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Sample records for plasma hepcidin assays

  1. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization.

    Science.gov (United States)

    Kroot, Joyce J C; Kemna, Erwin H J M; Bansal, Sukhvinder S; Busbridge, Mark; Campostrini, Natascia; Girelli, Domenico; Hider, Robert C; Koliaraki, Vasiliki; Mamalaki, Avgi; Olbina, Gordana; Tomosugi, Naohisa; Tselepis, Chris; Ward, Douglas G; Ganz, Tomas; Hendriks, Jan C M; Swinkels, Dorine W

    2009-12-01

    The recently discovered iron regulatory peptide hormone hepcidin holds promise as a novel biomarker in iron metabolism disorders. To date, various mass spectrometry and immunochemical methods have been developed for its quantification in plasma and urine. Differences in methodology and analytical performance hinder the comparability of data. As a first step towards method harmonization, several hepcidin assays were compared. Worldwide eight laboratories participated in a urinary and plasma round robin in which hepcidin was analyzed. For both urine and plasma: (i) the absolute hepcidin concentrations differed widely between methods, (ii) the between-sample variation and the analytical variation of the methods are similar. Importantly, the analytical variation as percentage of the total variance is low for all methods, indicating their suitability to distinguish hepcidin levels of different samples. Spearman correlations between methods were generally high. The round robin results inform the scientific and medical community on the status and agreement of the current hepcidin methods. Ongoing initiatives should facilitate standardization by exchanging calibrators and representative samples.

  2. A novel immunological assay for hepcidin quantification in human serum.

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    Vasiliki Koliaraki

    Full Text Available BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L and 10 patients with iron deficiency anemia (15.7 microg/L and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L compared to 32 age-matched healthy controls (42.7 microg/L. CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

  3. Plasma Concentrations of Hepcidin in Anemic Zimbabwean Infants.

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    Tatenda G Mupfudze

    Full Text Available Anemia in infancy is a global public health problem. We evaluated the relative contributions of iron deficiency and inflammation to infant anemia.We measured plasma hepcidin, ferritin, soluble transferrin receptor (sTfR, alpha-1-acid glycoprotein and C-reactive protein (CRP by ELISA on archived plasma from 289 HIV-unexposed anemic or non-anemic Zimbabwean infants at ages 3 mo, 6 mo and 12 mo. Among anemic infants, we determined the proportion with iron-deficiency anemia (IDA and anemia of inflammation (AI. We undertook regression analyses of plasma hepcidin and anemia status, adjusting for sex, age and birthweight.Anemic infants at 3 mo were more stunted and had higher CRP (median 0.45 vs 0.21 mg/L; P = 0.037 and hepcidin (median 14.7 vs 9.7 ng/mL; P = 0.022 than non-anemic infants, but similar levels of ferritin and sTfR; 11% infants had IDA and 15% had AI. Anemic infants at 6 mo had higher hepcidin (median 7.9 vs 4.5 ng/mL; P = 0.016 and CRP (median 2.33 vs 0.32 mg/L; P<0.001, but lower ferritin (median 13.2 vs 25.1 μg/L; P<0.001 than non-anemic infants; 56% infants had IDA and 12% had AI. Anemic infants at 12 mo had lower ferritin (median 3.2 vs 22.2 μg/L; P<0.001 and hepcidin (median 0.9 vs 1.9 ng/mL; P = 0.019, but similar CRP levels; 48% infants had IDA and 8% had AI. Comparing anemic with non-anemic infants, plasma hepcidin was 568% higher, 405% higher and 64% lower at 3 mo, 6 mo and 12 mo, respectively, after adjusting for sex and birthweight (all p<0.01. Plasma hepcidin declined significantly with age among anemic but not non-anemic infants. Girls had 61% higher hepcidin than boys, after adjusting for age, anemia and birthweight (p<0.001.Anemia is driven partly by inflammation early in infancy, and by iron deficiency later in infancy, with plasma hepcidin concentrations reflecting the relative contribution of each. However, there is need to better characterize the drivers of hepcidin during infancy in developing countries.

  4. Second round robin for plasma hepcidin methods: first steps toward harmonization.

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    Kroot, Joyce J C; van Herwaarden, Antonius E; Tjalsma, Harold; Jansen, Rob T P; Hendriks, Jan C M; Swinkels, Dorine W

    2012-10-01

    Measurements of the iron regulatory hormone hepcidin by various methodologies and laboratories are not harmonized. As a result different numeric results are obtained for the same clinical sample. We investigated whether better agreement between plasma hepcidin methods can be achieved by harmonization. Native plasma pools (n = 11) of a variety of hepcidin concentrations and blank plasma spiked with three different quantities of synthetic hepcidin-25 purchased from two different commercial sources (n = 6), were distributed in duplicate among 21 methods worldwide. We assessed commutability by comparing results from synthetic hepcidin with those from native samples in various method couples by Bland-Altman plots. Methods differed substantially in absolute values and reproducibility. For the majority of methods we found that samples with synthetic hepcidin-25 were noncommutable with the native samples. In an attempt to harmonize by using native hepcidin results, we selected two methods that showed good mutual agreement of native results and calculated consensus values as the medians for the 11 duplicate native samples obtained by these two methods. Finally, we constructed algorithms enabling the laboratories to calculate the hepcidin consensus (HEPCON) value using their own native hepcidin results. We found that the use of these algorithms substantially reduced the between-method CV. Until commutable materials are defined, hepcidin harmonization can be achieved by exploiting specific algorithms, allowing each lab to report their native hepcidin concentrations in HEPCON values. This study represents the first step toward harmonization of plasma hepcidin methods and facilitates aggregation of hepcidin data from different research investigations.

  5. A Novel Immunological Assay for Hepcidin Quantification in Human Serum

    OpenAIRE

    Vasiliki Koliaraki; Martha Marinou; Vassilakopoulos, Theodoros P.; Eustathios Vavourakis; Emmanuel Tsochatzis; Pangalis, Gerassimos A.; George Papatheodoridis; Alexandra Stamoulakatou; Dorine W Swinkels; George Papanikolaou; Avgi Mamalaki

    2009-01-01

    BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a v...

  6. A novel immunological assay for hepcidin quantification in human serum.

    NARCIS (Netherlands)

    Koliaraki, V.; Marinou, M.; Vassilakopoulos, T.P.; Vavourakis, E.; Tsochatzis, E.; Pangalis, G.A.; Papatheodoridis, G.; Stamoulakatou, A.; Swinkels, D.W.; Papanikolaou, G.; Mamalaki, A.

    2009-01-01

    BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overloa

  7. Plasma hepcidin levels and anemia in old age. The Leiden 85-Plus Study

    NARCIS (Netherlands)

    Elzen, W.P. den; Craen, A.J. de; Wiegerinck, E.T.G.; Westendorp, R.G.J.; Swinkels, D.W.; Gussekloo, J.

    2013-01-01

    Hepcidin, an important regulator of iron homeostasis, is suggested to be causally related to anemia of inflammation. The aim of this study was to explore the role of plasma hepcidin in anemia among older persons from the general population. The Leiden 85-Plus Study is a population-based study of

  8. Plasma hepcidin levels and anemia in old age. The Leiden 85-Plus Study

    NARCIS (Netherlands)

    Elzen, W.P. den; Craen, A.J. de; Wiegerinck, E.T.G.; Westendorp, R.G.J.; Swinkels, D.W.; Gussekloo, J.

    2013-01-01

    Hepcidin, an important regulator of iron homeostasis, is suggested to be causally related to anemia of inflammation. The aim of this study was to explore the role of plasma hepcidin in anemia among older persons from the general population. The Leiden 85-Plus Study is a population-based study of 85-

  9. Plasma hepcidin levels and anemia in old age. The Leiden 85-Plus Study

    NARCIS (Netherlands)

    Elzen, W.P. den; Craen, A.J. de; Wiegerinck, E.T.G.; Westendorp, R.G.J.; Swinkels, D.W.; Gussekloo, J.

    2013-01-01

    Hepcidin, an important regulator of iron homeostasis, is suggested to be causally related to anemia of inflammation. The aim of this study was to explore the role of plasma hepcidin in anemia among older persons from the general population. The Leiden 85-Plus Study is a population-based study of 85-

  10. Targeting the Hepcidin-Ferroportin Axis in the Diagnosis and Treatment of Anemias

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    Elizabeta Nemeth

    2010-01-01

    Full Text Available The hepatic peptide hormone hepcidin regulates dietary iron absorption, plasma iron concentrations, and tissue iron distribution. Hepcidin acts by causing the degradation of its receptor, the cellular iron exporter ferroportin. The loss of ferroportin decreases iron flow into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron, thereby lowering plasma iron concentrations. Malfunctions of the hepcidin-ferroportin axis contribute to the pathogenesis of different anemias. Deficient production of hepcidin causes systemic iron overload in iron-loading anemias such as beta-thalassemia; whereas hepcidin excess contributes to the development of anemia in inflammatory disorders and chronic kidney disease, and may cause erythropoietin resistance. The diagnosis of different forms of anemia will be facilitated by improved hepcidin assays, and the treatment will be enhanced by the development of hepcidin agonists and antagonists.

  11. Advances in quantitative hepcidin measurements by time-of-flight mass spectrometry.

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    Dorine W Swinkels

    Full Text Available Assays for the detection of the iron regulatory hormone hepcidin in plasma or urine have not yet been widely available, whereas quantitative comparisons between hepcidin levels in these different matrices were thus far even impossible due to technical restrictions. To circumvent these limitations, we here describe several advances in time-of flight mass spectrometry (TOF MS, the most important of which concerned spiking of a synthetic hepcidin analogue as internal standard into serum and urine samples. This serves both as a control for experimental variation, such as recovery and matrix-dependent ionization and ion suppression, and at the same time allows value assignment to the measured hepcidin peak intensities. The assay improvements were clinically evaluated using samples from various patients groups and its relevance was further underscored by the significant correlation of serum hepcidin levels with serum iron indices in healthy individuals. Most importantly, this approach allowed kinetic studies as illustrated by the paired analyses of serum and urine samples, showing that more than 97% of the freely filtered serum hepcidin can be reabsorbed in the kidney. Thus, the here reported advances in TOF MS-based hepcidin measurements represent critical steps in the accurate quantification of hepcidin in various body fluids and pave the way for clinical studies on the kinetic behavior of hepcidin in both healthy and diseased states.

  12. Combinatorial effects of malaria season, iron deficiency, and inflammation determine plasma hepcidin concentration in African children.

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    Atkinson, Sarah H; Armitage, Andrew E; Khandwala, Shivani; Mwangi, Tabitha W; Uyoga, Sophie; Bejon, Philip A; Williams, Thomas N; Prentice, Andrew M; Drakesmith, Hal

    2014-05-22

    Hepcidin is the master regulatory hormone that governs iron homeostasis and has a role in innate immunity. Although hepcidin has been studied extensively in model systems, there is less information on hepcidin regulation in global health contexts where iron deficiency (ID), anemia, and high infectious burdens (including malaria) all coexist but fluctuate over time. We evaluated iron status, hepcidin levels, and determinants of hepcidin in 2 populations of rural children aged ≤8 years, in the Gambia and Kenya (total n = 848), at the start and end of a malaria season. Regression analyses and structural equation modeling demonstrated, for both populations, similar combinatorial effects of upregulating stimuli (iron stores and to a lesser extent inflammation) and downregulating stimuli (erythropoietic drive) on hepcidin levels. However, malaria season was also a significant factor and was associated with an altered balance of these opposing factors. Consistent with these changes, hepcidin levels were reduced whereas the prevalence of ID was increased at the end of the malaria season. More prevalent ID and lower hepcidin likely reflect an enhanced requirement for iron and an ability to efficiently absorb it at the end of the malaria season. These results, therefore, have implications for ID and malaria control programs. © 2014 by The American Society of Hematology.

  13. Hepcidin-Induced Iron Deficiency Is Related to Transient Anemia and Hypoferremia in Kawasaki Disease Patients

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    Huang, Ying-Hsien; Kuo, Ho-Chang; Huang, Fu-Chen; Yu, Hong-Ren; Hsieh, Kai-Sheng; Yang, Ya-Ling; Sheen, Jiunn-Ming; Li, Sung-Chou; Kuo, Hsing-Chun

    2016-01-01

    Kawasaki disease (KD) is a type of systemic vasculitis that primarily affects children under the age of five years old. For sufferers of KD, intravenous immunoglobulin (IVIG) has been found to successfully diminish the occurrence of coronary artery lesions. Anemia is commonly found in KD patients, and we have shown that in appropriately elevated hepcidin levels are related to decreased hemoglobin levels in these patients. In this study, we investigated the time period of anemia and iron metabolism during different stages of KD. A total of 100 patients with KD and 20 control subjects were enrolled in this study for red blood cell and hemoglobin analysis. Furthermore, plasma, urine hepcidin, and plasma IL-6 levels were evaluated using enzyme-linked immunosorbent assay in 20 KD patients and controls. Changes in hemoglobin, plasma iron levels, and total iron binding capacity (TIBC) were also measured in patients with KD. Hemoglobin, iron levels, and TIBC were lower (p < 0.001, p = 0.009, and p < 0.001, respectively) while plasma IL-6 and hepcidin levels (both p < 0.001) were higher in patients with KD than in the controls prior to IVIG administration. Moreover, plasma hepcidin levels were positively and significantly correlated with urine hepcidin levels (p < 0.001) prior to IVIG administration. After IVIG treatment, plasma hepcidin and hemoglobin levels significantly decreased (both p < 0.001). Of particular note was a subsequent gradual increase in hemoglobin levels during the three weeks after IVIG treatment; nevertheless, the hemoglobin levels stayed lower in KD patients than in the controls (p = 0.045). These findings provide a longitudinal study of hemoglobin changes and among the first evidence that hepcidin induces transient anemia and hypoferremia during KD’s acute inflammatory phase. PMID:27187366

  14. SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

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    Johnson Philip J

    2008-10-01

    Full Text Available Abstract Background Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS. Results We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin. Conclusion We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

  15. Cellular catabolism of the iron-regulatory peptide hormone hepcidin.

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    Gloria Cuevas Preza

    Full Text Available Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep was added to ferroportin-GFP (Fpn-GFP expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.

  16. Correlation of serum hepcidin levels with disease progression in hepatitis B virus-related disease assessed by nanopore film based assay

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    Wang, Jing; Dong, Ailian; Liu, Gang; Anderson, Gregory J.; Hu, Tony Y.; Shi, Jian; Hu, Yulin; Nie, Guangjun

    2016-01-01

    Chronic hepatitis B virus (HBV) infection often develop into cirrhosis, and both are major risk factors of hepatocellular carcinoma. However, effective approaches for the monitoring of HBV-related disease progress are still in need. Increased iron storage has an important role in HBV-related diseases. Hepcidin is a key regulator of iron homeostasis whose expression changes are often indicative of abnormal iron metabolism. There are few reports of hepcidin levels in patients with HBV infections, and the available results are inconsistent. In this study, using a recently validated nanopore silica film based method, we measured serum hepcidin levels in 46 HBV-related patients and 20 healthy controls. Patients were divided into three groups: chronic hepatitis B without cirrhosis; HBV-related cirrhosis; and HBV-related cirrhosis with hepatocellular carcinoma. Compared to healthy controls, the mean serum hepcidin level was significantly higher in CHB patients without cirrhosis, and in those with hepatocellular carcinoma, but not in those with cirrhosis. Iron-loading, viral infection and liver dysfunction are determined to be the major regulators of hepcidin in these patients. These observations suggest correlations between serum hepcidin and progression of chronic HBV infection, and may shed a new light on the development of biomarkers for HBV-related disease surveillance. PMID:27694815

  17. Hepcidin modulation in human diseases: From research to clinic

    Institute of Scientific and Technical Information of China (English)

    Alberto Piperno; Raffaella Mariani; Paola Trombini; Domenico Girelli

    2009-01-01

    By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake and mobilization from stores to meet body iron need. In recent years there has been important advancement in our knowledge of hepcidin regulation that also has implications for understanding the physiopathology of some human disorders. Since the discovery of hepcidin and the demonstration of its pivotal role in iron homeostasis, there has been a substantial interest in developing a reliable assay of the hormone in biological fluids. Measurement of hepcidin in biological fluids can improve our understanding of iron diseases and be a useful tool for diagnosis and clinical management of these disorders. We reviewed the literature and our own research on hepcidin to give an updated status of the situation in this rapidly evolving field.

  18. Renal Handling of Circulating and Renal-Synthesized Hepcidin and Its Protective Effects against Hemoglobin-Mediated Kidney Injury.

    NARCIS (Netherlands)

    Swelm, R.P. van; Wetzels, J.F.; Verweij, V.G.; Laarakkers, C.M.; Pertijs, J.C.; Wijst, J.A. van der; Thevenod, F.; Masereeuw, R.; Swinkels, D.W.

    2016-01-01

    Urinary hepcidin may have protective effects against AKI. However, renal handling and the potential protective mechanisms of hepcidin are not fully understood. By measuring hepcidin levels in plasma and urine using mass spectrometry and the kidney using immunohistochemistry after intraperitoneal adm

  19. Subcutaneous Adipose Tissue from Obese and Lean Adults Does Not Release Hepcidin In Vivo

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    Lisa Tussing-Humphreys

    2011-01-01

    Full Text Available Hepcidin is the main regulator of systemic iron homeostasis and is primarily produced by the liver but is also expressed, at the mRNA-level, in periphery tissues including the subcutaneous and visceral adipose tissue. Obesity is associated with elevated hepcidin concentrations and iron depletion suggesting that the exaggerated fat mass in obesity could contribute significantly to circulating hepcidin levels consequently altering iron homeostasis. The objective of this study was to determine if abdominal subcutaneous adipose tissue (AbScAT releases hepcidin in vivo and if release is modified by obesity. Arterio-venous differences in concentrations of hepcidin were measured across AbScAT in 9 obese and 9 lean adults. Overall (n=18, mean plasma hepcidin concentrations were significantly higher in arterialized compared to AbScAT venous samples [mean difference (arterialized-AbScAT venous plasma hepcidin = 4.9±9.6 ng/mL, P=0.04]. Net regional release was not calculated because mean venous plasma hepcidin concentrations were lower than mean arterialized concentrations indicating no net release. Significant correlations between AbScAT venous and arterialized plasma hepcidin concentrations with anthropometric variables were not observed. Findings from this vein drainage study suggest there is no net release of hepcidin from the AbScAT depot and thereby no ability to signal systemically, even in obesity.

  20. Is hepcidin-25 a predictor of atherosclerosis in hemodialysis patients?

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    Kali, Alaaddin; Yayar, Ozlem; Erdogan, Bulent; Eser, Baris; Buyukbakkal, Mehmet; Ercan, Zafer; Merhametsiz, Ozgur; Haspulat, Ayhan; Gök Oğuz, Ebru; Canbakan, Basol; Ayli, Mehmet D

    2016-04-01

    Atherosclerotic cardiovascular disease is an important cause of mortality and morbidity in hemodialysis patients. Iron accumulation in arterial wall macrophages is increased in atherosclerotic lesions. Hepcidin is a key hepatic hormone regulating iron balance. It inhibits iron release from macrophages and iron absorption from enterocytes by binding and inactivating the cellular iron exporter ferroportin. The aim of this study is to investigate the relation of hepcidin-25, iron parameters, and atherosclerosis measured by carotid intima media thickness (CIMT) in hemodialysis patients. Eighty-two hemodialysis patients were enrolled in this cross-sectional study. Predialysis blood samples were centrifuged at 1500 g and 4°C for 10 minutes and stored at -80°C for the measurement of hepcidin-25. DRG hepcidin enzyme-linked immunosorbent assay kit was used for the measurement of hepcidin-25. Ultrasonographical B-mode imaging of bilateral carotid arteries was performed with a high-resolution real-time ultrasonography (Mindray DC7). Mean age of the study population was 57.90 ± 16.08 years and 43.9% were men. Total study population was grouped into two according to median value of hepcidin-25. There was no difference between groups with respect to age, dialysis vintage, and C-reactive protein. CIMT was found to be statistically significantly higher in low hepcidin-25 group. In correlation analysis, CIMT was found to be correlated with age (P < 0.01, R = 0.33) and hepcidin-25 (P < 0.01, R = 0.46). In linear regression analysis, age (β = 0.31) and hepcidin-25 (β = 0.44) were found to be the determinants of CIMT in hemodialysis patients. Our results implicate that hepcidin may take part in pathophysiology of atherosclerosis and cardiovascular disease in hemodialysis patients.

  1. Liquid chromatographic assay for dicloxacillin in plasma.

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    Alderete, Oscar; González-Esquivel, Dinora F; Del Rivero, L Misael; Castro Torres, Nelly

    2004-06-15

    A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.

  2. Hepcidin antagonists for potential treatments of disorders with hepcidin excess

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    Poli eMaura

    2014-04-01

    Full Text Available The discovery of hepcidin clarified the basic mechanism of the control of systemic iron homeostasis. Hepcidin is mainly produced by the liver as a propeptide and processed by furin into the mature active peptide. Hepcidin binds ferroportin, the only cellular iron exporter, causing the internalization and degradation of both. Thus hepcidin blocks iron export from the key cells for dietary iron absorption (enterocytes, recycling of haemoglobin iron (the macrophages and the release of storage iron from hepatocytes, resulting in the reduction of systemic iron availability. The BMP/HJV/SMAD pathway is the major regulator of hepcidin expression that responds to iron status. Also inflammation stimulates hepcidin via the IL6/STAT3 pathway with a support of an active BMP/HJV/SMAD pathway. In some pathological conditions hepcidin level is inadequately elevated and reduces iron availability in the body, resulting in anemia. These conditions occur in the genetic Iron Refractory Iron Deficiency Anemia (IRIDA and the common Anemia of Chronic Disease (ACD or Anemia of Inflammation. Currently, there is no definite treatment for ACD. Erythropoiesis stimulating agents and intravenous iron have been proposed in some cases but they are scarcely effective and may have adverse effects. Alternative approaches aimed to a pharmacological control of hepcidin expression have been attempted, targeting different regulatory steps. They include hepcidin sequestering agents (antibodies, anticalins and aptamers, inhibitors of BMP/SMAD or of IL6/STAT3 pathway or of hepcidin transduction (siRNA/shRNA or ferroportin stabilizers. In this review we summarized the biochemical interactions of the proteins involved in the BMP/HJV/SMAD pathway and its natural inhibitors, the murine and rat models with high hepcidin levels currently available and finally the progresses in the development of hepcidin antagonists, with particular attention to the role of heparins and heparin sulphate

  3. Gas Plasma Surface Chemistry for Biological Assays.

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    Sahagian, Khoren; Larner, Mikki

    2015-01-01

    Biological systems respond to and interact with surfaces. Gas plasma provides a scalable surface treatment method for designing interactive surfaces. There are many commercial examples of plasma-modified products. These include well plates, filtration membranes, dispensing tools, and medical devices. This chapter presents an overview of gas plasma technology and provides a guide to using gas plasma for modifying surfaces for research or product development.

  4. Improved immunoradiometric assay for plasma renin

    NARCIS (Netherlands)

    J. Deinum (Jacob); F.H.M. Derkx (Frans); M.A.D.H. Schalekamp (Maarten)

    1999-01-01

    textabstractBACKGROUND: Our renin IRMA overestimated renin in plasmas with high prorenin-to-renin ratios. We suspected that the overestimation of renin was caused less by cross-reactivity of the renin-specific antibody with prorenin than by a conformational change of pr

  5. Development of robotic plasma radiochemical assays for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J. [Brookhaven National Lab., Upton, NY (United States). Dept. of Chemistry

    1995-12-01

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [{sup 11}C]Benztropine, [{sup 11}C]cocaine, [{sup 11}C]clorgyline, [{sup 11}C]deprenyl, [{sup 11}C]methadone, [{sup 11}C]methylphenidate, [{sup 11}C]raclorpride, and [{sup 11}C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers.

  6. Development of a rapid, simple assay of plasma total carotenoids

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    Donaldson Michael

    2012-09-01

    Full Text Available Abstract Background Plasma total carotenoids can be used as an indicator of risk of chronic disease. Laboratory analysis of individual carotenoids by high performance liquid chromatography (HPLC is time consuming, expensive, and not amenable to use beyond a research laboratory. The aim of this research is to establish a rapid, simple, and inexpensive spectrophotometric assay of plasma total carotenoids that has a very strong correlation with HPLC carotenoid profile analysis. Results Plasma total carotenoids from 29 volunteers ranged in concentration from 1.2 to 7.4 μM, as analyzed by HPLC. A linear correlation was found between the absorbance at 448 nm of an alcohol / heptane extract of the plasma and plasma total carotenoids analyzed by HPLC, with a Pearson correlation coefficient of 0.989. The average coefficient of variation for the spectrophotometric assay was 6.5% for the plasma samples. The limit of detection was about 0.3 μM and was linear up to about 34 μM without dilution. Correlations between the integrals of the absorption spectra in the range of carotenoid absorption and total plasma carotenoid concentration gave similar results to the absorbance correlation. Spectrophotometric assay results also agreed with the calculated expected absorbance based on published extinction coefficients for the individual carotenoids, with a Pearson correlation coefficient of 0.988. Conclusion The spectrophotometric assay of total carotenoids strongly correlated with HPLC analysis of carotenoids of the same plasma samples and expected absorbance values based on extinction coefficients. This rapid, simple, inexpensive assay, when coupled with the carotenoid health index, may be useful for nutrition intervention studies, population cohort studies, and public health interventions.

  7. An Evaluation of the Correlation between Hepcidin Serum Levels and Disease Activity in Inflammatory Bowel Disease

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    Zehra Betül Paköz

    2015-01-01

    Full Text Available Aim. While there are many well-defined serological markers for inflammatory bowel disease (IBD, there is limited evidence that they positively affect clinical outcomes. This study aimed to evaluate the correlation between hepcidin serum levels and disease activity in IBD. Materials and Methods. Eighty-five consecutive IBD patients were enrolled in the study. Hepcidin serum levels were assessed using an enzyme-linked immunosorbent assay (ELISA and were compared with disease activity as well as the interleukin-6 (IL-6 and C-reactive protein (CRP levels. Results. The mean hepcidin serum levels in Crohn’s disease (CD patients in remission and in the active phase were 3837±1436 and 3752±1274 pg/mL, respectively P=0.613. The mean hepcidin serum levels in ulcerative colitis (UC patients in remission and in the active phase were 4285±8623 and 3727±1176 pg/mL, respectively P=0.241. Correlation analysis between inflammatory markers and hepcidin serum levels indicated that there was no correlation between hepcidin levels and IL-6 P=0.582 or CRP P=0.783. Conclusion. As an acute-phase protein, hepcidin seems to have a lower efficacy than other parameters in the detection of activation in IBD.

  8. Serum Hepcidin Predicts Uremic Accelerated Atherosclerosis in Chronic Hemodialysis Patients with Diabetic Nephropathy

    Institute of Scientific and Technical Information of China (English)

    Han Li; Su-Juan Feng; Lu-Lu Su; Wei Wang; Xiao-Dong Zhang; Shi-Xiang Wang

    2015-01-01

    Background:Hepcidin,as a regulator of body iron stores,has been recently discovered to play a critical role in the pathogenesis of anemia of chronic disease.Atherosclerotic cardiovascular disease is the most common complication and the leading cause of death in chronic hemodialysis (CHD) patients.In the current study,we aimed to explore the relationship between serum hepcidin and uremic accelerated atherosclerosis (UAAS) in CHD patients with diabetic nephropathy (CHD/DN).Methods:A total of 78 CHD/DN and 86 chronic hemodialyzed nondiabetic patients with chronic glomerulonephritis (CHD/non-DN) were recruited in this study.The level of serum hepcidin-25 was specifically measured by liquid chromatography-tandem mass spectrometry.Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay.Results:High serum level ofhepcidin-25 was seen in CHD patients.Serum hepcidin-25 in CHD/DN was significantly higher than that in CHD/non-DN patients.Serum hepcidin-25 was positively correlated with ferritin,high-sensitivity C-reactive protein (hs-CRP),TNF-α,and IL-6 in CHD/DN patients.CHD/DN patients exhibited higher common carotid artery intima media thickness (CCA-IMT),hs-CRP,and hepcidin-25 levels than that in CHD/non-DN patients.Moreover,in CHD/DN patients,CCA-IMT was positively correlated with serum hepcidin,hs-CRP,and low-density lipoprotein-cholesterol.On multiple regression analysis,serum hepcidin and hs-CRP level exhibited independent association with IMT in CHD/DN patients.Conclusions:These findings suggest possible linkage between iron metabolism and hepcidin modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients.

  9. SERUM HEPCIDIN REFERENCE RANGE, GENDER DIFFERENCES, MENOPAUSAL DEPENDENCE AND BIOCHEMICAL CORRELATES IN HEALTHY SUBJECTS

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    Biljana Ilkovska

    2016-05-01

    Full Text Available Background: Hepcidin has emerged as the central regulatory molecule of iron homeostasis. Iron deficiency and iron overload play a major role in molecular insights of many disease states and serum hepcidin normal values and biochemical correlations are of substantial importance. Objective: The aim of this study is to examine the serum hepcidin reference range, gender and age differences, menopausal dependence and biochemical correlates in healthy subjects. Methods: Serum hepcidin concentration was measured with a competitive enzyme-linked immunosorbent assay (DRG Hepcidin-25 ELISA Kit together with hemoglobin, hematocrit, serum iron, transferrin and C-reactive protein in 120 healthy subjects both men and pre- and post-menopausal women. Results: Normal serum hepcidin values were found in the range of 1,23 – 36,46 ng/mL (mean 9,25 ± 6,45 ng/mL.There were statistically significant differences in measured hepcidin levels between men (12,34 ± 7,37 ng/mL and women (6,16 ± 3,2 ng/mL (p<0.01 and between pre- menopausal (5,51 ± 2,8 ng/mL and post-menopausal women (7,29 ± 3,59 ng/mL ( p<0,05. Strong correlations were found with serum ferritin and hemoglobin but not with serum iron, transferrin and CRP. No 5-year age interval differences were deemed significant. Conclusion: Serum hepcidin concentration varied substantially between subjects, which is reflected in wide reference ranges. Serum hepcidin levels were gender and menopausal status related and were in correlation with hemoglobin and serum ferritin in healthy subjects

  10. The A736V TMPRSS6 polymorphism influences hepcidin and iron metabolism in chronic hemodialysis patients: TMPRSS6 and hepcidin in hemodialysis

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    Pelusi Serena

    2013-02-01

    Full Text Available Abstract Background Aim of this study was to evaluate whether the A736V TMPRSS6 polymorphism, a major genetic determinant of iron metabolism in healthy subjects, influences serum levels of hepcidin, the hormone regulating iron metabolism, and erythropoiesis in chronic hemodialysis (CHD. Methods To this end, we considered 199 CHD patients from Northern Italy (157 with hepcidin evaluation, and 188 healthy controls without iron deficiency, matched for age and gender. Genetic polymorphisms were evaluated by allele specific polymerase chain reaction assays, and hepcidin quantified by mass spectrometry. Results Serum hepcidin levels were not different between the whole CHD population and controls (median 7.1, interquartile range (IQR 0.55-17.1 vs. 7.4, 4.5-17.9 nM, respectively, but were higher in the CHD subgroup after exclusion of subjects with relative iron deficiency (p = 0.04. In CHD patients, the A736V TMPRSS6 polymorphism influenced serum hepcidin levels in individuals positive for mutations in the HFE gene of hereditary hemochromatosis (p 30 ng/ml; n = 86, hepcidin was associated with lower mean corpuscular volume (p = 0.002, suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the “high hepcidin” 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p = 0.016, independently of subclinical inflammation (p = 0.02. Conclusions The A736V TMPRSS6 genotype influences hepcidin levels, erythropoiesis, and anemia management in CHD patients. Evaluation of the effect of TMPRSS6 genotype on clinical outcomes in prospective studies in CHD may be useful to predict the outcomes of hepcidin manipulation, and to guide treatment personalization by optimizing anemia management.

  11. Plasma drug activity assay for treatment optimization in tuberculosis patients.

    Science.gov (United States)

    Heysell, Scott K; Mtabho, Charles; Mpagama, Stellah; Mwaigwisya, Solomon; Pholwat, Suporn; Ndusilo, Norah; Gratz, Jean; Aarnoutse, Rob E; Kibiki, Gibson S; Houpt, Eric R

    2011-12-01

    Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C(2 h)). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C(2 h) plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C(2 h) rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ~1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (≤ 2.0) TDA was significantly associated with both lower isoniazid and rifampin C(2 h) levels, and very low (≤ 1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.

  12. [Plasma assay of methadone enantiomers with high performance liquid chromatography].

    Science.gov (United States)

    Batista, R; Badré-Sentenac, S; Bardin, C; Chast, F

    2004-05-01

    6-Dimethylamino-4,4-diphenylheptane-3-one (methadone) is a synthetic opioid. Presence of an assymetrical carbon in its structure explains existence of two enantiomers. Levogyral enantiomer or R-methadone exhibits an 25 fold superior analgesic activity to the dextrogyral enantiomer S-methadone. In order to run separately a plasma assay of these two enantiomers, a chromatographic chiral separation method coupled with an ultraviolet detection has been performed. It allows selective assay of each enantiomer. This method, although an analytical interference with d-propoxyphene, is sensitive, reproductible, specific and shows a convenient resolution for the analysis of both the stereospecific and racemic forms. This method can be applied for pharmacokinetic study of the drug in patients treated by methadone.

  13. Iron Supplementation during Three Consecutive Days of Endurance Training Augmented Hepcidin Levels

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    Aya Ishibashi

    2017-07-01

    Full Text Available Iron supplementation contributes an effort to improving iron status among athletes, but it does not always prevent iron deficiency. In the present study, we explored the effect of three consecutive days of endurance training (twice daily on the hepcidin-25 (hepcidin level. The effect of iron supplementation during this period was also determined. Fourteen male endurance athletes were enrolled and randomly assigned to either an iron-treated condition (Fe condition, n = 7 or a placebo condition (Control condition; CON, n = 7. They engaged in two 75-min sessions of treadmill running at 75% of maximal oxygen uptake on three consecutive days (days 1–3. The Fe condition took 12 mg of iron twice daily (24 mg/day, and the CON condition did not. On day 1, both conditions exhibited significant increases in serum hepcidin and plasma interleukin-6 levels after exercise (p < 0.05. In the CON condition, the hepcidin level did not change significantly throughout the training period. However, in the Fe condition, the serum hepcidin level on day 4 was significantly higher than that of the CON condition (p < 0.05. In conclusion, the hepcidin level was significantly elevated following three consecutive days of endurance training when moderate doses of iron were taken.

  14. Increased hepcidin expression in colorectal carcinogenesis

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    Douglas G Ward; Keith Roberts; Matthew J Brookes; Howard Joy; Ashley Martin; Tariq Ismail; Robert Spychal; Tariq Iqbal; Chris Tselepis

    2008-01-01

    AIM:To investigate whether the iron stores regulator hepcidin is implicated in colon cancer-associated anae-mia and whether it might have a role in colorectal car-cinogenesis.METHODS:Mass spectrometry (MALDI-TOF MS and SELDI-TOF MS) was employed to measure hepcidin in urine collected from 56 patients with colorectal cancer.Quantitative Real Time RT-PCR was utilised to determine hepcidin mRNA expression in colorectal cancer tissue.Hepcidin cellular localisation was determined using im-munohistochemistry.RESULTS:We demonstrate that whilst urinary hepcidin expression was not correlated with anaemia it was posi-tively associated with increasing T-stage of colorectal cancer (P<0.05).Furthermore,we report that hepcidin mRNA is expressed in 34% of colorectal cancer tissue specimens and was correlated with ferroportin repres-sion.This was supported by hepcidin immunoreactivity in colorectal cancer tissue.CONCLUSION:We demonstrate that systemic hepcidin expression is unlikely to be the cause of the systemic anaemia associated with colorectal cancer.However,we demonstrate for the first time that hepcidin is expressed by colorectal cancer tissue and that this may represent a novel oncogenic signalling mechanism.

  15. An insight into the relationships between hepcidin, anemia, infections and inflammatory cytokines in pediatric refugees: a cross-sectional study.

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    Sarah Cherian

    Full Text Available BACKGROUND: Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA is unclear. We investigated the relationships between hepcidin, cytokines and iron status in a pediatric population with a high prevalence of both anemia and co-morbid infections. METHODOLOGY/PRINCIPAL FINDINGS: African refugee children <16 years were consecutively recruited at the initial post-resettlement health check with 181 children meeting inclusion criteria. Data on hematological parameters, cytokine levels and co-morbid infections (Helicobacter pylori, helminth and malaria were obtained and urinary hepcidin assays performed. The primary outcome measure was urinary hepcidin levels in children with and without iron deficiency (ID and/or ID anaemia (IDA. The secondary outcome measures included were the relationship between co-morbid infections and (i ID and IDA, (ii urinary hepcidin levels and (iii cytokine levels. IDA was present in 25/181 (13.8%. Children with IDA had significantly lower hepcidin levels (IDA median hepcidin 0.14 nmol/mmol Cr (interquartile range 0.05-0.061 versus non-IDA 2.96 nmol/mmol Cr, (IQR 0.95-6.72, p<0.001. Hemoglobin, log-ferritin, iron, mean cell volume (MCV and transferrin saturation were positively associated with log-hepcidin levels (log-ferritin beta coefficient (beta: 1.30, 95% CI 1.02 to 1.57 and transferrin was inversely associated (beta: -0.12, 95% CI -0.15 to -0.08. Cytokine levels (including IL-6 and co-morbid infections were not associated with IDA or hepcidin levels. CONCLUSIONS/SIGNIFICANCE: This is the largest pediatric study of the in vivo associations between hepcidin, iron status and cytokines. Gastro-intestinal infections (H. pylori and helminths did not elevate urinary hepcidin or IL-6 levels in refugee children, nor were they associated with IDA. Longitudinal and mechanistic studies

  16. Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt.

    Science.gov (United States)

    Han, Chang Yeob; Koo, Ja Hyun; Kim, Sung Hoon; Gardenghi, Sara; Rivella, Stefano; Strnad, Pavel; Hwang, Se Jin; Kim, Sang Geon

    2016-12-22

    Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.

  17. Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

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    José P. Oliveira-Filho

    2014-01-01

    Full Text Available Hypoferremia observed during systemic inflammatory disorders is regulated by hepcidin. Hepcidin up-regulation is particularly important during acute inflammation, as it restricts the availability of iron, which is necessary for pathogenic microorganism growth before adaptive immunity occurs. The aim of this study was to evaluate the clinical findings and hepatic hepcidin mRNA expression in horses using a Freund's complete adjuvant (FCA model of inflammation. The expression of hepcidin mRNA in the liver was determined in healthy horses following two intramuscular injections of FCA at 0 h and 12 h. Plasma iron and fibrinogen concentrations were measured at multiple time points between 0 h and 240 h post-FCA injection (PI. Hepcidin mRNA expression was determined by RT-qPCR using liver biopsy samples performed at 0 h (control, 6 h and 18 h PI. The mean plasma fibrinogen level was significantly different from the control values only between 120 and 216 h PI. The mean plasma iron level was significantly lower than the control between 16 and 72 h PI, reaching the lowest levels at 30 h PI (33 % of the initial value, and returned to the reference value from 96 h PI to the end of the experiment. Hepcidin mRNA expression increased at 6 h PI and remained high at 18 h PI. The iron plasma concentration was an earlier indicator of inflammatory processes in horses when compared with fibrinogen and might be useful for the early detection of inflammation in the horse. FCA administration caused the rapid onset of hypoferremia, and this effect was likely the result of up-regulated hepatic hepcidin gene expression. This study emphasizes the importance of hepcidin and iron metabolism during inflammation in horses.

  18. Hepcidin and Iron Homeostasis during Pregnancy

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    Mary Dawn Koenig

    2014-08-01

    Full Text Available Hepcidin is the master regulator of systemic iron bioavailability in humans. This review examines primary research articles that assessed hepcidin during pregnancy and postpartum and report its relationship to maternal and infant iron status and birth outcomes; areas for future research are also discussed. A systematic search of the databases Medline and Cumulative Index to Nursing and Allied Health returned 16 primary research articles including 10 human and six animal studies. Collectively, the results indicate that hepcidin is lower during pregnancy than in a non-pregnant state, presumably to ensure greater iron bioavailability to the mother and fetus. Pregnant women with undetectable serum hepcidin transferred a greater quantity of maternally ingested iron to their fetus compared to women with detectable hepcidin, indicating that maternal hepcidin in part determines the iron bioavailability to the fetus. However, inflammatory states, including preeclampsia, malaria infection, and obesity were associated with higher hepcidin during pregnancy compared to healthy controls, suggesting that maternal and fetal iron bioavailability could be compromised in such conditions. Future studies should examine the relative contribution of maternal versus fetal hepcidin to the control of placental iron transfer as well as optimizing maternal and fetal iron bioavailability in pregnancies complicated by inflammation.

  19. Rapidly Escalating Hepcidin and Associated Serum Iron Starvation Are Features of the Acute Response to Typhoid Infection in Humans

    Science.gov (United States)

    Darton, Thomas C.; Blohmke, Christoph J.; Giannoulatou, Eleni; Waddington, Claire S.; Jones, Claire; Sturges, Pamela; Webster, Craig; Drakesmith, Hal; Pollard, Andrew J.; Armitage, Andrew E.

    2015-01-01

    Background Iron is a key pathogenic determinant of many infectious diseases. Hepcidin, the hormone responsible for governing systemic iron homeostasis, is widely hypothesized to represent a key component of nutritional immunity through regulating the accessibility of iron to invading microorganisms during infection. However, the deployment of hepcidin in human bacterial infections remains poorly characterized. Typhoid fever is a globally significant, human-restricted bacterial infection, but understanding of its pathogenesis, especially during the critical early phases, likewise is poorly understood. Here, we investigate alterations in hepcidin and iron/inflammatory indices following experimental human typhoid challenge. Methodology/Principal Findings Fifty study participants were challenged with Salmonella enterica serovar Typhi and monitored for evidence of typhoid fever. Serum hepcidin, ferritin, serum iron parameters, C-reactive protein (CRP), and plasma IL-6 and TNF-alpha concentrations were measured during the 14 days following challenge. We found that hepcidin concentrations were markedly higher during acute typhoid infection than at baseline. Hepcidin elevations mirrored the kinetics of fever, and were accompanied by profound hypoferremia, increased CRP and ferritin, despite only modest elevations in IL-6 and TNF-alpha in some individuals. During inflammation, the extent of hepcidin upregulation associated with the degree of hypoferremia. Conclusions/Significance We demonstrate that strong hepcidin upregulation and hypoferremia, coincident with fever and systemic inflammation, are hallmarks of the early innate response to acute typhoid infection. We hypothesize that hepcidin-mediated iron redistribution into macrophages may contribute to S. Typhi pathogenesis by increasing iron availability for macrophage-tropic bacteria, and that targeting macrophage iron retention may represent a strategy for limiting infections with macrophage-tropic pathogens such as S

  20. Serum Hepcidin Levels, Iron Dyshomeostasis and Cognitive Loss in Alzheimer’s Disease

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    Sternberg, Zohara; Hu, Zihua; Sternberg, Daniel; Waseh, Shayan; Quinn, Joseph F.; Wild, Katharine; Jeffrey, Kaye; Zhao, Lin; Garrick, Michael

    2017-01-01

    This pilot study examined the status of the master iron regulatory peptide, hepcidin, and peripheral related iron parameters in Alzheimer’s disease (AD) and mild cognitive impairment patients, and evaluated the relationship between iron dyshomeostasis and amyloid-beta (Aβ), cognitive assessment tests, neuroimaging and clinical data. Frozen serum samples from the Oregon Tissue Bank were used to measure serum levels of hepcidin, ferritin, Aβ40, Aβ42 using enzyme-linked immunosorbent assay. Serum transferrin levels were determined indirectly as total iron binding capacity, serum iron was measured and the percent saturation of transferrin calculated. The study variables were correlated with the patients’ existing cognitive assessment tests, neuroimaging, and clinical data. Hepcidin, and iron-related proteins tended to be higher in AD patients than controls, reaching statistical significance for ferritin, whereas Aβ40, Aβ42 serum levels tended to be lower. Patients with pure AD had three times higher serum hepcidin levels than controls; gender differences in hepcidin and iron-related proteins were observed. Patient stratification based on clinical dementia rating-sum of boxes revealed significantly higher levels of iron and iron-related proteins in AD patients in the upper 50% as compared to controls, suggesting that iron dyshomeostasis worsens as cognitive impairment increases. Unlike Aβ peptides, iron and iron-related proteins showed significant association with cognitive assessment tests, neuroimaging, and clinical data. Hepcidin and iron-related proteins comprise a group of serum biomarkers that relate to AD diagnosis and AD disease progression. Future studies should determine whether strategies targeted to diminishing hepcidin synthesis/secretion and improving iron homeostasis could have a beneficial impact on AD progression. PMID:28400987

  1. Relationship between the Ingestion of a Polyphenol-Rich Drink, Hepcidin Hormone, and Long-Term Training

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    Débora Villaño

    2016-10-01

    Full Text Available The effects of polyphenol-rich foods on the iron status of athletes, as well as the effect of physical training on the hormone hepcidin, implicated in iron metabolism, are not clear. We investigated the influence on iron metabolism of a long-term training intervention of 120 days, measuring the hepcidin concentration in the plasma of 16 elite triathletes, and the effect of the ingestion of 200 mL of either aronia-citrus juice or a placebo drink for 45 days, in a crossover design. The highest plasma hepcidin concentrations were observed at the beginning of the study (116 ± 63 nM and levels steadily decreased until the end of the intervention (final value 10 ± 7.5 nM. Long-term training might reduce inflammation and, hence, could be responsible for the decrease in hepcidin in triathletes. Polyphenols from aronia-citrus juice did not interfere in iron absorption, as we did not observe significant differences between the intake of the placebo drink or juice with regard to hepcidin levels. Further studies are required to ascertain the time and conditions necessary to restore hepcidin levels, which reflect the iron status of triathletes.

  2. Molecular Mechanisms Regulating Hepcidin Revealed by Hepcidin Disorders

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    Clara Camaschella

    2011-01-01

    Full Text Available Iron is essential for human life, but toxic if present in excess. To avoid iron overload and maintain iron homeostasis, all cells are able to regulate their iron content through the post-transcriptional control of iron genes operated by the cytosolic iron regulatory proteins that interact with iron responsive elements on iron gene mRNA. At the systemic level, iron homeostasis is regulated by the liver peptide hepcidin. Disruption of these regulatory loops leads to genetic diseases characterized by iron deficiency (iron-refractory iron-deficiency anemia or iron overload (hemochromatosis. Alterations of the same systems are also found in acquired disorders, such as iron-loading anemias characterized by ineffective erythropoiesis and anemia of chronic diseases (ACD associated with common inflammatory conditions. In ACD, iron is present in the body, but maldistributed, being deficient for erythropoiesis, but sequestered in macrophages. Studies of the hepcidin regulation by iron and inflammatory cytokines are revealing new pathways that might become targets of new therapeutic intervention in iron disorders.

