indicates that there is no a relationship between the degree of physical activity and Q10 levels when the general population is studied. However, very interestingly, we have found a different tendency between Q10 levels and physical activity depending on the age of individuals. In young people, higher activity correlates with lower Q10 levels in plasma whereas in older adults this ratio changes and higher activity is related to higher plasma Q10 levels and higher Q10/Chol ratios. Higher Q10 levels in plasma are related to lower lipoperoxidation and oxidized LDL levels in elderly people. Our results highlight the importance of life habits in the analysis of Q10 in plasma and indicate that the practice of physical activity at old age can improve antioxidant capacity in plasma and help to prevent cardiovascular diseases.

  13. Study on Effect of Gd (III) Speciation on Ca (II) Speciation in Human Blood Plasma by Computer Simulation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Ca (II) speciation and effect of Gd (III) speciation on Ca (II) speciation in human blood plasma were studied by computer simulation. [CaHCO3]+ is a predominant compound species of Ca (II). Gd (III) can compete with Ca (II) for biological molecules. The presence of Gd (III) results in a increase of concentration of free Ca (II) and a decrease of concentration of Ca (II) compounds.

  14. A New Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Bisoprolol in Human Plasma Samples

    OpenAIRE

    Gabriela Peste; Nela Bibire; Mihai Apostu; Aurel Vlase; Corneliu Oniscu

    2009-01-01

    Liquid chromatography (LC) coupled with mass spectrometry (MS) detection is one of the most powerful analytical tools for organic compound analysis. The advantages of using LC/MS methods over HPLC methods include: selectivity, chromatographic integrity, peak assignment, structural information, and rapid method development. In this paper, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of bisoprolol in human plasma s...

  15. Quantitation of drugs via molecularly imprinted polymer solid phase extraction and electrospray ionization mass spectrometry: benzodiazepines in human plasma

    OpenAIRE

    2011-01-01

    The association of solid phase extraction with molecularly imprinted polymers (MIP) and electrospray ionization mass spectrometry (ESI-MS) is applied to the direct extraction and quantitation of benzodiazepines in human plasma. The target analytes are sequestered by MIP and directly analyzed by ESI-MS. Due to the MIP highly selective extraction, ionic suppression during ESI is minimized; hence no separation is necessary prior to ESI-MS, which greatly increases analytical speed. Benzodiazepine...

  16. Determination of anticonvulsants in human plasma using SPME in a heated interface coupled online to liquid chromatography (SPME-LC)

    OpenAIRE

    Alves, Claudete; Gomes, Paulo Clairmont Feitosa de Lima; Neto, Álvaro José dos Santos; Rodrigues, Jose Carlos; Lanças, Fernando Mauro

    2012-01-01

    A simple and sensitive method using solid phase microextraction (SPME) and liquid chromatography (LC) with heated online desorption (SPME-LC) was developed and validated to analyze anticonvulsants (AEDs) in human plasma samples. A heated lab-made interface chamber was used in the desorption procedure, which allowed the transference of the whole extracted sample. The SPME conditions were optimized by applying an experimental design. Important factors are discussed such as fiber coating types, ...

  17. The time course of metabolites in human plasma after 6-[(18)F]fluoro-l-m-tyrosine administration.

    Science.gov (United States)

    Wahl, L M; Chen, J J; Thompson, M; Chirakal, R; Nahmias, C

    1999-11-01

    For the investigation of intracerebral dopamine metabolism, 6-[(18)F]fluoro-l-m-tyrosine (FmT) has been proposed as an alternative tracer that, unlike 6-[(18)F]fluoro-l-dopa, is not subjected to O-methylation. We have studied the time course of FmT and its labelled metabolites in plasma after intravenous injection into humans, employing a method that we have developed and validated to analyze labelled metabolites of FmT in plasma. The study population comprised 38 subjects, 14 of whom were taking an aromatic amino acid decarboxylase inhibitor (carbidopa), and obtained arterialized venous blood samples at various times after an intravenous injection of between 185 and 370 MBq of FmT. The major metabolite of FmT present in plasma was 6-[(18)F]fluoro-3-hydroxyphenylacetic acid. The time course of the fraction of radioactivity in plasma attributable to FmT was fitted well by the decay of two exponential functions. The fast component of the decay accounted for 40%-50% of the radioactivity and had a half-life of about 5 min. The slow component of the decay had a half-life of about 6 h in the subjects not taking carbidopa and 20 h in the patients taking carbidopa. When the total available FmT was calculated for each individual subject and expressed as a proportion of total radioactivity, this quantity did not differ significantly from that determined from the corresponding population mean. There was significantly more (15% on average) FmT available over the course of the 2-h experiment in the group pretreated with carbidopa. Our results demonstrate that the major metabolite in plasma after an intravenous injection of FmT is 6-[(18)F]fluoro-3-hydroxyphenylacetic acid. The clearance of FmT from plasma compares well with the clearance of intravenously administered levodopa. Carbidopa increases significantly the availability of FmT in plasma. These results verify, in a large number of human subjects, earlier analyses of FmT metabolism in other species. We validate a