  3. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation.

    Science.gov (United States)

    Ye, Zhengqi; Zetterberg, Craig; Gao, Hong

    2017-03-14

    Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

  4. Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression

    Institute of Scientific and Technical Information of China (English)

    Sizhao; Lu; Emily; Zmijewski; John; Gollan; Duygu; Dee; Harrison-Findik

    2014-01-01

    AIM: To determine the regulation of human hepcidin(HAMP) and mouse hepcidin(hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. METHODS: For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR(qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation(ChIP) assays.RESULTS: The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3(P < 0.001), but did not cause any significant changes in HAMP expression. Short-term(1 h) Jo2 treatment(0.2 μg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis(P < 0.001) and an increase in SAA3(P < 0.023) and IL-6(P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57

  5. Hepcidin: regulation of the master iron regulator

    Science.gov (United States)

    Rishi, Gautam; Wallace, Daniel F.; Subramaniam, V. Nathan

    2015-01-01

    Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors. PMID:26182354

  6. Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma

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    Cumin, F.; de Gasparo, M.; Wood, J.M.; Schnell, C.; Frueh, F.; Graf, P. (Ciba-Geigy Limited, Basel (Switzerland))

    1989-10-01

    A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r = 0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 microM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with CGP 38 560 A (50 micrograms/kg). Similar values for the concentrations of renin inhibitor in plasma were obtained with the radioinhibitor binding assay and the enzyme inhibitor assay, and there was a significant correlation between values obtained with the two different methodologies (r = 0.94). The plasma levels of renin inhibitor reached a maximum at the end of infusion and then decreased rapidly, indicating a short plasma half-life. The changes in biochemical parameters, plasma renin activity, and plasma concentration of active renin could be related to the concentrations of CGP 38 560 measured in the plasma.

  7. Hepcidin as a potential biomarker for blood doping.

    Science.gov (United States)

    Leuenberger, Nicolas; Bulla, Emanuele; Salamin, Olivier; Nicoli, Raul; Robinson, Neil; Baume, Norbert; Saugy, Martial

    2017-07-01

    The concentration of hepcidin, a key regulator of iron metabolism, is suppressed during periods of increased erythropoietic activity. The present study obtained blood samples from 109 elite athletes and examined the correlations between hepcidin and markers of erythropoiesis and iron metabolism (i.e., haemoglobin, erythropoietin (EPO), ferritin, erythroferrone (ERFE), and iron concentration). Furthermore, an administration study was undertaken to examine the effect of recombinant human EPO (rhEPO) delta (Dynepo™) on hepcidin concentrations in healthy male volunteers. The effects on hepcidin were then compared with those on reticulocyte percentage (Ret%) and ferritin concentration. There was a significant positive correlation between hepcidin and ferritin, iron, and haemoglobin levels in athletes, whereas hepcidin showed an inverse correlation with ERFE. Administration of rhEPO delta reduced hepcidin levels, suggesting that monitoring hepcidin may increase the sensitivity of the Athlete Biological Passport (ABP) for detecting rhEPO abuse. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  8. Association of Hepcidin-25 with survival after kidney transplantation

    NARCIS (Netherlands)

    Eisenga, Michele F.; Dullaart, Robin P. F.; Berger, Stefan P.; Sloan, John H.; de Vries, Aiko P. J.; Bakker, Stephan J. L.; Gaillard, Carlo A. J. M.

    2016-01-01

    Background Hepcidin is considered the master regulator of iron homoeostasis. Novel hepcidin antagonists have recently been introduced as potential treatment for iron-restricted anaemia. Meanwhile, serum hepcidin has been shown to be positively associated with cardiovascular disease and inversely wit

  9. Fibrin and fibrinogen degradation products in plasma : clinical and methodological studies using enzyme immuno assays

    NARCIS (Netherlands)

    H. Kroneman (Herman)

    1992-01-01

    textabstractThis thesis comprises studies with monoclonal antibody-based plasma assays for derivatives of fibrin and fibrinogen in patients with diseases and conditions characterized by an activated state of coagulation and fibrinolysis

  10. Low Serum Hepcidin in Patients with Autoimmune Liver Diseases.

    Directory of Open Access Journals (Sweden)

    Aggeliki Lyberopoulou

    Full Text Available Hepcidin, a liver hormone, is important for both innate immunity and iron metabolism regulation. As dysfunction of the hepcidin pathway may contribute to liver pathology, we analysed liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases. Hepcidin mRNA levels were determined in liver biopsies obtained from 126 patients with HCV (n = 21, HBV (n = 23, autoimmune cholestatic disease (primary biliary cirrhosis and primary sclerosing cholangitis; PBC/PSC; n = 34, autoimmune hepatitis (AIH; n = 16 and non-alcoholic fatty liver disease (NAFLD; n = 32. Sera sampled on the biopsy day from the same patients were investigated for serum hepcidin levels. Hepatic hepcidin mRNA levels correlated positively with ferritin and negatively with serum γ-GT levels. However, no correlation was found between serum hepcidin and either ferritin or liver hepcidin mRNA. Both serum hepcidin and the serum hepcidin/ferritin ratio were significantly lower in AIH and PBC/PSC patients' sera compared to HBV, HCV or NAFLD (P<0.001 for each comparison and correlated negatively with serum ALP levels. PBC/PSC and AIH patients maintained low serum hepcidin during the course of their two-year long treatment. In summary, parallel determination of liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases shows that circulating hepcidin and its respective ratio to ferritin are significantly diminished in patients with autoimmune liver diseases. These novel findings, once confirmed by follow-up studies involving bigger size and better-matched disease subgroups, should be taken into consideration during diagnosis and treatment of autoimmune liver diseases.

  11. Low Serum Hepcidin in Patients with Autoimmune Liver Diseases.

    Science.gov (United States)

    Lyberopoulou, Aggeliki; Chachami, Georgia; Gatselis, Nikolaos K; Kyratzopoulou, Eleni; Saitis, Asterios; Gabeta, Stella; Eliades, Petros; Paraskeva, Efrosini; Zachou, Kalliopi; Koukoulis, George K; Mamalaki, Avgi; Dalekos, George N; Simos, George

    2015-01-01

    Hepcidin, a liver hormone, is important for both innate immunity and iron metabolism regulation. As dysfunction of the hepcidin pathway may contribute to liver pathology, we analysed liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases. Hepcidin mRNA levels were determined in liver biopsies obtained from 126 patients with HCV (n = 21), HBV (n = 23), autoimmune cholestatic disease (primary biliary cirrhosis and primary sclerosing cholangitis; PBC/PSC; n = 34), autoimmune hepatitis (AIH; n = 16) and non-alcoholic fatty liver disease (NAFLD; n = 32). Sera sampled on the biopsy day from the same patients were investigated for serum hepcidin levels. Hepatic hepcidin mRNA levels correlated positively with ferritin and negatively with serum γ-GT levels. However, no correlation was found between serum hepcidin and either ferritin or liver hepcidin mRNA. Both serum hepcidin and the serum hepcidin/ferritin ratio were significantly lower in AIH and PBC/PSC patients' sera compared to HBV, HCV or NAFLD (Pserum ALP levels. PBC/PSC and AIH patients maintained low serum hepcidin during the course of their two-year long treatment. In summary, parallel determination of liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases shows that circulating hepcidin and its respective ratio to ferritin are significantly diminished in patients with autoimmune liver diseases. These novel findings, once confirmed by follow-up studies involving bigger size and better-matched disease subgroups, should be taken into consideration during diagnosis and treatment of autoimmune liver diseases.

  12. Serum hepcidin concentrations and type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Hepcidin is a peptide hormone with both paracrine andendocrine functions that help in maintaining body ironstores. Type 2 diabetes (T2D) is one of the sequelae ofexcess body iron stores; thus, iron regulatory hormonehepcidin may have a direct or at least an indirect role inthe aetiopathogenesis of T2D. Both human and animalstudies at molecular and genetic levels have attemptedto establish a role for hepcidin in the development ofT2D, and a few epidemiologic studies have also showeda link between hepcidin and T2D at population level,but the findings are still inconclusive. Recent data havesuggested different pathways in which hepcidin could beassociated with T2D with much emphasis on its primaryor secondary role in insulin resistance. Some of thesuggested pathways are via transcription modulator ofhepcidin (STAT3); ferroportin 1 expression on the cellsinvolved in iron transport; transmembrane protease 6enzyme; and pro-inflammatory cytokines, interleukin(IL)-1, IL-6, tumor necrosis factor-α and IL-10. Thisreview briefly reports the existing evidence on thepossible links between hepcidin and T2D and concludesthat more data are needed to confirm or refute hepcidin'srole in the development of T2D. Examining this rolecould provide a further evidence base for iron in theaetiopathogenesis of T2D.

  13. Silkworm larvae plasma (SLP) assay for detection of bacteria: False positives secondary to inflammation in vivo.

    Science.gov (United States)

    Ma, Michelle; Rice, Tyler A; Percopo, Caroline M; Rosenberg, Helene F

    2017-01-01

    The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count.

  14. Hepcidin and Hfe in iron overload in beta-thalassemia.

    Science.gov (United States)

    Gardenghi, Sara; Ramos, Pedro; Follenzi, Antonia; Rao, Niva; Rachmilewitz, Eliezer A; Giardina, Patricia J; Grady, Robert W; Rivella, Stefano

    2010-08-01

    Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with beta-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in beta-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in beta-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.

  15. Hepcidin and Hfe in iron overload in β-thalassemia

    Science.gov (United States)

    Gardenghi, Sara; Ramos, Pedro; Follenzi, Antonia; Rao, Niva; Rachmilewitz, Eliezer A.; Giardina, Patricia J.; Grady, Robert W.; Rivella, Stefano

    2013-01-01

    Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with β-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in β-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in β-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries. PMID:20712796

  16. High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair

    Directory of Open Access Journals (Sweden)

    Sarika Pathak-Sharma

    2017-07-01

    Full Text Available The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.

  17. The influence of platelets, plasma and red blood cells on functional haemostatic assays.

    Science.gov (United States)

    Bochsen, Louise; Johansson, Pär I; Kristensen, Annemarie T; Daugaard, Gedske; Ostrowski, Sisse R

    2011-04-01

    Functional whole blood haemostatic assays are used increasingly to guide transfusion therapy and monitor medical treatment and are also applied for in-vitro evaluations of the haemostatic potential of stored platelets. We investigated how the cellular and plasmatic elements, both isolated and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing plasma from platelet concentrates eliminated the TEG response and diminished the Multiplate aggregation response, but readding plasma to the pure platelet concentrates restored the response. Each of the elements in whole blood, plasma, platelets and RBC, affected the Multiplate and TEG results differently. The results emphasize that the concentrations of all cellular and plasmatic components in whole blood should be taken into account when interpreting results obtained by TEG and multiplate.

  18. Methodology for determination of plasma cortisol in fish using Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

    DEFF Research Database (Denmark)

    Velasco-Santamaría, Yohana M.; Cruz-Casallas, Pablo E.

    2007-01-01

    Objective. To determine plasma cortisol procedure in fish using competitive enzymelinked immunosorbent assay (ELISA). Materials and methods. Two plasma samples of juveniles rainbow trout Oncorhynchus mykiss were analized by using ELISA human kit for cortisol assay. For standard curve calibration...... seven standard solutions of cortisol in human plasma (0, 20, 50, 100, 200, 400 and 800 ng.ml-1) were used. For the recovery test 50, 100 and 200 ng.ml-1 standard solutions of cortisol were used; for the linearity test four dilutions of the fish plasma samples (1/2, 1/4, 1/8 and 1/16) were prepared....... To each well fish plasma samples and standard solutions were added and its content were conjugated to peroxidase and subsequently enzyme substrate was added. The enzymatic reaction was stopped by addition of phosphoric acid 0.5 M and the absorbance was read at 450 nm. The accuracy of the pipetting...

  19. The influence of platelets, plasma and red blood cells on functional haemostatic assays

    DEFF Research Database (Denmark)

    Bochsen, Louise; Johansson, Pär I.; Kristensen, Annemarie Thuri

    2011-01-01

    and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet...... concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG...... and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing...

  20. 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

  1. Anemia in diffuse large B-cell non-Hodgkin lymphoma: the role of interleukin-6, hepcidin and erythropoietin

    NARCIS (Netherlands)

    Tisi, M.C.; Bozzoli, V.; Giachelia, M.; Massini, G.; Ricerca, B.M.; Maiolo, E.; D'Alo, F.; Larocca, L.M.; Piciocchi, A.; Tjalsma, H.; Swinkels, D.W.; Voso, M.T.; Leone, G.; Hohaus, S.

    2014-01-01

    Anemia is a frequent sign in patients with diffuse large B-cell lymphoma (DLBCL) at diagnosis. We determined erythropoietin, hepcidin and interleukin-6 (IL-6) in plasma samples of 53 patients with DLBCL. The majority of patients (40/53, 75%) showed defective endogenous erythropoietin production, in

  2. Hepcidin Response to Exercise: A Review

    Directory of Open Access Journals (Sweden)

    Raúl Domínguez

    2014-09-01

    Full Text Available Given the multiple functions of iron in the body, any state of iron deficiency will induce a series of secondary effects that could compromise sports performance. Low serum iron levels are commonly observed in athletes during the course of a training period, especially in those performing aerobic exercises and resistance training. Sometimes, body iron levels will even fall below those detected in sedentary individuals, and we could go as far as to say that iron deficiency is the most frequently observed nutrition disorder among athletes of any sport. Hepcidin, a hormone secreted by hepatocytes whose principal mechanism of action is the degradation of ferroportin (the main iron exporter from macrophages and the basolateral membrane of duodenal enterocytes, has been proposed as the main regulator of the body’s iron reserves. Thus, elevated serum hepcidin levels lead to diminished iron absorption and recycling, while lower levels of the hormone will cause greater iron absorption. Among the factors that affect the hepcidin response produced, we should highlight an individual’s total iron levels, erythropoietic demands, state of hypoxia, dietary iron, inflammation and physical exercise. Given the important role played by iron regulatory mechanisms in physical performance, this report reviews our current understanding of the physiological response of hepcidin to different sports intensities and modalities. Turk Jem 2014; 18: 84-91

  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. Identification of centrarchid hepcidins and evidence that 17β-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Robertson, L.S.; Iwanowicz, L.R.; Marranca, J.M.

    2009-01-01

    Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17β-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.

  5. Evolution of the hepcidin gene in primates

    Directory of Open Access Journals (Sweden)

    Tossi Alessandro

    2008-03-01

    Full Text Available Abstract Background Hepcidin/LEAP-1 is an iron regulatory hormone originally identified as an antimicrobial peptide. As part of a systematic analysis of the evolution of host defense peptides in primates, we have sequenced the orthologous gene from 14 species of non-human primates. Results The sequence of the mature peptide is highly conserved amongst all the analyzed species, being identical to the human one in great apes and gibbons, with a single residue conservative variation in Old-World monkeys and with few substitutions in New-World monkeys. Conclusion Our analysis indicates that hepcidin's role as a regulatory hormone, which involves interaction with a conserved receptor (ferroportin, may result in conservation over most of its sequence, with the exception of the stretch between residues 15 and 18, which in New-World monkeys (as well as in other mammals shows a significant variation, possibly indicating that this structural region is involved in other functions.

  6. Validated enzyme-linked immunosorbent assay for determination of rosuvastatin in plasma at picogram level.

    Science.gov (United States)

    Darwish, Ibrahim A; Al-Obaid, Abdul-Rahman M; Al-Malaq, Hamoud A

    2013-05-01

    In this study, a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of rosuvastatin (ROS) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes ROS with high affinity, and ROS conjugate of bovine serum albumin (ROS-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labelled second anti-rabbit IgG antibody (HRP-IgG) and 3,3`,5,5`-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the colour intensity in the assay wells. The assay limit of detection was 25 pg ml(-1) and the effective working range at relative standard deviations (RSD) of ≤ 5% was 40-2000 pg ml(-1). Analytical recovery of ROS from spiked plasma was 96.2 - 104.8 ± 2.12 - 5.42%. The precision of the assay was satisfactory; RSD was 2.47 - 4.46 and 3.24 - 5.27% for the intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ~ 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed ELISA has a great value in routine analysis of ROS for its pharmacokinetic studies.

  7. 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.

  8. The effects of acute exercise bouts on hepcidin in women.

    NARCIS (Netherlands)

    Newlin, M.K.; Williams, S.; McNamara, T.; Tjalsma, H.; Swinkels, D.W.; Haymes, E.M.

    2012-01-01

    PURPOSE: To investigate the effects of acute exercise on serum hepcidin and iron (sFe) in active women. Changes in interleukin-6 (IL-6), hepcidin, ferritin, and sFe in response to 2 different exercise durations were compared. METHODS: Twelve women age 19-32 yr performed 2 treadmill runs (60 and 120

  9. The role of hepcidin-25 in kidney transplantation

    NARCIS (Netherlands)

    Chan, W.; Ward, D.G.; McClean, A.; Bosch, J.A.; Jones, D.; Kaur, O.; Drayson, M.; Whitelegg, A.; Iqbal, T.; McTernan, P.G.; Tselepis, C.; Borrows, R.

    2013-01-01

    Background: Hepcidin-25 is a peptide hormone involved in iron absorption and homeostasis and found at increased serum levels in conditions involving systemic inflammation, renal dysfunction, and increased adiposity. Hepcidin may play a role in the pathogenesis of anemia, but its role in kidney trans

  10. The effects of acute exercise bouts on hepcidin in women.

    NARCIS (Netherlands)

    Newlin, M.K.; Williams, S.; McNamara, T.; Tjalsma, H.; Swinkels, D.W.; Haymes, E.M.

    2012-01-01

    PURPOSE: To investigate the effects of acute exercise on serum hepcidin and iron (sFe) in active women. Changes in interleukin-6 (IL-6), hepcidin, ferritin, and sFe in response to 2 different exercise durations were compared. METHODS: Twelve women age 19-32 yr performed 2 treadmill runs (60 and 120

  11. A direct, automated, immuno-turbidimetric assay of free protein S antigen in plasma.

    Science.gov (United States)

    Deffert, C; Esteve, F; Grimaux, M; Gouault-Heilmann, M

    2001-03-01

    A new, fully automated, one-step, immuno-turbidimetric assay of free protein S (fPS) in plasma (STA Liatest Free Protein S; Diagnostica Stago, Asnières, France) has been developed for STA analysers. This technique combines the advantages of a direct assay of fPS using two monoclonal antibodies, which specifically recognize fPS but not protein S (PS)-C4b-binding protein complexes, and the advantages of automation. The assay has good analytical performances, with intra- and inter-assay variation coefficients below 5% for normal values, and slightly higher for abnormal values. In a comparison study with a one-step enzyme-linked immunosorbent assay for fPS (Asserachrom Free Protein S; Diagnostica Stago), a correlation coefficient of 0.93 with a regression line close to 1 was found between the two techniques (n = 166 normal or PS-deficient plasma samples collected from healthy subjects and individuals with a personal or family history of thrombosis). This new technique is specific, reproducible, easy to perform, and provides a useful tool in the diagnosis of PS deficiency.

  12. Hepcidin: an important iron metabolism regulator in chronic kidney disease

    Directory of Open Access Journals (Sweden)

    Sandra Azevedo Antunes

    Full Text Available Abstract Anemia is a common complication and its impact on morbimortality in patients with chronic kidney disease (CKD is well known. The discovery of hepcidin and its functions has contributed to a better understanding of iron metabolism disorders in CKD anemia. Hepcidin is a peptide mainly produced by hepatocytes and, through a connection with ferroportin, it regulates iron absorption in the duodenum and its release of stock cells. High hepcidin concentrations described in patients with CKD, especially in more advanced stages are attributed to decreased renal excretion and increased production. The elevation of hepcidin has been associated with infection, inflammation, atherosclerosis, insulin resistance and oxidative stress. Some strategies were tested to reduce the effects of hepcidin in patients with CKD, however more studies are necessary to assess the impact of its modulation in the management of anemia in this population.

  13. Mass spectrometry analysis of hepcidin peptides in experimental mouse models.

    Directory of Open Access Journals (Sweden)

    Harold Tjalsma

    Full Text Available The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1 and its paralogue Hepcidin-2 (Hep-2 at the peptide level. To this purpose, Fourier transform ion cyclotron resonance (FTICR and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i 3 mouse strains (C57Bl/6; DBA/2 and BABL/c upon stimulation with intravenous iron and LPS, ii homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X mutated mice and double affected mice, and iii mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.

  14. Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay

    Directory of Open Access Journals (Sweden)

    Marta E. Martinuzzo

    2017-02-01

    Full Text Available Dabigatran etexilate (direct thrombin inhibitor is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc, correlate cc with classic coagulation assays, prothrombin time (PT and activated partial thromboplastin time (APTT, and evaluate them according to the creatinine clearance (CLCr. Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation receiving dabigatran at 150 (n = 19 or 110 (n = 21 mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL. PT and APTT (IL were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL. DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients' cc: median 83 (4-945 ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min showed significantly higher median cc: 308 (49-945, compared to the average 72 (12-190 and highest tertile, 60 (4-118 ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies

  15. Multiplex DNA assay based on nanoparticle probes by single particle inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Zhang, Shixi; Han, Guojun; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

    2014-04-01

    A multiplex DNA assay based on nanoparticle (NP) tags detection utilizing single particle mode inductively coupled plasma mass spectrometry (SP-ICP-MS) as ultrasensitive readout has been demonstrated in the article. Three DNA targets associated with clinical diseases (HIV, HAV, and HBV) down to 1 pM were detected by DNA probes labeled with AuNPs, AgNPs, and PtNPs via DNA sandwich assay. Single nucleotide polymorphisms in genes can also be effectively discriminated. Since our method is unaffected by the sample matrix, it is well-suited for diagnostic applications. Moreover, with the high sensitivity of SP-ICP-MS and the variety of NPs detectable by SP-ICP-MS, high-throughput DNA assay could be achieved without signal amplification or chain reaction amplification.

  16. Expression of Hepcidin and Growth Differentiation Factor 15 (GDF-15 Levels in Thalassemia Patients with Iron Overload and Positive Anti Hepatitis C Virus

    Directory of Open Access Journals (Sweden)

    Nuri Dyah Indrasari

    2016-09-01

    Full Text Available Background: Thalassemia patients who undergo life-long recurrent blood transfusion will experience iron overload in various organs including the liver and possibly suffer from chronic hepatitis C infection which may lead to liver impairment. The liver produces hepcidin, a hormone which plays role in the regulation of iron level in the blood. Various factors may influence hepcidin level in the blood. Chronic hepatitis C causes iron overload and liver impairment. Liver impairment and haemolytic anaemia due to haemoglobinopathy will suppress hepcidin production. Anaemia stimulates growth differentiation factor 15 (GDF-15 to increase erythropoiesis and suppress hepcidin production. Iron overload causes increase in hepcidin level. Presence of factors which decrease or increase hepcidin production will express various levels of hepcidin. This study aimed to identify the expression of hepcidin and GDF-15 levels in thalassemia patients with iron overload and positive anti-HCV. Information on hepcidin and GDF-15 levels are beneficial in the management of iron overload in thalassemia with positive anti-HCV. Method: This study was a descriptive analytic study in thalassemia patients who had received recurrent blood transfusion ≥ 12 times, suffered from iron overload (transferrin saturation > 55% and ferritin > 1,000 ng/mL, which consisted of 31 individuals with positive anti-HCV and 27 individuals with negative anti-HCV. This study was performed in Thalassemia Centre Department of Child Health and Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, in October 2011–January 2012. Serum hepcidin and GDF-15 examinations were performed using enzyme-linked immunosorbent assay (ELISA method. Aspartate aminotransferase (AST and alanine aminotransferase (ALT examinations were performed using colorimetry method. Data on ferritin and transferrin saturation were obtained from medical records in the last 3

  17. 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.

  18. Iron, anemia and hepcidin in malaria

    Directory of Open Access Journals (Sweden)

    Natasha eSpottiswoode

    2014-05-01

    Full Text Available Malaria and iron have a complex but important relationship. Plasmodium proliferation requires iron, both during the clinically silent liver stage of growth and in the disease-associated phase of erythrocyte infection. Precisely how the protozoan acquires its iron from its mammalian host remains unclear, but iron chelators can inhibit pathogen growth in vitro and in animal models. In humans, iron deficiency appears to protect against severe malaria, while iron supplementation increases risks of infection and disease. Malaria itself causes profound disturbances in physiological iron distribution and utilization, through mechanisms that include hemolysis, release of heme, dyserythropoiesis, anemia, deposition of iron in macrophages, and inhibition of dietary iron absorption. These effects have significant consequences. Malarial anemia is a major global health problem, especially in children, that remains incompletely understood and is not straightforward to treat. Furthermore, the changes in iron metabolism during a malaria infection may modulate susceptibility to coinfections. The release of heme and accumulation of iron in granulocytes may explain increased vulnerability to non-typhoidal Salmonella during malaria. The redistribution of iron away from hepatocytes and into macrophages may confer host resistance to superinfection, whereby blood-stage parasitemia prevents the development of a second liver-stage Plasmodium infection in the same organism. Key to understanding the pathophysiology of iron metabolism in malaria is the activity of the iron regulatory hormone hepcidin. Hepcidin is upregulated during blood-stage parasitemia and likely mediates much of the iron redistribution that accompanies disease. Understanding the regulation and role of hepcidin may offer new opportunities to combat malaria and formulate better approaches to treat anemia in the developing world.

  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. Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement

    DEFF Research Database (Denmark)

    Campbell, Duncan J; Nussberger, Juerg; Stowasser, Michael

    2009-01-01

    BACKGROUND: Measurement of plasma renin is important for the clinical assessment of hypertensive patients. The most common methods for measuring plasma renin are the plasma renin activity (PRA) assay and the renin immunoassay. The clinical application of renin inhibitor therapy has thrown...... are susceptible to renin overestimation due to prorenin activation. In addition, activity assays performed with peptidase inhibitors may overestimate the degree of inhibition of PRA by renin inhibitor therapy. Moreover, immunoassays may overestimate the reactive increase in plasma renin concentration in response...

  1. Hepcidin: A useful marker in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Serap Duru

    2012-01-01

    Full Text Available Purpose: This study was designed to evaluate the levels of hepcidin in the serum of patients with chronic obstructive pulmonary disease (COPD. Methods: In the study, 74 male patients (ages 45-75 in a stable period for COPD were grouped as Group I: Mild COPD (n:25, Group II: Moderate COPD (n:24, and Group III: Severe COPD (n:25. Healthy non-smoker males were included in Group IV (n:35 as a control group. The differences of hepcidin level among all the groups were examined. Also, in the patient groups with COPD, hepcidin level was compared with age, body mass index, cigarette (package/year, blood parameters (iron, total iron binding capacity, ferritin, hemoglobin, hematocrit [hct], respiratory function tests, and arterial blood gas results. Results: Although there was no difference between the healthy control group and the mild COPD patient group (P=0.781 in terms of hepcidin level, there was a difference between the moderate (P=0.004 and the severe COPD patient groups (P=0.002. The hepcidin level of the control group was found to be higher than the moderate and severe COPD patient groups. In the severe COPD patients, hepcidin level increased with the increase in serum iron (P=0.000, hct (P=0.009, ferritin levels (P=0.012, and arterial oxygen saturation (SaO2, P=0.000. Conclusion: The serum hepcidin level that is decreased in severe COPD brings into mind that it may play a role in the mechanism to prevent hypoxemia. The results suggest that serum hepcidin level may be a useful marker in COPD. Larger prospective studies are needed to confirm our findings between hepcidin and COPD.

  2. Development of a Fibrinogen-Specific Sandwich Enzyme-Linked Immunosorbent Assay Microarray Assay for Distinguishing Between Blood Plasma and Serum Samples

    Energy Technology Data Exchange (ETDEWEB)

    Gonzales, Rachel M.; Zhang, Qibin; Zangar, Richard C.; Smith, Richard D.; Metz, Thomas O.

    2011-07-01

    We have developed a fibrinogen-specific sandwich ELISA microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies, 49D2, HPA001900, and F8512, were evaluated in conjunction with 1D6 as detection antibody, and the data show that 49D2 and, to a lesser extent, F8512 successfully identify previously unknown plasma and serum samples based upon a ~28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high throughput manner prior to proteomics analyses.

  3. [Clinical usefulness of plasma PIVKA-II assay and its limitations in patients with hepatocellular carcinoma].

    Science.gov (United States)

    Fujiyama, S; Morishita, T; Shibata, J; Sato, T

    1989-04-01

    Plasma abnormal prothrombin (protein induced by vitamin K absence or antagonist-II: PIVKA-II) was evaluated as a serological marker for hepatocellular carcinoma (HCC). Its plasma levels were measured by enzyme immunoassay using an anti-PIVKA-II monoclonal antibody in 1010 patients with various diseases. Of 192 patients with HCC, 116 (60%) had abnormal PIVKA-II levels greater than 0.1 AU/ml. Elevation of PIVAK-II levels was observed rarely in chronic hepatitis, liver cirrhosis and other malignant tumors. Plasma PIVKA-II levels in HCC increased with tumor size. Normal levels were observed in patients with tumors measuring 2 cm or less in diameter. As a result, diagnostic application of plasma PIVKA-II levels to small liver tumors is limited. The sensitivity of PIVKA-II in the diagnosis and monitoring of HCC was increased by serial and simultaneous determinations of AFP, because high PIVKA-II levels were observed more often in low AFP-producing HCC patients. In some patients with HCC, plasma PIVKA-II levels decreased after surgical resection of the tumor or chemoembolization with cisplatin suspended in Lipiodol (LPS), but later rose again with recurrence of the disease. Elevated plasma PIVKA-II levels were not related to low vitamin K concentration in the serum. In fact, in many patients vitamin K administration resulted in only a moderate reduction of PIVKA-II levels. From these results, plasma PIVKA-II assay by the EIA method using a monoclonal antibody is a useful tool for the diagnosis and monitoring of HCC, particularly in HCC patients with low AFP levels.

  4. [New insights on hepcidin in anemia of chronic disease].

    Science.gov (United States)

    Wang, Feng-Dan; Zhou, Dao-Bin

    2009-12-01

    Anemia of chronic disease is normocytic and normochromic. One of the mechanisms is misbalance of iron metabolism. Hepcidin, a kind of protein secreted by liver is considered to be the hormone regulating iron metabolism. It binds to ferroportin and induces the latter one's internalization. Thus, iron transportation from iron storage cells to serum is reduced. Cytokines are elevated in chronic disease. They stimulate hepcidin expression in liver through JAK2/STAT3 pathway. As a result, iron absorption and reabsorption is blocked, which leads to the misbalance of iron metabolism in anemia of chronic disease. In this article, the hepcidin and its relation to iron metabolism and anemia in chronic disease are reviewed.

  5. High-performance liquid chromatographic assay for metamizol metabolites in rat plasma: application to pharmacokinetic studies.

    Science.gov (United States)

    Domínguez-Ramírez, Adriana Miriam; Calzadilla, Patricia Carrillo; Cortés-Arroyo, Alma Rosa; Hurtado Y de la Peña, Marcela; López, José Raúl Medina; Gómez-Hernández, Martín; López-Muñoz, Francisco Javier

    2012-12-01

    In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma samples (50-100μl) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were linear within a range of 1-100μg/ml (r(2)≥0.99). The intra-day coefficients of variation (CV) were in the range of 1.3-8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the range of 0.6-9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1μg/ml for all metabolites using a plasma sample of 100μl. Plasma samples were stable at least for 4 weeks at -20°C. This method was found to be suitable for studying metamizol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and morphine, in single dose. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma.

    Science.gov (United States)

    O'Leary, Margaret A; Isbister, Geoffrey K

    2010-01-01

    Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10-20% for both methods, while within-day CV <10%. The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms. Crown Copyright 2009. Published by Elsevier Inc. 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. Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Ornatsky, Olga I; Kinach, Robert; Bandura, Dmitry R; Lou, Xudong; Tanner, Scott D; Baranov, Vladimir I; Nitz, Mark; Winnik, Mitchell A

    2008-01-01

    Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

  9. 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.

  10. A study of the conditions and accuracy of the thrombin time assay of plasma fibrinogen

    DEFF Research Database (Denmark)

    Jespersen, J; Sidelmann, Johannes Jakobsen

    1982-01-01

    The conditions, accuracy, precision and possible error of the thrombin time assay of plasma fibrinogen are determined. Comparison with an estimation of clottable protein by absorbance at 280 nm gave a correlation coefficient of 0.96 and the regression line y = 1.00 x + 0.56 (n = 34). Comparison...... with a radial immunodiffusion method yielded the correlation coefficient 0.97 and the regression line y = 1.18 x = 2.47 (n = 26). The presence of heparin in clinically applied concentrations produced a slight shortening of the clotting times. The resulting error in the estimated concentrations of fibrinogen...

  11. A rapid and simple assay for growth hormone-binding protein activity in human plasma.

    Science.gov (United States)

    Baumann, G; Shaw, M A; Amburn, K

    1988-12-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

  12. A modified Plasmodium falciparum growth inhibition assay (GIA) to assess activity of plasma from malaria endemic areas.

    Science.gov (United States)

    Mlambo, Godfree; Kumar, Nirbhay

    2007-02-01

    Plasma samples from patients undergoing treatment in malaria endemic countries often contain anti-malaria drugs, that may overstate effects of specific antibodies in growth inhibition assays (GIA). We describe a modified assay that uses drug resistant P. falciparum parasites (W2) that circumvents the requirement for dialyzing samples that may likely contain drugs such as chloroquine and sulfadoxine/pyrimethamine (SP).

  13. Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma

    DEFF Research Database (Denmark)

    Munkvad, S; Jespersen, J; Sidelmann, Johannes Jakobsen;

    1990-01-01

    by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes...

  14. 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...

  15. ROLE OF HEPCIDIN IN MECHANISM OF ANEMIA CHRONIC DISEASE PATIENTS

    Directory of Open Access Journals (Sweden)

    Ketut Suega

    2014-08-01

    Full Text Available Background: Anemia chronic disease (ACD is an anemia found in certain chronic disease states, typically marked by the disturbance of iron homeostasis or hypoferremia. This condition leads to shortage of iron for hemoglobin synthesis but the iron storage in bone marrow is left undisturbed. The discovery of hepcidin and its role in iron metabolism has given new insights in anemia chronic disease management. Consecutive sampling method was applied to choose ACD patients at Sanglah General Hospital, Bali-Indonesia. Questionnaire was constructed to note demographic aspect and disease or clinical condition underlies ACD (inflammation, infection, malignancy and others. Hepcidin, Serum IL-6 and CRP level were measured. Sample size and Path analysis mediation method were used to define hepcidin’s role on mechanism how anemia develop in ACD patients in which the direct and indirect effects of IL-6 and CRP to hemoglobin (Hb  were counted partially or combined through hepcidin mediation variable. The cumulative influence of IL-6, CRP and hepcidin on anemia (Hb was only 0.12 or about 12% of hemoglobin level was influenced by IL-6, CRP and hepcidin together whereas the other 93% was influenced by another unknown and unclear factors. Hepcidin could be used as a mediation variable for the development of anemia because the direct influence of IL-6 as exogenous factor was less than its indirect influence through hepcidin. It was not proven for CRP as exogenous variable because the direct influence of CRP to hemoglobin was stronger than the influence of CRP through hepcidin.

  16. Characterization of Putative Erythroid Regulators of Hepcidin in Mouse Models of Anemia

    Science.gov (United States)

    Mirciov, Cornel S. G.; Wilkins, Sarah J.; Dunn, Linda A.; Anderson, Gregory J.; Frazer, David M.

    2017-01-01

    Iron is crucial for many biological functions, but quantitatively the most important use of iron is in the production of hemoglobin in red blood cell precursors. The amount of iron in the plasma, and hence its availability for hemoglobin synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When the iron supply to erythroid precursors is limited, as often occurs during stimulated erythropoiesis, these cells produce signals to inhibit hepatic hepcidin production, thereby increasing the amount of iron that enters the plasma. How stimulated erythropoiesis suppresses hepcidin production is incompletely understood, but erythroferrone, Gdf15 and Twsg1 have emerged as candidate regulatory molecules. To further examine the relationship between erythropoiesis and the candidate erythroid regulators, we have studied five mouse models of anemia, including two models of β-thalassemia (Hbbth3/+ and RBC14), the hemoglobin deficit mouse (hbd), dietary iron deficient mice and mice treated with phenylhydrazine to induce acute hemolysis. Hematological parameters, iron status and the expression of Erfe (the gene encoding erythroferrone), Gdf15 and Twsg1 in the bone marrow and spleen were examined. Erfe expression was the most consistently upregulated of the candidate erythroid regulators in all of the mouse models examined. Gene expression was particularly high in the bone marrow and spleen of iron deficient animals, making erythroferrone an ideal candidate erythroid regulator, as its influence is strongest when iron supply to developing erythroid cells is limited. Gdf15 expression was also upregulated in most of the anemia models studied although the magnitude of the increase was generally less than that of Erfe. In contrast, very little regulation of Twsg1 was observed. These results support the prevailing hypothesis that erythroferrone is a promising erythroid regulator and demonstrate that Erfe expression is stimulated most strongly when the iron supply

  17. Interleukin-10 regulates hepcidin in Plasmodium falciparum malaria

    KAUST Repository

    Huang, Honglei

    2014-02-10

    Background: Acute malarial anemia remains a major public health problem. Hepcidin, the major hormone controlling the availability of iron, is raised during acute and asymptomatic parasitemia. Understanding the role and mechanism of raised hepcidin and so reduced iron availability during infection is critical to establish evidence-based guidelines for management of malaria anemia. Our recent clinical evidence suggests a potential role of IL-10 in the regulation of hepcidin in patients with acute P. falciparum malaria. Methods: We have measured secretion of hepcidin by primary macrophages and the hepatoma cell line HepG2 stimulated with IL-10, IL-6 and Plasmodium falciparum-infected erythrocytes. Findings: We have observed that IL-10 and IL-6 production increased in primary macrophages when these cells were co-cultured with Plasmodium falciparum-infected erythrocytes. We found that IL-10 induced hepcidin secretion in primary macrophages in a dose-dependent manner but not in HepG2 cells. These effects were mediated through signal transducer and activator of transcription (STAT) 3-phosphorylation and completely abrogated by a specific STAT3 inhibitor. Conclusion: IL-10 can directly regulate hepcidin in primary macrophages but not in HepG2 cells. This effect can be modulated by Plasmodium falciparum. The results are consistent with a role for IL-10 in modulating iron metabolism during acute phase of infection. 2014 Huang et al.

  18. [Effect of hepcidin on iron metabolism in athletes].

    Science.gov (United States)

    Domínguez, Raúl; Garnacho-Castaño, Manuel Vicente; Maté-Muñoz, José Luis

    2014-12-01

    The role of iron in the human body is essential, and athletes must always try to keep an adequate iron status. Hepcidin is proposed as the main hormone responsible for the control of iron reserves in the body, given its ability to induce degradation of ferroportin. The action of hepcidin on ferroportin leads to a decreased dietary iron absorption, as well as to a decrease in macrophages. Several factors such as the iron status, the amount of dietary iron, the inflammation, the hypoxia, the testosterone and the physical exercise have been pointed out as affecting the synthesis of hepcidin. This study has aimed at analysing the researches on hepcidin response to exercise, as well as designing a specific strategy to prevent a potential ferropenic status in athletes. The main findings are an association between exercise at an intensity over 65% VO2max and transient increases in the synthesis of hepcidin, and a possible regulatory effect of intermittent hypoxic stimuli in the early post-exercise recovery. Other factors such as the training volume, sex, kind of exercise or the type of surface where the training takes place do not seem to affect the response of hepcidin to exercise.

  19. The use of cold plasma technology to reduce carryover in screening assays.

    Science.gov (United States)

    Akhlaq, Mohammed; Rosethorne, Elizabeth M; Sattikar, Afrah; Kent, Toby C

    2013-08-01

    The accurate transfer of biological reagents represents a fundamental step in the drug screening process, and the elimination of carryover is critical for the generation of accurate measurements of biological activity. The introduction of automated liquid robotics into screening laboratories has transformed the drug screening process, enabling accurate and reproducible transfer of liquids to become a high-throughput activity, but has also introduced a new challenge for drug discoverers: to establish screening workflows that limit analyte carryover for the generation of high-quality screening data. The widespread use of pipetting tips on automated liquid handlers often necessitates the use of optimized wash protocols for removing contaminants and frequently requires the use and disposal of large quantities of organic solvents. Furthermore, many chemical and biological reagents are recalcitrant to removal from pipetting tips by treatment with organic solvents. The use of cold atmospheric plasma technology provides an alternative approach for removal of contaminants and offers many advantages over traditional decontamination protocols commonly used during biological screening. This report describes the evaluation of a cold plasma tip-cleaning system for reducing carryover in a range of biological screening assays requiring the transfer of low molecular weight compound, nucleic acid, and bacterial liquid transfers. The validation of this technology for biological screening assays is presented, and the impact of this technology for screening workflows is discussed.

  20. Solid-phase extraction and HPLC assay of nicotine and cotinine in plasma and brain.

    Science.gov (United States)

    Dawson, Ralph; Messina, S M; Stokes, C; Salyani, S; Alcalay, N; De Fiebre, N C; De Fiebre, C M

    2002-01-01

    The aim of this study was to develop a simple and reliable assay for nicotine (NIC) and its major metabolite, cotinine (COT), in plasma and brain. A method was developed that uses an extraction method compatible with reverse-phase high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection. Sequential solid-phase extraction on silica columns followed by extraction using octadecyl (C18) columns resulted in mean percent recovery (n = 5) of 51 +/- 5, 64 +/- 10, and 52 +/- 10% for NIC, COT, and phenylimidazole (PI), respectively, in spiked 1-mL serum samples. Recovery (mean +/- SEM) of the internal standard (PI) from spiked samples of nicotine-injected rats averaged 64.1 +/- 1.5% (n = 138) from plasma, and 20.7+/-0.8% (n = 128) from brain. The limits of detection of NIC in plasma samples were approximately 8 ng per mL, and of COT, 13.6 ng per mL. Further optimization of our extraction method, using slower flow rates and solid-phase extraction on silica columns, followed by C18 column extraction, yielded somewhat better recoveries (38 +/-3%) for 1-mL brain homogenates. Interassay precision (coefficient of variation) was determined on the basis of daily calibrations for 2 months and was found to be 7%, 9%, and 9% for NIC, COT, and PI, respectively, whereas intra-assay variability was 3.9% for both NIC and COT. Limited studies were performed on analytical columns for comparison of retention, resolution, asymmetry, and column capacity. We concluded that a simple two-step solid-phase extraction method, coupled with HPLC separation and UV detection, can be used routinely to measure NIC and COT in biological fluids and tissues.

  1. Radiometric assay of red cell and plasma cholinesterase in pesticide appliers from Minnesota.

    Science.gov (United States)

    Potter, W T; Garry, V F; Kelly, J T; Tarone, R; Griffith, J; Nelson, R L

    1993-03-01

    In this study we demonstrate the uses of radiometric assay to detect anticholinesterases in a human population (N = 80) exposed to a broad spectrum of pesticides. The assay is nondilutional. Therefore, anticholinesterase (AChE) agents with low binding affinity can be detected. Our initial results show statistically significant exposure-related decreases in either red cell (AChE) or plasma cholinesterase activity ((butyrl)cholinesterase; BuChE) occurred not only among pesticide appliers who use organophosphates, but also among appliers of the fumigant phosphine. These data extend earlier observations made in laboratory animals exposed to this fumigant. Significant exposure-related decreases in AChE activity were seen in herbicide appliers and appear to be associated with exposure to the herbicide 2-methoxy-3,6-dichlorobenzoic acid. There was no evidence of exposure-related decreases in BuChE activity in herbicide appliers. Our in vivo data, coupled with preliminary in vitro studies of phosphine (50% AChE inhibition, 10 ppm) and 2-methoxy-3,6-chlorobenzoic acid (50% AChE and BuChE inhibition, 70 ppm), suggest that the radiometric assay may be used to detect a broader spectrum of biologically active anticholinesterase agents.

  2. Liquid chromatography-tandem mass spectrometric assay for the PARP inhibitor rucaparib in plasma.

    Science.gov (United States)

    Sparidans, Rolf W; Durmus, Selvi; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

    2014-01-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the poly(ADP-ribose) polymerase-1 inhibitor rucaparib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing gefitinib as internal standard. Diluted extract was directly injected into the reversed-phase chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 1.25-2000ng/ml calibration range with r(2)=0.9958±0.0012 for linear regression with quadratic weighting (n=6). Within day precisions (n=18) were 2.0-5.4%, between day (3 days; n=18) precisions 3.2-8.0% and accuracies (n=18) were 89.7-93.2%. At the lower limit of quantification (1.25ng/ml) these parameters were 9.6%, 13.7% and 85.3%, respectively. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine drug pharmacokinetics in female FVB wild type mice. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

    Science.gov (United States)

    Lerche, Mathilde H; Meier, Sebastian; Jensen, Pernille R; Hustvedt, Svein-Olaf; Karlsson, Magnus; Duus, Jens Ø; Ardenkjaer-Larsen, Jan H

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.

  4. 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.

  5. Is Serum Hepcidin Causative in Hemochromatosis? Novel Analysis from a Liver Transplant with Hemochromatosis

    Directory of Open Access Journals (Sweden)

    Paul C Adams

    2008-01-01

    Full Text Available BACKGROUND: Hepcidin is a circulating hepatic hormone that regulates iron balance. It has been speculated that hepcidin insufficiency or dysregulation may be the primary defect in genetic hemochromatosis.

  6. Iron, hepcidin and the metal connection.

    Directory of Open Access Journals (Sweden)

    Olivier eLoréal

    2014-06-01

    Full Text Available Identification of new players in iron metabolism, such as hepcidin, which regulates ferroportin and divalent metal transporter 1 expression, has improved our knowledge of iron metabolism and iron-related diseases. However, from both experimental data and clinical findings, iron-related proteins appear to also be involved in the metabolism of other metals, especially divalent cations. Reports have demonstrated that some metals may affect, directly or indirectly, the expression of proteins involved in iron metabolism. Throughout their lives, individuals are exposed to various metals during personal and/or occupational activities. Therefore, better knowledge of the connections between iron and other metals could improve our understanding of iron-related diseases, especially the variability in phenotypic expression, as well as a variety of diseases in which iron metabolism is secondarily affected. Controlling the metabolism of other metals could represent a promising innovative therapeutic approach.

  7. Evaluation of a New Fully Automated Assay for Plasma Intact FGF23.

    Science.gov (United States)

    Souberbielle, Jean-Claude; Prié, Dominique; Piketty, Marie-Liesse; Rothenbuhler, Anya; Delanaye, Pierre; Chanson, Philippe; Cavalier, Etienne

    2017-07-31

    Several FGF23 immunoassays are available. However, they are reserved for research purposes as none have been approved for clinical use. We evaluated the performances of a new automated assay for intact FGF23 on the DiaSorin Liaison platform which is approved for clinical use. We established reference values in 908 healthy French subjects aged 18-89 years, and measured iFGF23 in patients with disorders of phosphate metabolism and in patients with chronic kidney disease (CKD). Intra-assay CV was 1.04-2.86% and inter-assay CV was 4.01-6.3%. The limit of quantification was <10 ng/L. Serum iFGF23 concentrations were considerably lower than EDTA values highlighting the importance of using exclusively EDTA plasma. Liaison iFGF23 values were approximately 25% higher than Immutopics values. In the 908 healthy subjects, distribution of the Liaison iFGF23 values was Gaussian with a mean ± 2SD interval of 22.7-93.1 ng/L. Men had a slightly higher level than women (60.3 ± 17.6 and 55.2 ± 17.2 ng/L, respectively). Plasma iFGF23 concentration in 11 patients with tumour-induced osteomalacia, 8 patients with X-linked hypophosphatemic rickets, 43 stage 3a, 43 stage 3b, 43 stage 4, 44 stage 5 CKD patients, and 44 dialysis patients were 217.2 ± 144.0, 150.9 ± 28.6, 98.5 ± 42.0, 130.8 ± 88.6, 130.8 ± 88.6, 331.7 ± 468.2, 788.8 ± 1306.6 and 6103.9 ± 11,178.8 ng/L, respectively. This new iFGF23 assay available on a platform that already allows the measurement of other important parameters of the mineral metabolism is a real improvement for the laboratories and clinicians/researchers involved in this field.

  8. In anemia of multiple myeloma hepcidin is induced by increased bone-morphogenetic protein-2

    Science.gov (United States)

    Hepcidin is the principal iron-regulatory hormone and pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contrib...

  9. The determinants of hepcidin level in chronic kidney disease and hemodialysis Saudi patients

    Directory of Open Access Journals (Sweden)

    Tarek Mohamed Ali

    2014-06-01

    Conclusions: Hepcidin levels are correlated to the glycemic status in CKD and HD patients and hepcidin levels in hemodialysed patients were significantly correlated with eGFR but it is not considered as an independent predictor for hepcidin level in these patients.

  10. Hepcidin levels in children with chronic liver disease

    Directory of Open Access Journals (Sweden)

    Murat Cakir

    2015-01-01

    Full Text Available Background/Aim: We aimed to analyze serum hepcidin level in children with chronic liver disease (CLD and its relationship with serum cytokines level, liver function tests, hepatic iron content, and liver fibrosis. Patients and Methods: The study included 34 children with CLD, and 15 age- and gender-matched healthy children. Serum hepcidin, ferritin, iron level, interleukin-6 (IL-6, transforming growth factor-β (TGF-β , total oxidant status (TOS, and antioxidant status (TAS were studied in all patients and in the control group. Liver iron content (LIC was measured from the liver biopsy specimen. Results: Serum ferritin levels were higher in patients with CLD than control group (100.1 ± 98.2 ng/mL vs 50.5 ± 32.2 ng/mL, P = 0.016. No significant difference was found in hepcidin levels. Hepcidin levels in children with CLD was positively correlated with ferritin (r = 0.75, P = 0.001, pediatric end-stage liver disease (PELD score (r = 0.56, P = 0.001, TAS (r = 0.42,P = 0.02, but negatively correlated with albumin level (r = −0.45,P = 0.008. Transferrin saturation and hepcidin:ferritin ratio were significantly low in patients with severe fibrosis compared with patients with mild/without fibrosis (15.5 ± 5.5 vs 34.3 ± 30.1, P = 0.017 and 1 ± 0.5 vs 1.9 ± 1.4,P = 0.04, respectively. Conclusion: Serum hepcidin levels in children with CLD reflect both liver functions and TAS, and severe fibrosis is associated with low hepcidin:ferritin ratio in children with CLD.

  11. Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods.

    Science.gov (United States)

    Yokotani, Kaori; Umegaki, Keizo

    2017-02-01

    The contribution of (-)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500 mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.

  12. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification.

    Science.gov (United States)

    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1). A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines (2). Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3)) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2

  13. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

    Directory of Open Access Journals (Sweden)

    Jean Ruelle

    2014-11-01

    Full Text Available Introduction: Low or undetectable plasma viral load (VL using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1. Materials and Methods: A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA in an accredited environment (ISO15189:2012 norm. Parameters tested were in line with the digital MIQE guidelines (2. Inter-run coefficient of variation (CV was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3 using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5’FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168 were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999 and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5. Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were

  14. High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Posaconazole and Vincristine in Rat Plasma

    Directory of Open Access Journals (Sweden)

    Hadeel A. Khalil

    2015-01-01

    Full Text Available Purpose. Developing a validated HPLC-DAD method for simultaneous determination of posaconazole (PSZ and vincristine (VCR in rat plasma. Methods. PSZ, VCR, and itraconazole (ITZ were extracted from 200 μL plasma using diethyl ether in the presence of 0.1 M sodium hydroxide solution. The organic layer was evaporated in vacuo and dried residue was reconstituted and injected through HC-C18 (4.6 × 250 mm, 5 μm column. In the mobile phase, acetonitrile and 0.015 M potassium dihydrogen orthophosphate (30 : 70 to 80 : 20, linear gradient over 7 minutes pumped at 1.5 mL/min. VCR and PSZ were measured at 220 and 262 nm, respectively. Two Sprague Dawley rats were orally dosed PSZ followed by iv dosing of VCR and serial blood sampling was performed. Results. VCR, PSZ, and ITZ were successfully separated within 11 min. Calibration curves were linear over the range of 50–5000 ng/mL for both drugs. The CV% and % error of the mean were ≤18% and limit of quantitation was 50 ng/mL for both drugs. Rat plasma concentrations of PSZ and VCR were simultaneously measured up to 72 h and their calculated pharmacokinetics parameters were comparable to the literature. Conclusion. The assay was validated as per ICH guidelines and is appropriate for pharmacokinetics drug-drug interaction studies.

  15. 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%.

  16. Advance in Hepcidin%Hepcidin研究进展

    Institute of Scientific and Technical Information of China (English)

    郑晖; 金悦; 方向明

    2009-01-01

    Hepcidin是一种主要南肝脏合成的富含半胱氨酸的小分子多肽.hepcidin的表达水平与机体铁负荷、缺氧、贫血、感染及炎症等应激有关.炎症和铁超负荷能上调hepcidin表达,而缺氧及贫血则抑制其表达.hepcidin通过调节肠道铁吸收、巨噬细胞铁释放以及肝脏储存铁的释放,维持机体铁稳态.新近研究发现,hepcidin表达水平或功能的异常与血色素沉着症、炎症性贫血及脓毒症的发生发展有关.%Hepcidin is a small cystine-rich cationic peptide produced mainly by hepatocytes.The expression levels of hepcidin correlate with iron,hypoxia,anemia,infection and inflammation.Inflammation and iron loading induce hepcidin synthesis,whereas anemia/hypoxia suppresses its expression.Hepcidin maintain the iron homeostasis via modulating intestinal iron absorption,iron recycling in macrophages,and iron release from liver.Recent studies demonstrated that hepcidin may play an important role in the pathophysiology of hemochromatosis,anemia of inflammation and sepsis.

  17. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Energy Technology Data Exchange (ETDEWEB)

    Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail: sd.tanner@utoronto.ca; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)

    2007-03-15

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  18. Effect of testosterone on hepcidin, ferroportin, ferritin and iron binding capacity in patients with hypogonadotropic hypogonadism and type 2 diabetes.

    Science.gov (United States)

    Dhindsa, Sandeep; Ghanim, Husam; Batra, Manav; Kuhadiya, Nitesh D; Abuaysheh, Sanaa; Green, Kelly; Makdissi, Antoine; Chaudhuri, Ajay; Dandona, Paresh

    2016-11-01

    As the syndrome of hypogonadotropic hypogonadism (HH) is associated with anaemia and the administration of testosterone restores haematocrit to normal, we investigated the potential underlying mechanisms. Randomized, double-blind, placebo-controlled trial. We measured basal serum concentrations of erythropoietin, iron, iron binding capacity, transferrin (saturated and unsaturated), ferritin and hepcidin and the expression of ferroportin and transferrin receptor (TR) in peripheral blood mononuclear cells (MNC) of 94 men with type 2 diabetes. Forty-four men had HH (defined as subnormal free testosterone along with low or normal LH concentrations) while 50 were eugonadal. Men with HH were randomized to testosterone or placebo treatment every 2 weeks for 15 weeks. Blood samples were collected at baseline, 3 and 15 weeks after starting treatment. Twenty men in testosterone group and 14 men in placebo group completed the study. Haematocrit levels were lower in men with HH (41·1 ± 3·9% vs 43·8 ± 3·4%, P = 0·001). There were no differences in plasma concentrations of hepcidin, ferritin, erythropoietin, transferrin or iron, or in the expression of ferroportin or TR in MNC among HH and eugonadal men. Haematocrit increased to 45·3 ± 4·5%, hepcidin decreased by 28 ± 7% and erythropoietin increased by 21 ± 7% after testosterone therapy (P ferritin concentrations, but transferrin concentration increased while transferrin saturation and iron concentrations decreased (P < 0·05). Ferroportin and TR mRNA expression in MNC increased by 70 ± 13% and 43 ± 10%, respectively (P < 0·01), after testosterone therapy. The increase in haematocrit following testosterone therapy is associated with an increase in erythropoietin, the suppression of hepcidin, and an increase in the expression of ferroportin and TR. © 2016 John Wiley & Sons Ltd.

  19. Cationic PEGylated liposomes incorporating an antimicrobial peptide tilapia hepcidin 2-3: an adjuvant of epirubicin to overcome multidrug resistance in cervical cancer cells.

    Science.gov (United States)

    Juang, Vivian; Lee, Hsin-Pin; Lin, Anya Maan-Yuh; Lo, Yu-Li

    Antimicrobial peptides (AMPs) have been recently evaluated as a new generation of adjuvants in cancer chemotherapy. In this study, we designed PEGylated liposomes encapsulating epirubicin as an antineoplastic agent and tilapia hepcidin 2-3, an AMP, as a multidrug resistance (MDR) transporter suppressor and an apoptosis/autophagy modulator in human cervical cancer HeLa cells. Cotreatment of HeLa cells with PEGylated liposomal formulation of epirubicin and hepcidin 2-3 significantly increased the cytotoxicity of epirubicin. The liposomal formulations of epirubicin and/or hepcidin 2-3 were found to noticeably escalate the intracellular H2O2 and O2(-) levels of cancer cells. Furthermore, these treatments considerably reduced the mRNA expressions of MDR protein 1, MDR-associated protein (MRP) 1, and MRP2. The addition of hepcidin 2-3 in liposomes was shown to markedly enhance the intracellular epirubicin uptake and mainly localized into the nucleus. Moreover, this formulation was also found to trigger apoptosis and autophagy in HeLa cells, as validated by significant increases in the expressions of cleaved poly ADP ribose polymerase, caspase-3, caspase-9, and light chain 3 (LC3)-II, as well as a decrease in mitochondrial membrane potential. The apoptosis induction was also confirmed by the rise in sub-G1 phase of cell cycle assay and apoptosis percentage of annexin V/propidium iodide assay. We found that liposomal epirubicin and hepcidin 2-3 augmented the accumulation of GFP-LC3 puncta as amplified by chloroquine, implying the involvement of autophagy. Interestingly, the partial inhibition of necroptosis and the epithelial-mesenchymal transition by this combination was also verified. Altogether, our results provide evidence that coincubation with PEGylated liposomes of hepcidin 2-3 and epirubicin caused programmed cell death in cervical cancer cells through modulation of multiple signaling pathways, including MDR transporters, apoptosis, autophagy, and/or necroptosis

  20. Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus liver

    Directory of Open Access Journals (Sweden)

    José P. Oliveira-Filho

    2012-10-01

    Full Text Available The hypoferremia that is observed during systemic inflammatory processes is mediated by hepcidin, which is a peptide that is mainly synthesized in the livers of several mammalian species. Hepcidin plays a key role in iron metabolism and in the innate immune system. It's up-regulation is particularly useful during acute inflammation, and it restricts the iron availability that is necessary for the growth of pathogenic microorganisms. In this study, the hepcidin mRNA of Equus asinus has been characterized, and the expression of donkey hepcidin in the liver has been determined. The donkey hepcidin sequence has an open reading frame (ORF of 261 nucleotides, and the deduced corresponding protein sequence has 86 amino acids. The amino acid sequence of donkey hepcidin was most homologous to Equus caballus (98%. The mature donkey hepcidin sequence (25 amino acids was 100% homologous to the equine mature hepcidin and has eight conserved cysteine residues that are found in all of the investigated hepcidin sequences. The expression profile of donkey hepcidin in the liver was high and was similar to the reference gene expression. The donkey hepcidin sequence was deposited in GenBankTM (HQ902884 and may be useful for additional studies on iron metabolism and the inflammatory process in this species.

  1. Is iron overload in alcohol-related cirrhosis mediated by hepcidin?

    Institute of Scientific and Technical Information of China (English)

    Tariq Iqbal; Azzam Diab; Douglas G Ward; Matthew J Brookes; Chris Tselepis; Jim Murray; Elwyn Elias

    2009-01-01

    In this case report we describe the relationship between ferritin levels and hepcidin in a patient with alcohol-related spur cell anemia who underwent liver transplantation. We demonstrate a reciprocal relationship between serum or urinary hepcidin and serum ferritin, which indicates that inadequate hepcidin production by the diseased liver is associated with elevated serum ferritin. The ferritin level falls with increasing hepcidin production after transplantation. Neither inflammatory indices (IL6) nor erythropoietin appear to be related to hepcidin expression in this case. We suggest that inappropriately low hepcidin production by the cirrhotic liver may contribute substantially to elevated tissue iron stores in cirrhosis and speculate that hepcidin replacement in these patients may be of therapeutic benefit in the future.

  2. Iron and hepcidin as risk factors in atherosclerosis

    DEFF Research Database (Denmark)

    Galesloot, Tessel E; Janss, Luc L; Burgess, Stephen;

    2015-01-01

    -wide association meta-analysis on iron status, and assessed associations of individual SNPs and quartiles of a multi-SNP score with NIMA. Quartile 4 versus quartile 1 of the multi-SNP score showed directionally consistent associations with the hypothesized direction of effect for all NIMA in women, indicating...... that increased body iron status is a risk factor for atherosclerosis in women. We observed no single SNP associations that fit the hypothesized directions of effect between iron and NIMA, except for rs651007, associated with decreased ferritin concentration and decreased atherosclerosis risk. Two of six NIMA......-related SNPs showed association with the ratio hepcidin/ferritin, suggesting that an increased hepcidin/ferritin ratio increases atherosclerosis risk. Genomic correlations were close to zero, except for hepcidin and ferritin with ABI at rest [-0.27 (SE 0.34) and -0.22 (SE 0.35), respectively] and ABI after...

  3. Exercise as a mediator of hepcidin activity in athletes.

    Science.gov (United States)

    Peeling, Peter

    2010-11-01

    Iron is a trace mineral used by the body in many physiological processes that are essential for athletic performance. However, it is common that an athlete's iron stores are compromised via several well-established exercise-related mechanisms such as hemolysis, hematuria, sweating and gastrointestinal bleeding. Recently, however, a new mechanism for athletics-induced iron deficiency has been proposed, involving the influence of physical activity on the post-exercise hepcidin response. Hepcidin is a liver-produced hormone that regulates iron metabolism in the gut and macrophages. This hormone has become the focus of recent investigations into altered iron metabolism in athletes, and may be a mitigating factor implicated in athletics-induced iron deficiency. This review attempts to summarize and disseminate the collective knowledge currently held regarding exercise and hepcidin expression, in addition to suggesting the direction for future research in this area.

  4. Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model

    Science.gov (United States)

    Millot, Sarah; Delaby, Constance; Moulouel, Boualem; Lefebvre, Thibaud; Pilard, Nathalie; Ducrot, Nicolas; Ged, Cécile; Lettéron, Philippe; de Franceschi, Lucia; Deybach, Jean Charles; Beaumont, Carole; Gouya, Laurent; De Verneuil, Hubert; Lyoumi, Saïd; Puy, Hervé; Karim, Zoubida

    2017-01-01

    Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis. PMID:28143953

  5. A sensitive and robust HPLC assay with fluorescence detection for the quantification of pomalidomide in human plasma for pharmacokinetic analyses.

    Science.gov (United States)

    Shahbazi, Shandiz; Peer, Cody J; Polizzotto, Mark N; Uldrick, Thomas S; Roth, Jeffrey; Wyvill, Kathleen M; Aleman, Karen; Zeldis, Jerome B; Yarchoan, Robert; Figg, William D

    2014-04-01

    Pomalidomide is a second generation IMiD (immunomodulatory agent) that has recently been granted approval by the Food and Drug Administration for treatment of relapsed multiple myeloma after prior treatment with two antimyeloma agents, including lenalidomide and bortezomib. A simple and robust HPLC assay with fluorescence detection for pomalidomide over the range of 1-500ng/mL has been developed for application to pharmacokinetic studies in ongoing clinical trials in various other malignancies. A liquid-liquid extraction from human plasma alone or pre-stabilized with 0.1% HCl was performed, using propyl paraben as the internal standard. From plasma either pre-stabilized with 0.1% HCl or not, the assay was shown to be selective, sensitive, accurate, precise, and have minimal matrix effects (HPLC-FL assay allows a broader range of laboratories to measure pomalidomide for application to clinical pharmacokinetics.

  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. Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance.

    Science.gov (United States)

    Welch, Nicholas G; Madiona, Robert M T; Easton, Christopher D; Scoble, Judith A; Jones, Robert T; Muir, Benjamin W; Pigram, Paul J

    2016-11-10

    Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.

  8. Serum Hepcidin Levels Are Associated With Obesity but Not Liver Disease

    Science.gov (United States)

    Vuppalanchi, Raj; Troutt, Jason S; Konrad, Robert J.; Ghabril, Marwan; Saxena, Romil; Bell, Lauren N; Kowdley, Kris V.; Chalasani, Naga

    2013-01-01

    Objective Hepcidin is regulated by anemia and inflammation. It is primarily expressed in the liver but studies have reported its expression in adipose tissue. We investigated the relationship between BMI and serum hepcidin and also examined the relationship between liver histology and serum hepcidin in the morbidly obese. Design and Methods Serum and liver tissue from patients undergoing bariatric surgery (bariatric cohort, n=105) and serum from healthy blood donors (n=60) were used to conduct this study. Serum hepcidin was measured using sandwich ELISA, highly specific for hepcidin-25. Serum ferritin, IL-6, IL-1β and liver function biochemistries were also measured. Results After controlling for covariates, BMI ≥ 35 kg/m2 was significantly associated with higher serum hepcidin level compared to individuals with lower BMI groups (17.7 ± 11.5 vs. 3.3 ± 4.7 ng/ml, P=0.002). Presence of NAFLD was not associated with higher serum levels of hepcidin (multivariate P=0.37) There was no association between serum hepcidin levels and liver histology (presence of steatohepatitis, advanced fibrosis, or NAFLD activity score) in the bariatric cohort. Conclusions Obesity, but not the presence of NAFLD was associated with serum hepcidin levels. There was no association between serum hepcidin and liver histology in the morbidly obese undergoing bariatric surgery. PMID:23512600

  9. Hepcidin is suppressed by erythropoiesis in hemoglobin E β-thalassemia and β-thalassemia trait

    Science.gov (United States)

    Jones, Emma; Pasricha, Sant-Rayn; Allen, Angela; Evans, Patricia; Fisher, Chris A.; Wray, Katherine; Premawardhena, Anuja; Bandara, Dyananda; Perera, Ashok; Webster, Craig; Sturges, Pamela; Olivieri, Nancy F.; St. Pierre, Timothy; Armitage, Andrew E.; Porter, John B.; Weatherall, David J.

    2015-01-01

    Hemoglobin E (HbE) β-thalassemia is the most common severe thalassemia syndrome across Asia, and millions of people are carriers. Clinical heterogeneity in HbE β-thalassemia is incompletely explained by genotype, and the interaction of phenotypic variation with hepcidin is unknown. The effect of thalassemia carriage on hepcidin is also unknown, but it could be relevant for iron supplementation programs aimed at combating anemia. In 62 of 69 Sri Lankan patients with HbE β-thalassemia with moderate or severe phenotype, hepcidin was suppressed, and overall hepcidin inversely correlated with iron accumulation. On segregating by phenotype, there were no differences in hepcidin, erythropoiesis, or hemoglobin between severe or moderate disease, but multiple linear regression showed that erythropoiesis inversely correlated with hepcidin only in severe phenotypes. In moderate disease, no independent predictors of hepcidin were identifiable; nevertheless, the low hepcidin levels indicate a significant risk for iron overload. In a population survey of Sri Lankan schoolchildren, β-thalassemia (but not HbE) trait was associated with increased erythropoiesis and mildly suppressed hepcidin, suggesting an enhanced propensity to accumulate iron. In summary, the influence of erythropoiesis on hepcidin suppression associates with phenotypic disease variation and pathogenesis in HbE β-thalassemia and indicates that the epidemiology of β-thalassemia trait requires consideration when planning public health iron interventions. PMID:25519750

  10. [Peripheral blood monocyte hepcidin in patients with multiple myeloma is associated with anemia of chronic disease].

    Science.gov (United States)

    Han, Xiao; Zhou, Dao-Bin; Duan, Ming-Hui; Wang, Xuan; Zhang, Jie-Ping; Zhao, Yong-Qiang; Shen, Ti; Wu, Yong-Ji

    2013-04-01

    Disorders of iron utilization caused by abnormal elevation of hepcidin levels are the main mechanism of anemia of chronic disease. Hepcidin is mainly produced by the liver. Recently it has been found that monocytes are another source of hepcidin. The increased hepcidin in serum and urine of multiple myeloma patients may be one cause of anemia of chronic disease (ACD). However it is unclear whether the peripheral blood monocyte hepcidin is involved in the pathogenesis of anemia of chronic disease. This study was purposed to investigate the role of monocyte hepcidin in multiple myeloma patients with anemia of chronic disease. The clinical data and peripheral venous blood of multiple myeloma patients were collected.Serum concentration of IL-6 and TNF-α was detected by ELISA. Peripheral blood monocytes were isolated by CD14(+) magnetic beads. Hepcidin, IL-6 and TNF-α mRNA of monocytes were detected by real time quantitative PCR. The results showed that the expression level of monocyte hepcidin mRNA in myeloma patients was higher than that in normal controls. In untreated patients, the expression level of monocyte hepcidin mRNA was negatively correlated with hemoglobin, and positively correlated with serum ferritin and IL-6 levels, but unrelated with TNF-α levels.It is concluded that the increased monocyte hepcidin levels in multiple myeloma patients may play an etiologic role in ACD.

  11. Combined treatment of 3-hydroxypyridine-4-one derivatives and green tea extract to induce hepcidin expression in iron-overloaded β-thalassemic mice

    Directory of Open Access Journals (Sweden)

    Supranee Upanan

    2015-12-01

    Conclusions: The GTE + DFP treatment could ameliorate iron overload and liver oxidative damage in non-transfusion dependent β-thalassemic mice, by chelating toxic iron in plasma and tissues, and increasing hepcidin expression to inhibit duodenal iron absorption and iron release from hepatocytes and macrophages in the spleen. There is probably an advantage in giving GTE with DFP when treating patients with iron overload.

  12. Training surface and intensity: inflammation, hemolysis, and hepcidin expression.

    NARCIS (Netherlands)

    Peeling, P.; Dawson, B.; Goodman, C.; Landers, G.; Wiegerinck, E.T.G.; Swinkels, D.W.; Trinder, D.

    2009-01-01

    PURPOSE: This investigation assessed the effects of training intensity and ground surface type on hemolysis, inflammation, and hepcidin activity during running. METHODS: Ten highly trained male endurance athletes completed a graded exercise test, two continuous 10-km runs on a grass (GRASS) and a bi

  13. Training surface and intensity: inflammation, hemolysis, and hepcidin expression.

    NARCIS (Netherlands)

    Peeling, P.; Dawson, B.; Goodman, C.; Landers, G.; Wiegerinck, E.T.G.; Swinkels, D.W.; Trinder, D.

    2009-01-01

    PURPOSE: This investigation assessed the effects of training intensity and ground surface type on hemolysis, inflammation, and hepcidin activity during running. METHODS: Ten highly trained male endurance athletes completed a graded exercise test, two continuous 10-km runs on a grass (GRASS) and a bi

  14. Mass fragmentographic assay of nanogram amounts of the antidepressant drug mianserin hydrochloride (Org GB 94) in human plasma.

    Science.gov (United States)

    de Ridder, J J; Koppens, P C; van Hal, H J

    1977-05-01

    For the assay of the antidepressant compound mianserin hydrochloride (Org GB 94) in human plasma, a mass fragmentographic method, using the deuterated analogue as internal standard and a high-performance liquid chromatographie sample clean-up procedure has been developed. The assay specifications obtained are a lower limit for reliable measurements of 1 ng/ml, and accuracy of ca. 0.01 ng/ml, a precision of 6--7% and a capacity of about 60 samples per day. The applicability of the assay method is illustrated by measurements of single-dose and steady-state plasma levels in clinical experiments, demonstrating the possibility of monitoring plasma levels during at least 24 h after a single dose of 15 mg of Org GB 94. The mean steady-state plasma levels after a daily dose of 3 X 20 mg of Org GB 94 appeared to be remarkably constant with time: 38, 36 and 34 ng/ml after 2, 4, and 6 weeks of treatment of 18 depressed patients.

  15. Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype-specific PRM assays.

    Science.gov (United States)

    Kim, Yeoun Jin; Gallien, Sebastien; El-Khoury, Victoria; Goswami, Panchali; Sertamo, Katriina; Schlesser, Marc; Berchem, Guy; Domon, Bruno

    2015-09-01

    The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α,  SAA 1β,  SAA1γ,  SAA2α,  SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer.

  16. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    Science.gov (United States)

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  17. Study of anemia in nondialysis dependent chronic kidney disease with special reference to serum hepcidin

    Science.gov (United States)

    Goyal, H.; Mohanty, S.; Sharma, M.; Rani, A.

    2017-01-01

    We studied the role of serum hepcidin in anemia of chronic kidney disease (CKD) in a hospital-based cross-sectional study. Serum hepcidin, ferritin, and high-sensitivity C-reactive protein (hsCRP) levels were evaluated in patients of CKD. Hepcidin levels were increased in patients as compared to healthy adults. Hepcidin levels increased as CKD progressed through stage 3–5 (P trend = 0.015) but did not correlate with estimated glomerular filtration rate. Hepcidin correlated positively with ferritin (P EPO) levels (P = 0.0258) but did not correlate with either hsCRP or estimated glomerular filtration rate. Iron status influenced hepcidin levels of patients. Patients were divided according to iron status on the basis of TSAT and serum ferritin levels. We observed that while absolute iron deficiency (transferrin saturation EPO to overcome iron-restricted erythropoiesis. PMID:28182038

  18. Hepcidin serum levels and resistance to recombinant human erythropoietin therapy in hemodialysis patients

    Directory of Open Access Journals (Sweden)

    Kristina Petrulienė

    2017-01-01

    Conclusions: CRP, albumin, BMI, and hospitalization rate per year were found to be significant determinants of ERI in MHD patients. Inadequate dialysis was associated with higher epoetin requirements. There were no difference in patient mortality by ERI, but a significant difference in hospitalization rates and mean length of one hospitalization was revealed. A significant positive relation between hepcidin and ERI was revealed. ERI and ferritin were found to be significant determinants of hepcidin in MHD patients. Hepcidin was not related to mortality.

  19. Iron-regulatory protein hepcidin is increased in female athletes after a marathon.

    Science.gov (United States)

    Roecker, L; Meier-Buttermilch, R; Brechtel, L; Nemeth, E; Ganz, T

    2005-12-01

    The propose of this study was to determine the influence of marathon race on hepcidin excretion in female athletes (age 26-45 years). Urine samples were taken before, immediately after, 1 and 3 days after the race. In the average, hepcidin transiently increased at day 1 from 32 to 85 ng/mg creatinine. We propose that the frequently observed iron deficiency of females runners is caused by elevated hepcidin levels.

  20. A rapid UPLC-MS/MS assay for eicosanoids in human plasma: Application to evaluate niacin responsivity.

    Science.gov (United States)

    Miller, Tricia M; Poloyac, Samuel M; Anderson, Kacey B; Waddell, Brooke L; Messamore, Erik; Yao, Jeffrey K

    2017-01-18

    A rapid and sensitive method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to simultaneously quantify hydroxyeicosatetraenoic (HETE), dihydroxyeicosatrienoic (DiHETrE), epoxyeicosatrienoic acid (EET), and prostaglandin metabolites of arachidonic acid in human plasma. Sample preparation consisted of solid phase extraction with Oasis HLB (30mg) cartridges for all metabolites. Separation of HETEs, EETs, and DiHETrEs was achieved on an Acquity UPLC BEH C18, 1.7µm (100×2.1mm) reversed-phase column (Waters Corp, Millford, MA) with negative electrospray ionization mass spectrometric detection. A second injection of the same extracted sample allowed for separation and assessment of prostaglandin metabolites under optimized UPLC-MS/MS conditions. Additionally, the endogenous levels of these metabolites in five different matrices were determined in order to select the optimal matrix for assay development. Human serum albumin was shown to have the least amount of endogenous metabolites, a recovery efficiency of 79-100% and a matrix effect of 71 - 100%. Linear calibration curves ranging from 0.416 to 66.67ng/ml were validated. Inter-assay and intra-assay variance was less than 15% at most concentrations. This method was successfully applied to quantify metabolite levels in plasma samples of healthy control subjects receiving niacin administration to evaluate the association between niacin administration and eicosanoid plasma level response.

  1. Liquid chromatography-tandem mass spectrometric assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma.

    Science.gov (United States)

    Dolman, M Emmy M; den Hartog, Ilona J M; Molenaar, Jan J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2014-01-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing rucaparib as internal standard. After dilution with water, the extract was directly injected into the reversed-phase LC system. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 5-10,000ng/ml calibration range using double logarithmic calibration, 5ng/ml was the lower limit of quantification. Within day precisions (n=6) were 2.9-5.6%, between day (3 days; n=18) precisions 3.2-7.2%. Accuracies were between 95.9 and 99.0% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to determine plasma pharmacokinetics after intraperitoneal administration of AT7519 in mice with neuroblastoma xenografts. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. A comparison of serum and plasma cytokine values using a multiplexed assay in cats.

    Science.gov (United States)

    Gruen, Margaret E; Messenger, Kristen M; Thomson, Andrea E; Griffith, Emily H; Paradise, Hayley; Vaden, Shelly; Lascelles, B D X

    2016-12-01

    Degenerative joint disease (DJD) is highly prevalent in cats, and pain contributes to morbidity. In humans, alterations of cytokine concentrations have been associated with joint deterioration and pain. Similar changes have not been investigated in cats. Cytokine concentrations can be measured using multiplex technology with small samples of serum or plasma, however, serum and plasma are not interchangeable for most bioassays. Correlations for cytokine concentrations between serum and plasma have not been evaluated in cats. To evaluate the levels of detection and agreement between serum and plasma samples in cats. Paired serum and plasma samples obtained from 38 cats. Blood was collected into anti-coagulant free and EDTA Vacutainer(®) tubes, serum or plasma extracted, and samples frozen at -80°C until testing. Duplicate samples were tested using a 19-plex feline cytokine/chemokine magnetic bead panel. Agreement between serum and plasma for many analytes was high, however correlation coefficients ranged from -0.01 to 0.97. Results from >50% of samples were below the lower limit of quantification for both serum and plasma for nine analytes, and for an additional three analytes for plasma only. While serum and plasma agreement was generally good, detection was improved using serum samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

    Science.gov (United States)

    Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.

    2017-01-01

    Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

  4. Bioanalytical qualification of clinical biomarker assays in plasma using a novel multi-analyte Simple Plex(™) platform.

    Science.gov (United States)

    Gupta, Vinita; Davancaze, Teresa; Good, Jeremy; Kalia, Navdeep; Anderson, Michael; Wallin, Jeffrey J; Brady, Ann; Song, An; Xu, Wenfeng

    2016-12-01

    Immune-checkpoint inhibitors are presumed to break down the tolerogenic state of immune cells by activating T-lymphocytes that release cytokines and enhance effector cell function for elimination of tumors. Measurement of cytokines is being pursued for better understanding of the mechanism of action of immune-checkpoint inhibitors, as well as to identify potential predictive biomarkers. In this study, we show bioanalytical qualification of cytokine assays in plasma on a novel multi-analyte immunoassay platform, Simple Plex(™). The qualified assays exhibited excellent sensitivity as evidenced by measurement of all samples within the quantifiable range. The accuracy and precision were 80-120% and 10%, respectively. The qualified assays will be useful in assessing mechanism of action cancer immunotherapies.

  5. Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli.

    Science.gov (United States)

    Houamel, Dounia; Ducrot, Nicolas; Lefebvre, Thibaud; Daher, Raed; Moulouel, Boualem; Sari, Marie-Agnes; Letteron, Philippe; Lyoumi, Said; Millot, Sarah; Tourret, Jerome; Bouvet, Odile; Vaulont, Sophie; Vandewalle, Alain; Denamur, Erick; Puy, Hervé; Beaumont, Carole; Gouya, Laurent; Karim, Zoubida

    2016-03-01

    The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.

  6. Production of Recombinant Vector Containing the Coding Sequence of Human Hepcidin

    Directory of Open Access Journals (Sweden)

    Keyhanvar, N.

    2013-01-01

    Full Text Available Background and objective: Hepcidin is a cystein-rich antimicrobial peptide,which is secreted by the liver. It fights against wide spectrum of bacteria, virusesand fungi and it is a major regulator of iron homeostasis. Today, scientists havemade many efforts on the production of hepcidin. Baculovirus expression systemis one of the best eukaryotic expression systems for production of recombinanthepcidin and production of the recombinant vector is one of the most importantsteps in this expression system.Material & Methods: First, the total RNA was separated from HepG2 cell lineas a source of hepcidin expression. Then, after synthesis of total cDNA, humanhepcidin sequence was amplified, using specific primers by PCR method. Next,hepcidin sequence was cloned into pTZ57R/T vector. After digestion ofrecombinant vector using ECoRI and BamHI restriction enzymes, recombinantpFastBac HT B vector containing human hepcidin cDNA was produced.Results: Coding sequence of human hepcidin is correctly cloned into pTZ57R/Tvector and sub cloning into pFastBac HT B vector is performed successfully. Thepresence of a clear band near 274 bp resulted from PCR amplification andrestriction enzyme are the confirmation of the cloning of human hepcidin.Conclusion: According to our knowledge, the present study is the first work thatfocuses on recombinant vector containing coding sequence of human prohepcidin.This recombinant vector can be used for human hepcidin production.Keywords: Vector; Hepcidin; Iron

  7. EDTA-treated cotton-thread microfluidic device used for one-step whole blood plasma separation and assay.

    Science.gov (United States)

    Ulum, Mokhamad Fakhrul; Maylina, Leni; Noviana, Deni; Wicaksono, Dedy Hermawan Bagus

    2016-04-21

    This study aims to observe the wicking and separation characteristics of blood plasma in a cotton thread matrix functioning as a microfluidic thread-based analytical device (μTAD). We investigated several cotton thread treatment methods using ethylenediaminetetraacetic acid (EDTA) anticoagulant solution for wicking whole blood samples and separating its plasma. The blood of healthy Indonesian thin tailed sheep was used in this study to understand the properties of horizontal wicking and separation on the EDTA-treated μTAD. The wicking distance and blood cell separation from its plasma was observed for 120 s and documented using a digital phone camera. The results show that untreated cotton-threads stopped the blood wicking process on the μTAD. On the other hand, the deposition of EDTA anticoagulant followed by its drying on the thread at room temperature for 10 s provides the longest blood wicking with gradual blood plasma separation. Furthermore, the best results in terms of the longest wicking and the clearest on-thread separation boundary between blood cells and its plasma were obtained using the μTAD treated with EDTA deposition followed by 60 min drying at refrigerated temperature (2-8 °C). The separation length of blood plasma in the μTADs treated with dried-EDTA at both room and refrigerated temperatures was not statistically different (P > 0.05). This separation occurs through the synergy of three factors, cotton fiber, EDTA anticoagulant and blood platelets, which induce the formation of a fibrin-filter via a partial coagulation process in the EDTA-treated μTAD. An albumin assay was employed to demonstrate the efficiency of this plasma separation method during a one-step assay on the μTAD. Albumin in blood is an important biomarker for kidney and heart disease. The μTAD has a slightly better limit of detection (LOD) than conventional blood analysis, with an LOD of 114 mg L(-1) compared to 133 mg L(-1), respectively. However, the μTAD performed

  8. 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.

  9. Clinical value of plasma B-type natriuretic peptide assay in pediatric pneumonia accompanied by heart failure

    Science.gov (United States)

    HU, DAN; LIU, YANG; TAO, HUIXIAN; GAO, JINPING

    2015-01-01

    Previous studies have shown that B-type natriuretic peptide (BNP) is useful in differentiating cardiac from pulmonary causes of dyspnea in adults. To date, international guidelines have recommended measurements of circulating BNP as a biomarker for diagnostic and prognostic purposes, as well as therapeutic monitoring, in adults with cardiac diseases, particularly those suffering from acute and chronic heart failure (HF). The aim of the present study was to investigate the differential diagnostic and therapeutic analysis of BNP levels assayed in pediatric pneumonia accompanied by HF. The clinical data of 80 patients with pneumonia, aged 1–3 years, were analyzed. The patients were divided into two groups: Simple pneumonia (46 cases) and pneumonia accompanied by HF (34 cases). All patients underwent two plasma BNP assays: The first one upon admission to the hospital and the second one prior to discharge. The plasma BNP levels of 20 healthy children were used as the negative control. Plasma BNP levels were measured using the Triage® BNP automated immunoassay systems and reagents. Statistical analysis showed that the plasma BNP levels of the patients upon admission were higher in the pneumonia accompanied by HF group compared with those in the simple pneumonia group (750±120 vs. 135±50 pg/ml; P<0.05). In addition, in the pneumonia accompanied by HF group, the plasma BNP levels of the patients were higher upon admission to the hospital than they were prior to discharge (750±120 vs. 115±45 pg/ml; P<0.05); therefore, plasma BNP may comprise a sensitive diagnostic and therapeutic evaluative marker for pediatric patients with pneumonia accompanied by HF. This finding could prove invaluable in the clinical diagnosis and treatment of the disease. PMID:26668612

  10. Fluorogenic MMP activity assay for plasma including MMPs complexed to α2-macroglobulin

    NARCIS (Netherlands)

    Beekman, B.; Drijfhout, J.W.; Ronday, H.K.; TeKoppele, J.M.

    1999-01-01

    Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the prote

  11. Fluorogenic MMP activity assay for plasma including MMPs complexed to α2-macroglobulin

    NARCIS (Netherlands)

    Beekman, B.; Drijfhout, J.W.; Ronday, H.K.; TeKoppele, J.M.

    1999-01-01

    Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the

  12. Application of a design of experiment approach in the development of a sensitive bioanalytical assay in human plasma.

    Science.gov (United States)

    Dawes, Michelle L; Bergum, James S; Schuster, Alan E; Aubry, Anne-Françoise

    2012-11-01

    To support a first-in-human (FIH) clinical study in healthy volunteers, a human plasma assay, a 20-fold more sensitive method than the validated non-clinical LC-MS/MS assays, was requested. For the clinical assay, a LLOQ of 0.050 ng/mL for Compound A and 0.100 ng/mL for Compound B was desired to accurately determine the analyte concentrations in human plasma samples across all treatment groups. A design of experiment (DOE) investigation was performed in an effort to optimize the extraction procedure of the bioanalytical assay used to support the first in human study and future clinical studies. Three factors, extraction buffer pH (two pHs), volume ratio of organic solvent to plasma (two ratios), and extraction shake time (three times), were selected for the DOE. Both analytes were analyzed at a low concentration, 0.150 ng/mL, and a stable isotope label internal standard was used for each analyte. To estimate the recovery of each analyte from the extraction, the response ratio of each analyte over the respective internal standard was used, and to estimate matrix effects, the absolute response (peak area) of each analyte was used. The results of the DOE indicated that the three factors tested had a more significant effect on the extraction of the metabolite, Compound B, compared to that of the parent, Compound A. The extraction buffer pH had the greatest influence on Compound B and the volume of extraction solvent had an influence on both analytes. Unexpectedly, a longer extraction time caused an apparent decrease in the overall recovery for both analytes. This was presumably due to an increased extraction of interfering matrix components. Optimal conditions were achieved for the combined analysis of both compounds using the DOE approach.

  13. A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

    Directory of Open Access Journals (Sweden)

    M.F. Alves

    2005-06-01

    Full Text Available A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(DnpP-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl was optimized for the measurement of angiotensin I-converting enzyme (ACE in human plasma and rat tissues. Abz-FRK(DnpP-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(DnpP-OH correlated closely (r = 0.90, P < 0.001. The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(DnpP-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

  14. Hepcidin attenuates amyloid beta-induced inflammatory and pro-oxidant responses in astrocytes and microglia.

    Science.gov (United States)

    Urrutia, Pamela J; Hirsch, Etienne C; González-Billault, Christian; Núñez, Marco T

    2017-07-01

    Alzheimer's disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and neuronal death. Aggregated amyloid-β (Aβ) induces inflammation and oxidative stress, which have pivotal roles in the pathogenesis of AD. Hepcidin is a key regulator of systemic iron homeostasis. Recently, an anti-inflammatory response to hepcidin was reported in macrophages. Under the hypothesis that hepcidin mediates anti-inflammatory response in the brain, in this study, we evaluated the putative anti-inflammatory role of hepcidin on Aβ-activated astrocytes and microglia. Primary culture of astrocytes and microglia were treated with Aβ, with or without hepcidin, and cytokine levels were then evaluated. In addition, the toxicity of Aβ-treated astrocyte- or microglia-conditioned media was tested on neurons, evaluating cellular death and oxidative stress generation. Finally, mice were injected in the right lateral ventricle with Aβ, with or without hepcidin, and hippocampus glial activation and oxidative stress were evaluated. Pre-treatment with hepcidin reduced the expression and secretion of TNF-α and IL-6 in astrocytes and microglia treated with Aβ. Hepcidin also reduced neurotoxicity and oxidative damage triggered by conditioned media obtained from astrocytes and microglia treated with Aβ. Stereotaxic intracerebral injection of hepcidin reduced glial activation and oxidative damage triggered by Aβ injection in mice. Overall, these results are consistent with the hypothesis that in astrocytes and microglia hepcidin down-regulates the inflammatory and pro-oxidant processes induced by Aβ, thus protecting neighboring neurons. This is a newly described property of hepcidin in the central nervous system, which may be relevant for the development of strategies to prevent the neurodegenerative process associated with AD. © 2017 International Society for Neurochemistry.

  15. Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue.

    Science.gov (United States)

    Gotardo, Érica Martins Ferreira; dos Santos, Aline Noronha; Miyashiro, Renan Akira; Gambero, Sheley; Rocha, Thalita; Ribeiro, Marcelo Lima; Gambero, Alessandra

    2013-01-01

    Since the discovery that hepcidin is expressed in the adipose tissue of obese subjects, attention has been increasingly focused on alterations in iron homeostasis that are associated with adiposity. We examined the production of hepcidin, the expression of hepcidin-related genes and the iron content of the adipose tissue in obesity using Swiss mice fed a high-fat diet (HFD). The mice were maintained on a control diet or HFD for 12 or 24 wk, and body weight, adiposity and glucose homeostasis were evaluated. The expression of several genes (hepcidin, TfR1, TfR2, DMT1, FT-heavy, ferroportin, IRP-1, IRP-2 and HIF-1) and the protein expression of hepcidin and IL-6 were quantified. The iron level was assessed using a Prussian blue reaction in paraffin-embedded tissue. After 24 wk on the HFD, we observed increases in the levels of hepcidin in the serum and the visceral adipose tissue. The IL-6 levels also increased in the visceral adipose tissue. Adipocytes isolated from the visceral adipose tissues of lean and obese mice expressed hepcidin at comparable levels; however, isolated macrophages from the stromal vascular fraction expressed higher hepcidin levels. Adipose tissues from obese mice displayed increased tfR2 expression and the presence of iron. Our results indicate that IL-6 and iron may affect the signaling pathways governing hepcidin expression. Thus, the mice fed HFD for 24 wk represent a suitable model for the study of obesity-linked hepcidin alterations. In addition, hepcidin may play local roles in controlling iron availability and interfering with inflammation in adipose tissue.

  16. An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI).

    Science.gov (United States)

    Popp, Robert; Malmström, David; Chambers, Andrew G; Lin, David; Camenzind, Alexander G; van der Gugten, J Grace; Holmes, Daniel T; Pugia, Michael; Jaremek, Marta; Cornett, Shannon; Suckau, Detlev; Borchers, Christoph H

    2015-06-01

    Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC-MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC-MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC-MS/MS. The assay is performed on 350μL of plasma, and has a linear range from 0.08 to 5.3ng/L/s in the reflector mode, and 0.04 to 5.3ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC-MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R(2) value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics.

  17. Assessment of apixaban plasma levels by laboratory tests: suitability of three anti-Xa assays. A multicentre French GEHT study.

    Science.gov (United States)

    Gouin-Thibault, Isabelle; Flaujac, Claire; Delavenne, Xavier; Quenet, Sara; Horellou, Marie-Hélenè; Laporte, Silvy; Siguret, V; Lecompte, T

    2014-02-01

    While laboratory monitoring is not required in patients treated with apixaban, a direct factor-Xa inhibitor, assessment of its concentration is useful in some critical situations. However, few data are available on its effect on coagulation tests and on the suitability of anti-Xa assays for its quantification. It was the objective of this study to identify laboratory tests suitable for apixaban concentration assessment. Coagulation tests - PT and aPTT- and anti-Xa assays were performed in apixaban-spiked plasma samples. To evaluate the sensitivity of PT and aPTT to apixaban, we conducted a first monocenter part, with a wide range of concentrations (50-1,000 ng/ml), a large panel of reagents (20 reagents), and two coagulometers (STAR®, Stago and ACL TOP®, IL), and a second multicenter part involving 13 laboratories using either a common PT reagent (RecombiPlastin2G®) or the local PT and aPTT reagents. In the multicentre part, five blinded apixaban-spiked plasma samples (0/100/200/400/800 ng/ml - checked by HPLC-MS/MS) were used; apixaban concentrations were measured with three anti-Xa assays, apixaban calibrators and controls (Stago). PT and aPTT tests using a large panel of reagents displayed a low sensitivity to a wide range of apixaban concentrations. The concentrations to double PT ranged from 400 to >1,000 ng/ml with the 10 reagents. With the three anti-Xa assays, inter-laboratory precision and accuracy were below 11% and 12%, respectively. In conclusion, whereas PT and aPTT tests were not sensitive enough to detect apixaban, the three anti-Xa assays tested using lyophilised apixaban calibrators and controls allowed to reliably quantify a wide range of apixaban concentrations.