  18. Simultaneous determination of piroxicam, meloxicam and tenoxicam in human plasma by liquid chromatography with tandem mass spectrometry.

    Science.gov (United States)

    Ji, Hye Young; Lee, Hye Won; Kim, Young Hoon; Jeong, Dong Won; Lee, Hye Suk

    2005-11-05

    A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of piroxicam, meloxicam and tenoxicam in human plasma was developed. Piroxicam, meloxicam, tenoxicam and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analyzed on a Sunfire column with the mobile phase of methanol:ammonium formate (15 mM, pH 3.0) (60:40, v/v). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring (MRM) mode. The standard curve was linear (r=1.000) over the concentration range of 0.50-200 ng/ml. The coefficient of variation (CV) and relative error (RE) for intra- and inter-assay statistics at three QC levels were 1.0-5.4% and -5.9 to 2.8%, respectively. The recoveries of piroxicam, meloxicam and tenoxicam ranged from 78.3 to 87.1%, with that of isoxicam being 59.7%. The lower limit of quantification for piroxicam, meloxicam and tenoxicam was 0.50 ng/ml using a 100 microl plasma sample. This method was successfully applied to a pharmacokinetic study of piroxicam after application of transdermal piroxicam patches to humans.

  19. 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.

  20. Simultaneous determination of telmisartan and amlodipine in human plasma by LC-MS]MS and its application in a human pharmacokinetic study

    Institute of Scientific and Technical Information of China (English)

    Vasu Babu Ravi; Jaswanth Kumar Inamadugu; Nageswara Rao Pilli; Vudagandla Sreenivasulu; Venkateswarlu Ponnerid

    2012-01-01

    A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of telmisartan and amlodipine in human plasma. Carbamazepine was used as an internal standard. Analytes and the internal standard were extracted from human plasma by solid-phase extraction technique using Waters Oasis HLB 1 cm3 (30 mg) extraction cartridge. The reconstituted samples were chromatographed on a Hypurity advance C18 column (50mm × 4.6mm, 5 gm) using a mixture of acetonitrile -5 mM ammonium acetate buffer (pH-4.0) (50:50, v/v) as the mobile phase at a flow rate of 0.8mL/min. The calibration curve obtained was linear (r_〉0.99) over the concentration range of 2.01-400.06 ng/mL for telmisartan and 0.05 -10.01 ng/mL for amlodipine. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.

  1. 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...

  2. 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.

  3. High hydrostatic pressure treatment for the inactivation of Staphylococcus aureus in human blood plasma.

    Science.gov (United States)

    Rivalain, Nolwennig; Roquain, Jean; Boiron, Jean-Michel; Maurel, Jean-Paul; Largeteau, Alain; Ivanovic, Zoran; Demazeau, Gérard

    2012-02-15

    For the past 30years, pressure inactivation of microorganisms has been developed in biosciences, in particular for foods and more recently for biological products, including pharmaceutical ones. In many past studies, the effect of high hydrostatic pressure (HHP) processes on pathogens focused mainly on the effect of an increase of the pressure value. To assure the safety of pharmaceutical products containing fragile therapeutic components, development of new decontamination processes at the lowest pressure value is needed to maintain their therapeutic properties. The aim of this study was therefore to evaluate the impact of the process parameters characterizing high-pressure treatments [such as the pressurization rate (PR) and the application mode (AM)] on the inactivation of pathogens, in particular to determine how these parameters values could help decrease the pressure value necessary to reach the same inactivation level. The effect of these physical parameters was evaluated on the inactivation of Staphylococcus aureus ATCC 6538 which is an opportunistic pathogen of important relevance in the medical, pharmaceutical and food domains. Human blood plasma was chosen as the suspension medium because of its physiological importance in the transfusion field. It was shown that the optimization of all the selected parameters could lead to a high inactivation level (≈5log(10) decrease of the initial bacterial load) at a pressure level as low as 200MPa, underlining some synergistic effects among these parameters. Complete inactivation of the initial bacterial population was achieved for the following conditions: PR=50MPas(-1), AM=5×2min, T≈-5°C and P=300MPa.