  18. An insight into the relationships between hepcidin, anemia, infections and inflammatory cytokines in pediatric refugees: a cross-sectional study.

    NARCIS (Netherlands)

    Cherian, S.; Forbes, D.A.; Cook, A.G.; Sanfilippo, F.M.; Kemna, E.H.J.M.; Swinkels, D.W.; Burgner, D.P.

    2008-01-01

    BACKGROUND: Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA) is unclear. We investigated the relationships between hepcidin, cytokines and iron sta

  19. Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young woman

    Science.gov (United States)

    Hepcidin is a key regulator of iron homeostasis, but to date no studies have examined the effect of hepcidin on iron absorption in humans. Our objective was to assess relations between both serum hepcidin and serum prohepcidin with nonheme-iron absorption in the presence and absence of food with the...

  20. Hepcidin: A Critical Regulator Of Iron Metabolism During Hypoxia

    Science.gov (United States)

    2011-01-01

    Critical Regulator of IronMetabolism during Hypoxia Korry J. Hintze1 and James P. McClung2 1Department of Nutrition , Dietetics & Food Sciences, Utah State...University, Logan, UT 84322, USA 2Military Nutrition Division, US Army Research Institute of Environmental Medicine (USARIEM), Natick, MA 01760, USA...through a series of cellular transport proteins that are sensitive to the recently discovered iron regulatory hormone, hepcidin. The discovery of

  1. Application of the Technicon Chem 1+ chemistry analyzer to the Syva Emit ethyl alcohol assay in plasma and urine.

    Science.gov (United States)

    Urry, F M; Kralik, M; Wozniak, E; Crockett, H; Jennison, T A

    1993-09-01

    The performance of the Technicon Chem 1+ chemistry analyzer with the Syva Emit ethyl alcohol assay in plasma and urine was evaluated. Spiked specimens from 0 to 600 mg/dL were tested, and expected versus measured concentrations were monitored. Linear regression line equations of y = 0.9314x + 5.4 and y = 0.9005x + 4.6, and correlation coefficients (r) of 0.9997 and 0.9995, were obtained for plasma and urine, respectively. A limit of detection of 5 mg/dL for plasma and urine, and a limit of quantitation of 20 mg/dL for plasma and 15 mg/dL for urine were obtained. Recovery was within 10% of expected concentration from 20 to 600 mg/dL. Precision was evaluated, giving the following coefficients of variation: within-run precision: plasma, 1.31-2.20; urine, 1.16-1.21; total precision: plasma, 2.72-3.38; urine, 2.98-4.64. No carry-over was detected when alternating 600 mg/dL and negative specimens. No interference from acetone, isopropanol, or methanol was detected. No significant differences in evaporation of alcohol at two concentrations, or from the two matrices were observed. Evaporation from a small cup (200 microL) was more than twice as great as from a large cup (2 mL). The Chem 1+ was compared to a gas chromatographic method. Plasma specimens of 0-352 mg/dL produced a linear regression line of y = 1.0112x + 6.0, r = 0.9859; urine specimens of 0-313 mg/dL produced a line of y = 1.0493x - 0.3, r = 0.9910. The capability to separate positive and negative specimens at 20% around a cutoff concentration of 20 mg/dL was examined. Four hundred specimens were analyzed, with only one specimen incorrectly classified (a false positive). The Chem 1+ chemistry analyzer demonstrated reliable performance of the Emit ethyl alcohol assay of plasma and urine specimens.

  2. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    Science.gov (United States)

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-07-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

  3. LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.

    Science.gov (United States)

    Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H

    2017-02-05

    Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib.

  4. Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera.

    Science.gov (United States)

    Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Levin, Carina; Koren, Ariel; Breuer, William; Rachmilewitz, Eliezer; Breda, Laura; Rivella, Stefano; Cabantchik, Z Ioav; Rechavi, Gideon

    2006-10-01

    Beta-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of beta-thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in beta-thalassaemia might suppress liver hepcidin expression. Sera from beta-thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from beta-thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by beta-thalassaemia major sera (r = 0.852, P < 0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from beta-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0.765, P < 0.0099). Our results suggest that, in beta-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

  5. A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

    DEFF Research Database (Denmark)

    Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune

    2015-01-01

    PURPOSE: To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony...... plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. RESULTS: The assay detected specific and saturable immunoglobulin G (IgG) binding to each...... cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients...

  6. In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2

    Science.gov (United States)

    Maes, Ken; Nemeth, Elizabeta; Roodman, G. David; Huston, Alissa; Esteve, Flavia; Freytes, Cesar; Callander, Natalie; Katodritou, Eirini; Tussing-Humphreys, Lisa; Rivera, Seth; Vanderkerken, Karin; Lichtenstein, Alan

    2010-01-01

    Hepcidin is the principal iron-regulatory hormone and a pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contributes to MM-related anemia. Searching for hepcidin-inducing cytokines in MM, we quantified the stimulation of hepcidin promoter-luciferase activity in HuH7 cells by MM sera. MM sera activated the hepcidin promoter significantly more than did normal sera. We then examined the role of bone morphogenetic proteins (BMPs) and interleukin-6 (IL-6), the major transcriptional regulators of hepcidin. Mutations in both BMP-responsive elements abrogated the activation dramatically, while mutations in the IL-6–responsive signal transducer and activator of transcription 3-binding site (STAT3-BS) had only a minor effect. Cotreatment with anti–BMP-2/4 or noggin-Fc blocked the promoter induction with all MM sera, anti–IL-6 blocked it with a minority of sera, whereas anti–BMP-4, -6, or -9 antibodies had no effect. BMP-2–immunodepleted MM sera had decreased promoter stimulatory capacity, and BMP-2 concentrations in MM sera were significantly higher than in normal sera. Our results demonstrate that BMP-2 is a major mediator of the hepcidin stimulatory activity of MM sera. PMID:20679527

  7. Non-Anticoagulant Heparins Are Hepcidin Antagonists for the Treatment of Anemia

    Directory of Open Access Journals (Sweden)

    Maura Poli

    2017-04-01

    Full Text Available The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs, which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.

  8. The dietary flavonoid myricetin regulates iron homeostasis by suppressing hepcidin expression.

    Science.gov (United States)

    Mu, Mingdao; An, Peng; Wu, Qian; Shen, Xiaoyun; Shao, Dandan; Wang, Hao; Zhang, Yingqi; Zhang, Shenshen; Yao, Hui; Min, Junxia; Wang, Fudi

    2016-04-01

    Hepcidin, a master regulator of iron homeostasis, is a promising target in treatment of iron disorders such as hemochromatosis, anemia of inflammation and iron-deficiency anemia. We previously reported that black soybean seed coat extract could inhibit hepcidin expression. Based on this finding, we performed a screen in cultured cells in order to identify the compounds in black soybeans that inhibit hepcidin expression. We found that the dietary flavonoid myricetin significantly inhibited the expression of hepcidin both in vitro and in vivo. Treating cultured cells with myricetin decreased both HAMP mRNA levels and promoter activity by reducing SMAD1/5/8 phosphorylation. This effect was observed even in the presence of bone morphogenic protein-6 (BMP6) and interleukin-6 (IL-6), two factors that stimulate hepcidin expression. Furthermore, mice that were treated with myricetin (either orally or systemically) had reduced hepatic hepcidin expression, decreased splenic iron levels and increased serum iron levels. Notably, myricetin-treated mice increased red blood cell counts and hemoglobin levels. In addition, pretreating mice with myricetin prevented LPS-induced hypoferremia. We conclude that myricetin potently inhibits hepcidin expression both in vitro and in vivo, and this effect is mediated by altering BMP/SMAD signaling. These experiments highlight the feasibility of identifying and characterizing bioactive phytochemicals to suppress hepcidin expression. These results also suggest that myricetin may represent a novel therapy for treating iron deficiency-related diseases.

  9. The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study

    NARCIS (Netherlands)

    Santen, S. van; Kroot, J.J.C.; Zijderveld, G.; Wiegerinck, E.T.G.; Spaanderman, M.E.A.; Swinkels, D.W.

    2013-01-01

    Abstract Background: Iron deficiency is a commonly encountered problem in pregnancy and a frequently observed cause of pregnancy-associated anemia. We longitudinally assessed the iron regulatory hormone hepcidin during gestation and postpartum and related hepcidin to conventional indicators of iron

  10. The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study

    NARCIS (Netherlands)

    Santen, S. van; Kroot, J.J.C.; Zijderveld, G.; Wiegerinck, E.T.G.; Spaanderman, M.E.A.; Swinkels, D.W.

    2013-01-01

    Abstract Background: Iron deficiency is a commonly encountered problem in pregnancy and a frequently observed cause of pregnancy-associated anemia. We longitudinally assessed the iron regulatory hormone hepcidin during gestation and postpartum and related hepcidin to conventional indicators of iron

  11. Expression of the iron hormone hepcidin distinguished different types of anemia in African children

    NARCIS (Netherlands)

    Pasricha, S.R.; Atkinson, S.H.; Armitage, A.E.; Khandwala, S.; Veenemans, J.; Cox, S.E.; Eddowes, L.A.; Hayes, T.; Doherty, C.P.; Demir, A.Y.; Tijhaar, E.J.; Verhoef, H.; Prentice, A.M.; Drakesmith, H.

    2014-01-01

    Childhood anemia is a major global health problem resulting from multiple causes. Iron supplementation addresses iron deficiency anemia but is undesirable for other types of anemia and may exacerbate infections. The peptide hormone hepcidin governs iron absorption; hepcidin transcription is mediated

  12. Serum hepcidin: reference ranges and biochemical correlates in the general population

    NARCIS (Netherlands)

    Galesloot, T.E.; Vermeulen, S.; Geurts-Moespot, A.; Klaver, S.M.; Kroot, J.J.C.; Tienoven, D. van; Wetzels, J.F.M.; Kiemeney, L.A.L.M.; Sweep, F.C.; Heijer, M. den; Swinkels, D.W.

    2011-01-01

    To date, concentrations of the promising biomarker hepcidin have only been assessed in serum of relatively small series of healthy volunteers and patients. We assessed age- and sex-stratified reference ranges of serum hepcidin concentration in a selected reference set and performed regression

  13. Maternal and Cord Blood Hepcidin Concentrations in Severe Iron Deficiency Anemia

    Directory of Open Access Journals (Sweden)

    Sriparna Basu

    2016-10-01

    Conclusion: Even in the presence of low serum iron and ferritin, maternal and cord blood hepcidin concentrations remained high in severe anemia. Failure of this proportional suppression of hepcidin indicates poor systemic bioavailability of iron to the mother and poor placental transport.

  14. Expression of the iron hormone hepcidin distinguished different types of anemia in African children

    NARCIS (Netherlands)

    Pasricha, S.R.; Atkinson, S.H.; Armitage, A.E.; Khandwala, S.; Veenemans, J.; Cox, S.E.; Eddowes, L.A.; Hayes, T.; Doherty, C.P.; Demir, A.Y.; Tijhaar, E.J.; Verhoef, H.; Prentice, A.M.; Drakesmith, H.

    2014-01-01

    Childhood anemia is a major global health problem resulting from multiple causes. Iron supplementation addresses iron deficiency anemia but is undesirable for other types of anemia and may exacerbate infections. The peptide hormone hepcidin governs iron absorption; hepcidin transcription is mediated

  15. Correlates of hepcidin and NTBI according to HFE status in patients referred to a liver centre

    NARCIS (Netherlands)

    Ryan, E.; Ryan, J.D.; Russell, J.; Coughlan, B.; Tjalsma, H.; Swinkels, D.W.; Stewart, S.; Crowe, J.P.

    2015-01-01

    BACKGROUND/AIMS: Innately low hepcidin levels lead to iron overload in HFE-associated hereditary haemochromatosis. METHODS: This study compared hepcidin and non-transferrin bound iron (NTBI) levels in untreated iron-loaded and non-iron-loaded C282Y homozygotes to levels in C282Y/H63D compound

  16. The effects of fat loss after bariatric surgery on inflammation, serum hepcidin, and iron absorption

    NARCIS (Netherlands)

    Cepeda-Lopez, Ana C.; Allende-Labastida, Javier; Melse-Boonstra, Alida; Osendarp, Saskia J.M.; Herter-Aeberli, Isabelle; Moretti, Diego; Rodriguez-Lastra, Ramiro; Gonzalez-Salazar, Francisco; Villalpando, Salvador; Zimmermann, Michael B.

    2016-01-01

    Background: Iron deficiency is common in obese subjects. This may be due to an increase in serum hepcidin and a decrease in iron absorption from adiposity-related inflammation. Objective: We evaluated whether weight and fat loss in obese subjects would decrease inflammation and serum hepcidin and

  17. Liver congestion in heart failure contributes to inappropriately increased serum hepcidin despite anemia.

    Science.gov (United States)

    Ohno, Yukako; Hanawa, Haruo; Jiao, Shuang; Hayashi, Yuka; Yoshida, Kaori; Suzuki, Tomoyasu; Kashimura, Takeshi; Obata, Hiroaki; Tanaka, Komei; Watanabe, Tohru; Minamino, Tohru

    2015-01-01

    Hepcidin is a key regulator of mammalian iron metabolism and mainly produced by the liver. Hepcidin excess causes iron deficiency and anemia by inhibiting iron absorption from the intestine and iron release from macrophage stores. Anemia is frequently complicated with heart failure. In heart failure patients, the most frequent histologic appearance of liver is congestion. However, it remains unclear whether liver congestion associated with heart failure influences hepcidin production, thereby contributing to anemia and functional iron deficiency. In this study, we investigated this relationship in clinical and basic studies. In clinical studies of consecutive heart failure patients (n = 320), anemia was a common comorbidity (41%). In heart failure patients without active infection and ongoing cancer (n = 30), log-serum hepcidin concentration of patients with liver congestion was higher than those without liver congestion (p = 0.0316). Moreover, in heart failure patients with liver congestion (n = 19), the anemia was associated with the higher serum hepcidin concentrations, which is a type of anemia characterized by induction of hepcidin. Subsequently, we produced a rat model of heart failure with liver congestion by injecting monocrotaline that causes pulmonary hypertension. The monocrotaline-treated rats displayed liver congestion with increase of hepcidin expression at 4 weeks after monocrotaline injection, followed by anemia and functional iron deficiency observed at 5 weeks. We conclude that liver congestion induces hepcidin production, which may result in anemia and functional iron deficiency in some patients with heart failure.

  18. Correlates of hepcidin and NTBI according to HFE status in patients referred to a liver centre

    NARCIS (Netherlands)

    Ryan, E.; Ryan, J.D.; Russell, J.; Coughlan, B.; Tjalsma, H.; Swinkels, D.W.; Stewart, S.; Crowe, J.P.

    2015-01-01

    BACKGROUND/AIMS: Innately low hepcidin levels lead to iron overload in HFE-associated hereditary haemochromatosis. METHODS: This study compared hepcidin and non-transferrin bound iron (NTBI) levels in untreated iron-loaded and non-iron-loaded C282Y homozygotes to levels in C282Y/H63D compound hetero

  19. Hepcidin: an important iron metabolism regulator in chronic kidney disease.

    Science.gov (United States)

    Antunes, Sandra Azevedo; Canziani, Maria Eugênia Fernandes

    2016-01-01

    Anemia is a common complication and its impact on morbimortality in patients with chronic kidney disease (CKD) is well known. The discovery of hepcidin and its functions has contributed to a better understanding of iron metabolism disorders in CKD anemia. Hepcidin is a peptide mainly produced by hepatocytes and, through a connection with ferroportin, it regulates iron absorption in the duodenum and its release of stock cells. High hepcidin concentrations described in patients with CKD, especially in more advanced stages are attributed to decreased renal excretion and increased production. The elevation of hepcidin has been associated with infection, inflammation, atherosclerosis, insulin resistance and oxidative stress. Some strategies were tested to reduce the effects of hepcidin in patients with CKD, however more studies are necessary to assess the impact of its modulation in the management of anemia in this population. Resumo Anemia é uma complicação frequente e seu impacto na morbimortalidade é bem conhecido em pacientes com doença renal crônica (DRC). A descoberta da hepcidina e de suas funções contribuíram para melhor compreensão dos distúrbios do metabolismo de ferro na anemia da DRC. Hepcidina é um peptídeo produzido principalmente pelos hepatócitos, e através de sua ligação com a ferroportina, regula a absorção de ferro no duodeno e sua liberação das células de estoque. Altas concentrações de hepcidina descritas em pacientes com DRC, principalmente em estádios mais avançados, são atribuídas à diminuição da excreção renal e ao aumento de sua produção. Elevação de hepcidina tem sido associada à ocorrência de infecção, inflamação, aterosclerose, resistência à insulina e estresse oxidativo. Algumas estratégias foram testadas para diminuir os efeitos da hepcidina em pacientes com DRC, entretanto, serão necessários mais estudos para avaliar o impacto de sua modulação no manejo da anemia nessa população.

  20. Enzymatic assay of total cholesterol in serum or plasma by amperometric measurement of rate of oxygen depletion following saponification.

    Science.gov (United States)

    Kumar, A; Christian, G D

    1977-01-17

    A method for serum or plasma cholesterol assay involving amperometric measurement of the rate of oxygen depletion in the cholesterol oxidase-catalyzed oxidation of cholesterol is described. The hydrolysis of the serum cholesterol esters is accomplished by saponification of 50 mul of sample with 0.2 ml of ethanolic KOH (1.0 mol/1) containing 0.5% Triton X-100 for 5 min at 75 degrees C. The rate of oxygen consumption in a 25-mul aliquot of this is measured with a Clark electrode in a Beckman Glucose Analyzer and the assay takes about one minute after incubation; results are read digitally on the instrument. The analyzer cell contains 1 ml of 1 M phosphate buffer, pH 7.4, with 100 mg sodium cholate/100 ml and 0.1-0.2 U cholesterol oxidase.

  1. A rapid and simple assay for lamotrigine in serum/plasma by HPLC, and comparison with an immunoassay.

    Science.gov (United States)

    Morgan, Phillip E; Fisher, Danielle S; Evers, Richard; Flanagan, Robert J

    2011-07-01

    Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average.

  2. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    Science.gov (United States)

    Miura, Taichi; Ando, Ayumi; Hirano, Kazumi; Ogura, Chika; Kanazawa, Tatsuya; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Hamaguchi, Satoshi

    2014-11-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells.

  3. Hepcidin levels are low during pregnancy and increase around delivery in women without iron deficiency - a prospective cohort study

    DEFF Research Database (Denmark)

    Hedengran, Katrine K; Nelson, Dick; Andersen, Malene R;

    2015-01-01

    OBJECTIVE: To investigate hepcidin during pregnancy, delivery and postpartum in women with sufficient iron supplementation. METHODS: Hepcidin was measured using LC-MS spectroscopy in 37 women during pregnancy, delivery and postpartum period in this longitudinal study. RESULTS: Hepcidin was low...... during pregnancy and increased at delivery and postpartum. No correlations with inflammatory markers or iron metabolism were observed during pregnancy; at delivery a correlation with inflammatory markers was observed. CONCLUSION: During pregnancy, in women with sufficient iron supplementation, hepcidin...... is low and does not reflect iron status. During delivery and the postpartum period, hepcidin functions as a marker of inflammation....

  4. Characterization of equine vitamin D–binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease

    DEFF Research Database (Denmark)

    Pihl, Tina Holberg; Jacobsen, Stine; Olsen, Dorthe T.

    2017-01-01

    spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed...

  5. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    Science.gov (United States)

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  6. Hepcidin Suppresses Brain Iron Accumulation by Downregulating Iron Transport Proteins in Iron-Overloaded Rats.

    Science.gov (United States)

    Du, Fang; Qian, Zhong-Ming; Luo, Qianqian; Yung, Wing-Ho; Ke, Ya

    2015-08-01

    Iron accumulates progressively in the brain with age, and iron-induced oxidative stress has been considered as one of the initial causes for Alzheimer's disease (AD) and Parkinson's disease (PD). Based on the role of hepcidin in peripheral organs and its expression in the brain, we hypothesized that this peptide has a role to reduce iron in the brain and hence has the potential to prevent or delay brain iron accumulation in iron-associated neurodegenerative disorders. Here, we investigated the effects of hepcidin expression adenovirus (ad-hepcidin) and hepcidin peptide on brain iron contents, iron transport across the brain-blood barrier, iron uptake and release, and also the expression of transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin 1 (Fpn1) in cultured microvascular endothelial cells and neurons. We demonstrated that hepcidin significantly reduced brain iron in iron-overloaded rats and suppressed transport of transferrin-bound iron (Tf-Fe) from the periphery into the brain. Also, the peptide significantly inhibited expression of TfR1, DMT1, and Fpn1 as well as reduced Tf-Fe and non-transferrin-bound iron uptake and iron release in cultured microvascular endothelial cells and neurons, while downregulation of hepcidin with hepcidin siRNA retrovirus generated opposite results. We concluded that, under iron-overload, hepcidin functions to reduce iron in the brain by downregulating iron transport proteins. Upregulation of brain hepcidin by ad-hepcidin emerges as a new pharmacological treatment and prevention for iron-associated neurodegenerative disorders.

  7. Are serum hepcidin levels chronically elevated in collegiate female distance runners?

    Science.gov (United States)

    Ma, Xiaoya; Patterson, Kaitlyn J; Gieschen, Kayla M; Bodary, Peter F

    2013-10-01

    The prevalence of iron deficiency tends to be higher in athletic populations, especially among endurance-trained females. Recent studies have provided evidence that the iron-regulating hormone hepcidin is transiently increased with acute exercise and suggest that this may contribute to iron deficiency anemia in athletes. The purpose of this study was to determine whether resting serum hepcidin is significantly elevated in highly trained female distance runners compared with a low exercise control group. Due to the importance of the monocyte in the process of iron recycling, monocyte expression of hepcidin was also measured. A single fasted blood sample was collected midseason from twenty female distance runners averaging 81.9 ± 14.2 km of running per week. Ten age-, gender-, and BMI-matched low-exercise control subjects provided samples during the same period using identical collection procedures. There was no difference between the runners (RUN) and control subjects (CON) for serum hepcidin levels (p = .159). In addition, monocyte hepcidin gene expression was not different between the two groups (p = .635). Furthermore, no relationship between weekly training volume and serum hepcidin concentration was evident among the trained runners. The results suggest that hepcidin is not chronically elevated with sustained training in competitive collegiate runners. This is an important finding because the current clinical conditions that link hepcidin to anemia include a sustained elevation in serum hepcidin. Nevertheless, additional studies are needed to determine the clinical relevance of the well-documented, transient rise in hepcidin that follows acute sessions of exercise.

  8. Depleted iron stores and iron deficiency anemia associated with reduced ferritin and hepcidin and elevated soluble transferrin receptors in a multiethnic group of preschool-age children.

    Science.gov (United States)

    Weiler, Hope A; Jean-Philippe, Sonia; Cohen, Tamara R; Vanstone, Catherine A; Agellon, Sherry

    2015-09-01

    Iron deficiency anemia is prevalent in subgroups of the Canadian population. The objective of this study was to examine iron status and anemia in preschool-age children. Healthy children (n = 430, 2-5 years old, Montreal, Quebec, Canada) were sampled from randomly selected daycares. Anthropometry, demographics, and diet were assessed. Biochemistry included hemoglobin, ferritin, soluble transferrin receptors (sTfR), ferritin index, markers of inflammation (C-reactive protein, interleukin 6 (IL-6), and tumour necrosis factor alpha (TNFα)), and hepcidin. Iron deficiency and anemia cutoffs conformed to the World Health Organization criteria. Differences among categories were tested using mixed-model ANOVA or χ(2) tests. Children were 3.8 ± 1.0 years of age, with a body mass index z score of 0.48 ± 0.97, and 51% were white. Adjusted intakes of iron indicated deficiency. Hemoglobin was higher in white children, whereas ferritin was higher with greater age and female sex. Inflammatory markers and hepcidin did not vary with any demographic variable. The prevalence of iron deficiency was 16.5% (95% confidence interval (CI), 13.0-20.0). Three percent (95% CI, 1.4-4.6) of children had iron deficiency anemia and 12.8% (95% CI, 9.6-16.0) had unexplained anemia. Children with iron deficiency, with and without anemia, had lower plasma ferritin and hepcidin but higher sTfR, ferritin index, and IL-6, whereas those with unexplained anemia had elevated TNFα. We conclude that iron deficiency anemia is not very common in young children in Montreal. While iron deficiency without anemia is more common than iron deficiency with anemia, the correspondingly reduced circulating hepcidin would have enabled heightened absorption of dietary iron in support of erythropoiesis.

  9. The iron-regulatory peptide hepcidin is upregulated in the ischemic and in the remote myocardium after myocardial infarction.

    Science.gov (United States)

    Simonis, Gregor; Mueller, Katrin; Schwarz, Peggy; Wiedemann, Stephan; Adler, Guido; Strasser, Ruth H; Kulaksiz, Hasan

    2010-09-01

    Recent evidence suggests that iron metabolism contributes to the ischemic damage after myocardial infarction. Hepcidin, a recently discovered peptide hormone, regulates iron uptake and metabolism, protecting the body from iron overload. In this study we analyzed the regulation of hepcidin in the heart and blood of rats after myocardial infarction. To induce a myocardial infarction in the rats, left anterior descending coronary artery ligation was performed. After 1-24h, biopsies from the ischemic and the non-ischemic myocardium were taken. In these biopsies, the mRNA levels and the protein expression of hepcidin were analyzed by quantitative RT-PCR and immunoblot analysis, respectively. In parallel, the serum levels of prohepcidin were measured by ELISA. Six hours after myocardial infarction, the hepcidin mRNA expression was temporally upregulated in the ischemic and in the non-ischemic myocardium. The upregulation was specific for hepcidin, since other iron-related genes (hemojuvelin, IREG-1) remained unchanged. Furthermore, the alteration of the hepcidin protein expression in the ischemic area was connected to the level of hepcidin in the serum of the infarcted rats, where hepcidin also raised up. Angiotensin receptor blockade with candesartan did not influence the mRNA regulation of hepcidin. Together, these data show a particular upregulation of the iron-regulatory peptide hepcidin in the ischemic and the non-ischemic myocardium after myocardial infarction. It is speculated that upregulation of hepcidin may reduce iron toxicity and thus infarct size expansion in an infarcted heart.

  10. High Performance Liquid Chromatography Tandem Mass Spectrometry Assay for the Determination of Cobinamide in Pig Plasma

    Science.gov (United States)

    McCracken, Brent A.; Brittain, Matthew K.

    2015-01-01

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely utilized for the analysis of compounds in biological matrices due to its selectivity and sensitivity. This study describes the application of an LC-MS/MS-based approach toward the analysis of cobinamide in Yorkshire pig plasma. The selectivity, accuracy, precision, recovery, linearity, range, carryover, sensitivity, matrix effect, interference, stability, reproducibility, and ruggedness of the method were investigated in pig plasma. The accuracy and precision of the method was determined to be within 10% over three different days over a range of concentrations (25–10,000 ng/mL) that spanned more than two orders of magnitude. The lower limit of quantitation (LLOQ) for dicyanocobinamide was determined to be 25 ng/mL in pig plasma. Carryover was acceptable, as the area response of the carryover blanks were ≤15% of the area response of the nearest LLOQ standard for the analyte, while it was nonexistent for the internal standard. Specificity was ensured using six different lots of pig plasma. While the matrix effects of dicyanocobinamide in plasma were enhanced, ginsenoside Rb1 experienced signal suppression under the described conditions. The absolute recovery results for both compounds were consistent, precise, and reproducibly lower than expected at ~60% for dicyanocobinamide and ~22% for ginsenoside Rb1, confirming that a matrix standard curve was required for accurate quantitation. Cobinamide was shown to be very stable in matrix at various storage conditions including room temperature, refrigerated, and frozen at time intervals of 20 hours, 4 days, and 60 days respectively. This method was demonstrated to be sensitive, reproducible, stable, and rugged, and it should be applicable to the analysis of cobinamide in other biological matrices and species. PMID:26540437

  11. A hepcidina como parâmetro bioquímico na avaliação da anemia por deficiência de ferro Hepcidin as a biochemical parameter for the assessment of iron deficiency anemia

    Directory of Open Access Journals (Sweden)

    Andrea dos Reis Lemos

    2010-01-01

    Full Text Available A anemia por deficiência de ferro caracteriza-se como o mais prevalente problema nutricional em todo o mundo. Nesta revisão reuniu-se informações a respeito do metabolismo da hepcidina, avaliando-se seu valor como parâmetro bioquímico na anemia por deficiência de ferro. Realizou-se um levantamento bibliográfico nas bases de dados PUBMED e LILACS, período 2006-2010, referentes à hepcidina como um biomarcador para a regulação do metabolismo do ferro. Foram localizados 35 estudos publicados em revistas internacionais e um estudo sobre o assunto em revista nacional. A produção de hepcidina é regulada homeostaticamente pela anemia e hipóxia. Quando a oferta de oxigênio está inadequada ocorre diminuição do nível de hepcidina. Consequentemente, maior quantidade de ferro proveniente da dieta e dos estoques dos macrófagos e hepatócitos se tornam disponíveis. A hepcidina possui a função de se ligar à ferroportina, regulando a liberação do ferro para o plasma. Quando as concentrações de hepcidina estão baixas, as moléculas de ferroportina são expostas na membrana plasmática e liberam o ferro. Quando os níveis de hepcidina aumentam, a hepcidina liga-se às moléculas de ferroportina induzindo sua internalização e degradação, e o ferro liberado diminui progressivamente. Aparentemente o desenvolvimento do diagnóstico e terapia da anemia baseados no bioindicador hepcidina pode oferecer uma abordagem mais efetiva. Estudos epidemiológicos são necessários para comprovar o valor da hepcidina no diagnóstico diferencial das anemias, incluindo protocolos de amostragem para análise, com padronização similar às utilizadas em outras avaliações bioquímicas, e estabelecimento de pontos de corte para a expressão urinária e plasmática desse peptídeo.Iron deficiency anemia is the most prevalent nutritional problem in the world. Information on the metabolism of hepcidin and its possible significance as a biochemical

  12. LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

    Directory of Open Access Journals (Sweden)

    Dinesh S. Patel

    2012-10-01

    Full Text Available This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS. The analyte and quetiapine as internal standard (IS were extracted from 200 μL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm×4.6 mm, 5 μm under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation and relative recovery were 90%, respectively. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range −7.43 to 8.07%.

  13. Measurement of intact insulin-like growth factor-binding protein-3 in human plasma using a ligand immunofunctional assay.

    Science.gov (United States)

    Lassarre, C; Binoux, M

    2001-03-01

    Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with (125)I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1-160 and 1-95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind (125)I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of (125)I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable. The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 microg/mL. The dynamic range of the LIFA was 0.50-3.75 microL equivalent of the plasma pool in a total volume of 300 microL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively. In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 +/- 0.08 (SEM) mg/L, and that of

  14. Radioreceptor assay to study the affinity of benzodiazepines and their receptor binding activity in human plasma including their active metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Dorow, R.G.; Seidler, J.; Schneider, H.H. (Schering A.G., Berlin (Germany, F.R.))

    1982-04-01

    A radioreceptor assay has been established to measure the receptor affinities of numerous benzodiazepines in clinical use. The time course of receptor binding activity was studied by this method in the plasma of eight healthy subjects randomly treated with 1mg lormetazepam (Noctamid(R)), 2mg flunitrazepam (Rohypnol(R)), and 10mg diazepam (Valium(R)), and placebo on a cross-over basis. Blood samples were collected up to 154h after treatment. Receptor affinities of numerous benzodiazepines in vitro show good correlation with therapeutic human doses (r=0.96) and may be predictive of drug potency in man. Mean peak plasma levels of lormetazepam binding equivalents were 4.8+-1 ng/ml at 2h after lormetazepam, 7.2+-1.8 ng/ml at 8h after flunitrazepam, and 17.9+-2.7 ng/ml at 15h after diazepam. Plasma elimination halflives of benzodiazepine binding equivalents were 9.3, 23 and 63h, respectively. Slow elimination of benzodiazepine binding equivalents following flunitrazepam and diazepam may be due to persistent active metabolites.

  15. Malaria and Age Variably but Critically Control Hepcidin Throughout Childhood in Kenya.

    Science.gov (United States)

    Atkinson, Sarah H; Uyoga, Sophie M; Armitage, Andrew E; Khandwala, Shivani; Mugyenyi, Cleopatra K; Bejon, Philip; Marsh, Kevin; Beeson, James G; Prentice, Andrew M; Drakesmith, Hal; Williams, Thomas N

    2015-10-01

    Both iron deficiency (ID) and malaria are common among African children. Studies show that the iron-regulatory hormone hepcidin is induced by malaria, but few studies have investigated this relationship longitudinally. We measured hepcidin concentrations, markers of iron status, and antibodies to malaria antigens during two cross-sectional surveys within a cohort of 324 Kenyan children ≤ 8 years old who were under intensive surveillance for malaria and other febrile illnesses. Hepcidin concentrations were the highest in the youngest, and female infants, declined rapidly in infancy and more gradually thereafter. Asymptomatic malaria and malaria antibody titres were positively associated with hepcidin concentrations. Recent episodes of febrile malaria were associated with high hepcidin concentrations that fell over time. Hepcidin concentrations were not associated with the subsequent risk of either malaria or other febrile illnesses. Given that iron absorption is impaired by hepcidin, our data suggest that asymptomatic and febrile malaria contribute to the high burden of ID seen in African children. Further, the effectiveness of iron supplementation may be sub-optimal in the presence of asymptomatic malaria. Thus, strategies to prevent and eliminate malaria may have the added benefit of addressing an important cause of ID for African children.

  16. Analysis of hepcidin expression: in situ hybridization and quantitative polymerase chain reaction from paraffin sections.

    Science.gov (United States)

    Sakuraoka, Yuhki; Sawada, Tokihiko; Shiraki, Takayuki; Park, Kyunghwa; Sakurai, Yuhichiro; Tomosugi, Naohisa; Kubota, Keiichi

    2012-07-28

    To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

  17. Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

    Institute of Scientific and Technical Information of China (English)

    Yuhki Sakuraoka; Tokihiko Sawada; Takayuki Shiraki; Kyunghwa Park; Yuhichiro Sakurai; Naohisa Tomosugi; Keiichi Kubota

    2012-01-01

    AIM:TO establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC).METHODS:Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC.Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years.Quantitative PCR was performed.Immunohistochemistry and in situ hybridization for hepcidin were also performed.RESULTS:Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully.The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues.A method of in situ hybridization for hepcidin was established successfully,and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue.CONCLUSION:We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

  18. Phytoestrogens modulate hepcidin expression by Nrf2: Implications for dietary control of iron absorption.

    Science.gov (United States)

    Bayele, Henry K; Balesaria, Sara; Srai, Surjit K S

    2015-12-01

    Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis.

  19. Erythroferrone contributes to hepcidin repression in a mouse model of malarial anemia

    Science.gov (United States)

    Latour, Chloé; Wlodarczyk, Myriam F.; Jung, Grace; Gineste, Aurélie; Blanchard, Nicolas; Ganz, Tomas; Roth, Marie-Paule; Coppin, Hélène; Kautz, Léon

    2017-01-01

    Malaria, a major global health challenge worldwide, is accompanied by a severe anemia secondary to hemolysis and increased erythrophagocytosis. Iron is an essential functional component of erythrocyte hemoglobin and its availability is controlled by the liver-derived hormone hepcidin. We examined the regulation of hepcidin during malarial infection in mice using the rodent parasite Plasmodium berghei K173. Mice infected with Plasmodium berghei K173 develop a severe anemia and die after 18 to 22 days without cerebral malaria. During the early phase of blood-stage infection (days 1 to 5), a strong inflammatory signature was associated with an increased production of hepcidin. Between days 7 and 18, while infection progressed, red blood cell count, hemoglobin and hematocrit dramatically decreased. In the late phase of malarial infection, hepcidin production was reduced concomitantly to an increase in the messenger RNA expression of the hepcidin suppressor erythroferrone in the bone marrow and the spleen. Compared with wild-type mice, Erfe−/− mice failed to adequately suppress hepcidin expression after infection with Plasmodium berghei K173. Importantly, the sustained production of hepcidin allowed by erythroferrone ablation was associated with decreased parasitemia, providing further evidence that transient iron restriction could be beneficial in the treatment of malaria. PMID:27658439

  20. Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.

    Science.gov (United States)

    Sadr, Vahideh; Saffar, Behnaz; Emamzadeh, Rahman

    2017-04-30

    Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl(-)(1) of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150μgml(-1), 18.7μg/ml(-1) and 37.5μg/ml(-1), respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.

  1. Decreased serum hepcidin concentration correlates with brain iron deposition in patients with HBV-related cirrhosis.

    Directory of Open Access Journals (Sweden)

    Dong Lin

    Full Text Available PURPOSE: Excessive brain iron accumulation contributes to cognitive impairments in hepatitis B virus (HBV-related cirrhotic patients. The underlying mechanism remains unclear. Hepcidin, a liver-produced, 25-aminoacid peptide, is the major regulator of systemic iron metabolism. Abnormal hepcidin level is a key factor in some body iron accumulation or deficiency disorders, especially in those associated with liver diseases. Our study was aimed to explore the relationship between brain iron content in patients with HBV-related cirrhosis and serum hepcidin level. METHODS: Seventy HBV-related cirrhotic patients and forty age- sex-matched healthy controls were enrolled. Brain iron content was quantified by susceptibility weighted phase imaging technique. Serum hepcidin as well as serum iron, serum transferrin, ferritin, soluble transferrin receptor, total iron binding capacity, and transferrin saturation were tested in thirty cirrhotic patients and nineteen healthy controls. Pearson correlation analysis was performed to investigate correlation between brain iron concentrations and serum hepcidin, or other iron parameters. RESULTS: Cirrhotic patients had increased brain iron accumulation compared to controls in the left red nuclear, the bilateral substantia nigra, the bilateral thalamus, the right caudate, and the right putamen. Cirrhotic patients had significantly decreased serum hepcidin concentration, as well as lower serum transferring level, lower total iron binding capacity and higher transferrin saturation, compared to controls. Serum hepcidin level negatively correlated with the iron content in the right caudate, while serum ferritin level positively correlated with the iron content in the bilateral putamen in cirrhotic patients. CONCLUSIONS: Decreased serum hepcidin level correlated with excessive iron accumulation in the basal ganglia in HBV-related cirrhotic patients. Our results indicated that systemic iron overload underlined regional

  2. Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis.

    Science.gov (United States)

    Masaratana, Patarabutr; Latunde-Dada, Gladys O; Patel, Neeta; Simpson, Robert J; Vaulont, Sophie; McKie, Andrew T

    2012-08-15

    Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in

  3. Failure of corn trypsin inhibitor to affect the thrombin generation assay in plasma from severe hemophiliacs.

    Science.gov (United States)

    Mohammed, B M; Martin, E J; Salinas, V; Carmona, R; Young, G; Brophy, D F

    2014-09-01

    The thrombin generation assay (TGA) is an important global coagulation assay; however, it suffers from the lack of preanalytical standardization. The addition of corn trypsin inhibitor (CTI) to blood collection tubes before TGA has been previously advocated to block the contact activation pathway. Emerging data, however, suggest that CTI may only be necessary when minimal tissue factor (TF) concentrations < 1 pmol are used. To determine whether blood collection tubes containing CTI influenced TGA parameters. This cross-sectional, observational study performed the TGA using TF 1 pmol L(-1) in 15 healthy volunteers, 14 severely factor VIII (FVIII)-deficient patients, and 15 severely FVIII-deficient patients with documented FVIII inhibitors. TGA was conducted using blood tubes that contained CTI 33 μg mL(-1) and no CTI. CTI markedly reduced peak thrombin (P = 0.002) and endogenous thrombin potential (P < 0.001) in the healthy volunteers but had no significant effect on TGA parameters in severely FVIII-deficient patients or those with inhibitors. This lack of effect raises additional questions regarding the need for CTI as a preanalytical addition to blood collection tubes during TGA in severe hemophiliacs, particularly when activating samples with TF 1 pmol L(-1) . © 2014 International Society on Thrombosis and Haemostasis.

  4. Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients[S

    Science.gov (United States)

    Liu, Wei; Xu, Libin; Lamberson, Connor R.; Merkens, Louise S.; Steiner, Robert D.; Elias, Ellen R.; Haas, Dorothea; Porter, Ned A.

    2013-01-01

    Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3β-hydroxysterol-Δ7-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d7-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials. PMID:23072947

  5. Comparative biochemical studies of fresh frozen plasma and pooled solvent/detergent-treated plasma (octaplasLG(®) ) with focus on protein S and its impact in different thrombin generation assay set-ups.

    Science.gov (United States)

    Heger, A; Janisch, S; Pock, K; Römisch, J

    2016-10-01

    The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG(®) , in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Sixteen octaplasLG(®) batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Mean protein S levels were lower in octaplasLG(®) , but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found. © 2016 International Society of Blood Transfusion.

  6. Hepcidin levels in hereditary hyperferritinemia:Insights into the iron-sensing mechanism in hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Jayantha; Arnold; Arvind; Sangwaiya; Vijay; Manglam; Mark; Thursz; Caroline; Beaumont; Caroline; Kannengiesser; Mark; Busbridge

    2010-01-01

    AIM:To study the role of hepcidin in hereditary hyperferritinemia cataract syndrome(HHCS). METHODS:Six patients from two families with HHCS, confirmed by genetic analysis showing A to G mutation at position+40 in the L-ferritin gene,were recruited to undergo serum hepcidin and prohepcidin measurements using radioimmunoassay and enzyme linked immunoassay,respectively,and measurements were compared with levels in serum from 25 healthy volunteers(14 females),mean age 36±11.9 years.RESULTS:The serum hepcidin an...

  7. Hepcidin-25 in chronic hemodialysis patients is related to residual kidney function and not to treatment with erythropoiesis stimulating agents.

    Directory of Open Access Journals (Sweden)

    Neelke C van der Weerd

    Full Text Available Hepcidin-25, the bioactive form of hepcidin, is a key regulator of iron homeostasis as it induces internalization and degradation of ferroportin, a cellular iron exporter on enterocytes, macrophages and hepatocytes. Hepcidin levels are increased in chronic hemodialysis (HD patients, but as of yet, limited information on factors associated with hepcidin-25 in these patients is available. In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients. Baseline data from 405 patients (62% male; age 63.7 ± 13.9 [mean SD] enrolled in the CONvective TRAnsport STudy (CONTRAST; NCT00205556 were studied. Predialysis hepcidin concentrations were measured centrally with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patient-, laboratory- and treatment related characteristics were entered in a backward multivariable linear regression model. Hepcidin-25 levels were independently and positively associated with ferritin (p<0.001, hsCRP (p<0.001 and the presence of diabetes (p = 0.02 and inversely with the estimated glomerular filtration rate (p = 0.01, absolute reticulocyte count (p = 0.02 and soluble transferrin receptor (p<0.001. Men had lower hepcidin-25 levels as compared to women (p = 0.03. Hepcidin-25 was not associated with the maintenance dose of erythropoiesis stimulating agents (ESA or iron therapy. In conclusion, in the currently studied cohort of chronic HD patients, hepcidin-25 was a marker for iron stores and erythropoiesis and was associated with inflammation. Furthermore, hepcidin-25 levels were influenced by residual kidney function. Hepcidin-25 did not reflect ESA or iron dose in chronic stable HD patients on maintenance therapy. These results suggest that hepcidin is involved in the pathophysiological pathway of renal anemia and iron availability in these patients, but

  8. 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.

  9. High-sensitivity simultaneous liquid chromatography–tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma

    Directory of Open Access Journals (Sweden)

    Abhishek Gandhi

    2015-10-01

    Full Text Available A sensitive and simultaneous liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of ethinyl estradiol and levonorgestrel. The analytes were extracted with methyl-tert-butyl ether: n-hexane (50:50, v/v solvent mixture, followed by dansyl derivatization. The chromatographic separation was performed on a Kinetex C18 (50 mm×4.6 mm, 2.6 µm column with a mobile phase of 0.1% (v/v formic acid in water and acetonitrile in gradient composition. The mass transitions were monitored in electrospray positive ionization mode. The assay exhibited a linear range of 0.100–20.0 ng/mL for levonorgestrel and 4.00–500 pg/mL for ethinyl estradiol in human plasma. A run time of 9.0 min for each sample made it possible to analyze a throughput of more than 100 samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic and bioequivalence studies.

  10. Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva.

    Science.gov (United States)

    Mazzucchelli, Iolanda; Rapetti, Manuela; Fattore, Cinzia; Franco, Valentina; Gatti, Giuliana; Perucca, Emilio

    2011-08-01

    The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min(-1), with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r(2) = 0.998 ± 0.002 for plasma (n = 10) and r(2) = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25-20.0 μg ml(-1), with a limit of quantification at 0.25 μg ml(-1). Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.

  11. High-sensitivity simultaneous liquid chromatography-tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma

    Institute of Scientific and Technical Information of China (English)

    Abhishek Gandhi; Swati Guttikar; Priti Trivedi

    2015-01-01

    A sensitive and simultaneous liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of ethinyl estradiol and levonorgestrel. The analytes were extracted with methyl-tert-butyl ether: n-hexane (50:50, v/v) solvent mixture, followed by dansyl derivatization. The chromatographic separation was performed on a Kinetex C18 (50 mm × 4.6 mm, 2.6μm) column with a mobile phase of 0.1% (v/v) formic acid in water and acetonitrile in gradient composition. The mass transitions were monitored in electrospray positive ionization mode. The assay exhibited a linear range of 0.100-20.0 ng/mL for levonorgestrel and 4.00-500 pg/mL for ethinyl estradiol in human plasma. A run time of 9.0 min for each sample made it possible to analyze a throughput of more than 100 samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic and bioequivalence studies.

  12. Clinical indications for plasma protein assays: transthyretin (prealbumin) in inflammation and malnutrition.

    Science.gov (United States)

    Myron Johnson, A; Merlini, Giampaolo; Sheldon, Joanna; Ichihara, Kiyoshi

    2007-01-01

    A large number of circumstances are associated with reduced serum concentrations of transthyretin (TTR), or prealbumin. The most common of these is the acute phase response, which may be due to inflammation, malignancy, trauma, or many other disorders. Some studies have shown a decrease in hospital stay with nutritional therapy based on TTR concentrations, but many recent studies have shown that concentrations of albumin, transferrin, and transthyretin correlate with severity of the underlying disease rather than with anthropometric indicators of hypo- or malnutrition. There are few if any conditions in which the concentration of this protein by itself is more helpful in diagnosis, prognosis, or follow up than are other clinical findings. In the majority of cases, the serum concentration of C-reactive protein is adequate for detection and monitoring of acute phase responses and for prognosis. Although over diagnosis and treatment of presumed protein energy malnutrition is probably not detrimental to most patients, the failure to detect other causes of decreased concentrations (such as serious bacterial infections or malignancy) of the so-called visceral or hepatic proteins could possibly result in increased morbidity or even mortality. In addition to these caveats, assays for TTR have a relatively high level of uncertainty ("imprecision"). Clinical evaluation--history and physical examination--should remain the mainstay of nutritional assessment.

  13. Development and evaluation of an affordable real-time qualitative assay for determining HIV-1 virological failure in plasma and dried blood spots.

    Science.gov (United States)

    Aitken, Susan C; Kliphuis, Aletta; Bronze, Michelle; Wallis, Carole L; Kityo, Cissy; Balinda, Sheila; Stevens, Wendy; Spieker, Nicole; de Oliveira, Tulio; Rinke de Wit, Tobias F; Schuurman, Rob

    2013-06-01

    Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for "open platform" applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.

  14. Hepcidin is an antibacterial, stress-inducible peptide of the biliary system.

    Directory of Open Access Journals (Sweden)

    Pavel Strnad

    Full Text Available BACKGROUND/AIMS: Hepcidin (gene name HAMP, an IL-6-inducible acute phase peptide with antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of HAMP synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. While the role of hepcidin in biliary system is unknown, a recent study demonstrated that conditional gp130-knockout mice display diminished hepcidin levels and increased rate of biliary infections. METHODS: Expression and localization of HAMP in biliary system was analyzed by real time RT-PCR, in-situ hybridization, immunostaining and -blotting, while prohepcidin levels in human bile were determined by ELISA. RESULTS: Hepcidin was detected in mouse/human gallbladder and bile duct epithelia. Biliary HAMP is stress-inducible, in that it is increased in biliary cell lines upon IL-6 stimulation and in gallbladder mucosa of patients with acute cholecystitis. Hepcidin is also present in the bile and elevated prohepcidin levels were observed in bile of primary sclerosing cholangitis (PSC patients with concurrent bacterial cholangitis compared to PSC subjects without bacterial infection (median values 22.3 vs. 8.9; p = 0.03. In PSC-cholangitis subjects, bile prohepcidin levels positively correlated with C-reactive protein and bilirubin levels (r = 0.48 and r = 0.71, respectively. In vitro, hepcidin enhanced the antimicrobial capacity of human bile (p<0.05. CONCLUSION: Hepcidin is a stress-inducible peptide of the biliary epithelia and a potential marker of biliary stress. In the bile, hepcidin may serve local functions such as protection from bacterial infections.

  15. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

    2016-08-05

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  16. Joint Model of Iron and Hepcidin During the Menstrual Cycle in Healthy Women.

    Science.gov (United States)

    Angeli, Adeline; Lainé, Fabrice; Lavenu, Audrey; Ropert, Martine; Lacut, Karine; Gissot, Valérie; Sacher-Huvelin, Sylvie; Jezequel, Caroline; Moignet, Aline; Laviolle, Bruno; Comets, Emmanuelle

    2016-03-01

    Hepcidin regulates serum iron levels, and its dosage is used in differential diagnostic of iron-related pathologies. We used the data collected in the HEPMEN (named after HEPcidin during MENses) study to investigate the joint dynamics of serum hepcidin and iron during the menstrual cycle in healthy women. Ninety menstruating women were recruited after a screening visit. Six fasting blood samples for determination of iron-status variables were taken in the morning throughout the cycle, starting on the second day of the period. Non-linear mixed effect models were used to describe the evolution of iron and hepcidin. Demographic and medical covariates were tested for their effect on model parameters. Parameter estimation was performed using the SAEM algorithm implemented in the Monolix software. A general pattern was observed for both hepcidin and iron, consisting of an initial decrease during menstruation, followed by a rebound and stabilising during the second half of the cycle. We developed a joint model including a menstruation-induced decrease of both molecules at the beginning of the menses and a rebound effect after menses. Iron stimulated the release of hepcidin. Several covariates, including contraception, amount of blood loss and ferritin, were found to influence the parameters. The joint model of iron and hepcidin was able to describe the fluctuations induced by blood loss from menstruation in healthy non-menopausal women and the subsequent regulation. The HEPMEN study showed fluctuations of iron-status variables during the menstrual cycle, which should be considered when using hepcidin measurements for diagnostic purposes in women of child-bearing potential.

  17. Increased serum hepcidin levels in subjects with the metabolic syndrome: a population study.

    Directory of Open Access Journals (Sweden)

    Nicola Martinelli

    Full Text Available The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS. Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia--DHF, and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome--DIOS. However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.

  18. Detection of cord blood hepcidin levels as a biomarker for early-onset neonatal sepsis.

    Science.gov (United States)

    Cizmeci, Mehmet Nevzat; Kara, Semra; Kanburoglu, Mehmet Kenan; Simavli, Serap; Duvan, Candan Iltemur; Tatli, Mustafa Mansur

    2014-03-01

    Early-onset neonatal sepsis (EONS) continues to be a severe condition associated with a high mortality and morbidity. However, symptoms and laboratory markers of this serious condition are nonspecific and currently there are no available standard tests to provide perfect diagnostic accuracy. An early recognition and initiation of antimicrobial therapy are essential in order to prevent morbidity and mortality. Hepcidin, the key regulator of iron homeostasis, is also an acute-phase reactant, which has a critical role in inflammation and contributes to host defense by interfering with microorganism's access to iron. Since hepcidin expression is induced by interleukin-6 (IL-6), it also plays role in the innate immune system. Recently, endogenous expression of hepcidin by macrophages and neutrophils in response to bacterial pathogens confirmed its role in innate immunity. The clear link between the hepcidin molecule and innate immunity may be used for the detection of EONS. We hypothesized that an increased level of hepcidin in cord blood may be used as a reliable biological marker of EONS and designed a prospective cohort study to test this hypothesis and collected pilot data. Cord blood samples of all infants born between January 2009 and December 2010 at our university hospital were collected after parental consent and a total of 38 infants were enrolled in the study who fulfilled the sepsis criteria. The range of cord blood hepcidin was found to be significantly increased in newborns with EONS (min-max: 118.1-8400 ng/mL). To the best of our knowledge, this is the first study to investigate the pathophysiologic relevance of hepcidin in EONS and demonstrate increased levels of hepcidin in cord blood as an acute-phase reactant in response to sepsis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Hepcidin detects iron deficiency in Sri Lankan adolescents with a high burden of hemoglobinopathy: A diagnostic test accuracy study

    Science.gov (United States)

    Wray, Katherine; Allen, Angela; Evans, Emma; Fisher, Chris; Premawardhena, Anuja; Perera, Lakshman; Rodrigo, Rexan; Goonathilaka, Gayan; Ramees, Lebbe; Webster, Craig; Armitage, Andrew E; Prentice, Andrew M

    2017-01-01

    Abstract Anemia affects over 800 million women and children globally. Measurement of hepcidin as an index of iron status shows promise, but its diagnostic performance where hemoglobinopathies are prevalent is unclear. We evaluated the performance of hepcidin as a diagnostic test of iron deficiency in adolescents across Sri Lanka. We selected 2273 samples from a nationally representative cross‐sectional study of 7526 secondary schoolchildren across Sri Lanka and analyzed associations between hepcidin and participant characteristics, iron indices, inflammatory markers, and hemoglobinopathy states. We evaluated the diagnostic accuracy of hepcidin as a test for iron deficiency with estimation of the AUCROC, sensitivity/specificity at each hepcidin cutoff, and calculation of the Youden Index to find the optimal threshold. Hepcidin was associated with ferritin, sTfR, and hemoglobin. The AUCROC for hepcidin as a test of iron deficiency was 0.78; hepcidin outperformed Hb and sTfR. The Youden index‐predicted cutoff to detect iron deficiency (3.2 ng/mL) was similar to thresholds previously identified to predict iron utilization and identify deficiency in African populations. Neither age, sex, nor α‐ or β‐thalassemia trait affected diagnostic properties of hepcidin. Hepcidin pre‐screening would prevent most iron‐replete thalassemia carriers from receiving iron whilst still ensuring most iron deficient children were supplemented. Our data indicate that the physiological relationship between hepcidin and iron status transcends specific populations. Measurement of hepcidin in individuals or populations could establish the need for iron interventions. PMID:27883199

  20. An Insight into the Relationships between Hepcidin, Anemia, Infections and Inflammatory Cytokines in Pediatric Refugees: A Cross-Sectional Study

    OpenAIRE

    2008-01-01

    BACKGROUND: Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA) is unclear. We investigated the relationships between hepcidin, cytokines and iron status in a pediatric population with a high prevalence of both anemia and co-morbid infections. METHODOLOGY/PRINCIPAL FINDINGS: African refugee children

  1. Associations between serum hepcidin, ferritin and Hb concentrations and type 2 diabetes risks in a Han Chinese population.

    Science.gov (United States)

    Guo, Xin; Zhou, Daizhan; An, Peng; Wu, Qian; Wang, Hao; Wu, Aimin; Mu, Mingdao; Zhang, Di; Zhang, Zhou; Wang, Hui; He, Lin; Liu, Yun; Wang, Fudi

    2013-12-01

    Systemic Fe overload can contribute to abnormal glucose metabolism and the onset of type 2 diabetes (T2D). Although hepcidin is the master regulator of systemic Fe homeostasis, few studies have systematically evaluated the associations of serum hepcidin concentrations with Fe metabolism parameters and risks for the development of T2D. In this regard, whether hepcidin concentrations are associated with T2D remains controversial. We measured serum hepcidin and ferritin concentrations in a case-control study of 1259 Han Chinese participants to evaluate the possible associations of serum hepcidin concentrations with Fe metabolism parameters and risks of T2D. Individuals with diabetes (n 555) and control participants (n 704) were recruited and serum hepcidin and ferritin concentrations were quantified. Additionally, selected biochemical and anthropometric variables were determined. A logistic regression analysis was performed to evaluate the association of serum hepcidin and ferritin concentrations with T2D. A linear regression analysis was used to test for associations between serum hepcidin and ferritin concentrations and a number of clinical, demographic and diabetes-associated variables. We found that serum hepcidin concentrations correlated with Hb and serum ferritin concentrations. No differences in hepcidin concentrations were found between the group with diabetes and the control group. Hepcidin concentrations were not significantly correlated with T2D risk factors. We also found that serum ferritin concentrations were elevated in individuals with diabetes and were positively correlated with both Hb concentrations and T2D risk factors. The present findings suggest that serum ferritin concentrations correlate with T2D risk factors, while serum hepcidin concentrations are positively associated with Hb and serum ferritin concentrations, but do not correlate with T2D.

  2. Adaptation of the plasma inhibitory activity assay to detect Aurora, ABL and FLT3 kinase inhibition by AT9283 in pediatric leukemia.

    Science.gov (United States)

    Podesta, Jennifer E; Sugar, Richard; Squires, Matt; Linardopoulos, Spiros; Pearson, Andrew D J; Moore, Andrew S

    2011-09-01

    Non-invasive assessment of biomarker modulation is important for evaluating targeted therapeutics, particularly in pediatrics. The plasma inhibitory activity (PIA) assay is used clinically to assess FLT3 inhibition ex vivo and guide dosing. AT9283 is a novel Aurora kinase inhibitor with secondary activity against FLT3 and ABL. We adapted the PIA assay to simultaneously detect inhibition of Aurora and FLT3 in AML, and Aurora and ABL in CML by AT9283. Furthermore, we optimized the assay for children, where limited blood volumes are available for pharmacodynamic studies. Simultaneously detecting multiple kinase inhibition may identify important mechanisms of action for novel anti-leukemic drugs.

  3. High Sulfation and a High Molecular Weight Are Important for Anti-hepcidin Activity of Heparin

    Science.gov (United States)

    Asperti, Michela; Naggi, Annamaria; Esposito, Emiliano; Ruzzenenti, Paola; Di Somma, Margherita; Gryzik, Magdalena; Arosio, Paolo; Poli, Maura

    2016-01-01

    Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins) or by high sulfation (SS-Heparins), but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH) and separated them in fractions of molecular weight in the range 4–16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells and in mice. The two approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with high N-acetylation and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites. PMID:26955355

  4. Hepcidin and iron metabolism disorders in patients with chronic kidney disease

    Directory of Open Access Journals (Sweden)

    Jelić Marija

    2013-01-01

    Full Text Available Bacground/Aim. Hepcidin may play a pathogenetic role in iron metobolism disorders. The aim of this study was to determine the correlation between hepcidin concentration and parameters of iron metabolism in patients with different stage of chronic kidney disease (CKD. Methods. The study involved 104 patients with CKD: 64 on hemodialysis (HD and 40 patients in pre-dialysis stadium (pre-HD with adequate erythropoetin therapy and iron supplementation. The HD group was divided in four subgroups according to the level of serum ferritin (up to 100; 100-199; 200-499 and over 500 ng/mL. Parameters of anemia, iron status, inflamation and hepcidin level were evaluated. Results. The HD patients had a significantly lower eritrocyte count, erythrocytes indexes, hemoglobin and transferrin saturation and significantly higher iron, ferritin, hepcidin and total iron binding capacity (TIBC. The HD subgroups up to 199 ng/mL of serum feritin had lower high-sensitivity Creactive protein (hsCRP, iron and higher unbuffered iron binding capacity (UIBC, transferrin saturation and TIBC compared to the HD subgroups over 200 ng/mL. The lowest and the highest ferritin subgroups had the highest hepcidin level and it showed significant correlation with ferritin. Conclusion. Hepcidin may serve as a marker for better diagnosing and monitoring anemia and iron metabolism disorders in CKD.

  5. Expression of the iron hormone hepcidin distinguishes different types of anemia in African children.

    Science.gov (United States)

    Pasricha, Sant-Rayn; Atkinson, Sarah H; Armitage, Andrew E; Khandwala, Shivani; Veenemans, Jacobien; Cox, Sharon E; Eddowes, Lucy A; Hayes, Theodore; Doherty, Conor P; Demir, Ayse Y; Tijhaar, Edwin; Verhoef, Hans; Prentice, Andrew M; Drakesmith, Hal

    2014-05-01

    Childhood anemia is a major global health problem resulting from multiple causes. Iron supplementation addresses iron deficiency anemia but is undesirable for other types of anemia and may exacerbate infections. The peptide hormone hepcidin governs iron absorption; hepcidin transcription is mediated by iron, inflammation, and erythropoietic signals. However, the behavior of hepcidin in populations where anemia is prevalent is not well established. We show that hepcidin measurements in 1313 African children from The Gambia and Tanzania (samples taken in 2001 and 2008, respectively) could be used to identify iron deficiency anemia. A retrospective secondary analysis of published data from 25 Gambian children with either postmalarial or nonmalarial anemia demonstrated that hepcidin measurements identified individuals who incorporated >20% oral iron into their erythrocytes. Modeling showed that this sensitivity of hepcidin expression at the population level could potentially enable simple groupings of individuals with anemia into iron-responsive and non-iron-responsive subtypes and hence could guide iron supplementation for those who would most benefit.

  6. Evaluation of Hepcidin Isoforms in Hemodialysis Patients by a Proteomic Approach Based on SELDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Natascia Campostrini

    2010-01-01

    Full Text Available The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versus 4.69 nM, and 4.06 versus 1.76 nM, resp., P<.05 for both. The clearance effects of a single dialysis session by different dialysis techniques and membranes were also investigated, showing an average reduction by 51.3% ± 29.2% for hepcidin-25 and 34.2% ± 28.4% for hepcidin-20 but only minor differences among the different dialysis modalities. Measurement of hepcidin isoforms through MS-based techniques can be a useful tool for better understanding of their biological role in hemodialysis patients and other clinical conditions.

  7. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma...... samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  8. Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

    NARCIS (Netherlands)

    Sparidans, Rolf W; van Hoppe, Stephanie; Rood, Johannes J M; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

    2016-01-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out as

  9. Iron status and systemic inflammation, but not gut inflammation, strongly predict gender-specific concentrations of serum hepcidin in infants in rural kenya

    National Research Council Canada - National Science Library

    Jaeggi, T; Moretti, D; Kvalsvig, J; Holding, P.A; Tjalsma, H; Kortman, G.A.M; Joosten, I; Mwangi, A; Zimmermann, M.B

    2013-01-01

    Hepcidin regulation by competing stimuli such as infection and iron deficiency has not been studied in infants and it's yet unknown whether hepcidin regulatory pathways are fully functional in infants...

  10. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    Science.gov (United States)

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

  11. Serum hepcidin levels are innately low in HFE-related haemochromatosis but differ between C282Y-homozygotes with elevated and normal ferritin levels.

    NARCIS (Netherlands)

    Dijk, B.A.C. van; Laarakkers, C.M.; Klaver, S.M.; Jacobs, E.M.G.; Tits, L.J.H. van; Janssen, M.C.H.; Swinkels, D.W.

    2008-01-01

    HFE C282Y-homozygosity has been associated with low hepcidin expression, leading to increased ferritin levels. However, serum hepcidin protein levels have not been documented in humans. In the current study, we compared serum hepcidin levels of newly diagnosed HFE C282Y-homozygotes with (N = 15) and

  12. Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?

    Institute of Scientific and Technical Information of China (English)

    Duygu Dee Harrison-Findik

    2009-01-01

    Despite heavy consumption over a long period of time,only a small number of alcoholics develop alcoholic liver disease. This alludes to the possibility that other factors,besides alcohol, may be involved in the progression of the disease. Over the years, many such factors have indeed been identified, including iron. Despite being crucial for various important biological processes, iron can also be harmful due to its ability to catalyze Fenton chemistry. Alcohol and iron have been shown to interact synergistically to cause liver injury. Iron-mediated cell signaling has been reported to be involved in the pathogenesis of experimental alcoholic liver disease. Hepcidin is an iron-regulatory hormone synthesized by the liver,which plays a pivotal role in iron homeostasis. Both acute and chronic alcohol exposure suppress hepcidin expression in the liver. The sera of patients with alcoholic liver disease, particularly those exhibiting higher serum iron indices, have also been reported to display reduced prohepcidin levels. Alcohol-mediated oxidative stress is involved in the inhibition of hepcidin promoter activity and transcription in the liver. This in turn leads to an increase in intestinal iron transport and liver iron storage. Hepcidin is expressed primarily in hepatocytes.It is noteworthy that both hepatocytes and Kupffer cells are involved in the progression of alcoholic liver disease. However, the activation of Kupffer cells and TNF-α signaling has been reported not to be involved in the down-regulation of hepcidin expression by alcohol in the liver. Alcohol acts within the parenchymal cells of the liver to suppress the synthesis of hepcidin. Due to its crucial role in the regulation of body iron stores, hepcidin may act as a secondary risk factor in the progression of alcoholic liver disease. The clarification of the mechanisms by which alcohol disrupts iron homeostasis will allow for further understanding of the pathogenesis of alcoholic liver disease.

  13. Testosterone Administration Inhibits Hepcidin Transcription and is Associated with Increased Iron Incorporation into Red Blood Cells

    Science.gov (United States)

    Guo, Wen; Bachman, Eric; Li, Michelle; Roy, Cindy N.; Blusztajn, Jerzy; Wong, Siu; Chan, Stephen Y.; Serra, Carlo; Jasuja, Ravi; Travison, Thomas G.; Muckenthaler, Martina U.; Nemeth, Elizabeta; Bhasin, Shalender

    2013-01-01

    Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferring saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound 58Fe, the amount of 58Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to BMP-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. Conclusion: Testosterone inhibits hepcidin transcription through its interaction with BMP-Smad signaling. Testosterone administration is associated with increased iron

  14. Decreased serum hepcidin, inflammation, and improved functional iron status six-months post-restrictive bariatric surgery.

    Science.gov (United States)

    Excess adiposity is associated with low-grade inflammation and decreased iron status. Iron depletion (ID) in obesity is thought to be mediated by an inflammation-induced increase in the body’s main regulator of iron homeostasis, hepcidin. Elevated hepcidin can result in ID as it prevents the release...

  15. The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

    NARCIS (Netherlands)

    Schwoebel, F.; Eijk, L.T.; Zboralski, D.; Sell, S.; Buchner, K.; Maasch, C.; Purschke, W.G.; Humphrey, M.; Zollner, S.; Eulberg, D.; Morich, F.; Pickkers, P.; Klussmann, S.

    2013-01-01

    Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only

  16. Meta-GWAS and Meta-Analysis of Exome Array Studies Do Not Reveal Genetic Determinants of Serum Hepcidin

    NARCIS (Netherlands)

    Galesloot, Tessel E; Verweij, Niek; Traglia, Michela; Barbieri, Caterina; van Dijk, Freerk; Geurts-Moespot, Anneke J; Girelli, Domenico; Kiemeney, Lambertus A L M; Sweep, Fred C G J; Swertz, Morris A; van der Meer, Peter; Camaschella, Clara; Toniolo, Daniela; Vermeulen, Sita H; van der Harst, Pim; Swinkels, Dorine W

    2016-01-01

    Serum hepcidin concentration is regulated by iron status, inflammation, erythropoiesis and numerous other factors, but underlying processes are incompletely understood. We studied the association of common and rare single nucleotide variants (SNVs) with serum hepcidin in one Italian study and two la

  17. The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

    NARCIS (Netherlands)

    Schwoebel, F.; Eijk, L.T.; Zboralski, D.; Sell, S.; Buchner, K.; Maasch, C.; Purschke, W.G.; Humphrey, M.; Zollner, S.; Eulberg, D.; Morich, F.; Pickkers, P.; Klussmann, S.

    2013-01-01

    Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only kno

  18. Iron and hepcidin as risk factors in atherosclerosis: what do the genes say?

    Science.gov (United States)

    Galesloot, Tessel E; Janss, Luc L; Burgess, Stephen; Kiemeney, Lambertus A L M; den Heijer, Martin; de Graaf, Jacqueline; Holewijn, Suzanne; Benyamin, Beben; Whitfield, John B; Swinkels, Dorine W; Vermeulen, Sita H

    2015-07-11

    Previous reports suggested a role for iron and hepcidin in atherosclerosis. Here, we evaluated the causality of these associations from a genetic perspective via (i) a Mendelian randomization (MR) approach, (ii) study of association of atherosclerosis-related single nucleotide polymorphisms (SNPs) with iron and hepcidin, and (iii) estimation of genomic correlations between hepcidin, iron and atherosclerosis. Analyses were performed in a general population sample. Iron parameters (serum iron, serum ferritin, total iron-binding capacity and transferrin saturation), serum hepcidin and genome-wide SNP data were available for N = 1,819; non-invasive measurements of atherosclerosis (NIMA), i.e., presence of plaque, intima media thickness and ankle-brachial index (ABI), for N = 549. For the MR, we used 12 iron-related SNPs that were previously identified in a genome-wide association meta-analysis on iron status, and assessed associations of individual SNPs and quartiles of a multi-SNP score with NIMA. Quartile 4 versus quartile 1 of the multi-SNP score showed directionally consistent associations with the hypothesized direction of effect for all NIMA in women, indicating that increased body iron status is a risk factor for atherosclerosis in women. We observed no single SNP associations that fit the hypothesized directions of effect between iron and NIMA, except for rs651007, associated with decreased ferritin concentration and decreased atherosclerosis risk. Two of six NIMA-related SNPs showed association with the ratio hepcidin/ferritin, suggesting that an increased hepcidin/ferritin ratio increases atherosclerosis risk. Genomic correlations were close to zero, except for hepcidin and ferritin with ABI at rest [-0.27 (SE 0.34) and -0.22 (SE 0.35), respectively] and ABI after exercise [-0.29 (SE 0.34) and -0.30 (0.35), respectively]. The negative sign indicates an increased atherosclerosis risk with increased hepcidin and ferritin concentrations. Our results suggest a

  19. Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax).

    Science.gov (United States)

    Álvarez, Claudio Andrés; Acosta, Félix; Montero, Daniel; Guzmán, Fanny; Torres, Elisa; Vega, Belinda; Mercado, Luis

    2016-08-01

    The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species.

  20. Hepcidin expression in the liver of rats fed a magnesium-deficient diet.

    Science.gov (United States)

    Ishizaki, Natsumi; Kotani, Megumi; Funaba, Masayuki; Matsui, Tohru

    2011-10-01

    Mg deficiency accelerates Fe accumulation in the liver, which may induce various metabolic disturbances. In the present study, we examined the gene expression of Hepcidin, a peptide hormone produced in the liver to regulate intestinal Fe absorption negatively, in Mg-deficient rats. Although liver Fe concentration was significantly higher in rats fed an Mg-deficient diet for 4 weeks than in rats fed a control diet, Hepcidin expression in the liver was comparable between the dietary groups. Previous studies revealed that Fe overload up-regulated Hepcidin expression through transcriptional activation by Fe-induced bone morphogenetic protein (Bmp) 6, a growth/differentiation factor belonging to the transforming growth factor-β family, in the liver. Mg deficiency up-regulated the expression of Bmp6 but did not affect the expression of inhibition of DNA binding 1, a sensitive Bmp-responsive gene. In addition, the expression of Bmp receptors such as activin receptor-like kinase 2 (Alk2), activin receptor type IIA (Actr2a), activin receptor type IIB (Actr2b) and Bmp type II receptor (Bmpr2) was lower in the liver of Mg-deficient rats than in that of control rats. The present study indicates that accumulation of hepatic Fe by Mg deficiency is a stimulant inducing Bmp6 expression but not Hepcidin expression by blunting Bmp signalling possibly resulting from down-regulation of the receptor expression. Unresponsive Hepcidin expression may have a role in Mg deficiency-induced changes related to increased liver Fe.

  1. Iron status and the acute post-exercise hepcidin response in athletes.

    Directory of Open Access Journals (Sweden)

    Peter Peeling

    Full Text Available This study explored the relationship between serum ferritin and hepcidin in athletes. Baseline serum ferritin levels of 54 athletes from the control trial of five investigations conducted in our laboratory were considered; athletes were grouped according to values 100 μg/L (SF>100. Data pooling resulted in each athlete completing one of five running sessions: (1 8 × 3 min at 85% vVO2peak; (2 5 × 4 min at 90% vVO2peak; (3 90 min continuous at 75% vVO2peak; (4 40 min continuous at 75% vVO2peak; (5 40 min continuous at 65% vVO2peak. Athletes from each running session were represented amongst all four groups; hence, the mean exercise duration and intensity were not different (p>0.05. Venous blood samples were collected pre-, post- and 3 h post-exercise, and were analysed for serum ferritin, iron, interleukin-6 (IL-6 and hepcidin-25. Baseline and post-exercise serum ferritin levels were different between groups (p0.05. Post-exercise IL-6 was significantly elevated compared to baseline within each group (p100; p<0.05. An athlete's iron stores may dictate the baseline hepcidin levels and the magnitude of post-exercise hepcidin response. Low iron stores suppressed post-exercise hepcidin, seemingly overriding any inflammatory-driven increases.

  2. Iron status and the acute post-exercise hepcidin response in athletes.

    Science.gov (United States)

    Peeling, Peter; Sim, Marc; Badenhorst, Claire E; Dawson, Brian; Govus, Andrew D; Abbiss, Chris R; Swinkels, Dorine W; Trinder, Debbie

    2014-01-01

    This study explored the relationship between serum ferritin and hepcidin in athletes. Baseline serum ferritin levels of 54 athletes from the control trial of five investigations conducted in our laboratory were considered; athletes were grouped according to values 100 μg/L (SF>100). Data pooling resulted in each athlete completing one of five running sessions: (1) 8 × 3 min at 85% vVO2peak; (2) 5 × 4 min at 90% vVO2peak; (3) 90 min continuous at 75% vVO2peak; (4) 40 min continuous at 75% vVO2peak; (5) 40 min continuous at 65% vVO2peak. Athletes from each running session were represented amongst all four groups; hence, the mean exercise duration and intensity were not different (p>0.05). Venous blood samples were collected pre-, post- and 3 h post-exercise, and were analysed for serum ferritin, iron, interleukin-6 (IL-6) and hepcidin-25. Baseline and post-exercise serum ferritin levels were different between groups (piron or IL-6 (p>0.05). Post-exercise IL-6 was significantly elevated compared to baseline within each group (p100; pathlete's iron stores may dictate the baseline hepcidin levels and the magnitude of post-exercise hepcidin response. Low iron stores suppressed post-exercise hepcidin, seemingly overriding any inflammatory-driven increases.

  3. Hepcidin and Iron Metabolism in Pregnancy: Correlation with Smoking and Birth Weight and Length.

    Science.gov (United States)

    Chełchowska, Magdalena; Ambroszkiewicz, Jadwiga; Gajewska, Joanna; Jabłońska-Głąb, Ewa; Maciejewski, Tomasz M; Ołtarzewski, Mariusz

    2016-09-01

    To estimate the effect of tobacco smoking on iron homeostasis and the possible association between hepcidin and the neonatal birth weight and length, concentrations of serum hepcidin and selected iron markers were measured in 81 healthy pregnant women (41 smokers and 40 nonsmokers). The smoking mothers had significantly lower concentrations of serum hepcidin (p smoking pregnant women. Logistic regression analysis showed the highest negative impact of the number of cigarettes smoked per day (β = -0.46; p smoking mothers' infants were significantly lower than in tobacco abstinent group (p smoking affected hepcidin level in serum of pregnant women in a dose-dependent manner. Low concentrations of iron and hemoglobin in maternal serum coexisting with high level of erythropoietin suggest that smoking could lead to subclinical iron deficiency and chronic hypoxia not only in mothers but also in fetus. Low serum hepcidin concentration in smoking pregnant women might be associated with lower fetal birth weight and length.

  4. Assay of calcium borogluconate veterinary medicines for calcium gluconate, boric acid, phosphorus, and magnesium by using inductively coupled plasma emission spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lyons, D.J.; Spann, K.P.

    1985-03-01

    An inductively coupled plasma spectrometric method is described for the determination of 4 elements (Ca, B, P, and Mg) in calcium borogluconate veterinary medicines. Samples are diluted, acidified, and sprayed directly into the plasma. Reproducibility relative confidence intervals for a single sample assay are +/- 1.4% (calcium), +/- 1.8% (boron), +/- 2.6% (phosphorus), and +/- 1.4% (magnesium). The total element concentrations for each of 4 elements compared favorably with concentrations determined by alternative methods. Formulation estimates of levels of calcium gluconate, boric acid, phosphorus, and magnesium salts can be made from the analytical data.

  5. Comparison of the artus Epstein-Barr virus (EBV) PCR kit and the Abbott RealTime EBV assay for measuring plasma EBV DNA loads in allogeneic stem cell transplant recipients.

    Science.gov (United States)

    Vinuesa, Víctor; Solano, Carlos; Giménez, Estela; Navarro, David

    2017-02-24

    The ability of the artus Epstein-Barr virus (EBV) PCR kit and the Abbott RealTime EBV PCR assay to detect and quantify plasma EBV DNAemia was compared. The agreement between these assays was 95.8%. The EBV DNA loads measured by the two assays significantly correlated (P=< 0.0001).

  6. Combined treatment of 3-hydroxypyridine-4-one derivatives and green tea extract to induce hepcidin expression in iron-overloaded b-thalassemic mice

    Institute of Scientific and Technical Information of China (English)

    Supranee; Upanan; Kanjana; Pangjit; Chairat; Uthaipibull; Suthat; Fucharoen; Andrew; T.Mc; Kie; Somdet; Srichairatanakool

    2015-01-01

    Objective:To evaluate the efficacy of deferiprone(DFP),1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one(CM1)or green tea extract(GTE)in enhancing expression of hepatic hepcidin1(Hamp1)m RNA and relieving iron overload in b-globin knockout thalassemic mice.Methods:The b-globin knockout thalassemic mice were fed with a ferrocenesupplemented diet for 2 months and oral administration of deionized water,DFP(50 mg/kg),CM1(50 mg/kg),GTE(50 mg epigallocatechin 3-gallate equivalent/kg),GTE along with DFP(50 mg/kg),and GTE along with CM1(50 mg/kg)every day for 3months.Levels of hepatic Hamp1 m RNA,plasma non-transferrin bound iron,plasma alanine aminotransferase activity and tissue iron content were determined.Results:All chelation treatments could reduce plasma non-transferrin bound iron concentrations.Additionally,hepatic Hamp1 m RNA expression was significantly upregulated in the mice in a GTE+DFP combined treatment,correlating with a decrease in the plasma alanine aminotransferase activity and tissue iron deposition.Conclusions:The GTE+DFP treatment could ameliorate iron overload and liver oxidative damage in non-transfusion dependent b-thalassemic mice,by chelating toxic iron in plasma and tissues,and increasing hepcidin expression to inhibit duodenal iron absorption and iron release from hepatocytes and macrophages in the spleen.There is probably an advantage in giving GTE with DFP when treating patients with iron overload.

  7. Combined treatment of 3-hydroxypyridine-4-one derivatives and green tea extract to induce hepcidin expression in iron-overloaded b-thalassemic mice

    Institute of Scientific and Technical Information of China (English)

    Supranee Upanan; Kanjana Pangjit; Chairat Uthaipibull; Suthat Fucharoen; Andrew T McKie; Somdet Srichairatanakool

    2015-01-01

    Objective: To evaluate the efficacy of deferiprone (DFP), 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) or green tea extract (GTE) in enhancing expres-sion of hepatic hepcidin1 (Hamp1) mRNA and relieving iron overload in b-globin knockout thalassemic mice. Methods: The b-globin knockout thalassemic mice were fed with a ferrocene-supplemented diet for 2 months and oral administration of deionized water, DFP (50 mg/kg), CM1 (50 mg/kg), GTE (50 mg epigallocatechin 3-gallate equivalent/kg), GTE along with DFP (50 mg/kg), and GTE along with CM1 (50 mg/kg) every day for 3 months. Levels of hepatic Hamp1 mRNA, plasma non-transferrin bound iron, plasma alanine aminotransferase activity and tissue iron content were determined. Results: All chelation treatments could reduce plasma non-transferrin bound iron con-centrations. Additionally, hepatic Hamp1 mRNA expression was significantly up-regulated in the mice in a GTE+DFP combined treatment, correlating with a decrease in the plasma alanine aminotransferase activity and tissue iron deposition. Conclusions: The GTE + DFP treatment could ameliorate iron overload and liver oxidative damage in non-transfusion dependent b-thalassemic mice, by chelating toxic iron in plasma and tissues, and increasing hepcidin expression to inhibit duodenal iron absorption and iron release from hepatocytes and macrophages in the spleen. There is probably an advantage in giving GTE with DFP when treating patients with iron overload.

  8. The diagnostic value of neutrophil gelatinase-associated lipocalin and hepcidin in bacteria translocation of liver cirrhosis.

    Science.gov (United States)

    Zhang, Jiangguo; Gong, Fengyun; Li, Ling; Zhao, Manzhi; Wu, Zhuhua; Song, Jianxin

    2015-01-01

    Bacterial translocation (BT) or bacterial DNA (bactDNA) translocation is a critical pathogenesis mechanism of spontaneous bacterial peritonitis. Studies of BT or bactDNA translocation are limited in humans. Neutrophil gelatinase associated lipocalin (NGAL) can efficiently distinguish bacterial and nonbacterial ascites in ascitic patients. Hepcidin is a useful marker of bacterial infection in the late-onset sepsis. However, the relationship between NGAL, hepcidin and BT was still unclear. In present study, the levels of NGAL, hepcidin and their relationship with BT or bactDNA translocation were investigated. Weekly doses of carbon tetrachloride (CCl4) were given to induce liver cirrhosis in Sprague-Dawley rats. Trypticase (blood) soy agars were used to culture bacteria. BactDNA was sequenced by ABIPRISM 310 automated sequencer. The levels of NGAL and hepcidin were assessed by ELISA. Receiver operating characteristic (ROC) curve was used to determine the cut-off values and compare the diagnostic performance of NGAL and hepcidin. 56 cirrhotic and 10 normal rats were included in this study. The levels of both two biomarkers were significantly higher in BT or bactDNA translocation group compared to non-translocation group. The area under ROC curve for the diagnosis of BT was 0.910 for serum NGAL, 0.858 for serum hepcidin and 0.940 for their combination, whereas that for the diagnosis of bactDNA translocation was 0.906 for NGAL, 0.779 for hepcidin and 0.950 for their combination, respectively. The combination of NGAL and hepcidin improved the ability to detect BT or bactDNA presence in MLNs and ascites. BT and the presence of bactDNA in MLNs were observed in a rat cirrhotic model. Serum NGAL and hepcidin can serve as sensitive and specific tests for diagnosis of BT or bactDNA translocation. NGAL in combination with hepcidin can improve the accuracy of diagnosis.

  9. Noninvasive Personalization of Lung Cancer Therapy Using a New, Clinical-Grade Assay for Plasma-Based Measurement and Monitoring of Tumor Genotype

    Science.gov (United States)

    2016-12-01

    AWARD NUMBER: W81XWH-14-1-0128 TITLE:Noninvasive Personalization of Lung Cancer Therapy Using a New, Clinical-Grade Assay for Plasma-Based...Measurement and Monitoring of Tumor Genotype PRINCIPAL INVESTIGATOR: Geoffrey Oxnard, MD CONTRACTING ORGANIZATION: Dana-Farber Cancer Institute...2. REPORT TYPE Final 3. DATES COVERED 30 Sep 2014 – 29 Sep 2016 4. TITLE AND SUBTITLE Noninvasive Personalization of Lung Cancer Therapy Using a

  10. Hepcidin and Ferritin Levels in Fever of Unknown Origin: Is There a New Biomarker?

    Directory of Open Access Journals (Sweden)

    Ana Lopez Aparicio

    2015-09-01

    Full Text Available Background and objectives: Significantly elevated serum ferritin levels are associated with both iron overload and some inflammatory conditions. Hepcidin is a protein that interferes with iron absorption in inflammatory states and acts as an acute-phase reactant. Materials and methods: Here we report the case a 33-year-old patient who presented with high fever, skin lesions and arthralgia lasting for 2 weeks. His ferritin level was 13,800 µg/l and his hepcidin level was 61 ng/dl. Results: The final diagnosis was adult onset Still's disease. The condition evolved satisfactorily with steroid treatment, but after several weeks the patient presented with an unexpected recurrence. Conclusions: Hepcidin is a good inflammatory marker that could be useful in the differential diagnosis of hyperferritinaemia.

  11. Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major.

    Science.gov (United States)

    Andreani, Marco; Radio, Francesca Clementina; Testi, Manuela; De Bernardo, Carmelilia; Troiano, Maria; Majore, Silvia; Bertucci, Pierfrancesco; Polchi, Paola; Rosati, Renata; Grammatico, Paola

    2009-09-01

    Hepcidin is a 25-amino acid peptide, derived from cleavage of an 84 amino acid pro-peptide produced predominantly by hepatocytes. This molecule, encoded by the hepcidin antimicrobial peptide (HAMP) gene shows structural and functional properties consistent with a role in innate immunity. Moreover, as demonstrated in mice and humans, hepcidin is a major regulator of iron metabolism, and acts by binding to ferroportin and controlling its concentration and trafficking. In this study we investigated the influence that mutations in HAMP and/or hemocromatosis (HFE) genes might exert on iron metabolism in a group of poly-transfused thalassemic patients in preparation for bone marrow transplantation. Our results showed that the presence of the c.-582 A>G polymorphism (rs10421768) placed in HAMP promoter (HAMP-P) might play a role in iron metabolism, perhaps varying the transcriptional activation that occurs through E-boxes located within the promoter.

  12. Development of a liquid chromatography-mass spectrometry (LC/MS) assay method for the quantification of PSC 833 (Valspodar) in rat plasma.

    Science.gov (United States)

    Binkhathlan, Ziyad; Somayaji, Vishwa; Brocks, Dion R; Lavasanifar, Afsaneh

    2008-06-15

    A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.

  13. The Role of Insulin Therapy in Correcting Hepcidin Levels in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Driton Vela

    2017-05-01

    Full Text Available Objectives: Iron overload can cause or contribute to the pathogenesis of type 2 diabetes mellitus (T2DM, but how the major parameters of iron metabolism change in different settings of diabetes are still unclear. The aim of this study was to determine the relationship between iron, ferritin, and hepcidin levels in diabetic patients and the effect of insulin treatment. Methods: The study included 80 subjects, 60 with T2DM and 20 without (control group. Serum hepcidin, insulin, ferritin, and iron levels were determined as well as other clinical parameters. The associations between these parameters were analyzed between both groups. Results: Hepcidin levels expressed as mean± standard deviation between groups showed no significant changes (14.4±6.7 ng/mL for the control group, and 18.4±7.9 ng/mL for patients with diabetes, p = 0.069. Parameters of iron metabolism showed modest correlation with the parameters of glucose metabolism. However, the correlation between ferritin and insulin in both groups was statistically significant (p = 0.032; ρ = 0.480 vs. p = 0.011; ρ = 0.328. Conclusions: Our study showed that hepcidin levels in patients with T2DM on insulin therapy do not change, which might be a result of treatment with insulin. In this context, insulin treatment can be used as a novel method for correction of hepcidin levels. By correcting hepcidin levels, we can prevent cellular iron overload and reduce the risk of diabetes.

  14. Genetics, Genetic Testing, and Management of Hemochromatosis: 15 Years Since Hepcidin.

    Science.gov (United States)

    Pietrangelo, Antonello

    2015-10-01

    The discovery of hepcidin in 2000 and the subsequent unprecedented explosion of research and discoveries in the iron field have dramatically changed our understanding of human disorders of iron metabolism. Today, hereditary hemochromatosis, the paradigmatic iron-loading disorder, is recognized as an endocrine disease due to the genetic loss of hepcidin, the iron hormone produced by the liver. This syndrome is due to unchecked transfer of iron into the bloodstream in the absence of increased erythropoietic needs and its toxic effects in parenchymatous organs. It is caused by mutations that affect any of the proteins that help hepcidin to monitor serum iron, including HFE and, in rarer instances, transferrin-receptor 2 and hemojuvelin, or make its receptor ferroportin, resistant to the hormone. In Caucasians, C282Y HFE homozygotes are numerous, but they are only predisposed to hemochromatosis; complete organ disease develops in a minority, due to alcohol abuse or concurrent genetic modifiers that are now being identified. HFE gene testing can be used to diagnose hemochromatosis in symptomatic patients, but analyses of liver histology and full gene sequencing are required to identify patients with rare, non-HFE forms of the disease. Due to the central pathogenic role of hepcidin, it is anticipated that nongenetic causes of hepcidin loss (eg, end-stage liver disease) can cause acquired forms of hemochromatosis. The mainstay of hemochromatosis management is still removal of iron by phlebotomy, first introduced in 1950s, but identification of hepcidin has not only shed new light on the pathogenesis of the disease and the approach to diagnosis, but etiologic therapeutic applications from these advances are now foreseen.

  15. High sulfation and a high molecular weight are important for anti-hepcidin activity of heparin

    Directory of Open Access Journals (Sweden)

    Michela eAsperti

    2016-01-01

    Full Text Available Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins or by high sulfation (SS-Heparins, but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH and separated them in fractions of molecular weight in the range 4-16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells, in mice, and also for the capacity to bind an Heparin Binding Domain peptide. The three approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with an increase of the N-acetylation level and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites.

  16. Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6.

    Science.gov (United States)

    Truksa, Jaroslav; Gelbart, Terri; Peng, Hongfan; Beutler, Ernest; Beutler, Bruce; Lee, Pauline

    2009-11-01

    Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo, we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.

  17. HPLC Plasma Assay of a Novel Anti-MRSA Compound, Kaempferol-3-O-Alpha-L-(2",3"-di-p-coumaroyl)rhamnoside, from Sycamore Leaves.

    Science.gov (United States)

    Zhang, Yiguan; Valeriote, Frederick; Swartz, Kenneth; Chen, Ben; Hamann, Mark T; Rodenburg, Douglas L; McChesney, James D; Shaw, Jiajiu

    2015-08-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that is resistant to current antibiotic therapy. Thus, there is an urgent need for novel antimicrobial agents that can effectively combat these new strains of drug-resistant "superbugs". Recently, fractionation of an extract from Platanus occidentalis (American sycamore) leaves produced an active kaempferol molecule, 3-O-alpha-L-(2",3"-di-p-coumaroyl)rhamnoside (KCR), in four isomeric forms; all four isomers exhibit potent anti-MRSA activity. In order to further the preclinical development of KCR as a new antibiotic class, we developed and validated a simple analytical method for assaying KCR plasma concentration. Because KCR will be developed as a new drug, although comprising four stereoisomers, the analytical method was devised to assay the total amount of all four isomers. In the present work, both a plasma processing procedure and an HPLC method have been developed and validated. Mouse plasma containing KCR was first treated with ethanol and then centrifuged. The supernatant was dried, suspended in ethanol, centrifuged, and the supernatant was injected into an HPLC system comprising a Waters C18, a mobile phase composing methanol, acetonitrile, and trifluoroacetic acid and monitored at 313 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 0.27 microg/mL, and high accuracy. In summary, this method allows a rapid analysis of KCR in the plasma samples for pharmacokinetics studies.

  18. UPLC-MS/MS assay of riluzole in human plasma and cerebrospinal fluid (CSF): Application in samples from spinal cord injured patients.

    Science.gov (United States)

    Sarkar, Mahua; Grossman, Robert G; Toups, Elizabeth G; Chow, Diana S-L

    2017-09-01

    In the present study, a sensitive and robust LC-MS/MS method has been developed and validated for the quantification of riluzole in human plasma and cerebrospinal fluid (CSF) in clinical samples from patients with spinal cord injury (SCI). Riluzole and its labeled internal standard (IS) were isolated from plasma and CSF by liquid-liquid extraction using ethyl acetate. Riluzole (m/z 235→166) and IS (m/z 238→169) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode. The assay was linear in the concentration range of 0.5 (LLOQ, signal/noise ratio>10)-800ng/ml in plasma, and 1.0 (LLOQ)-800ng/ml in CSF samples. The intra- and inter-day accuracy in plasma were 94.2-110.0% and 97.8-102.0%, respectively, and those in CSF were 87.6-105.1% and 91.9-98.8%, respectively. The intra- and inter-day precision were 2.2-7.2% and 4.0-9.1%, respectively, in plasma, and 1.4-14.1% and 2.6-11.5%, respectively in CSF. Matrix effect was negligible from both matrices with signal percentages of 97.6-100.6% in plasma and 99.4-106.4% in CSF. The recoveries were >75% in plasma, >84% in CSF with low protein (53.9mg/dl), and >68% in CSF with high protein (348.2mg/dl). This method was successfully applied to quantify riluzole concentrations in plasma and CSF from patients with SCI. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. A fish antimicrobial peptide, tilapia hepcidin TH2-3, shows potent antitumor activity against human fibrosarcoma cells.

    Science.gov (United States)

    Chen, Jyh-Yih; Lin, Wei-Ju; Lin, Tai-Lang

    2009-09-01

    As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48-96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.