  4. Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates.

    Science.gov (United States)

    Stremler, K E; Stafforini, D M; Prescott, S M; McIntyre, T M

    1991-06-15

    Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.

  5. 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.

  6. 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.

  7. Validation of a rapid, automated method for the measurement of ethylene glycol in human plasma.

    Science.gov (United States)

    Robson, Anna F; Lawson, Alexander J; Lewis, Laura; Jones, Alan; George, Stephen

    2017-07-01

    Background Ethylene glycol is a highly toxic compound found in various household products. Cases of poisoning are rare but may be fatal unless diagnosed and treated promptly. Early recognition of poisoning is critical for the management and recovery of patients. Indirect testing is not specific for the presence of ethylene glycol. Therefore, urgent and accurate measurement should be sought if ingestion is suspected in order to determine the need for treatment with an antidote. Here, we present the validation of an automated assay for measurement of ethylene glycol on an Abbott Architect using a commercially available kit (Catachem). Methods Analytical parameters of imprecision, linearity, stability and bias were determined using spiked human plasma samples processed on both the Catachem assay and on an in-house gas chromatography-mass spectrometry method. Interference was assessed using samples collected into a variety of sample collection tubes and spiked with a number of alcohols. Results Excellent agreement was observed between the two methodologies with the enzymatic assay demonstrating linearity and precision across the relevant clinical range (50-3000 mg/L). In addition, the Catachem assay displayed no interference from a number of different sample tubes and alcohols. However, propylene glycol interference was observed at concentrations associated with excessive use (>1 g/L) and 2,3-butanediol interference observed at concentrations associated with butanone ingestion. Inspection of the enzymatic reaction profile was found to differentiate between alcohols. Conclusions This automated assay is suitable for the diagnosis of ethylene glycol poisoning and is now in routine use, enabling the laboratory to provide a rapid 24 h service with support by gas chromatography-mass spectrometry as necessary.

  8. 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.

  9. Circulating Plasma microRNAs can differentiate Human Sepsis and Systemic Inflammatory Response Syndrome (SIRS).

    Science.gov (United States)

    Caserta, Stefano; Kern, Florian; Cohen, Jonathan; Drage, Stephen; Newbury, Sarah F; Llewelyn, Martin J

    2016-06-20

    Systemic inflammation in humans may be triggered by infection, termed sepsis, or non-infective processes, termed non-infective systemic inflammatory response syndrome (SIRS). MicroRNAs regulate cellular processes including inflammation and may be detected in blood. We aimed to establish definitive proof-of-principle that circulating microRNAs are differentially affected during sepsis and non-infective SIRS. Critically ill patients with severe (n = 21) or non-severe (n = 8) intra-abdominal sepsis; severe (n = 23) or non-severe (n = 21) non-infective SIRS; or no SIRS (n = 16) were studied. Next-generation sequencing and qRT-PCR were used to measure plasma microRNAs. Detectable blood miRNAs (n = 116) were generally up-regulated in SIRS compared to no-SIRS patients. Levels of these 'circulating inflammation-related microRNAs' (CIR-miRNAs) were 2.64 (IQR: 2.10-3.29) and 1.52 (IQR: 1.15-1.92) fold higher for non-infective SIRS and sepsis respectively (p SIRS. Six CIR-miRNAs (miR-30d-5p, miR-30a-5p, miR-192-5p, miR-26a-5p, miR-23a-5p, miR-191-5p) provided good-to-excellent discrimination of severe sepsis from severe SIRS (0.742-0.917 AUC of ROC curves). CIR-miRNA levels inversely correlated with pro-inflammatory cytokines (IL-1, IL-6 and others). Thus, among critically ill patients, sepsis and non-infective SIRS are associated with substantial, differential changes in CIR-miRNAs. CIR-miRNAs may be regulators of inflammation and warrant thorough evaluation as diagnostic and therapeutic targets.

  10. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

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