  20. 阻塞性睡眠呼吸暂停低通气综合征患者血清铁蛋白、铁调素的变化及其临床意义%The changes and clinical significance of serum ferritin and hepcidin in patients with obstructive sleep apnea hypopnea syndrome

    Institute of Scientific and Technical Information of China (English)

    董世荣; 张兰兰; 王蓓

    2013-01-01

    目的 探讨阻塞性睡眠呼吸暂停低通气综合征(OSAS)患者血清铁蛋白(SF)、铁调素的变化及其临床意义.方法 选取经多导睡眠监测确诊为OSAS的男性患者51例,其中单纯OSAS组36例(轻度OSAS组13例,中重度OSAS组23例),OSAS合并糖尿病组15例;另选取单纯2型糖尿病(T2DM)患者14例作为T2DM组;设立单纯肥胖男性18例为对照组.分别检测各组受试者空腹血糖、HbA1c及空腹胰岛素水平,并用ELISA法检测SF、铁调素水平,观察OSAS患者SF、铁调素对糖代谢的影响.结果 (1)轻度OSAS组、中重度OSAS组、OSAS合并糖尿病组和T2DM组的SF水平升高,分别为(268.24±47.94),(316.45 ±49.36),(377.66±49.95),(286.01 ±45.53) μg/L,均高于对照组(203.28±32.83) μg/L,差异有统计学意义(F =39.38,P <0.01);铁调素水平下降,分别为(66.41 ±12.00),(47.34 ±13.35),(34.52±8.49),(54.45±8.25) μg/L,均低于对照组(88.26±12.34)μg/L,差异有统计学意义(F=67.80,P<0.01).(2)SF、铁调素与睡眠呼吸紊乱指数、最长呼吸暂停时间、夜间血氧饱和度低于90%的时间占总睡眠时间的百分比、最低血氧饱和度存在相关性(r=-0.65 ~0.34,P均<0.01),且SF与铁调素之间存在线性相关(F=22.40,P<0.01).结论 OSAS患者存在SF及铁调素水平的异常,可能在T2DM的发生中发挥一定的作用.%Objective To explore the changes and clinical significance of serum ferritin (SF)and hepcidin in patients with obstructive sleep apnea hypopnea syndrome (OSAS).Methods 51 cases of male patients with OSAS,diagnosed by polysomnography were selected,which included simple OSAS group 36 cases (mild OSAS group 13 cases,moderate and severe OSAS group 23 cases),OSAS with diabetes group 15 cases.14 patients with simple type 2 diabetes mellitus(T2DM)were selected as T2DM group.And selected 18 obese men as control group.Measured the levels of fasting plasma glucose(FPG),HbA1c and fasting insulin of each group.SF and hepcidin

  1. Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice.

    Science.gov (United States)

    Gardenghi, Sara; Ramos, Pedro; Marongiu, Maria Franca; Melchiori, Luca; Breda, Laura; Guy, Ella; Muirhead, Kristen; Rao, Niva; Roy, Cindy N; Andrews, Nancy C; Nemeth, Elizabeta; Follenzi, Antonia; An, Xiuli; Mohandas, Narla; Ginzburg, Yelena; Rachmilewitz, Eliezer A; Giardina, Patricia J; Grady, Robert W; Rivella, Stefano

    2010-12-01

    Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

  2. Evaluation of the Relationship of Hepcidin Levels with Anemia and Inflammatory Markers in Patients on Peritoneal Dialysis: A Controlled Study

    Directory of Open Access Journals (Sweden)

    Zeki AYDIN

    2012-01-01

    Full Text Available OBJECTIVE: Hepcidin, a small peptide hormone synthesized in the liver, plays a central role in the regulation of iron metabolism. In addition, it acts as an intermediary in body defense and inflammation. Our aim in this study was to investigate the relationship of hepsidin levels with inflammation and iron indices in patients on peritoneal dialysis (PD.MATERIAL and METHODS: Nondiabetic PD patients were involved. Primary kidney disease, biochemical parameters, complete blood count, iron, total iron binding capacity (TIBC, ferritin, high sensitive C-reactive protein (hsCRP, fibrinogen, parathormone, interleukin (IL-6 and hepcidin levels were recorded as well as demographic parameters.RESULTS: Twenty-one PD patients (mean age 47.7±12.1 years and 17 healthy volunteers (mean age 54.0±7.2 years were involved. HepCidin levels were higher in the PD group (148.2±35.0 vs. 93.8±21.9; p<0.001. There was a positive correlation of hepcidin with urea, creatinine, phosphorus, ferritin, fibrinogen, IL-6 and parathormone; and a negative correlation with albumin, transaminases, calcium, TIBC, GFR, hemoglobin and hematocrit levels.CONCLUSION: Hepcidin levels increase with deepening anemia and show a positive correlation with inflammatory markers. Theurapeutic interventions regarding the effects of hepcidin on inflammatory status may play a role in the treatment of anemia due to inflammation. It may be beneficial to measure hepcidin levels together with ferritin, especially in patients with functional iron defficiency.

  3. Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma.

    Science.gov (United States)

    Teitelbaum, Aaron M; Flaker, Alicia M; Kharasch, Evan D

    2016-04-01

    A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing an α1-acid glycoprotein column within a 12-min run time. Chromatograph separation was significantly influenced by mobile phase pH and variability between columns. Analytes were quantified by positive ion electrospray tandem mass spectrometry following plasma protein precipitation with 20% trichloroacetic acid. Identification of erythrohydrobupropion enantiomer peaks and threohydrobupropion enantiomer peaks was achieved by sodium borohydride reduction of enantiopure (R)- and (S)-bupropion. Initial assay validation and sensitivity determination was on AB Sciex 3200, 4000 QTRAP, and 6500 mass spectrometers. Accuracy and precision were within 15% for each analyte. The assay was fully validated over analyte-specific concentrations using an AB Sciex 3200 mass spectrometer. Intra- and inter-assay precision and accuracy were within 12% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), and erythrohydrobupropion (1R,2S and 1S,2R) were 0.5, 2, 1, and 1ng/mL, respectively. All analytes were stable following freeze thaw cycles at -80°C and while stored at 4°C in the instrument autosampler. This method was applicable to clinical pharmacokinetic investigations of bupropion in patients. This is the first chromatographic method to resolve erythrohydrobupropion and threohydrobupropion enantiomers, and the first stereoselective LC-MS/MS assay to quantify bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion

  4. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

    Science.gov (United States)

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E; Ginzburg, Yelena Z

    2016-03-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

  5. HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells.

    Science.gov (United States)

    Shi, Wencai; Wang, Heyang; Zheng, Xuan; Jiang, Xin; Xu, Zheng; Shen, Hui; Li, Min

    2016-09-23

    Hepcidin synthesis is reported to be inadequate according to the body iron store in patients with non-alcoholic fatty liver disease (NAFLD) undergoing hepatic iron overload (HIO). However, the underlying mechanisms remain unclear. We hypothesize that hepatocyte nuclear factor-4α (HNF-4α) may negatively regulate hepcidin expression and contribute to hepcidin deficiency in NAFLD patients. The effect of HNF-4α on hepcidin expression was observed by transfecting specific HNF-4α small interfering RNA (siRNA) or plasmids into HepG2 cells. Both direct and indirect mechanisms involved in the regulation of HNF-4α on hepcidin were detected by real-time PCR, Western blotting, chromatin immunoprecipitation (chIP), and reporter genes. It was found that HNF-4α suppressed hepcidin messenger RNA (mRNA) and protein expressions in HepG2 cells, and this suppressive effect was independent of the potential HNF-4α response elements. Phosphorylation of SMAD1 but not STAT3 was inactivated by HNF-4α, and the SMAD4 response element was found essential to HNF-4α-induced hepcidin reduction. Neither inhibitory SMADs, SMAD6, and SMAD7 nor BMPR ligands, BMP2, BMP4, BMP6, and BMP7 were regulated by HNF-4α in HepG2 cells. BMPR1A, but not BMPR1B, BMPR2, ActR2A, ActR2B, or HJV, was decreased by HNF-4α, and HNF4α-knockdown-induced stimulation of hepcidin could be entirely blocked when BMPR1A was interfered with at the same time. In conclusion, the present study suggests that HNF-4α has a suppressive effect on hepcidin expression by inactivating the BMP pathway, specifically via BMPR1A, in HepG2 cells.

  6. A new automated method for the determination of the Total Antioxidant Capacity (TAC of human plasma, based on the crocin bleaching assay

    Directory of Open Access Journals (Sweden)

    Notas George

    2002-08-01

    Full Text Available Abstract Background Antioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (polyaromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. Methods In the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay, based on the crocin bleaching (oxidation method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L, and performed using an end point measurement. Results The TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM, while a diet rich in antioxidants more than doubled this value. Conclusions The proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and

  7. Hepcidin Plays a Key Role in 6-OHDA Induced Iron Overload and Apoptotic Cell Death in a Cell Culture Model of Parkinson’s Disease

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    Qi Xu

    2016-01-01

    Full Text Available Background. Elevated brain iron levels have been implicated in the pathogenesis of Parkinson’s disease (PD. However, the precise mechanism underlying abnormal iron accumulation in PD is not clear. Hepcidin, a hormone primarily produced by hepatocytes, acts as a key regulator in both systemic and cellular iron homeostasis. Objective. We investigated the role of hepcidin in 6-hydroxydopamine (6-OHDA induced apoptosis in a cell culture model of PD. Methods. We downregulated hepcidin using siRNA interference in N27 dopaminergic neuronal cells and made a comparison with control siRNA transfected cells to investigate the role of hepcidin in 6-OHDA induced neurodegeneration. Results. Hepcidin knockdown (32.3%, P<0.0001 upregulated ferroportin 1 expression and significantly (P<0.05 decreased intracellular iron by 25%. Hepcidin knockdown also reduced 6-OHDA induced caspase-3 activity by 42% (P<0.05 and DNA fragmentation by 29% (P=0.086 and increased cell viability by 22% (P<0.05. In addition, hepcidin knockdown significantly attenuated 6-OHDA induced protein carbonyls by 52% (P<0.05 and intracellular iron by 28% (P<0.01, indicating the role of hepcidin in oxidative stress. Conclusions. Our results demonstrate that hepcidin knockdown protected N27 cells from 6-OHDA induced apoptosis and that hepcidin plays a major role in reducing cellular iron burden and oxidative damage by possibly regulating cellular iron export mediated by ferroportin 1.

  8. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Directory of Open Access Journals (Sweden)

    Malin Berggrund

    2016-01-01

    Full Text Available The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA. Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  9. A seven day running training period increases basal urinary hepcidin levels as compared to cycling

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.J.; Swinkels, D.W.; Tjalsma, H.; Wiegerinck, E.T.G.; Trinder, D.; Peeling, P.

    2014-01-01

    BACKGROUND: This investigation compared the effects of an extended period of weight-bearing (running) vs. non-weight-bearing (cycling) exercise on hepcidin production and its implications for iron status. METHODS: Ten active males performed two separate exercise training blocks with either running (

  10. A multi-scale model of hepcidin promoter regulation reveals factors controlling systemic iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Guillem Casanovas

    2014-01-01

    Full Text Available Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades. Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation. We quantitatively analyzed how BMP6 and IL6 control hepcidin expression. Transcription factor (TF phosphorylation and reporter gene expression were measured under co-stimulation conditions, and the promoter was perturbed by mutagenesis. Using mathematical modeling, we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors, and experimentally validated the model predictions. Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signaling crosstalk is insignificant. We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis, which promotes iron homeostasis in vivo. IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal, because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site. This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation. Taken together, our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease.

  11. Hepcidin in obese children as a potential mediator of the association between obesity and iron deficiency.

    NARCIS (Netherlands)

    Giudice, E. Del; Santoro, N.; Amato, A.; Brienza, C.; Calabro, P.; Wiegerinck, E.T.G.; Cirillo, G.; Tartaglione, N.; Grandone, A.; Swinkels, D.W.; Perrone, L.

    2009-01-01

    CONTEXT: Obesity and iron deficiency are two of the most common nutritional disorders worldwide. Several studies found higher rates of iron deficiency in obese than in normal-weight children. Hepcidin represents the main inhibitor of intestinal iron absorption, and its expression is increased in adi

  12. Effect of exercise modality and intensity on post-exercise interleukin-6 and hepcidin levels

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.; Swinkels, D.W.; Tjalsma, H.; Trinder, D.; Peeling, P.

    2013-01-01

    The effect of exercise modality and intensity on Interleukin-6 (IL-6), iron status, and hepcidin levels was investigated. Ten trained male triathletes performed 4 exercise trials including low-intensity continuous running (L-R), low-intensity continuous cycling (L-C), high-intensity interval running

  13. Iron and hepcidin as risk factors in atherosclerosis: what do the genes say?

    NARCIS (Netherlands)

    Galesloot, T.E.; Janss, L.L.; Burgess, S.; Kiemeney, L.A.; Heijer, M. den; Graaf, J. de; Holewijn, S.; Benyamin, B.; Whitfield, J.B.; Swinkels, D.W.; Vermeulen, S.H.

    2015-01-01

    BACKGROUND: Previous reports suggested a role for iron and hepcidin in atherosclerosis. Here, we evaluated the causality of these associations from a genetic perspective via (i) a Mendelian randomization (MR) approach, (ii) study of association of atherosclerosis-related single nucleotide

  14. Hepcidin Response to Iron Therapy in Patients with Non-Dialysis Dependent CKD

    NARCIS (Netherlands)

    Gaillard, Carlo A.; Bock, Andreas H.; Carrera, Fernando; Eckardt, Kai-Uwe; Van Wyck, David B.; Bansal, Sukhvinder S.; Cronin, Maureen; Meier, Yvonne; Larroque, Sylvain; Roger, Simon D.; Macdougall, Iain C.

    2016-01-01

    Hepcidin is the key regulator of iron homeostasis but data are limited regarding its temporal response to iron therapy, and response to intravenous versus oral iron. In the 56-week, open-label, multicenter, prospective, randomized FIND-CKD study, 626 anemic patients with non-dialysis dependent

  15. Iron status and the acute post-exercise hepcidin response in athletes

    NARCIS (Netherlands)

    Peeling, P.; Sim, M.; Badenhorst, C.E.; Dawson, B.; Govus, A.D.; Abbiss, C.R.; Swinkels, D.W.; Trinder, D.

    2014-01-01

    This study explored the relationship between serum ferritin and hepcidin in athletes. Baseline serum ferritin levels of 54 athletes from the control trial of five investigations conducted in our laboratory were considered; athletes were grouped according to values <30 mug/L (SF<30), 30-50

  16. The effects of carbohydrate ingestion during endurance running on post-exercise inflammation and hepcidin levels.

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.; Wiegerinck, E.T.G.; Swinkels, D.W.; Townsend, M.A.; Trinder, D.; Peeling, P.

    2012-01-01

    The effect of carbohydrate (CHO) consumption during prolonged endurance running on post-exercise inflammation and hepcidin levels was investigated. Eleven well-trained male endurance athletes completed a graded exercise test, followed by two experimental running trials in a randomized order. The two

  17. Effect of exercise modality and intensity on post-exercise interleukin-6 and hepcidin levels

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.; Swinkels, D.W.; Tjalsma, H.; Trinder, D.; Peeling, P.

    2013-01-01

    The effect of exercise modality and intensity on Interleukin-6 (IL-6), iron status, and hepcidin levels was investigated. Ten trained male triathletes performed 4 exercise trials including low-intensity continuous running (L-R), low-intensity continuous cycling (L-C), high-intensity interval running

  18. A seven day running training period increases basal urinary hepcidin levels as compared to cycling

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.J.; Swinkels, D.W.; Tjalsma, H.; Wiegerinck, E.T.G.; Trinder, D.; Peeling, P.

    2014-01-01

    BACKGROUND: This investigation compared the effects of an extended period of weight-bearing (running) vs. non-weight-bearing (cycling) exercise on hepcidin production and its implications for iron status. METHODS: Ten active males performed two separate exercise training blocks with either running

  19. Hepcidin in obese children as a potential mediator of the association between obesity and iron deficiency.

    NARCIS (Netherlands)

    Giudice, E. Del; Santoro, N.; Amato, A.; Brienza, C.; Calabro, P.; Wiegerinck, E.T.G.; Cirillo, G.; Tartaglione, N.; Grandone, A.; Swinkels, D.W.; Perrone, L.

    2009-01-01

    CONTEXT: Obesity and iron deficiency are two of the most common nutritional disorders worldwide. Several studies found higher rates of iron deficiency in obese than in normal-weight children. Hepcidin represents the main inhibitor of intestinal iron absorption, and its expression is increased in

  20. Influence of post-exercise hypoxic exposure on hepcidin response in athletes

    NARCIS (Netherlands)

    Badenhorst, C.E.; Dawson, B.; Goodman, C.; Sim, M.; Cox, G.R.; Gore, C.J.; Tjalsma, H.; Swinkels, D.W.; Peeling, P.

    2014-01-01

    PURPOSE: To assess the influence of a simulated altitude exposure (~2,900 m above sea level) for a 3 h recovery period following intense interval running on post-exercise inflammation, serum iron, ferritin, erythropoietin, and hepcidin response. METHODS: In a cross-over design, ten well-trained male

  1. Cumulative effects of consecutive running sessions on hemolysis, inflammation and hepcidin activity.

    NARCIS (Netherlands)

    Peeling, P.; Dawson, B.; Goodman, C.; Landers, G.; Wiegerinck, E.T.G.; Swinkels, D.W.; Trinder, D.

    2009-01-01

    The effect of two running sessions completed within a 12-h period on hemolysis, inflammation, and hepcidin activity in endurance athletes was investigated. Ten males completed two experimental trials in a randomized, counterbalanced order. The two trials included (a) a one-running-session trial (T1)

  2. Iron status and the acute post-exercise hepcidin response in athletes

    NARCIS (Netherlands)

    Peeling, P.; Sim, M.; Badenhorst, C.E.; Dawson, B.; Govus, A.D.; Abbiss, C.R.; Swinkels, D.W.; Trinder, D.

    2014-01-01

    This study explored the relationship between serum ferritin and hepcidin in athletes. Baseline serum ferritin levels of 54 athletes from the control trial of five investigations conducted in our laboratory were considered; athletes were grouped according to values <30 mug/L (SF<30), 30-50 mug/

  3. Exercise-induced changes in iron status and hepcidin response in female runners.

    Directory of Open Access Journals (Sweden)

    Irena Auersperger

    Full Text Available BACKGROUND AND AIMS: Exercise-induced iron deficiency is a common finding in endurance athletes. It has been suggested recently that hepcidin may be an important mediator in this process. OBJECTIVE: To determine hepcidin levels and markers of iron status during long-term exercise training in female runners with depleted and normal iron stores. METHODS: Fourteen runners were divided into two groups according to iron status. Blood samples were taken during a period of eight weeks at baseline, after training and after ten days' recovery phase. RESULTS: Of 14 runners, 7 were iron deficient at baseline and 10 after training. Hepcidin was lower at recovery compared with baseline (p<0.05. The mean cell haemoglobin content, haemoglobin content per reticulocyte and total iron binding capacity all decreased, whereas soluble transferrin receptor and hypochromic red cells increased after training and recovery (p<0.05 for all. CONCLUSION: The prevalence of depleted iron stores was 71% at the end of the training phase. Hepcidin and iron stores decreased during long-term running training and did not recover after ten days, regardless of baseline iron status.

  4. The effects of carbohydrate ingestion during endurance running on post-exercise inflammation and hepcidin levels.

    NARCIS (Netherlands)

    Sim, M.; Dawson, B.; Landers, G.; Wiegerinck, E.T.G.; Swinkels, D.W.; Townsend, M.A.; Trinder, D.; Peeling, P.

    2012-01-01

    The effect of carbohydrate (CHO) consumption during prolonged endurance running on post-exercise inflammation and hepcidin levels was investigated. Eleven well-trained male endurance athletes completed a graded exercise test, followed by two experimental running trials in a randomized order. The two

  5. A novel assay method for the trace determination of Th and U in copper and lead using inductively coupled plasma mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    LaFerriere, Brian D.; Maiti, Tapas C.; Arnquist, Isaac J.; Hoppe, Eric W.

    2015-03-01

    This study describes a novel sample preparation and assay method developed in support of the MAJORANA DEMONSTRATOR experiment for the determination of thorium and uranium levels in copper and lead shielding components. Meticulously clean sample preparation methods combined with novel anion exchange separations for analyte pre-concentration and matrix removal were developed. Quantification was performed by inductively coupled plasma mass spectrometry. Detection limits of 0.0084 pg 232Th/g and 0.0106 pg 238U/g were determined for copper, while detection limits of 0.23 pg 232Th/g and 0.46 pg 238U/g were achieved for lead. These methods allow the Majorana Collaboration to accurately assay detector components and ensure that the experiment’s stringent radiopurity requirements are met.

  6. RAP-011, an activin receptor ligand trap, increases hemoglobin concentration in hepcidin transgenic mice.

    Science.gov (United States)

    Langdon, Jacqueline M; Barkataki, Sangjucta; Berger, Alan E; Cheadle, Chris; Xue, Qian-Li; Sung, Victoria; Roy, Cindy N

    2015-01-01

    Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-β superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis. © 2014 Wiley Periodicals, Inc.

  7. The various assays for measuring activity states of factor VIIa in plasma and therapeutic products: Diagnostic value and analytical usefulness in various pathophysiological states.

    Science.gov (United States)

    Amiral, Jean; Dunois, Claire; Amiral, Cédric; Seghatchian, Jerard

    2016-12-29

    The key coagulation factor FVII, and its activated form FVIIa, present a major interest for their role at the initiation phase of blood coagulation, and because they can activate all blood coagulation cascade, through the extrinsic, but also the intrinsic pathway. Blood activation initiated through FVII is first presented, as it is understood nowadays. Measurement of FVII and FVIIa were of main interest for epidemiological studies, but FVIIa contribution to assay results was only deduced. The introduction of specific FVIIa assays, functional or immunoassays, allowed measuring directly FVIIa without any interference of non-activated FVII, or other coagulation factors or their activated forms. The various methods available, and their characteristics are presented, with a special focus on two assays developed by our group for FVIIa (a clotting one and a chromogenic one). The FVIIa clotting assay shows evident superiority for measuring its activity in plasma, in pathophysiological conditions. The normal range is products, or for following recoveries in treated patients, including hemophiliacs with inhibitors, patients with severe bleeding risk (liver diseases, surgery, trauma, …), and lastly for measurement of its activity in therapeutic products.

  8. Use of the Treponema pallidum-specific captia syphilis IgG assay in conjunction with the rapid plasma reagin to test for syphilis.

    OpenAIRE

    1997-01-01

    The Captia Syphilis IgG enzyme immunoassay (EIA) was evaluated for use in conjunction with the rapid plasma reagin test (RPR) as a method to test for syphilis. A total of 1,288 serum specimens were tested by the routine laboratory protocol of the RPR followed by microhemagluttination assay for Treponema pallidum (MHA-TP) testing of RPR-reactive sera as well as the EIA-RPR protocol in which the automated EIA followed by a manual RPR test for EIA-positive specimens is used. When using the routi...

  9. A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers.

    Science.gov (United States)

    Sidler-Moix, Anne-Laure; Mercier, Thomas; Decosterd, Laurent A; Di Paolo, Ermindo R; Berger-Gryllaki, Markoulina; Cotting, Jacques; Pannatier, André

    2012-05-01

    The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented.

  10. Highly sensitive HPLC-DAD method for the assay of gefitinib in patient plasma and cerebrospinal fluid: application to a blood-brain barrier penetration study.

    Science.gov (United States)

    Fang, Luo; Song, Yu; Weng, Xu; Li, Fanzhu; Xu, Yaping; Lin, Nengming

    2015-12-01

    The quantification of intracranial gefitinib (GEF) exposure is limited owing to the sensitivity of analytical equipment. Although mass spectrometry (MS) is the preferred method because of its high sensitivity, the equipment is not available in many laboratories, especially in developing Asian countries. In this paper, we developed a highly sensitive high performance liquid chromatography-diode array detector (HPLC-DAD) method for the assay of GEF in human cerebrospinal fluid (CSF) and plasma. GEF was extracted from CSF and plasma by solid-phase extraction and liquid-liquid extraction, respectively. The chromatographic separation was performed on a C18 column with gradient elution of 0.1% triethylamine solution and acetonitrile, then finally detected at 344 nm. This method was validated and proved to be highly sensitive with a lower limit of quantitation value of 0.11 ng/mL in CSF and 11 ng/mL in plasma. The blood-brain barrier penetration ratio of GEF ranged from 1.48 to 2.41%. This method provides a reliable MS-independent solution for the quantitation of GEF in patients' CSF and plasma.

  11. Hepcidin-25 in chronic hemodialysis patients is related to residual kidney function and not to treatment with erythropoiesis stimulating agents

    National Research Council Canada - National Science Library

    Weerd, Neelke; Grooteman, Muriel; Bots, Michiel; Dorpel, Marinus; Hoedt, Claire; Mazairac, Albert; Nubé, Menso; Penne, Lars; Gaillard, Carlo; Wetzels, Jack; Wiegerinck, Erwin; Swinkels, Dorine; Blankestijn, Peter; Wee, Piet

    2012-01-01

    .... In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients...

  12. Increased serum hepcidin and alterations in blood iron parameters associated with asymptomatic P. falciparum and P. vivax malaria.

    NARCIS (Netherlands)

    Mast, Q. de; Syafruddin, D.; Keijmel, S.; Riekerink, T.O.; Deky, O.; Asih, P.B.; Swinkels, D.W.; Ven, A.J.A.M. van der

    2010-01-01

    BACKGROUND: Asymptomatic Plasmodium spp. infections and anemia are highly prevalent conditions in tropical regions. We studied whether asymptomatic parasitemia induces hepcidin- and/or cytokine-mediated iron maldistribution and anemia. DESIGN AND METHODS: A group of 1197 Indonesian schoolchildren,

  13. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    of the Aptima HIV-1 Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 (CAPCTMv2) for the quantification of HIV-1 RNA in plasma samples. STUDY DESIGN: The performance of the two tests was compared on 216 clinical plasma samples, on dilutions series in seven replicates of five clinical...

  14. Effect of oxygen plasma treatment on surface charge and wettability of PVC blood bag—In vitro assay

    Science.gov (United States)

    Khorasani, M. T.; Mirzadeh, H.

    2007-06-01

    Wettability and zeta potential studies were performed to characterize the hydrophobicity and surface charge of PVC blood bag samples and evaluate the effect of these properties on fibroblast cells growth. The surface properties of PVC and plasma treated PVC were compared by water drop contact angle and zeta potential measurement. Light microscopy was used to study the behavior of cell attachment and growth on these surfaces. Water drop contact angle measurement shows that the plasma treated PVC becomes more hydrophilic and wettability increased. Zeta potential and in vitro cell culture measurements noticed that the plasma treated PVC surface is more negatively charge and consequently attachment of the L929 fibroblast cells decreased on this surface.

  15. Effect of oxygen plasma treatment on surface charge and wettability of PVC blood bag-In vitro assay

    Energy Technology Data Exchange (ETDEWEB)

    Khorasani, M.T. [Biomaterial Department of Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of)]. E-mail: M.Khorasani@ippi.ac.ir; Mirzadeh, H. [Biomaterial Department of Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of)

    2007-06-15

    Wettability and zeta potential studies were performed to characterize the hydrophobicity and surface charge of PVC blood bag samples and evaluate the effect of these properties on fibroblast cells growth. The surface properties of PVC and plasma treated PVC were compared by water drop contact angle and zeta potential measurement. Light microscopy was used to study the behavior of cell attachment and growth on these surfaces. Water drop contact angle measurement shows that the plasma treated PVC becomes more hydrophilic and wettability increased. Zeta potential and in vitro cell culture measurements noticed that the plasma treated PVC surface is more negatively charge and consequently attachment of the L929 fibroblast cells decreased on this surface.

  16. Plasma erythropoietin by high-detectability immunoradiometric assay in untreated and treated patients with polycythaemia vera and essential thrombocythaemia

    Energy Technology Data Exchange (ETDEWEB)

    Carneskog, J.; Kutti, J.; Wadenvik, H. [Univ. of Goeteborg, Sahlgrenska Univ. Hospital, Dept. of Medicine, Haematology Section (Sweden); Lundberg, P.A.; Lindstedt, G. [Univ. of Goeteborg, Sahlgrenska Univ. Hospital, Dept. of Clinical Chemistry and Transfusion Medicine (Sweden)

    1998-12-31

    By using an immunoradiometric method with a stated detection limit of {<=}1 IU/l (stated normal reference limit in adults 3.7-16 IU/l) we determined EDTA-plasma erythropoietin (EPO) in 58 patients with polycythaemia vera (PV) and 49 patients with essential thrombocythaemia (ET). At the time of blood sampling, 20 of the PV patients were newly diagnosed and untreated, 23 were treated by phlebotomy only, and 30 also received myelosuppressive treatment (with 32P, hydroxyurea of alpha-interferon). Of the ET patients 24 were untreated and 28 received myelosuppressive therapy. For comparison plasma EPO was also determined in 10 patients with pseudopolycythaemia (PP). In this latter group the results for plasma EPO agreed well with the cited normal reference limits. The majority of untreated PV patients (12/20) had undetectable plasma EPO concentration, and the remainder all had values below the lower normal reference limit. Plasma EPO in PV was not significantly influenced by phlebotomy therapy. Twelve of the 24 untreated ET patients (50%) had plasma EPO values below the reference interval (undetectable in 2 patients). The mean EPO concentration was significantly lower in PV patients receiving phlebotomy therapy than in patients with untreated ET. In the total material of PV and ET treated with myelosuppressive agents the PV patients showed significantly lower values for EPO concentration than did patients with ET. The present results support the view that EPO measurements by high-detectability methods are diagnostically useful and should be included in the panel of new criteria for the diagnosis of PV. (au) 20 refs.

  17. Hepcidin-25 in diabetic chronic kidney disease is predictive for mortality and progression to end stage renal disease.

    Directory of Open Access Journals (Sweden)

    Martin Wagner

    Full Text Available Anemia is common and is associated with impaired clinical outcomes in diabetic chronic kidney disease (CKD. It may be explained by reduced erythropoietin (EPO synthesis, but recent data suggest that EPO-resistance and diminished iron availability due to inflammation contribute significantly. In this cohort study, we evaluated the impact of hepcidin-25--the key hormone of iron-metabolism--on clinical outcomes in diabetic patients with CKD along with endogenous EPO levels.249 diabetic patients with CKD of any stage, excluding end-stage renal disease (ESRD, were enrolled (2003-2005, if they were not on EPO-stimulating agent and iron therapy. Hepcidin-25 levels were measured by radioimmunoassay. The association of hepcidin-25 at baseline with clinical variables was investigated using linear regression models. All-cause mortality and a composite endpoint of CKD progression (ESRD or doubling of serum creatinine were analyzed by Cox proportional hazards models.Patients (age 67 yrs, 53% male, GFR 51 ml/min, hemoglobin 131 g/L, EPO 13.5 U/L, hepcidin-25 62.0 ng/ml were followed for a median time of 4.2 yrs. Forty-nine patients died (19.7% and forty (16.1% patients reached the composite endpoint. Elevated hepcidin levels were independently associated with higher ferritin-levels, lower EPO-levels and impaired kidney function (all p<0.05. Hepcidin was related to mortality, along with its interaction with EPO, older age, greater proteinuria and elevated CRP (all p<0.05. Hepcidin was also predictive for progression of CKD, aside from baseline GFR, proteinuria, low albumin- and hemoglobin-levels and a history of CVD (all p<0.05.We found hepcidin-25 to be associated with EPO and impaired kidney function in diabetic CKD. Elevated hepcidin-25 and EPO-levels were independent predictors of mortality, while hepcidin-25 was also predictive for progression of CKD. Both hepcidin-25 and EPO may represent important prognostic factors of clinical outcome and have the

  18. Meta-GWAS and Meta-Analysis of Exome Array Studies Do Not Reveal Genetic Determinants of Serum Hepcidin

    Science.gov (United States)

    Galesloot, Tessel E.; van Dijk, Freerk; Geurts-Moespot, Anneke J.; Girelli, Domenico; Kiemeney, Lambertus A. L. M.; Sweep, Fred C. G. J.; Swertz, Morris A.; van der Meer, Peter; Camaschella, Clara; Toniolo, Daniela; Vermeulen, Sita H.; van der Harst, Pim; Swinkels, Dorine W.

    2016-01-01

    Serum hepcidin concentration is regulated by iron status, inflammation, erythropoiesis and numerous other factors, but underlying processes are incompletely understood. We studied the association of common and rare single nucleotide variants (SNVs) with serum hepcidin in one Italian study and two large Dutch population-based studies. We genotyped common SNVs with genome-wide association study (GWAS) arrays and subsequently performed imputation using the 1000 Genomes reference panel. Cohort-specific GWAS were performed for log-transformed serum hepcidin, adjusted for age and gender, and results were combined in a fixed-effects meta-analysis (total N 6,096). Six top SNVs (p<5x10-6) were genotyped in 3,821 additional samples, but associations were not replicated. Furthermore, we meta-analyzed cohort-specific exome array association results of rare SNVs with serum hepcidin that were available for two of the three cohorts (total N 3,226), but no exome-wide significant signal (p<1.4x10-6) was identified. Gene-based meta-analyses revealed 19 genes that showed significant association with hepcidin. Our results suggest the absence of common SNVs and rare exonic SNVs explaining a large proportion of phenotypic variation in serum hepcidin. We recommend extension of our study once additional substantial cohorts with hepcidin measurements, GWAS and/or exome array data become available in order to increase power to identify variants that explain a smaller proportion of hepcidin variation. In addition, we encourage follow-up of the potentially interesting genes that resulted from the gene-based analysis of low-frequency and rare variants. PMID:27846281

  19. Comparison of the artus HIV-1 QS-RGQ and VERSANT HIV-1 RNA 1.0 assays for quantitative detection of human immunodeficiency virus type 1 in plasma samples.

    Science.gov (United States)

    Dvir, Roee; Canducci, Filippo; Racca, Sara; Rolla, Serena; Stucchi, Sonia; Clementi, Massimo

    2015-07-01

    Several integrated diagnostic platforms to quantify human immunodeficiency virus type-1 viremia have been developed in recent years. We evaluated the performances of the Artus HIV-1 QS-RGQ assay, using the complete QIAsymphony RGQ workflow. 192 clinical plasma specimens and external control panel samples were analyzed, using the Artus assay and the routine Siemens VERSANT HIV-1 RNA 1.0 assay. Three samples were excluded due to amplification inhibition. Among the remaining 189 specimens, 130 samples were detected as positive (above the limit of detection by both assays; median log10 difference: 0.01) and 18 samples were detected as negative. Eight samples (4.2%), all slightly above the limit of detection of the Versant assay, were negative with the Artus assay. The remaining 33 samples (beside 3 negative by Artus assay) were positive by both assays, but below the limit of detection at least in one of them. Results from the external panel samples showed a mean Log10 variation of -0.18 and -0.45 for the Versant and the Artus assays, respectively. As both assays showed highly correlated results, the QIAsymphony RGQ system, using the Artus HIV-1 QS-RGQ assay, could be considered a potential platform for HIV-1 RNA quantification in plasma.

  20. Serum hepcidin levels in Helicobacter pylori-infected children with iron-deficiency anemia: a case-control study.

    Science.gov (United States)

    Azab, Seham F A; Esh, Asmaa M H

    2013-11-01

    Recently, hepcidin, an antimicrobial-like peptide hormone, has evolved as the master regulator of systemic iron homeostasis. Hepcidin integrates signals from diverse physiological inputs, forming a key connection between iron trafficking and response to infection. In this study, we aimed to investigate whether Helicobacter pylori infection modulates serum hepcidin level and response to oral iron therapy in children with iron-deficiency anemia. This was a case-control study including 60 children with iron-deficiency anemia (IDA; 30 H. pylori infected and 30 H. pylori noninfected) and 30 healthy children with comparable age and gender as the control group. Iron parameters including serum iron, ferritin, transferrin, total iron binding capacity, and transferrin saturation and serum hepcidin levels were assessed initially and after 3 months of oral iron therapy for IDA. Compared to the control group, serum hepcidin was significantly lower in H. pylori-noninfected children with IDA (P iron therapy (P iron therapy (P > 0.05). Although hepcidin showed significant positive correlations with serum ferritin, hemoglobin (Hb), iron, and transferrin saturation in noninfected children with IDA (P iron, and transferrin saturation in H. pylori-infected children with IDA (P iron therapy in children with iron-deficiency anemia.

  1. Obesity modulate serum hepcidin and treatment outcome of iron deficiency anemia in children: a case control study.

    Science.gov (United States)

    Sanad, Mohammed; Osman, Mohammed; Gharib, Amal

    2011-07-19

    Recently, hepcidin expression in adipose tissue has been described and shown to be increased in patients with severe obesity. We tried to assess the effect of obesity on hepcidin serum levels and treatment outcome of iron deficiency anemia in children. This was a case control study included 70 children with iron deficiency anemia "IDA" (35 obese and 35 non-obese) and 30 healthy non-obese children with comparable age and sex(control group). Parameters of iron status (Serum iron, ferritin, transferrin, total iron binding capacity and transferrin saturation) and serum hepcidin levels were assessed initially and after 3 months of oral iron therapy for IDA. Compared to the control group, serum hepcidin was significantly lower in non-obese children with IDA(p children with IDA (p children with IDA after 3 months of iron therapy (P children showed non-significant change in hepcidin level after iron therapy (p > 0.05). Although hepcidin showed significant positive correlations with Hb, serum iron and transferrin saturation in non-obese children with IDA, it showed significant negative correlations with Hb, serum iron and transferrin saturation in obese children with IDA (P iron therapy in childhood iron deficiency anemia.

  2. Low hepcidin levels in severely anemic malawian children with high incidence of infectious diseases and bone marrow iron deficiency.

    Science.gov (United States)

    Jonker, Femkje A M; Calis, Job C J; Phiri, Kamija; Kraaijenhagen, Rob J; Brabin, Bernard J; Faragher, Brian; Wiegerinck, Erwin T; Tjalsma, Harold; Swinkels, Dorine W; van Hensbroek, Michael Boele

    2013-01-01

    A reliable diagnostic biomarker of iron status is required for severely anemic children living in malarious areas because presumptive treatment with iron may increase their infection risk if they are not iron deficient. Current biomarkers are limited because they are altered by host inflammation. In this study hepcidin concentrations were assessed in severely anemic children living in a highly malarious area of Malawi and evaluated against bone marrow iron in order to determine the usefulness of hepcidin as a point of care test. 207 severely anemic children were assessed for levels of hepcidin, ferritin, serum transferrin receptor, erythropoietin, hematological indices, C-reactive protein, interleukin-6, malaria parasites and HIV infection. Deficiency of bone marrow iron stores was graded and erythroblast iron incorporation estimated. Interaction of covariates was assessed by structural-equation-modeling. Hepcidin was a poor predictor of bone marrow iron deficiency (sensitivity 66.7%; specificity 48.5%), and of iron incorporation (sensitivity 54.2%; specificity 61.8%), and therefore would have limitations as a point of care test in this category of children. As upregulation of hepcidin by inflammation and iron status was blunted by erythropoietin in this population, enhanced iron absorption through the low hepcidin values may increase infection risk. Current recommendations to treat all severely anemic children living in malarious areas with iron should therefore be reconsidered.

  3. Iron regulation in athletes: exploring the menstrual cycle and effects of different exercise modalities on hepcidin production.

    Science.gov (United States)

    Sim, Marc; Dawson, Brian; Landers, Grant; Trinder, Debbie; Peeling, Peter

    2014-04-01

    The trace element iron plays a number of crucial physiological roles within the body. Despite its importance, iron deficiency remains a common problem among athletes. As an individual's iron stores become depleted, it can affect their well-being and athletic capacity. Recently, altered iron metabolism in athletes has been attributed to postexercise increases in the iron regulatory hormone hepcidin, which has been reported to be upregulated by exercise-induced increases in the inflammatory cytokine interleukin-6. As such, when hepcidin levels are elevated, iron absorption and recycling may be compromised. To date, however, most studies have explored the acute postexercise hepcidin response, with limited research seeking to minimize/attenuate these increases. This review summarizes the current knowledge regarding the postexercise hepcidin response under a variety of exercise scenarios and highlights potential areas for future research-such as: a) the use of hormones though the female oral contraceptive pill to manipulate the postexercise hepcidin response, b) comparing the use of different exercise modes (e.g., cycling vs. running) on hepcidin regulation.

  4. A fast reverse-phase high performance liquid chromatographic tandem mass spectrometry assay for the quantification of clindamycin in plasma and saliva using a rapid resolution package.

    Science.gov (United States)

    Catena, Esther; Perez, Guiomar; Sadaba, Belen; Azanza, Jose Ramón; Campanero, Miguel Angel

    2009-11-01

    A new method for the quantitative analysis of clindamycin in human plasma and saliva by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) has been developed using a rapid resolution C18 column (2.1 mm x 30 mm x 3.5 microm). A simple deproteinization procedure was applied to the samples before analysis. Multiple reaction monitoring (MRM) mode of precursor-product ion transitions for clindamycin (425.1/126.1) and the internal standard, lincomycin (407.2/126.0) was used. Chromatographic separation was achieved at 0.6 ml/min in less than 1.5 min, with improved peak resolution and sensitivity between drug and internal standard. The assay exhibited a linear dynamic range between 0.05 and 15.0 microg/ml and gave a determination coefficient of 0.991 or better. The limit of quantification of the method was 10 ng/ml in both biological samples. Intra-day and inter-day precision ranged from 7.5% to 11.5%. Good accuracy was observed for both the intra-day and inter-day assays (R.S.D. below +/-4%). The suitability of the developed method for the analysis of clindamycin in plasma and saliva samples was demonstrated by the measure of clindamycin in samples taken up to 6h after oral and intravenous administration of this drug in infectious patients.

  5. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA.

    Science.gov (United States)

    Elbeik, Tarek; Nassos, Patricia; Kipnis, Patricia; Haller, Barbara; Ng, Valerie L

    2005-08-01

    Separation and storage of plasma within 2 h of phlebotomy is required for the VACUTAINER PPT Plasma Preparation Tube (PPT) versus 4 h for the predecessor VACUTAINER EDTA tube for human immunodeficiency virus type 1 (HIV-1) viral load (HIVL) testing by the VERSANT HIV-1 RNA 3.0 assay (branched DNA). The 2-h limit for PPT imposes time constraints for handling and transporting to the testing laboratory. This study compares HIVL reproducibility from matched blood in EDTA tubes and PPTs and between PPT pairs following processing within 4 h of phlebotomy, stability of plasma HIV-1 RNA at 24- and 72-h room temperature storage in the tube, and comparative labor and supply requirements. Blood from 159 patients was collected in paired tubes (EDTA/PPT or PPT/PPT): 86 paired EDTA tubes and PPTs were processed 4 h following phlebotomy and their HIVLs were compared, 42 paired PPT/PPT pairs were analyzed for intertube HIVL reproducibility, and 31 PPT/PPT pairs were analyzed for HIV-1 RNA stability by HIVL. Labor and supply requirements were compared between PPT and EDTA tubes. PPTs produce results equivalent to standard EDTA tube results when processed 4 h after phlebotomy. PPT intertube analyte results are reproducible. An average decrease of 13% and 37% in HIVL was observed in PPT plasma after 24 and 72 h of room temperature storage, respectively; thus, plasma can be stored at room temperature up to 24 h in the original tube. PPTs offer labor and supply savings over EDTA tubes.

  6. Identification of Guanosine 5‧-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway

    Science.gov (United States)

    Angmo, Stanzin; Tripathi, Neha; Abbat, Sheenu; Sharma, Shailesh; Singh, Shelley Sardul; Halder, Avishek; Yadav, Kamalendra; Shukla, Geeta; Sandhir, Rajat; Rishi, Vikas; Bharatam, Prasad V.; Yadav, Hariom; Singhal, Nitin Kumar

    2017-01-01

    Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5‧-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI.

  7. UPLC-MRM Mass Spectrometry Method for Measurement of the Coagulation Inhibitors Dabigatran and Rivaroxaban in Human Plasma and Its Comparison with Functional Assays.

    Directory of Open Access Journals (Sweden)

    Joachim Kuhn

    least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs.Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma.

  8. Molecular characterization, phylogeny and expression of a hepcidin gene in the blotched snakehead Channa maculata.

    Science.gov (United States)

    Gong, Li-cai; Wang, Hao; Deng, Li

    2014-05-01

    A hepcidin-like gene (cmHep) was cloned and characterized from the liver of the blotched snakehead Channa maculata. The complete cmHep cDNA was 756 bp in length, containing an open reading frame of 270 bp (encoding 89 amino acids), flanked by 210 bp and 276 bp of 5' and 3' untranslated regions, respectively. The deduced peptide of 89 amino acids consisted of 24 aa, 40 aa and 25 aa for signal peptide, prodomain and mature peptide, respectively. The mature peptide had eight cysteines at the identical conserved positions in common with most of other known hepcidins in vertebrates. cmHepc gene displayed a tripartite structure (three exons interrupted by two introns), which organisation was conserved between the blotched snakehead and other fish species. Phylogenetic analysis of hepcidins from C. maculata and other vertebrates showed that major phylogenetic grouping of fish hepcidin coincided with the current euteleosts classification, indicating the multiphyletic evolution of hepcidin in the teleosts. In the Acanthopterygii subclade, there were two distinct additional subclades named as HAMP-Ac1 and HAMP-Ac2. The blotched snakehead hepcidin was in the group HAMP-Ac1, which has the hypothetical iron regulatory sequence [Q-S/I-H-L/I-S/A] motif in N-terminal of mature peptide. The RT-PCR showed cmHep mRNA transcripts were widely distributed in all tissues tested in the blotched snakehead including the liver, gill, intestine, spleen, head kidney and peripheral white blood cell. The most abundant of cmHep mRNA was detected in liver. A significant up-regulation of cmHep expression was detected only in head kidney at 24h post-challenge with Vibrio parahaemolyticus in blotched snakehead adults, no significant differences found in liver, gill, intestine and spleen. The cmHep expression was up-regulated in spleen, head kidney and intestine at 24h post-injection with LPS in blotched snakehead juveniles, liver cmHep expression was not altered. Iron overloading and poly I

  9. Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay for the determination of sanfetrinem in human plasma.

    Science.gov (United States)

    De Nardi, C; Braggio, S; Ferrari, L; Fontana, S

    2001-10-25

    A rapid, selective and accurate high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 microl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C18(2), 50x2.0 (5 microm) column with a mobile phase consisting of water-acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 microg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.

  10. Effects of Rutin and Hesperidin and Their Al(III and Cu(II Complexes on in Vitro Plasma Coagulation Assays

    Directory of Open Access Journals (Sweden)

    Vesna Kuntić

    2011-02-01

    Full Text Available Two flavonoids, rutin and hesperidin, were investigated in vitro for anticoagulant activity through coagulation tests: activated partial thromboplastin time (aPTT, prothrombin time (PT and thrombin time (TT. Only an ethanolic solution of rutin at the concentration of 830 µM prolonged aPTT, while TT and PT were unaffected. In order to evaluate whether the prolongation of aPTT was due to the decrease of coagulation factors, the experiment with deficient plasma was performed, showing the effects on factors VIII and IX. Since pharmacological activity of flavonoids is believed to increase when they are coordinated with metal ions, complexes of these flavonoids with Al(III and Cu(II ions were also tested. The results showed that complexes significantly prolonged aPTT and had no effects on PT and TT. Assay with deficient plasma (plasma having the investigated factor at less then 1% revealed that complexes could bind to the coagulation factors, what may lead to a non-specific inhibition and aPTT prolongation. An effort was made to correlate stability of complexes with their anticoagulant properties.

  11. Reliable LC-MS/MS assay for the estimation of rilpivirine in human plasma: application to a bioequivalence study and incurred sample reanalysis.

    Science.gov (United States)

    Gupta, Ajay; Guttikar, Swati; Patel, Yogesh; Shrivastav, Pranav S; Sanyal, Mallika

    2015-04-01

    A simple, precise, and rapid stable isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of rilpivirine, a non-nucleoside reverse transcriptase inhibitor in human plasma. Rilpivirine and its deuterated analogue, rilpivirine-d6, used as an internal standard (IS) were quantitatively extracted by liquid-liquid extraction with methyl-tert-butyl ether and diethyl ether solvent mixture from 50 μL plasma. The chromatography was achieved on Gemini C18 (150 × 4.6 mm, 5 µm) analytical column in a run time of 2.2 min. The precursor → product ion transitions for rilpivirine (m/z 367.1 → 128.0) and IS (m/z 373.2 → 134.2) were monitored on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL. The mean extraction recovery for rilpivirine (94.9%) and IS (99.9%) from spiked plasma samples was consistent and reproducible. The IS-normalized matrix factors for rilpivirine ranged from 0.98 to 1.02 across three quality controls. Bench top, freeze-thaw, wet extract, and long-term stability of rilpivirine was examined in spiked plasma samples. The application of the method was demonstrated by a bioequivalence study with 25 mg rilpivirine tablet formulation in 40 healthy subjects. The assay reproducibility was shown by reanalysis of 200 study samples and the % change in the concentration of repeat values from the original values was within ±15%.

  12. Associations of common variants in HFE and TMPRSS6 with iron parameters are independent of serum hepcidin in a general population: a replication study.

    Science.gov (United States)

    Galesloot, Tessel E; Geurts-Moespot, Anneke J; den Heijer, Martin; Sweep, Fred C G J; Fleming, Robert E; Kiemeney, Lambertus A L M; Vermeulen, Sita H; Swinkels, Dorine W

    2013-09-01

    Genome-wide association studies have convincingly shown that single nucleotide polymorphisms (SNPs) in HFE and TMPRSS6 are associated with iron parameters. It was commonly thought that these associations could be explained by the intermediate effect on hepcidin concentration. A recent study in an isolated Italian population, however, concluded that these associations were not exclusively dependent on hepcidin values. We report here the second study to investigate the role of hepcidin in the associations between common variants in HFE and TMPRSS6 with iron parameters. We extracted 101 SNPs in HFE and TMPRSS6 from genome-wide imputed SNP data of 1832 individuals from the general population (Nijmegen Biomedical Study). Single locus and haplotype associations with serum iron parameters and hepcidin were studied using linear regression analyses. We found that HFE rs1800562 and TMPRSS6 rs855791 are the main determinants of HFE and TMPRSS6 related variation in serum iron, ferritin, transferrin saturation, and total iron binding capacity. These SNPs are associated with the ratios hepcidin/ferritin (pserum hepcidin (p>0.2). Adjustment for hepcidin or the ratio hepcidin/ferritin did not decrease the strength of the SNP-iron parameter associations. Our results do not support an intermediate role for hepcidin in the SNP-iron parameter associations, which confirms previous findings, and indicate a pleiotropic SNP effect on the hepcidin ratios and the iron parameters. Taken together, this suggests that there might be other, yet unknown, serum hepcidin independent mechanisms which play a role in the association of HFE and TMPRSS6 variants with serum iron parameters.

  13. CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L.

    Science.gov (United States)

    Mobarrez, Fariborz; Sjövik, Carolina; Soop, Anne; Hållström, Lars; Frostell, Claes; Pisetsky, David S; Wallén, Håkan

    2015-01-01

    CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary

  14. Decreasing TfR1 expression reverses anemia and hepcidin suppression in β-thalassemic mice.

    Science.gov (United States)

    Li, Huihui; Choesang, Tenzin; Bao, Weili; Chen, Huiyong; Feola, Maria; Garcia-Santos, Daniel; Li, Jie; Sun, Shuming; Follenzi, Antonia; Pham, Petra; Liu, Jing; Zhang, Jinghua; Ponka, Prem; An, Xiuli; Mohandas, Narla; Fleming, Robert; Rivella, Stefano; Li, Guiyuan; Ginzburg, Yelena

    2017-02-01

    Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apo-transferrin leads to more effective erythropoiesis, decreasing erythroferrone and de-repressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apo-transferrin's effect is mediated via decreased TfR1 expression, and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apo-transferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect which can be reversed by the administration of exogenous apo-transferrin. In vitro experiments demonstrate that apo-transferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/- mice--themselves exhibiting iron-restricted erythropoiesis with increased hepcidin--with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin de-repression, and increased erythroid enucleation relative to β-thalassemic mice. Our data demonstrates for the first time that TfR1+/- haplo-insufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either via directly induced haplo-insufficiency or exogenous apo-transferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron

  15. Hepcidin研究的认识%Current status of research on hepcidin

    Institute of Scientific and Technical Information of China (English)

    李蓉生

    2010-01-01

    @@ 2000年,Krause等[1]首先从人血浆超滤液中发现hepcidin,2001年Park等[2]在尿液中发现,并证明其在体外对某些细菌及真菌有抑制作用.以后的研究证实人类的hepcidin基因位于19号染色体q13.1上.

  16. Is hepcidin the bridge linking Helicobacter pylori and anemia of chronic infection? A research proposal.

    Science.gov (United States)

    Pellicano, R; Rizzetto, M

    2004-09-01

    Since the last decade, several studies have reported on the link between chronic Helicobacter pylori (H. pylori) or Helicobacter species (H. species) infection and a variety of extragastric manifestations, comprising iron-deficiency anemia. A crucial question concerns which possible pathogenic mechanism of H. pylori infection may be involved in chronic anemia. Recent findings support the hypothesis that in subjects with H. pylori-positive gastritis, concomitant changes in intragastric pH and ascorbic acid are present that might play a role in impairing alimentary iron absorption with consequent sideropenic anemia. It has also been speculated that H. pylori infected antrum could act as a sequestering focus for iron. The bacterium enhances gastric lactoferrin, which captures iron from transferrin. The iron thus bound to lactoferrin is in turn picked up by the bacterium, by means of its outer membrane receptors, for its own growth. These models, however, are not able to answer why iron-deficiency anemia does not develop in all infected subjects. Recently, a new anti-microbial liver-made peptide, namely hepcidin, has been characterised. The link between hepcidin induction, inflammation and anemia both in humans and in animal models supports its key role as mediator of anemia of inflammation. In the present paper, we highlight the data available on the association between H. pylori and iron-deficiency anemia and, we propose to evaluate a possible mechanism involving hepcidin in a bridging role linking the infection to the anemia.

  17. Iron as the Key Modulator of Hepcidin Expression in Erythroid Antibody-Mediated Hypoplasia

    Directory of Open Access Journals (Sweden)

    J. C. Fernandes

    2014-01-01

    Full Text Available Erythroid hypoplasia (EH is a rare complication associated with recombinant human erythropoietin (rHuEPO therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy.

  18. Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

    DEFF Research Database (Denmark)

    Bisp, Marianne R; Bor, Mustafa Vakur; Heinsvig, Else-Marie

    2002-01-01

    Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6...... were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate...... buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all...

  19. Determination of platinum group elements by inductively coupled plasma-mass spectrometry combined with nickel sulfide fire assay and tellurium coprecipitation

    Science.gov (United States)

    Sun, Yali; Guan, Xiyun; Du, Andao

    1998-09-01

    A method was developed for the determination of trace platinum group elements (PGEs) by nickel sulfide fire assay inductively coupled plasma-mass spectrometry (ICP-MS). With isotope dilution, the improved technique gives precise Os content data. Through the purification of the reagent nickel oxide, reagent blank was greatly reduced. Results obtained for the standard reference materials (SRM) GPt-1-GPt-7(GBW 07288-07294, China), DZ Σ-2 (GBW 07102, China) and Guilin Cu-Ni Ore are in good agreement with the recommended values for platinum group elements. The detection limits ranged from 0.01 to 0.39 ng/g. The relative standard deviations for Ru, Rh, Pd and Ir were less than 5%, for Os less than 1%, and Pt less than 8% for SRM GPt-6.

  20. Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

    Science.gov (United States)

    Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

    2015-10-01

    Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

  1. SPE-UPLC-MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women$

    Institute of Scientific and Technical Information of China (English)

    Pravin G. Vanol; Puran Singhal; Priyanka A. Shah; Jaivik V. Shah; Pranav S. Shrivastav; Mallika Sanyal

    2016-01-01

    A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method is described for determination of letrozole in human plasma. Following solid phase ex-traction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was per-formed on Acquity UPLC BEH C18 (50 mm ? 2.1 mm, 1.7 mm) column using methanol-0.1%formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spec-trometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM) following the transitions at m/z 286.2-217.0 and m/z 290.2-221.0, respectively. The calibration plots were linear through the con-centration range of 0.10–100 ng/mL (r2Z0.9990) using 100 mL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was r 5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR) of 74 subject samples.

  2. Use of delayed gamma rays for active non-destructive assay of {sup 235}U irradiated by pulsed neutron source (plasma focus)

    Energy Technology Data Exchange (ETDEWEB)

    Andola, Sanjay; Niranjan, Ram [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kaushik, T.C., E-mail: tckk@barc.gov.in [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Rout, R.K. [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kumar, Ashwani; Paranjape, D.B.; Kumar, Pradeep; Tomar, B.S.; Ramakumar, K.L. [Radioanalytical Chemistry Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Gupta, S.C. [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

    2014-07-01

    A pulsed neutron source based on plasma focus device has been used for active interrogation and assay of {sup 235}U by monitoring its delayed high energy γ-rays. The method involves irradiation of fissile material by thermal neutrons obtained after moderation of a burst of neutrons emitted upon fusion of deuterium in plasma focus (PF) device. The delayed gamma rays emitted from the fissile material as a consequence of induced fission were detected by a large volume sodium iodide (NaI(Tl)) detector. The detector is coupled to a data acquisition system of 2k input size with 2k ADC conversion gain. Counting was carried out in pulse height analysis mode for time integrated counts up to 100 s while the temporal profile of delayed gamma has been obtained by counting in multichannel scaling mode with dwell time of 50 ms. To avoid the effect of passive (natural) and active (from surrounding materials) backgrounds, counts have been acquired for gamma energy between 3 and 10 MeV. The lower limit of detection of {sup 235}U in the oxide samples with this set-up is estimated to be 14 mg.

  3. High-throughput LC-MS/MS assay for 6-methoxy-2-naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study.

    Science.gov (United States)

    Patel, Bhavin N; Sharma, Naveen; Sanyal, Mallika; Prasad, Arpana; Shrivastav, Pranav S

    2008-11-01

    A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.

  4. High-throughput assay for quantification of the plasma concentrations of thiopental using automated solid phase extraction (SPE) directly coupled to LC-MS/MS instrumentation.

    Science.gov (United States)

    Moosavi, Seyed Mojtaba; Shekar, Kiran; Fraser, John; Smith, Maree T; Ghassabian, Sussan

    2016-12-01

    Most previous assays for thiopental are time-consuming due to laborious sample extraction steps prior to analysis using gas chromatography or high pressure liquid chromatography. Here, we describe the first high-throughput liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for quantification of thiopental concentrations in samples of human plasma. Robotic on-line solid phase extraction (SPE) was used to elute the analytes of interest from samples of human plasma (50μL) loaded onto C18 SPE cartridges to which were added aliquots (50μL) of internal standard solution (thiopental-d5 100ng/mL) and 0.5% formic acid in water (100μL). Cartridges were washed using 10% methanol in ammonium acetate buffer (50mM, pH 7) before elution with mobile phase comprising 0.1% formic acid in water and acetonitrile with a flow rate of 0.55mL/min using a 7.2min run time. The analytes were separated on a C18 XTerra(®) analytical column. Mass spectrometry detection was performed using a QTrap 5500 mass spectrometer (AB Sciex) with negative ionisation. The multiple reaction monitoring (MRM) transitions for thiopental and the internal standard were 241→58, and 246→58, respectively. The calibration curve was linear over a range of 6-600ng/mL. Thiopental was stable in human plasma samples for at least 36h in the autosampler, as well as after three cycles of freeze and thaw, and after 3h storage at room temperature. The absolute recovery and matrix effect were 102% and 6.9%, respectively, and the within-run and between-run precision and accuracy were ≤15%. Our method is fully-validated and satisfies the requirements of the 2012 European Medicines Agency (EMEA) guideline for Bioanalytical Method Validation. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP).

    Science.gov (United States)

    Wang, Ke-Jian; Bo, Jun; Yang, Ming; Hong, Hua-Sheng; Wang, Xin-Hong; Chen, Fang-Yi; Yuan, Jian-Jun

    2009-04-01

    Hepcidin is known to be expressed in fish with bacterial challenge and iron overload. Here we first report the hepcidin expression induced in the developmental stages from embryo to fry of red sea bream (Pagarus major) and in juvenile black porgy (Acanthopagrus schlegelii B.) upon continuous waterborne exposure to BaP. The gene expression of CYP1A1 and IgL (immunoglobulin light chain) were both measured. Expression of the Pagarus major hepcidin gene (PM-hepc) was increased in post hatch fry at 24 h and 120 h exposure to BaP at concentrations of 0.1, 0.5 and 1.0 microg/l, respectively. The gene expression pattern was comparable to that of CYP1A1 but different from that of IgL. In addition, a high number of AS-hepc2 transcripts (Acanthopagrus schlegelii B. hepcidin gene) were detected in the liver upon exposure to 1.0 microg/l BaP. This study demonstrates that hepcidin gene expression is significantly induced in BaP-exposed red sea bream and black porgy.

  6. Expression of hepcidin at the choroid plexus in normal aging rats is associated with IL-6/Stat3 signaling pathway.

    Science.gov (United States)

    Liu, Chong-Bin; Wang, Rui; Dong, Miao-Wu; Gao, Xi-Ren; Yu, Feng

    2014-12-25

    Accumulating evidence has revealed that brain iron concentrations increase with aging, and the choroid plexus (CP) may be at the basis of iron-mediated toxicity and the increase in inflammation and oxidative stress that occurs with aging. The mechanism involves not only hepcidin, the key hormone in iron metabolism, but also iron-related proteins and signaling-transduction molecules, such as IL-6 and signal transducer and activator of transcription 3 (Stat3). The aim of the present study was to investigate the correlation between the IL-6/Stat3 signaling pathway and hepcidin at the CP in normal aging. Quantitative real time PCR and Western blot were used to determine the alterations in specific mRNA and corresponding protein changes at the CP at ages of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months in Brown-Norway/Fischer (B-N/F) rats. The results demonstrated that hepcidin mRNA level at the CP kept stable in young rats (from 3 to 18 months), and increased with aging (from 21 to 36 months). The alterations of IL-6/p-Stat3 mRNA and protein expressions in normal aging were in accordance with that of hepcidin mRNA. Our data suggest that IL-6 may regulate hepcidin expression at the CP, upon interaction with the cognate cellular receptor, and through the Stat3 signaling transduction pathway.

  7. Genomic organization and tissue-specific expression of hepcidin in the pacific mutton hamlet, Alphestes immaculatus (Breder, 1936).

    Science.gov (United States)

    Masso-Silva, Jorge; Diamond, Gill; Macias-Rodriguez, Maria; Ascencio, Felipe

    2011-12-01

    Hepcidin is a cysteine-rich peptide involved in iron metabolism, inflammatory response and as antimicrobial peptide. Despite the fact that hepcidins have been identified in several fish species, only few have been completely characterized. This study, described the identification and complete molecular characterization of the hepcidin antimicrobial peptide 1 (HAMP1) gene of Alphestes immaculatus. Moreover, its specific expression level at both basal and lipopolysaccharide (LPS)-induced conditions in different tissues was also determined by real-time PCR. Results showed that the HAMP1gene consists of three exons and two introns encoding a preprohepcidin composed of 90 aa (24 aa for signal peptide, 40 aa for prodomain and 26 aa for mature peptide). The promoter region analysis revealed a TATA box sequence and several putative transcription factor binding sites. A comparative analysis showed CEBPα, CEBPβ, NF-kB, HNF3, GATA-1 and c-Rel as the most common found in fishes. The mature peptide possesses a pI of 8.34, which is the average among fish hepcidin. In addition, the structural modeling showed a hairpin structure with four putative disulfide bonds. A phylogenetic analysis revealed that this hepcidin gene is a HAMP1 class, and is clustered into the same group with the Serranid fish Epinephelus moara and the Antarctic fish Lycodichthys dearborni. Finally, the relative expression levels showed high basal values in liver and muscle, whereas in LPS-induced fish the relative expression tendency changed, with the highest values in spleen and head kidney tissues. This study describes the completely characterized HAMP1 gene of A. immaculatus and their patterns of expression level at different conditions and in different tissues, showing by first time muscle hepcidin expression could be relevant in the immune response in fish. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. The role of hepcidin and haemojuvelin in the pathogenesis of iron disorders in patients with severe malnutrition

    Directory of Open Access Journals (Sweden)

    Justyna Przybyszewska

    2014-06-01

    Full Text Available introduction and objective. The clinical consequences of malnutrition are multi-directional and result in dysfunctions of the majority of internal organs and systems. The results of recent studies suggest that a significant role is played by malnutrition in pathophysiology of iron homeostasis disorders, but the underlying mechanism is unclear. The study describes the potential role of hepcidin and hemojuvelin in the pathogenesis of disorders of iron metabolism during malnutrition. state of knowledge. The participation of hepcidin in regulating iron homeostasis encompasses inhibiting the absorption of food iron from enterocytes and inhibiting the release of stored iron from the reticuloendothelial system cells. One of the factors that increases the post-translational level is the expression of hepcidin is IL-6. In studies focused on malnutrition it was observed that in persons with protein and energy deficits the level of proinflammatory cytokine, i.e. interleukin-6, in the serum, increased. The involvement of haemojuvelin in the overall iron homeostasis is related with the regulation of expression of the hepcidin coding gene on the transcription level. The highest haemojuvelin expression was observed in humans in skeletal muscles. Observations and analyses conducted in vivo allowed the conclusion that soluble HJV and cell-related haemojuvelin regulate hepcidin expression in response to changes in iron concentration. The research also demonstrated that soluble HJV neutralizes the inductive effect of IL-6 action on hepcidin expression. summary. It can be claimed that in persons with protein and/or protein-energetic malnutrition the muscle mass deficit may lead to insufficient production of haemojuvelin and sideropenia.

  9. Serum hepcidin levels are associated with serum triglycerides and interleukin-6 concentrations in patients with end-stage renal disease.

    Science.gov (United States)

    Samouilidou, Elisabeth; Pantelias, Konstantinos; Petras, Dimitrios; Tsirpanlis, George; Bakirtzi, Joulia; Chatzivasileiou, George; Tzanatos, Helen; Grapsa, Eirini

    2014-06-01

    Hepcidin has emerged as a peptide with a key role in the regulation of iron homeostasis in patients with chronic kidney disease (CKD), having a strong dependence on inflammation. Recent studies reveal that hepcidin may be also associated with the progression of atherosclerosis. This study was performed to analyze the relation of hepcidin to markers of atherosclerosis and inflammation in patients on dialysis. A total of 90 individuals were enrolled. Sixty patients with end-stage renal disease, who were on hemodialysis (HD) (N = 30) and peritoneal dialysis (N = 30) were compared with 30 normal controls (NC). Age, body mass index, time on dialysis, serum lipids, C-reactive protein (CRP) and interleukin-6 (IL-6) were measured and analyzed in correlation with hepcidin concentration. It was found that patients on HD and peritoneal dialysis have significantly higher (P triglycerides (r = 0.401, P = 0.005), HDL-C (r = -0.268, P = 0.048), CRP (r = 0.436, P = 0.0007) and IL-6 (r = 0.569, P triglycerides (β = 0.402, P = 0.041) and IL-6 (β = 0.559, P = 0.006). Moreover, patients with high triglycerides in combination with high IL-6 levels have significantly increased concentrations of hepcidin than those with low triglycerides and low IL-6 levels (P triglycerides and high IL-6 serum concentrations. This probably suggests that hepcidin may play a role to the progression of atherosclerosis and inflammation, but this hypothesis should be further evaluated.

  10. The radial immunodiffusion assay for plasma Histidine-rich Glycoprotein (HRG) based on a polyclonal antibody, shows a different specificity towards the two variants of a common amino acid polymorphism

    NARCIS (Netherlands)

    Hennis, B.C.; Hoffmann, J.J.M.L.; Kluft, C.

    1996-01-01

    Recently, two molecular weight forms of Histidine-rich Glycoprotein have been described which explain 59% of the variation in plasma HRG levels. Here, we demonstrate that this can partly be ascribed to a difference in specificity of the immuno assay for HRG towards the two alleles of the polymorphis

  11. Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

    Science.gov (United States)

    Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.

    2005-01-01

    This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

  12. Heterogeneity of pituitary and plasma prolactin in man: decreased affinity of big prolactin in a radioreceptor assay and evidence for its secretion

    Energy Technology Data Exchange (ETDEWEB)

    Garnier, P.E.; Aubert, M.L.; Kaplan, S.L.; Grumbach, M.M.

    1978-12-01

    Molecular heterogeneity of immunoreactive human PRL (IR-hPRL) plasma was assessed by exclusion chromatography in blood from 4 normal adults, 3 newborn infants, 2 late gestational women, 3 patients with primary hypothyroidism and high PRL levels, 2 with functional hyperprolactinemia, 3 with acromegaly, and 10 with PRL-secreting tumors. Three forms of PRL were detected: big-big hPRL, big hPRL, and little hPRL. In normal subjects, the proportion of big-big, big, and little hPRL components was 5.1%, 9.1%, and 85.8%, respectively, without change in the distribution after TRF stimulation. In 8 of 10 patients with PRL-secreting tumors, we detected a significantly higher proportion of big PRL. In 2 additional patients with prolactinomas, the proportion of big PRL was much higher. In 3 of 10 patients, the molecular heterogeneity of the tumor PRL was similar to that in plasma. In 1 acromegalic, there was a very high proportion of big-big hPRL. The PRL fractions were tested in a radioreceptor assay (RRA) using membranes from rabbit mammary gland. Big PRL was much less active than little PRL in the RRA. The fractions were rechromatographed after storage. Big PRL partially distributed as little or big-big PRL, while little PRL remained unchanged. Big-big PRL from tumor extract partially converted into big and little PRL. The big PRL obtained by rechromatography had low activity in the RRA. These observations suggest at least part of the receptor activity of big PRL may arise from generation of or contamination by little PRL. The decreased binding affinity of big PRL in the RRA also indicates that big PRL has little, if any, biological activity. The evidence suggests big PRL is a native PRL dimer linked by intermolecular disulfide bonds which arises in the lactotrope as a postsynthetic product or derivative and is not a true precursor prohormone.

  13. SPE–UPLC–MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women

    Directory of Open Access Journals (Sweden)

    Pravin G. Vanol

    2016-08-01

    Full Text Available A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS method is described for determination of letrozole in human plasma. Following solid phase extraction (SPE of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was performed on Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm column using methanol-0.1% formic acid in water (85:15, v/v as the mobile phase. Detection was carried out on a triple quadrupole mass spectrometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM following the transitions at m/z 286.2→217.0 and m/z 290.2→221.0, respectively. The calibration plots were linear through the concentration range of 0.10–100 ng/mL (r2≥0.9990 using 100 µL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was ≤5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR of 74 subject samples.

  14. Lack of hepcidin expression attenuates steatosis and causesfibrosis in the liver

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    AIM To investigate the role of key iron-regulatoryprotein, hepcidin in non-alcoholic fatty liver disease(NAFLD).METHODS: Hepcidin (Hamp1 ) knockout and floxedcontrol mice were administered a high fat and highsucrose (HFS) or a regular control diet for 3 or 7 mo.Steatosis, triglycerides, fibrosis, protein and gene expressionin mice livers were determined by histologicaland biochemical techniques, western blotting and realtimepolymerase chain reaction.RESULTS: Knockout mice exhibited hepatic ironaccumulation. Despite similar weight gains, HFS feedinginduced hepatomegaly in floxed, but not knockout,mice. The livers of floxed mice exhibited higher levelsof steatosis, triglycerides and c-Jun N-terminal kinase(JNK) phosphorylation than knockout mice. In contrast,a significant increase in fibrosis was observed inknockout mice livers within 3 mo of HFS administration.The hepatic gene expression levels of sterol regulatory element-binding protein-1c and fat-specific protein-27,but not peroxisome proliferator-activated receptoralphaor microsomal triglyceride transfer protein, wereattenuated in HFS-fed knockout mice. Knockout micefed with regular diet displayed increased carnitinepalmitoyltransferase-1a and phosphoenolpyruvatecarboxykinase-1 but decreased glucose-6-phosphataseexpression in the liver. In summary, attenuated steatosiscorrelated with decreased expression of lipogenic andlipid storage genes, and JNK phosphorylation. Deletionof Hamp1 alleles per se modulated hepatic expressionof beta-oxidation and gluconeogenic genes.CONCLUSION: Lack of hepcidin expression inhibitshepatic lipid accumulation and induces early developmentof fibrosis following high fat intake. Hepcidinand iron may play a role in the regulation of metabolicpathways in the liver, which has implications for NAFLDpathogenesis.

  15. Development and validation of a high-performance liquid chromatography-mass spectrometric assay for the determination of 17alpha-hydroxyprogesterone caproate (17-OHPC) in human plasma.

    Science.gov (United States)

    Zhang, Shimin; Mada, Sripal Reddy; Mattison, Don; Caritis, Steve; Venkataramanan, Raman

    2007-09-01

    A sensitive and specific method for the determination of 17alpha-hydroxyprogesterone caproate (17-OHPC) in human plasma using high-performance liquid chromatography and mass spectrometry has been developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis HLB extraction cartridge prior to chromatography. Medroxyprogesterone acetate (MPA) was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 3.5 microm, 2.1 mm x 10 mm, using a gradient elusion with a mobile phase consisting of acetonitrile [A] and 5% acetonitrile in water [B], with 0.1% formic acid being added to both [A] and [B], at a flow rate 0.2 ml/min. The retention times of 17-OHPC and MPA were 8.1 and 5.0 min, respectively, with a total run time of 15 min. Analysis was performed on Thermo Electron Finnigan TSQ Quantum Ultra mass spectrometer in a selected reaction-monitoring (SRM), positive mode using electron spray ionization (ESI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ions following SRM transitions monitored were m/z 429.2-->313.13 and 429.2-->271.1, for 17-OHPC and m/z 385.1-->276 for MPA. The extraction recoveries at concentrations of 5, 10 and 50 ng/ml were 97.1, 92.6 and 88.7%, respectively. The assay was linear over the range 0.5-50 ng/ml for 17-OHPC. The analysis of standard samples for 17-OHPC 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml demonstrated a relative standard deviation of 16.7, 12.4, 13.7, 1.4, 5.2, 3.7 and 5.3%, respectively (n=6). This method is simple, adaptable to routine application, and allows easy and accurate measurement of 17-OHPC in human plasma.

  16. Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus.

    Science.gov (United States)

    Gardenghi, Sara; Renaud, Tom M; Meloni, Alessandra; Casu, Carla; Crielaard, Bart J; Bystrom, Laura M; Greenberg-Kushnir, Noa; Sasu, Barbra J; Cooke, Keegan S; Rivella, Stefano

    2014-02-20

    Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

  17. The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

    Science.gov (United States)

    Harrison-Findik, Duygu Dee; Lu, Sizhao

    2015-05-06

    This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

  18. Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression

    Institute of Scientific and Technical Information of China (English)

    Duygu; Dee; Harrison-Findik; Sizhao; Lu; Emily; M; Zmijewski; Jocelyn; Jones; Matthew; C; Zimmerman

    2013-01-01

    AIM: To understand the role of mitochondrial-produced superoxide(O 2 ?) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression(Sod2 +/) and age-matched littermate control mice(LMC), expressing Sod2 gene on both alleles, were exposed to either 10%(w/v) ethanol in the drinking water or plain water(control) for 7 d. Total cellular O 2 ? levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O 2 ?-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O 2 ? in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. RESULTS: Sod2 +/- mice expressed 40% less MnSOD protein(SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 +/ and LMC mice. O 2 ? levels in hepatocytes of untreated Sod2 +/ mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O 2 ? levels in mitochondria of Sod2 +/ mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O 2 ? levels in hepatocytes of LMC mice than in Sod2 +/ mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O 2 ? levels in both Sod2 +/- and control mice. CONCLUSION: Mitochondrial O2 ? is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly

  19. Presence of hepcidin-25 in biological fluids: Bile,ascitic and pleural fluids

    Institute of Scientific and Technical Information of China (English)

    Jayantha; Arnold; Arvind; Sangwaiya; Vijay; Manglam; Frank; Geoghegan; Mark; Thursz; Mark; Busbridge

    2010-01-01

    AIM: To examine body fluids such as ascitic fluid (AF),saliva,bile and pleural effusions for the presence of hepcidin using a novel radioimmunoassay (RIA).METHODS: Serum samples were collected from 25 healthy volunteers (mean age: 36 ± 11.9 years,11 males,14 females).In addition bile was obtained from 12 patients undergoing endoscopic retrograde cholangiopancreatography (mean age: 66.9 ± 16.7 years,M:F = 5:7).Saliva was collected from 17 healthy volunteers (mean age: 35 ± 9.9 years,M:F = 8:9).Pleural and AF...

  20. Fast RPLC-UV method on short sub-two microns particles packed column for the assay of tenoxicam in plasma samples.

    Science.gov (United States)

    Sora, Iulia; Galaon, Toma; Udrescu, Stefan; Negru, Jean; David, Victor; Medvedovici, Andrei

    2007-03-12

    An extraction-less sample preparation technique followed by a RPLC-UV method on sub-two microns particles packed short column were used for the assay of tenoxicam in plasma samples. Protein precipitation was made by means of trichloroacetic acid addition. Supernatant was injected to the chromatographic column without any further pH adjustment. The mobile phase consisted in a mixture of acetonitrile and aqueous 0.1% phosphoric acid, at 2 mL/min flow rate and gradient elution. The Zorbax SB-C18 column (50 mm length, 4.6 mm internal diameter and 1.8 microm particle size) was thermostated at 60 degrees C. The mobile phase gradient composition program allowed separation of tenoxicam and piroxicam (internal standard), column clean-up and re-equilibration within 4 min. UV detection was achieved at 368+/-10 nm. The method is characterized by a low limit of quantitation of 25 ng/mL for tenoxicam, with a linearity interval up to 5500 ng/mL. The use of a low volume detection cell and detector high frequency data acquisition rate produced high precision and accuracy through a whole bioequivalence study of tenoxicam in two commercially available tablet formulations, after a single oral administration dose. Full method validation is presented. The high throughput characteristic of the proposed method allowed full validation and bioanalytical study completion within a 96 h period.

  1. A validated enantioselective LC-MS/MS assay for quantification of a major chiral metabolite of an achiral 11-β-hydroxysteroid-dehydrogenase 1 inhibitor in human plasma: Application to a clinical pharmacokinetic study.

    Science.gov (United States)

    Furlong, Michael T; Ji, Qin C; Iacono, Lisa; Dang, Oanh; Noren, Marzena; Bruce, John; Aubry, Anne-Françoise; Arnold, Mark E

    2016-06-01

    BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid-liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC-MS/MS assay was validated over a concentration range of 0.200-200ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days at -20°C and -70°C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.

  2. Insights into the Antimicrobial Properties of Hepcidins: Advantages and Drawbacks as Potential Therapeutic Agents

    Directory of Open Access Journals (Sweden)

    Lisa Lombardi

    2015-04-01

    Full Text Available The increasing frequency of multi-drug resistant microorganisms has driven research into alternative therapeutic strategies. In this respect, natural antimicrobial peptides (AMPs hold much promise as candidates for the development of novel antibiotics. However, AMPs have some intrinsic drawbacks, such as partial degradation by host proteases or inhibition by host body fluid composition, potential toxicity, and high production costs. This review focuses on the hepcidins, which are peptides produced by the human liver with a known role in iron homeostasis, as well by numerous other organisms (including fish, reptiles, other mammals, and their potential as antibacterial and antifungal agents. Interestingly, the antimicrobial properties of human hepcidins are enhanced at acidic pH, rendering these peptides appealing for the design of new drugs targeting infections that occur in body areas with acidic physiological pH. This review not only considers current research on the direct killing activity of these peptides, but evaluates the potential application of these molecules as coating agents preventing biofilm formation and critically assesses technical obstacles preventing their therapeutic application.

  3. Convenient assaying of Amisulpride in plasma%人体血浆中氨磺必利含量的简便测定法

    Institute of Scientific and Technical Information of China (English)

    王海岭; 王传升; 师天元

    2013-01-01

    目的 建立反相高效液相色谱(RP-HPLC)法测定人体血浆中氨磺必利的含量.方法 色谱柱为美国DiamonsilR(250 mm×4.6 mm,5μm)C18反相柱;流动相为甲醇-乙腈-缓冲液(6ml三乙胺加入1 000 ml水中,冰醋酸调pH值为6.8) =30∶50∶20,流速为1.1 ml/min;检测波长275 nm.结果 氨磺必利血浆浓度在2.1~ 268.8 ng/ml范围内,线性关系良好,标准曲线为C=1.039 6 F-0.020 7(r=0.999 7,n=8),定量下限2.1 ng/ml.结论 RP-HPLC法具有快速、简便、灵敏度高、重现性好等优点,可以广泛用于氨磺必利临床研究和不良反应监测.%[ Objective] To establish assaying of Amisulpride in plasma by RP-HPLC. [Methods] Column was Diamonsil R (250 mm ×4.6 mm,5 μm) C 18;Mobile phase was MeOH: Acetonitrile:Buffer (30: 50: 20,V/V) with 6 ml of triethylamine in 1 000 ml water and glacial acetic acid pH of 6.8,the flow rate was 1. 1 ml/min;UV wavelength was 275 nm. [Results] Within a concentration of amisulpride plasma concentration in the range of 2.1-268.8 ng/ml,the standard curve of was C = 1.039 6 F-0.020 7 and had a good linearity (r =0.999 1 ,n =8). The low limit of quantization was 2. 1 ng/ml. [Conclusion]RP-HPLC appears to be a rapid, convenient, sensitive and reproducible method for clinical research and adverse reaction monitor of Amisulpride.

  4. Assays for calcitonin receptors

    Energy Technology Data Exchange (ETDEWEB)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  5. Native plasma-derived FVIII/VWF complex has lower sensitivity to FVIII inhibitors than the combination of isolated FVIII and VWF proteins. Impact on Bethesda assay titration of FVIII inhibitors.

    Science.gov (United States)

    Bravo, M I; Da Rocha-Souto, B; Grancha, S; Jorquera, J I

    2014-11-01

    Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4-1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6-1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.

  6. Evaluation of the performance of Abbott m2000 and Roche COBAS Ampliprep/COBAS Taqman assays for HIV-1 viral load determination using dried blood spots and dried plasma spots in Kenya.

    Science.gov (United States)

    Zeh, Clement; Ndiege, Kenneth; Inzaule, Seth; Achieng, Rebecca; Williamson, John; Chih-Wei Chang, Joy; Ellenberger, Dennis; Nkengasong, John

    2017-01-01

    Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2-99.2] and 98.1% [95%CI: 89.7-100]) and those for DBS (93.9% [95%CI: 88.8-97.2] and 88.0% [95%CI: 82.2-92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4-97.7) and 73.6 (95%CI: 51.6-86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6-100.0] vs. 94.7% [95%CI: 89.8-97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4-13.7] compared to DPS: 94.0%, [95% CI: 83.5-98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and specificity of 96.6% [95% CI 91.8-98.7] and 90

  7. Hepcidin as a predictive factor and therapeutic target in erythropoiesis-stimulating agent treatment for anemia of chronic disease in rats.

    Science.gov (United States)

    Theurl, Milan; Nairz, Manfred; Schroll, Andrea; Sonnweber, Thomas; Asshoff, Malte; Haschka, David; Seifert, Markus; Willenbacher, Wolfgang; Wilflingseder, Doris; Posch, Wilfried; Murphy, Anthony T; Witcher, Derrick R; Theurl, Igor; Weiss, Günter

    2014-09-01

    Anemia of chronic disease is a multifactorial disorder, resulting mainly from inflammation-driven reticuloendothelial iron retention, impaired erythropoiesis, and reduced biological activity of erythropoietin. Erythropoiesis-stimulating agents have been used for the treatment of anemia of chronic disease, although with varying response rates and potential adverse effects. Serum concentrations of hepcidin, a key regulator of iron homeostasis, are increased in patients with anemia of chronic disease and linked to the pathogenesis of this disease, because hepcidin blocks cellular iron egress, thus limiting availability of iron for erythropoiesis. We tested whether serum hepcidin levels can predict and affect the therapeutic efficacy of erythropoiesis-stimulating agent treatment using a well-established rat model of anemia of chronic disease. We found that high pre-treatment hepcidin levels correlated with an impaired hematologic response to an erythropoiesis-stimulating agent in rats with anemia of chronic disease. Combined treatment with an erythropoiesis-stimulating agent and an inhibitor of hepcidin expression, LDN-193189, significantly reduced serum hepcidin levels, mobilized iron from tissue stores, increased serum iron levels and improved hemoglobin levels more effectively than did the erythropoiesis-stimulating agent or LDN-193189 monotherapy. In parallel, both the erythropoiesis-stimulating agent and erythropoiesis-stimulating agent/LDN-193189 combined reduced the expression of cytokines known to inhibit erythropoiesis. We conclude that serum hepcidin levels can predict the hematologic responsiveness to erythropoiesis-stimulating agent therapy in anemia of chronic disease. Pharmacological inhibition of hepcidin formation improves the erythropoiesis-stimulating agent's therapeutic efficacy, which may favor a reduction of erythropoiesis-stimulating agent dosages, costs and side effects.

  8. Role of hepcidin in the development of anemia in patients with rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Elena Andreyevna Galushko

    2012-01-01

    Full Text Available Chronic disease anemia (CDA diagnosed in many patients with rheumatoid arthritis (RA was described in the early 1970s. As earlier noted, iron metabolic disturbances in CDA are its diagnostic feature and the discovery of hepcidin, an iron-regulatory acute-phase protein, could largely clarify an association between the immune mechanism of impaired iron homeostasis and the development of CDA. Objective: to define the role of hepcidin in the differential diagnosis of CDA and true iron deficiency in patients with RA. Subjects and methods. The investigation enrolled 76 patients with RA (1987 ACR criteria admitted to the Research Institute of Rheumatology, Russian Academy of Medical Sciences, to be treated. The patients were divided into two groups. A study group comprised anemic patients (n = 47. The WHO criteria for anemia were considered to be hemoglobin (Hb levels of below 120 g/l for women and below 130 g/l for men. A control group consisted of non-anemic patients (n = 29. The anemic and non-anemic patients were matched for age (45.5±14.3 and 49.8±14.3 years, respectively and disease duration (2 months to 20 years (p > 0.05. Iron metabolic parameters, such as serum iron, total serum iron-binding capacity (TSIBC, iron transferrin saturation (ITS, transferrin receptors, and serum ferritin (SF, were studied and the level of hepcidin prohormone was estimated by direct enzyme immunoassay (Hepcidin Prohormone Enzyme Immunoassay Kit, IBL, Germany in all the patients to be analyzed. Cytokines, such as interleukin 6, tumor necrosis factor-а were determined by enzyme immunoassay (Bender MedSystems, Austria. The Institute’s differential diagnostic algorithm involving SF, TSIBC, and ITS was used to diagnose iron deficiency. The diagnosis was based on two stages of estimating iron values: isolated iron-deficiency anemia (IDA was diagnosed if SF was below the normal value (< 40 μg/l. If the patient had SF of μ40 μg/l with a simultaneous rise of TSIBC

  9. The clinical utility of plasma Epstein-Barr virus DNA assays in nasopharyngeal carcinoma: the dawn of a new era?: a systematic review and meta-analysis of 7836 cases.

    Science.gov (United States)

    Zhang, Wenna; Chen, Yupei; Chen, Lei; Guo, Rui; Zhou, Guanqun; Tang, Linglong; Mao, Yanping; Li, Wenfei; Liu, Xu; Du, Xiaojing; Sun, Ying; Ma, Jun

    2015-05-01

    In this study, we assessed the potential of plasma Epstein-Barr virus (EBV) DNA assays to predict clinical outcomes in a large sample of nasopharyngeal carcinoma (NPC) patients and proposed a risk stratification model based on standardized EBV DNA load monitoring.We conducted a meta-analysis of 14 prospective and retrospective comparative studies (n = 7 836 patients) to evaluate the correlation between pretreatment plasma EBV DNA (pre-DNA), midtreatment plasma EBV DNA (mid-DNA), posttreatment plasma EBV DNA (post-DNA), the half-life value of plasma EBV DNA clearance rate (t1/2), and clinical outcomes. Our primary endpoint was overall survival (OS). Our secondary endpoints were progression-free survival (PFS), distant-metastasis-free survival (DMFS), and local-regional-failure-free survival (LRFS).High pre-DNA, detectable mid-DNA, detectable post-DNA, and slow EBV DNA clearance rates were all significantly associated with poorer OS, with hazard radios (HRs) equal to 2.81, 3.29, 4.26, and 3.58, respectively. Pre-DNA, mid-DNA, and post-DNA had the same effects on PFS, DMFS, and LRFS.Plasma EBV DNA assays are highly prognostic of long-term survival and distant metastasis in NPC patients. Based on the results of this meta-analysis, we propose a 4-grade systematic risk stratification model. Given the inherent limitations of the included studies, future well-designed randomized clinical trials are required to confirm to the findings of this analysis and to contribute to the development of individualized treatment strategies for NPC patients.

  10. Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM01183 (lurbinectedin), a novel antineoplastic agent, in mouse, rat, dog, Cynomolgus monkey and mini-pig plasma.

    Science.gov (United States)

    Pernice, Tiziana; Bishop, Alan G; Guillen, Maria Jose; Cuevas, Carmen; Aviles, Pablo

    2016-05-10

    Lurbinectedin (PM01183) is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected sequences in the minor groove of DNA, inducing PM01183-DNA adducts that stall replication, DNA repair and transcription and gives rise to double-strand breaks and finally, caspase-dependent apoptotic cell death. PM01183 has demonstrated clinical antitumor activity in platinum resistant/refractory ovarian cancer patients. A rapid and sensitive liquid chromatography/tandem mass spectrometry assay was developed and validated to quantify PM01183 in plasma from nonclinical species. The bioanalysis consisted of a supported liquid extraction, followed by a gradient phase chromatography and, detection by positive ion electrospray tandem mass spectrometry. The calibration range for PM01183 was established using PM01183 standards from 0.1 to 100 ng/mL in blank plasma. The multiple reaction monitoring, based on the transition m/z 767.3→273.0, was specific for PM01183, and that based on the transition m/z 771.4→277.0 was specific for the internal standard (deuterated PM01183). No endogenous material interfered with the analysis of PM01183 and the internal standard from blank plasma. The limit of detection (LOD) of the assay was calculated as 0.025 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9937 to 0.9987. The mean inter-day accuracies for all calibration standards ranged from 92 to 108% (≤8% bias), and the mean inter-day precision for calibration standards was always less than 12%. The mean intra and inter-day assay accuracy for all quality control replicates remained between 91 and 109%. The mean intra and inter-day assay precision was less than 10% for all QC levels. The method was validated to demonstrate the specificity, recovery, limit of quantification, accuracy and precision of measurements. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies of PM01183 in nonclinical species. The main PK

  11. Iron, hepcidin and inflammatory status of young healthy overweight and obese women in Australia.

    Directory of Open Access Journals (Sweden)

    Hoi Lun Cheng

    Full Text Available BACKGROUND AND AIMS: Evidence suggests obesity-related inflammation alters iron metabolism potentially increasing the risk of iron deficiency. This cross-sectional study aimed to investigate iron, hepcidin and inflammatory status in young, healthy overweight and obese women. METHODS: 114 young (18-25 years, healthy comorbidity-free women with a body mass index (BMI ≥27.5 kg/m(2 were recruited. Biochemical data were analysed using mean ± standard deviation or median (interquartile range and multivariate modelling. Biochemical markers were also stratified according to varying degrees of overweight and obesity. RESULTS: Anaemia (haemoglobin 35.0 kg/m(2. This indicates that obesity alone may not be sufficient to induce clinically significant disturbances to iron metabolism as previously described. This may be attributed to the lack of comorbidity in this cohort.

  12. Iron, Hepcidin and Inflammatory Status of Young Healthy Overweight and Obese Women in Australia

    Science.gov (United States)

    Cheng, Hoi Lun; Bryant, Christian E.; Rooney, Kieron B.; Steinbeck, Katharine S.; Griffin, Hayley J.; Petocz, Peter; O’Connor, Helen T.

    2013-01-01

    Background and Aims Evidence suggests obesity-related inflammation alters iron metabolism potentially increasing the risk of iron deficiency. This cross-sectional study aimed to investigate iron, hepcidin and inflammatory status in young, healthy overweight and obese women. Methods 114 young (18–25 years), healthy comorbidity-free women with a body mass index (BMI) ≥27.5 kg/m2 were recruited. Biochemical data were analysed using mean ± standard deviation or median (interquartile range) and multivariate modelling. Biochemical markers were also stratified according to varying degrees of overweight and obesity. Results Anaemia (haemoglobin 35.0 kg/m2. This indicates that obesity alone may not be sufficient to induce clinically significant disturbances to iron metabolism as previously described. This may be attributed to the lack of comorbidity in this cohort. PMID:23861932

  13. Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.

    Science.gov (United States)

    Wong, Cheryl J; Koch, Michael; Behling-Kelly, Erica L

    2016-11-08

    Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.

  14. Comparison of levels of human immunodeficiency virus type 1 RNA in plasma as measured by the NucliSens nucleic acid sequence-based amplification and Quantiplex branched-DNA assays.

    Science.gov (United States)

    Ginocchio, C C; Tetali, S; Washburn, D; Zhang, F; Kaplan, M H

    1999-04-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

  15. Serum Hepcidin and Soluble Transferrin Receptor in the Assessment of Iron Metabolism in Children on a Vegetarian Diet.

    Science.gov (United States)

    Ambroszkiewicz, Jadwiga; Klemarczyk, Witold; Mazur, Joanna; Gajewska, Joanna; Rowicka, Grażyna; Strucińska, Małgorzata; Chełchowska, Magdalena

    2017-03-24

    The aim of this study was to assess the effect of vegetarian diet on iron metabolism parameters paying special attention to serum hepcidin and soluble transferrin receptor (sTfR) concentrations in 43 prepubertal children (age range 4.5-9.0 years) on vegetarian and in 46 children on omnivorous diets. There were no significant differences according to age, weight, height, and body mass index (BMI) between vegetarian and omnivorous children. Vegetarians had similar intake of iron and vitamin B12 and a significantly higher intake of vitamin C (p vegetarians. Hematologic parameters and serum iron concentrations were within the reference range in both groups of children. Serum transferrin levels were similar in all subjects; however, ferritin concentrations were significantly (p vegetarians than in omnivores. In children on a vegetarian diet, median hepcidin levels were lower (p vegetarians. We did not find significant associations with concentration of sTfR and selected biochemical, anthropometric, and dietary parameters in any of the studied groups of children. As hematologic parameters and iron concentrations in vegetarians and omnivores were comparable and ferritin level was lower in vegetarians, we suggest that inclusion of novel markers, in particular sTfR (not cofounded by inflammation) and hepcidin, can better detect subclinical iron deficiency in children following vegetarian diets.

  16. Urinary hepcidin identifies a serum ferritin cut-off for iron supplementation in young athletes: a pilot study.

    Science.gov (United States)

    Borrione, P; Spaccamiglio, A; Rizzo, M; Termine, A; Chierto, E; Campostrini, N; Quaranta, F; Di Gianfrancesco, A; Pigozzi, F

    2011-01-01

    The use of iron supplements should be a judicious choice, primarily when considering the possible risks deriving from an unjustified treatment. In trained athletes, levels of ferritin between 15 and 30 microg/L are frequently observed. Within this ferritin range, the usefulness of iron supplementation is still controversial. The aim of the present study is to evaluate the clinical usefulness of hepcidin assessment in the analysis of the iron status of young non-anemic athletes. Fifty young athletes were enrolled. The subjects were divided into 4 groups according to their ferritin levels. No statistically significant difference was found regarding hepcidin levels between athletes with ferritin lower than 15 microg/L and those in the 15-30 microg/L range. Similarly, no difference was found between athletes with ferritin higher than 50 microg/L and those in the 30-50 microg/L range. On the contrary, statistically significant differences were found between athletes with ferritin levels ranging from 15 to 30 microg/L and those in the 30-50 microg/L range. The present study suggests that serum ferritin levels below 30 microg/L indicate an asymptomatic iron deficiency status inhibiting hepcidin expression and that 30 microg/L should be considered the ferritin cut-off when considering an iron supplementation in young athletes.

  17. ER stress-inducible factor CHOP affects the expression of hepcidin by modulating C/EBPalpha activity.

    Directory of Open Access Journals (Sweden)

    Susana J Oliveira

    Full Text Available Endoplasmic reticulum (ER stress induces a complex network of pathways collectively termed the unfolded protein response (UPR. The clarification of these pathways has linked the UPR to the regulation of several physiological processes. However, its crosstalk with cellular iron metabolism remains unclear, which prompted us to examine whether an UPR affects the expression of relevant iron-related genes. For that purpose, the HepG2 cell line was used as model and the UPR was activated by dithiothreitol (DTT and homocysteine (Hcys. Here, we report that hepcidin, a liver secreted hormone that shepherds iron homeostasis, exhibits a biphasic pattern of expression following UPR activation: its levels decreased in an early stage and increased with the maintenance of the stress response. Furthermore, we show that immediately after stressing the ER, the stress-inducible transcription factor CHOP depletes C/EBPalpha protein pool, which may in turn impact on the activation of hepcidin transcription. In the later period of the UPR, CHOP levels decreased progressively, enhancing C/EBPalpha-binding to the hepcidin promoter. In addition, analysis of ferroportin and ferritin H revealed that the transcript levels of these iron-genes are increased by the UPR signaling pathways. Taken together, our findings suggest that the UPR can have a broad impact on the maintenance of cellular iron homeostasis.

  18. A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma

    DEFF Research Database (Denmark)

    Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John;

    2008-01-01

    BACKGROUND: The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample...... of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1......-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both...

  19. 采用肝素钠抗凝血浆测定电解质可行性分析%The Feasibility Analysis Concerning Using Heparin Sodium Anticoagulation Plasma To Assay Electrolyte

    Institute of Scientific and Technical Information of China (English)

    许祖林; 易彩响; 鲁海平; 陈桃丽; 谭琼

    2012-01-01

    Objective: To Probe into the feasibility of using the heparin sodium anticoagnlation plasma instead of serum by comparing with direct electrode method to assay the difference of electrolyte concentration between in the serum and in the heparin sodium anticoagulation plasma. Methods : Using the 9886 type electrolyte analyzer made by Shenzhen Hangchuang to assay the electrolyte concentration of the patient' s serum and heparin sodium anticoagulation plasma, and doing statistic analysis. Result: The result differences of testing Potassium, Sodium between in serum and in plasma is statistically significant; and the result differences of testing Chlorine between in serum and in plasma is not statistically significant. But the results of testing Potassium, Sodium and Chlorine between in both has good correlation, and all of the coefficient of this correlation is over or equal to 0. 975, and the biases of measured value about Potassium, Sodium and Chlorine between both of the specimens are not beyond the allowable error based on 1/2 CLIA' 88. Conclusion: It is feasible to assay Potassium, Sodium, Chlorine with the heparin sodium anticoagulation plasma instead of the serum, but should establish a corresponding conversion system.%目的:比较用电极直接法测定血清和肝素钠抗凝血浆电解质浓度的差异,探讨肝素钠抗凝血浆代替血清检测电解质的可行性。方法:用深圳航创9886型电解质分析仪测定患者血清和肝素钠抗凝血浆的电解质浓度并进行统计分析。结果:血浆与血清钾、钠测定结果差异具有统计学意义,血浆与血清氯测定结果差异无统计学意义,但血浆与血清钾、钠、氯的测定结果具的很好的相关性,相关性系数(r)均〉10.975,且两种标本钾、钠、氯测定值平均偏倚均没有超出1/2CLIA’88允许误差。结论:行肝素钠抗凝血浆代替血清测定钾、钠、氯是可行的,但应建立相应的换算体系。

  20. Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitrios

    2011-01-01

    Full Text Available Abstract Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0 assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD difference of -0.206 (0.298 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD difference: 0.160 (0.287 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86% differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3

  1. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    Science.gov (United States)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  2. Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples

    Directory of Open Access Journals (Sweden)

    M Paryan

    2012-03-01

    Full Text Available Background and Objectives: HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT methods, especially in multiplex format, more precise detection is possible.Materials and Methods: We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed.Results: Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%.Conclusions: The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.

  3. Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV DNA in plasma

    Directory of Open Access Journals (Sweden)

    Pollara Caterina

    2007-03-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy and an in-house assay (EBV RQ-PCR. Results The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p Conclusion Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

  4. A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk

    NARCIS (Netherlands)

    Kramps, J.A.; Maanen, van C.; Wetering, van de G.; Stienstra, G.; Quak, S.; Brinkhof, J.; Ronsholt, L.; Nylin, B.

    1997-01-01

    To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-struct

  5. Cloning,expression and antimicrobial activity of Hepcidin from Hypophthalmichthys molitrix%鲢抗菌肽 Hepcidin 的基因克隆和表达及抑菌活性分析

    Institute of Scientific and Technical Information of China (English)

    周卫军; 刘振兴; 柯浩; 马艳平; 郝乐; 徐明芳

    2014-01-01

    The full-length Hepcidin cDNA sequence GenBank No. KF312213 was amplified from the liver of chub(Hypophthalmichthys molitrix)by semi-Nested PCR[rapid amplification of cDNA ends(RACE)]. According to prodomain and mature peptide of the Hepci-din cDNA sequence,we designed an upstream primer with EcoR I restriction site and downstream primers with Sal I restriction site,and cloned the target gene into the expression vector pET-32a( + ). The recombined plasmid was transformed into the expression stain-E. coli Rosetta and expressed induciblely at different temperatures(37 ℃,28 ℃ and 16 ℃)and of different IPTG concentrations(0. 5 mmol· L -1 and 1. 0 mmol·L -1 ). The protein was purified by Ni SepharoseTM affinity chromatography column. The full-length of the Hepcidin cD-NA gene was 755 bp,which included a 282-bp ORF encoding a 93-amino acid prepropeptide. The prepropeptide contained a signal pep-tide(24 amino acids),a prodomain(42 amino acids)and a mature peptide(27 amino acids). The purified product displayed a single protein band through 15% SDS-PAGE electrophoresis. The purified production of pET-Hep/ Rosetta had obvious antibacterial effect on Streptococcus agalactiae,Staphylococcus aureus and Aeromonas hydrophila,but the control group showed no inhibitory effect.%利用同源克隆的方法从鲢(Hypophthalmichthys molitrix)的肝脏中克隆出抗菌肽 Hepcidin cDNA(GenBank 登入号 KF312213)后,通过 pET32a(+)构建含抗菌肽 Hepcidin 的重组质粒的 pET-Hep/ Rosetta 菌株,在37℃、28℃和16℃下分别用0.5 mmol·L -1和1.0 mmol·L -1 IPTG 诱导表达,产物用 Ni SepharoseTM 亲和层析柱纯化并进行体外抑菌试验。鲢 Hepcidin cDNA 总长度755 bp,ORF 为282 bp,5′UTR 为108 bp,3′UTR 为365 bp,编码93氨基酸,信号肽24个氨基酸,前域42个氨基酸和成熟肽27个氨基酸。pET-Hep/ Rosetta 在28℃和16℃主要是可溶性表达,37℃主要是包涵体表达。纯化的产物经15

  6. Enzyme assays.

    Science.gov (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  7. Elevated hepcidin serum level in response to inflammatory and iron signals in exercising athletes is independent of moderate supplementation with vitamin C and E.

    Science.gov (United States)

    Díaz, Víctor; Peinado, Ana B; Barba-Moreno, Laura; Altamura, Sandro; Butragueño, Javier; González-Gross, Marcela; Alteheld, Birgit; Stehle, Peter; Zapico, Augusto G; Muckenthaler, Martina U; Gassmann, Max

    2015-08-01

    Iron deficiency among endurance athletes is of major concern for coaches, physicians, and nutritionists. Recently, it has been observed that hepcidin, the master regulator of iron metabolism, was upregulated after exercise and was found to be related to interleukin-6 (IL-6) elevation. In this study performed on noniron deficient and well-trained runners, we observed that hepcidin concentrations remain elevated in response to inflammatory and iron signals despite a 28-days supplementation period with vitamins C (500 mg/day) and E (400 IU/day).

  8. P202-S Expanding the Capabilities of Peptide MRM-Based Assays in Plasma Using a Hybrid Triple-Quadrupole Linear Ion-Trap Mass Spectrometer

    OpenAIRE

    Hunter, C.

    2007-01-01

    As the study of protein biomarkers increases in importance, technical limitations to the detection of low-abundance proteins and high-throughput, high-precision quantitation remain to be overcome. The complexity and dynamic range of the plasma proteome makes the task of specific, quantitative detection even more challenging. Multiple reaction monitoring (MRM) capabilities of triple quadrupole MS systems have been explored as solutions to this challenge due to their well-known sensitivity and ...

  9. Development and validation of a high-throughput stereoselective LC-MS/MS assay for bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion in human plasma

    OpenAIRE

    Teitelbaum, Aaron M.; Flaker, Alicia M.; Kharasch, Evan D.

    2016-01-01

    A stereoselective analytical method was developed and validated for the quantification of bupropion, and principle metabolites hydroxybupropion, erythrohydrobupropion and threohydrobupropion in human plasma. Separation of individual enantiomers (R)-bupropion, (S)-bupropion, (R,R)-hydroxybupropion, (S,S-hydroxybupropion), (1S,2S)-threohydrobupropion, (1R,2R)-threohydrobupropion, (1R,2S)-erythrohydrobupropion, and (1S,2R)-erythrohydrobupropion was achieved utilizing a...

  10. Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

    Science.gov (United States)

    Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2016-09-15

    A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.

  11. Development and validation of an UPLC-Q/TOF-MS assay for the quantitation of neopanaxadiol in beagle dog plasma: Application to a pharmacokinetic study.

    Science.gov (United States)

    Geng, Cong; Wang, Chun-Hong; Hu, Hong; Gao, Xiao-Ping; Gong, Ai-Hua; Lin, Ying-Wei; Fan, Xiu-Shuang; Li, Heng; Yin, Jian-Yuan

    2016-10-27

    Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid-liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C18 column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 → 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.

  12. A validated assay to quantitate serotonin in lamb plasma using ultrahigh-performance liquid chromatography-tandem mass spectrometry: applications with LC/MS3.

    Science.gov (United States)

    Szeitz, András; Nguyen, Tuan-Anh T; Riggs, K Wayne; Rurak, Dan

    2014-08-01

    An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed and validated for the quantification of serotonin (5-HT) in lamb plasma using [(2)d(4)]-serotonin ([(2)d(4)]-5-HT) as an internal standard. Charcoal-stripped human plasma was used as the blank matrix during validation, and 5-HT was quantitated using selected reaction monitoring. The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity ultrahigh-performance liquid chromatograph coupled with an AB SCIEX QTRAP(®) 5500 hybrid linear ion trap triple quadrupole mass spectrometer. The method was validated for accuracy, precision, linearity, lower limit of quantification (LLOQ), selectivity, and other parameters. The LLOQ was 1.0 ng/mL, requiring 100 μL of sample. The method was applied to monitor the 5-HT levels in lamb plasma after the administration of fluoxetine. Tandem mass spectrometry cubed (MS(3)) experiments were also performed to investigate the fragmentation pattern of 5-HT and [(2)d(4)]-5-HT. A liquid chromatography-MS(3) (LC/MS(3)) method was developed, and the UHPLC/MS/MS and the LC/MS(3) methods were compared for performance.

  13. Determination of newly synthesized lipoic acid-niacin dimer in rat plasma by UPLC/electrospray ionization tandem mass spectrometry: assay development, validation and application to a pharmacokinetic study.

    Science.gov (United States)

    Chen, Xiao; Gao, Jingwen; Jiang, Yiming; Huang, Ping; Xie, Yuhui; Pi, Rongbiao; Zhu, Shuzhen; Yao, Meicun

    2014-02-01

    A simple, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the newly synthesized compound lipoic acid-niacin dimer (N2L) in plasma. Plasma samples were precipitated by methanol using tetrahydropalmatine as internal standard. Chromatographic separation was achieved on an Acquity BEH C18 (2.1 × 50 mm i.d., 1.7 µm) column; the mobile phase contains methanol and buffer solution (water with 0.5% formic acid and 10 mmol/L ammonium acetate). Multiple reaction monitoring (m/z 353.9 → 148.6 for N2L and m/z 356.0 → 192.0 for internal standard) was performed for detection and quantification. The method was validated to be rapid, specific, accurate and precise over the concentration range of 1-750 ng/mL; N2L was not stable on the bench-top or during freeze-freeze-thaw cycles in plasma, but was stable in the stock solution and after preparation in the autosampler for 24 h. The utility of the assay was confirmed by pharmacokinetic study of N2L in rats. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Lack of hepcidin ameliorates anemia and improves growth in an adenine-induced mouse model of chronic kidney disease.

    Science.gov (United States)

    Akchurin, Oleh; Sureshbabu, Angara; Doty, Steve B; Zhu, Yuan-Shan; Patino, Edwin; Cunningham-Rundles, Susanna; Choi, Mary E; Boskey, Adele; Rivella, Stefano

    2016-11-01

    Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted.

  15. The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream.

    Science.gov (United States)

    Cuesta, Alberto; Meseguer, José; Esteban, Maria Angeles

    2008-04-01

    Antimicrobial peptides (AMPs) are important mediators of the immune response against bacteria and hepcidin is a 20-25 residues member with known functions in iron regulation and the innate immune response. Most studies have focused on mammalian organisms but very little is known about other vertebrate groups including teleost fish. Thus, based on the sequence of an EST database, we have characterized hepcidin gene organization, gene expression, distribution and in vitro and in vivo regulation, as well as the biological activity of a synthetic peptide in the teleost fish gilthead seabream (Sparus aurata L.). First, it was found that the seabream hep gene genomic organization is formed by 3 exons and 2 introns, while the mRNA transcript is constitutively detected in most of the fish tissues but mainly in peritoneal leucocytes, head-kidney, liver and skin. Moreover, we have identified for the first time that hep is much more highly expressed in acidophilic granulocytes than in monocyte-macrophages and lymphocytes. In vitro, hep expression is up-regulated by several mitogens, pathogen-associated molecular patterns (PAMPs) and particulated antigens. Not surprisingly, intraperitoneal injection of bacteria or virus led to a significant gene up-regulation in the liver, head-kidney, peritoneal exudate or spleen. These observations suggest a major role for seabream hepcidin in the immune response to bacteria and viruses. Furthermore, the synthetic seabream Hep exerted an important antimicrobial activity against several bacterial strains in vitro reducing their viability. To conclude, seabream hep gene expression, up-regulation after in vitro or in vivo treatment with mitogens, PAMPs or particulated antigens and the direct in vitro biological activity against bacteria demonstrate that it is an important antimicrobial peptide and probably plays an important role in the innate immune response of fish.

  16. Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia.

    Science.gov (United States)

    Breda, Laura; Gardenghi, Sara; Guy, Ella; Rachmilewitz, Eliezer A; Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Rechavi, Gideon; Giardina, Patricia J; Grady, Robert W; Rivella, Stefano

    2005-01-01

    To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

  17. A reversed-phase HPLC-UV assay for simultaneous analysis of EGCG and ECG of tea polyphenols in rat plasma

    Institute of Scientific and Technical Information of China (English)

    FU Ting; LIANG Jun; HAN Guo-zhu; L(U) Li; LI Nan

    2008-01-01

    Objective To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of (-) Epigallocatechin-3-gallate (EGCG) and (-) Epicatechin-3-gallate (ECG), the main active ingredients of tea polyphenols (TP), in rat plasma. Methods EGCG and ECG were eluted on a Kromasil C18 analytical column (150 mm×4.6 mm, 5μm) protected by a C18 pre-column (4.6 mm×20 mm, 10μm) with a linear gradient mobile phase composed of CH3CN (A)-0.1% citric acid (B), which was run from initial 14 % A and 86 % B to 20 % A and 80 % B at a flow rate of 1.0 mL·min-1 in 14 min, then changed to 14% A and 86% B at a gradient flow rate of 1.0-1.5 mL·min-1 during 14-18 min, and then maintained until 22 min at a gradient flow rate of 1.5-1.0 mL·min-1. The UV detector was set at 280 nm. Plasma samples (200 μL each) were prepared by liquid-liquid extraction procedure with double volumes of EtoAc and then evaporation of organic phase under N2 stream to dry, followed by reconstitution with 100 μL of 20 % CH3CN aqueous solution. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves. Results The HPLC resulted in base-line separation of vanillin, EGCG, ECG and other components; there was no interference from blank plasma. The linear range was 0.5-300 μg·mL-1 for EC, CG (r=0.9999) and 0.1-60 μg·mL-1 for ECG (r = 0.9999). The intra-and inter-day precision (RSD) was better than 6.1% and 12.6%, respectively, and the average accuracy was between 86.25%-103.14%. The extraction recovery of EGCG and ECG was 79.80%-84.64% and 75.22 %-91.39 %, respectively. The plasma samples were stable for at least 30 days at -20 ℃ and 8 h at room temperature;EGCG, ECG and IS stock solutions 2 months at -20 ℃, and the EtoAc-extracted plasma samples 24 h at 4 ℃. Application of the method to the determination of EGCG and ECG in plasma of rats receiving iv 100 mg·kg-1 of TP showed that these 2 compounds

  18. Accuracy of recommended sampling and assay methods for the determination of plasma-free and urinary fractionated metanephrines in the diagnosis of pheochromocytoma and paraganglioma: a systematic review.

    Science.gov (United States)

    Därr, Roland; Kuhn, Matthias; Bode, Christoph; Bornstein, Stefan R; Pacak, Karel; Lenders, Jacques W M; Eisenhofer, Graeme

    2017-06-01

    To determine the accuracy of biochemical tests for the diagnosis of pheochromocytoma and paraganglioma. A search of the PubMed database was conducted for English-language articles published between October 1958 and December 2016 on the biochemical diagnosis of pheochromocytoma and paraganglioma using immunoassay methods or high-performance liquid chromatography with coulometric/electrochemical or tandem mass spectrometric detection for measurement of fractionated metanephrines in 24-h urine collections or plasma-free metanephrines obtained under seated or supine blood sampling conditions. Application of the Standards for Reporting of Diagnostic Studies Accuracy Group criteria yielded 23 suitable articles. Summary receiver operating characteristic analysis revealed sensitivities/specificities of 94/93% and 91/93% for measurement of plasma-free metanephrines and urinary fractionated metanephrines using high-performance liquid chromatography or immunoassay methods, respectively. Partial areas under the curve were 0.947 vs. 0.911. Irrespective of the analytical method, sensitivity was significantly higher for supine compared with seated sampling, 95 vs. 89% (p sampling compared with 24-h urine, 95 vs. 90% (p sampling, seated sampling, and urine. Test accuracy increased linearly from 90 to 93% for 24-h urine at prevalence rates of 0.0-1.0, decreased linearly from 94 to 89% for seated sampling and was constant at 95% for supine conditions. Current tests for the biochemical diagnosis of pheochromocytoma and paraganglioma show excellent diagnostic accuracy. Supine sampling conditions and measurement of plasma-free metanephrines using high-performance liquid chromatography with coulometric/electrochemical or tandem mass spectrometric detection provides the highest accuracy at all prevalence rates.

  19. SPE–UPLC–MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women

    OpenAIRE

    2016-01-01

    A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method is described for determination of letrozole in human plasma. Following solid phase extraction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was performed on Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm) column using methanol-0.1% formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spectrometer with ...

  20. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  1. A rapid and sensitive UHPLC-MS/MS assay for the determination of trelagliptin in rat plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Hu, Xiao-Xia; Lan, Tian; Chen, Zhe; Yang, Cheng-Cheng; Tang, Peng-Fei; Yuan, Ling-Jing; Hu, Guo-Xin; Cai, Jian-Ping

    2016-10-15

    This study aims to develop and validate a simple, rapid and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for exploring pharmacokinetic characteristics of trelagliptin. Protein precipitation by acetonitrile was used to prepare plasma sample. A RRHD Eclipse Plus C18 (2.1×50mm, 1.8μ) column with gradient mobile phase (containing acetonitrile and 0.1% formic acid) help to achieve the separation of trelagliptin and carbamazepine (IS) with high selectivity. Detection of target fragment ions m/z 358.2→133.9 for trelagliptin, and m/z 237.1→194.0 for IS was performed in positive-ion electrospray ionization mass spectrometry by multiple reaction monitoring. Linear calibration plots were achieved in the range of 5-4000ng/mL for trelagliptin (R(2)=0.999) in rat plasma. The recovery of trelagliptin ranged from 87.8% to 93.7%. The method was showed to be accurate, precise and stable. No obvious matrix effect was found. It has been fully validated and successfully applied to pharmacokinetic study of trelagliptin.

  2. Age-Dependent Retinal Iron Accumulation and Degeneration in Hepcidin Knockout Mice

    Science.gov (United States)

    Hadziahmetovic, Majda; Song, Ying; Ponnuru, Padmavathi; Iacovelli, Jared; Hunter, Allan; Haddad, Nadine; Beard, John; Connor, James R.; Vaulont, Sophie

    2011-01-01

    Purpose. Iron dysregulation can cause retinal disease, yet retinal iron regulatory mechanisms are incompletely understood. The peptide hormone hepcidin (Hepc) limits iron uptake from the intestine by triggering degradation of the iron transporter ferroportin (Fpn). Given that Hepc is expressed in the retina and Fpn is expressed in cells constituting the blood-retinal barrier, the authors tested whether the retina may produce Hepc to limit retinal iron import. Methods. Retinas of Hepc−/− mice were analyzed by histology, autofluorescence spectral analysis, atomic absorption spectrophotometry, Perls' iron stain, and immunofluorescence to assess iron-handling proteins. Retinal Hepc mRNA was evaluated through qPCR after intravitreal iron injection. Mechanisms of retinal Hepc upregulation were tested by Western blot analysis. A retinal capillary endothelial cell culture system was used to assess the effect of exogenous Hepc on Fpn. Results. Hepc−/− mice experienced age-dependent increases in retinal iron followed by retinal degeneration with autofluorescent RPE, photoreceptor death, and subretinal neovascularization. Hepc−/− mice had increased Fpn immunoreactivity in vascular endothelial cells. Conversely, in cultured retinal capillary endothelial cells, exogenous Hepc decreased both Fpn levels and iron transport. The retina can sense increased iron levels, upregulating Hepc after phosphorylation of extracellular signal regulated kinases. Conclusions. These findings indicate that Hepc is essential for retinal iron regulation. In the absence of Hepc, retinal degeneration occurs. Increases in Hepc mRNA levels after intravitreal iron injection combined with Hepc-mediated decreases in iron export from cultured retinal capillary endothelial cells suggest that the retina may use Hepc for its tissue-specific iron regulation. PMID:20811044

  3. Overweight children have higher circulating hepcidin concentrations and lower iron status but have dietary iron intakes and bioavailability comparable with normal weight children

    NARCIS (Netherlands)

    Aeberli, I.; Hurrell, R.F.; Zimmermann, M.B.

    2009-01-01

    Background: Obesity increases the risk for iron deficiency, but the underlying mechanism is unclear. It is possible that overweight individuals may have lower dietary iron intake and/or bioavailability. Alternatively, obesity-related inflammation may increase hepcidin concentrations and reduce iron

  4. Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

    NARCIS (Netherlands)

    Gardenghi, Sara; Renaud, Tom M; Meloni, Alessandra; Casu, Carla; Crielaard, Bart J; Bystrom, Laura M; Greenberg-Kushnir, Noa; Sasu, Barbra J; Cooke, Keegan S; Rivella, Stefano

    2014-01-01

    Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidi

  5. S-Propargyl-Cysteine, a Novel Hydrogen Sulfide Donor, Inhibits Inflammatory Hepcidin and Relieves Anemia of Inflammation by Inhibiting IL-6/STAT3 Pathway

    Science.gov (United States)

    Xin, Hong; Zhu, Yi Zhun

    2016-01-01

    Anemia of inflammation (AI) is clinically prevalent and greatly threatens public health. Traditional remedies have raised controversy during clinical practice, calling for alternative therapies. We have recently found that hydrogen sulfide (H2S) inhibits inflammatory hepcidin, the critical mediator of AI. However, due to the chemical property of H2S, there remains an urgent need for a stable H2S donor in AI treatment. Here we reported that S-propargyl-cysteine (SPRC), a novel water-soluble H2S donor, suppressed hepatic hepcidin and corrected hypoferremia induced by lipopolysaccharide. The effects of SPRC were reversed by inhibition of cystathionine γ-lyase, one of the major endogenous H2S synthases. Moreover, SPRC reduced serum hepcidin, improved transferrin saturation, and maintained erythrocyte membrane integrity in a chronic mouse AI model. Consistently, splenomegaly was ameliorated and splenic iron accumulation relieved. Mechanism study indicated that serum IL-6 content and hepatic Il-6 mRNA were decreased by SPRC, in parallel with reduced hepatic JAK2/STAT3 activation. On the whole, our data reveal the inhibition of inflammatory hepcidin by SPRC, and suggest SPRC as a potential remedy against AI. PMID:27649298

  6. Alcohol Activates TGF-Beta but Inhibits BMP Receptor-Mediated Smad Signaling and Smad4 Binding to Hepcidin Promoter in the Liver

    Directory of Open Access Journals (Sweden)

    Lisa Nicole Gerjevic

    2012-01-01

    Full Text Available Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs. Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

  7. Aspirin down Regulates Hepcidin by Inhibiting NF-κB and IL6/JAK2/STAT3 Pathways in BV-2 Microglial Cells Treated with Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Wan-Ying Li

    2016-12-01

    Full Text Available Aspirin down regulates transferrin receptor 1 (TfR1 and up regulates ferroportin 1 (Fpn1 and ferritin expression in BV-2 microglial cells treated without lipopolysaccharides (LPS, as well as down regulates hepcidin and interleukin 6 (IL-6 in cells treated with LPS. However, the relevant mechanisms are unknown. Here, we investigate the effects of aspirin on expression of hepcidin and iron regulatory protein 1 (IRP1, phosphorylation of Janus kinase 2 (JAK2, signal transducer and activator of transcription 3 (STAT3 and P65 (nuclear factor-κB, and the production of nitric oxide (NO in BV-2 microglial cells treated with and without LPS. We demonstrated that aspirin inhibited hepcidin mRNA as well as NO production in cells treated with LPS, but not in cells without LPS, suppresses IL-6, JAK2, STAT3, and P65 (nuclear factor-κB phosphorylation and has no effect on IRP1 in cells treated with or without LPS. These findings provide evidence that aspirin down regulates hepcidin by inhibiting IL6/JAK2/STAT3 and P65 (nuclear factor-κB pathways in the cells under inflammatory conditions, and imply that an aspirin-induced reduction in TfR1 and an increase in ferritin are not associated with IRP1 and NO.

  8. Overweight children have higher circulating hepcidin concentrations and lower iron status but have dietary iron intakes and bioavailability comparable with normal weight children

    NARCIS (Netherlands)

    Aeberli, I.; Hurrell, R.F.; Zimmermann, M.B.

    2009-01-01

    Background: Obesity increases the risk for iron deficiency, but the underlying mechanism is unclear. It is possible that overweight individuals may have lower dietary iron intake and/or bioavailability. Alternatively, obesity-related inflammation may increase hepcidin concentrations and reduce iron

  9. Associations of common variants in HFE and TMPRSS6 with iron parameters are independent of serum hepcidin in a general population: a replication study

    NARCIS (Netherlands)

    Galesloot, T.E.; Geurts-Moespot, A.; Heijer, M. den; Sweep, F.C.; Fleming, R.E.; Kiemeney, L.A.L.M.; Vermeulen, S.; Swinkels, D.W.

    2013-01-01

    BACKGROUND: Genome-wide association studies have convincingly shown that single nucleotide polymorphisms (SNPs) in HFE and TMPRSS6 are associated with iron parameters. It was commonly thought that these associations could be explained by the intermediate effect on hepcidin concentration. A recent

  10. Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

    NARCIS (Netherlands)

    Gardenghi, Sara; Renaud, Tom M; Meloni, Alessandra; Casu, Carla; Crielaard, Bart J; Bystrom, Laura M; Greenberg-Kushnir, Noa; Sasu, Barbra J; Cooke, Keegan S; Rivella, Stefano

    2014-01-01

    Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing

  11. Pharmacokinetics of dilevalol and its conjugates in man. Assay method for plasma, blood, urine and bile samples and preliminary pharmacokinetic studies.

    Science.gov (United States)

    Neubeck, M; Becker, C; Henke, D; Rösch, W; Spahn-Langguth, H; Mutschler, E

    1993-09-01

    The renal and biliary excretion of the beta-adrenoceptor blocking agent dilevalol (CAS 75659-07-3) and its conjugates was examined in a preliminary pharmacokinetic study. Plasma, urine and bile dilevalol concentrations were determined with a simplified procedure that is based on alkaline liquid-liquid extraction using diethyl ether and subsequent reversed-phase HPLC separation of the reconstituted samples (on a PRP-1 stationary phase using a mixture of methanol and pH 9.8 carbonate buffer as mobile phase). Triamterene was used as internal standard. The quantification of the conjugates was accomplished indirectly via enzymatic hydrolysis (glusulase) with and without addition of the beta-glucuronidase inhibitor 1,4-saccharolactone (at a final concentration of 5.5 mmol/l). In the pharmacokinetic study healthy volunteers and cholecystectomised patients with a T-drain received a single oral dose of 200 mg dilevalol. Furthermore, to healthy volunteers an i.v. dose of 60 mg dilevalol was given in order to estimate the absolute bioavailability. From the obtained data the systemic plasma clearance was calculated to be 1708 ml/min. The oral bioavailability was calculated to be 16%. The log concentration-time curves of the metabolites paralleled those of dilevalol in the terminal section with average terminal half-lives of approx. 5 h. In volunteers the fractions of the dose excreted renally were 0.5% for parent drug, 23% for the glucuronide(s) and 8% for the sulfate. The corresponding values found for the patients were not significantly different. In the patients' bile only 1.2% of the total dose were found (0.03% dilevalol, 1.1% dilevalol glucuronide(s), 0.1% dilevalol sulfate).(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Hepcidin, Cathelicidin-1 and IL-8 as immunological markers of responsiveness in early developmental stages of rainbow trout.

    Science.gov (United States)

    Santana, Paula A; Guzmán, Fanny; Forero, Juan C; Luna, Omar F; Mercado, Luis

    2016-09-01

    During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.

  13. EPO-dependent induction of erythroferrone drives hepcidin suppression and systematic iron absorption under phenylhydrazine-induced hemolytic anemia.

    Science.gov (United States)

    Jiang, Xingkang; Gao, Ming; Chen, Yue; Liu, Jing; Qi, Shiyong; Ma, Juan; Zhang, Zhihong; Xu, Yong

    2016-05-01

    Hemolytic anemia is a common form of anemia due to hemolysis, resulting in disordered iron homeostasis. In this study, a dose of 40mg/kg phenylhydrazine (PHZ) was injected into mice to successfully establish a pronounced anemia animal model, which resulted in stress erythropoiesis and iron absorption. We found that serum erythropoietin (EPO) concentration was dramatically elevated by nearly 5000-fold for the first 2days, and then drop to the basal level on day 6 after PHZ injection. Mirrored with serum EPO concentration, the mRNA expression of erythroferrone (ERFE) was rapidly increased in the bone marrow and spleen 3days after injection of PHZ, and then gradually decreased but was still higher than baseline on day 6. In addition, we also found that the hepcidin mRNA levels were gradually reduced almost up to 8-fold on day 5, and then was ameliorated compared to the untreated control. Mechanistic investigation manifested that the increase of serum EPO essentially determined the induction of ERFE expression particular at the first 3days after PHZ treatment. Lentiviral mediated ERFE knockdown significantly restrained hepcidin suppression under PHZ treatment. Thus, our data unearthed EPO-dependent ERFE expression acts as an erythropoiesis-driven regulator of iron metabolism under PHZ-induced hemolytic anemia.

  14. Elevated Serum Hepcidin Levels during an Intensified Training Period in Well-Trained Female Long-Distance Runners

    Directory of Open Access Journals (Sweden)

    Aya Ishibashi

    2017-03-01

    Full Text Available Iron is essential for providing oxygen to working muscles during exercise, and iron deficiency leads to decreased exercise capacity during endurance events. However, the mechanism of iron deficiency among endurance athletes remains unclear. In this study, we compared iron status between two periods involving different training regimens. Sixteen female long-distance runners participated. Over a seven-month period, fasting blood samples were collected during their regular training period (LOW; middle of February and during an intensified training period (INT; late of August to determine blood hematological, iron, and inflammatory parameters. Three-day food diaries were also assessed. Body weight and lean body mass did not differ significantly between LOW and INT, while body fat and body fat percentage were significantly lower in INT (p < 0.05. Blood hemoglobin, serum ferritin, total protein, and iron levels, total iron-binding capacity, and transferrin saturation did not differ significantly between the two periods. Serum hepcidin levels were significantly higher during INT than LOW (p < 0.05. Carbohydrate and iron intakes from the daily diet were significantly higher during INT than LOW (p < 0.05. In conclusion, an elevated hepcidin level was observed during an intensified training period in long-distance runners, despite an apparently adequate daily intake of iron.

  15. Elevated Serum Hepcidin Levels during an Intensified Training Period in Well-Trained Female Long-Distance Runners

    Science.gov (United States)

    Ishibashi, Aya; Maeda, Naho; Sumi, Daichi; Goto, Kazushige

    2017-01-01

    Iron is essential for providing oxygen to working muscles during exercise, and iron deficiency leads to decreased exercise capacity during endurance events. However, the mechanism of iron deficiency among endurance athletes remains unclear. In this study, we compared iron status between two periods involving different training regimens. Sixteen female long-distance runners participated. Over a seven-month period, fasting blood samples were collected during their regular training period (LOW; middle of February) and during an intensified training period (INT; late of August) to determine blood hematological, iron, and inflammatory parameters. Three-day food diaries were also assessed. Body weight and lean body mass did not differ significantly between LOW and INT, while body fat and body fat percentage were significantly lower in INT (p < 0.05). Blood hemoglobin, serum ferritin, total protein, and iron levels, total iron-binding capacity, and transferrin saturation did not differ significantly between the two periods. Serum hepcidin levels were significantly higher during INT than LOW (p < 0.05). Carbohydrate and iron intakes from the daily diet were significantly higher during INT than LOW (p < 0.05). In conclusion, an elevated hepcidin level was observed during an intensified training period in long-distance runners, despite an apparently adequate daily intake of iron. PMID:28335426

  16. The Study of the Interaction Between Hepcidin and Fpn by FRET%利用FRET技术研究Hepcidin和Fpn相互作用

    Institute of Scientific and Technical Information of China (English)

    刘晓志; 赵会景; 陈伟斌; 常彦忠; 段相林

    2011-01-01

    Iron is an essential trace element of life, ferroportin (Fpn) is the intestinal absorption of iron release of important cell proteins. Newly discovered antimicrobial peptide hepcidin secreted by the liver can regulate the important role of intestinal iron absorption, but still play a role in the lack of Fpn and experimental basis for hepcidin. So fluorescence resonance energy transfer technology (fluorescence resonance energy transfer,FRET) on the role of hepcidin and Fpn relationship between the depth study. First of all, were hepcidin-CFP fusion protein expression vector and identification; then with YFP, Fpn-YFP gene in animal cell expression vector, expression, and FRET detection. The results confirmed that hepcidin and Fpn direct interaction between,and found that two proteins interact in the cytoplasm after the distribution of hepcidin also. The results for the treatment of disorders of iron metabolism to provide a new and important theoretical basis for therapeutic strategies.%铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白.新近发现肝脏分泌的抗菌多肽hepcidin具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据.应用荧光共振能量转移技术(fluorescence resonance energy transfer,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究.首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测.实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布.为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据.

  17. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

    Directory of Open Access Journals (Sweden)

    Paolo Gaibani

    2013-01-01

    Full Text Available Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

  18. Liquid chromatography-tandem mass spectrometric assay for aliskiren, a novel renin inhibitor in micro-volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects.

    Science.gov (United States)

    Adireddy, Vinayender; Pilli, Nageswara Rao; Derangula, Venkata Ramu; Satla, Shobha Rani; Ganguri, Chinna Veera Badraiah; Ponneri, Venkateswarlu

    2013-08-01

    This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via liquid-liquid extraction using tert-butyl methyl ether. The chromatographic separation was achieved on a C18 column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r(2) ≥ 0.99) over the concentration range of 0.10-1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies.

  19. Hepcidin and 1,25(OH)2D3 effectively restore Ca2+ transport in β-thalassemic mice: reciprocal phenomenon of Fe2+ and Ca2+ absorption.

    Science.gov (United States)

    Kraidith, Kamonshanok; Svasti, Saovaros; Teerapornpuntakit, Jarinthorn; Vadolas, Jim; Chaimana, Rattana; Lapmanee, Sarawut; Suntornsaratoon, Panan; Krishnamra, Nateetip; Fucharoen, Suthat; Charoenphandhu, Narattaphol

    2016-07-01

    Previously, β-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of β-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how β-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous β-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3 treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3 and hepcidin could be used to alleviate the β-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in β-thalassemia.

  20. Antimicrobial activity of human hepcidin 20 and 25 against clinically relevant bacterial strains: effect of copper and acidic pH.

    Science.gov (United States)

    Maisetta, Giuseppantonio; Petruzzelli, Raffaele; Brancatisano, Franca Lisa; Esin, Semih; Vitali, Alberto; Campa, Mario; Batoni, Giovanna

    2010-11-01

    Hepcidin 25 (hep-25) is a peptide primarily produced by human liver with a central role in iron homeostasis. Its isoform, hepcidin 20 (hep-20), has an unknown function and lacks the first five aminoacids of the amino-terminal portion. This sequence is crucial for iron regulation by hep-25 and contains a molecular motif able to bind metals. Aim of this study, was to evaluate the antibacterial properties of both peptides in vitro, against a wide range of bacterial clinical isolates and in different experimental conditions. Although both peptides were found to be bactericidal against a variety of clinical isolates with different antibiotic resistance profiles, hep-20 was active at lower concentrations than hep-25, in most of the cases. Killing kinetics, carried on in sodium-phosphate buffer at pH 7.4, demonstrated that bactericidal activity occurred not earlier than 30-90 min of incubation. Bactericidal activity of hep-25 was slightly enhanced in the presence of copper, while the same metal did not affect the activity of hep-20. Interestingly, bactericidal activity of both hepcidins was highly enhanced at acidic pH. Acidic pH (pH 5.0 and 6.6) not only reduced the microbicidal concentrations of hepcidins, but also shortened the killing times of both peptides, as compared to pH 7.4. Combining hep-20 and hep-25 at pH 5.0 a bactericidal effect could be obtained at very low concentrations of both peptides. These results render hepcidins interesting for the design of new drugs for the treatment of infections occurring in body districts with physiologic acidic pH. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. UPLC-MS/MS assay for the simultaneous quantification of carvedilol and its active metabolite 4'-hydroxyphenyl carvedilol in human plasma to support a bioequivalence study in healthy volunteers.

    Science.gov (United States)

    Patel, Daxesh P; Sharma, Primal; Sanyal, Mallika; Singhal, Puran; Shrivastav, Pranav S

    2013-08-01

    An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of carvedilol and its pharmacologically active metabolite 4'-hydroxyphenyl carvedilol in human plasma using their deuterated internal standards (IS). Samples were prepared by solid-phase extraction using 100 μL human plasma. Chromatographic separation of analytes was achieved on UPLC C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile-4.0 mM ammonium formate, pH 3.0 adjusted with 0.1% formic acid (78:22, v/v) as the mobile phase. The multiple reaction monitoring transitions for both the analytes and IS were monitored in the positive electrospray ionization mode. The method was validated over a concentration range of 0.05-50 ng/mL for carvedilol and 0.01-10 ng/mL for 4'-hydroxyphenyl carvedilol. Intra- and inter-batch precision (% CV) and accuracy for the analytes varied from 0.74 to 3.88 and 96.4 to 103.3% respectively. Matrix effect was assessed by post-column analyte infusion and by calculation of precision values (coefficient of variation) in the measurement of the slope of calibration curves from eight plasma batches. The assay recovery was within 94-99% for both the analytes and IS. The method was successfully applied to support a bioequivalence study of 12.5 mg carvedilol tablets in 34 healthy subjects.

  2. Evidence of relative iron deficiency in platelet- and plasma-pheresis donors correlates with donation frequency.

    Science.gov (United States)

    Li, Huihui; Condon, Frances; Kessler, Debra; Nandi, Vijay; Rebosa, Mark; Westerman, Mark; Shaz, Beth H; Ginzburg, Yelena

    2016-12-01

    The loss of iron stores and resulting iron deficiency is well documented in whole blood or red blood cell donors. We hypothesized that relative iron deficiency also occurs as a result of more frequent platelet- and plasma-pheresis (apheresis) donation. To test this hypothesis, we proposed a pilot cross-sectional study to analyze erythropoiesis- and iron-related parameters in white male apheresis donors: (1) relative to controls, (2) in correlation with apheresis donation frequency, and (3) in correlation with pre-donation platelet count. Fifty eligible apheresis donors and eight controls were enrolled in the study. Apheresis donors were found to have a lower serum ferritin and serum hepcidin and exhibited evidence of iron restricted erythropoiesis relative to controls. Furthermore, among donors, lower MCV, CH(r) , hepcidin concentration, and serum ferritin were observed in more frequent apheresis donors. Correlations between donation frequency and hepcidin and ferritin were noted in apheresis donors. This pilot study demonstrates that apheresis donors are relatively iron deficient compared to controls and supports the premise that frequent apheresis donation correlates with relatively iron restricted erythropoiesis. An analysis of iron- and erythropoiesis-related parameters in a broader population of frequent apheresis donors (i.e., female and non-white donors) may demonstrate larger deficits and an even greater potential benefit of iron replacement. J. Clin. Apheresis 31:551-558, 2016. © 2015 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Development of a rapid and sensitive UPLC-MS/MS assay for the determination of TM-2 in beagle dog plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Lin, Hongli; Zhao, Yunli; Men, Lei; Yang, Mingjing; Liu, Hui; Shao, Yanjie; Wang, Pei; Tang, Xing; Yu, Zhiguo

    2015-01-01

    A simple and sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of TM-2, which was a novel semi-synthetic taxane derivative in beagle dog plasma. Cabazitaxel was chosen as internal standard. Following extraction by methyl tert-butyl ether, the chromatographic separation was achieved on a Thermo Syncronis C18 column (50 × 2.1 mm, 1.7 µm) by gradient elution within a runtime of 3.5 min. The mobile phase consisted of (A) acetonitrile and (B) 2 mmol/L ammonium acetate in water. The detection was accomplished using positive ion electrospray ionization in multiple reaction monitoring mode. The MS/MS ion transitions were monitored at m/z 812.39 → 551.35 for TM-2 and 836.36 → 555.26 for IS, respectively. The method was linear for TM-2 (r = 0.9924) ranging from 2.5 to 1000 ng/mL. The intra-day and inter-day precisions (relative standard deviation) were within 8.0 and 17.6%, respectively, and the accuracy (relative error) was less than 2.3%. The extraction recovery ranged from 83.1 to 97.1%. The reliable method was successfully applied to a pharmacokinetic study of TM-2 in beagle dogs after intravenous drip with different doses of 0.6, 1.2, and 2.4 mg/kg, respectively.

  4. Liver iron is a major regulator of hepcidin gene expression via BMP/SMAD pathway in a rat model of chronic renal failure under treatment with high rHuEPO doses.

    Science.gov (United States)

    Ribeiro, Sandra; Garrido, Patrícia; Fernandes, João; Rocha-Pereira, Petronila; Costa, Elísio; Belo, Luís; Reis, Flávio; Santos-Silva, Alice

    2016-05-01

    Hepcidin is the major central regulator of iron metabolism, controlling iron absorption and mobilization. Considering its interaction with several factors that are altered in chronic kidney disease (CKD), particularly in hyporesponsive CKD patients under therapy with high recombinant human erythropoietin (rHuEPO) doses, it was aimed to study the impact of increasing rHuEPO doses on the regulation of iron-hepcidin metabolism. The blood, cellular, and tissue studies, using the remnant kidney rat model of CKD induced by 5/6 nephrectomy, under rHuEPO (100, 200, 400, and 600 IU/Kg body weight [BW]/week) treatment during 3 weeks were performed. It was found that the rHuEPO stimulus triggered a first wave to achieve correction of anemia, by inhibiting hepcidin synthesis, favoring erythropoiesis and iron absorption; this continuous stimulus enhanced iron absorption leading to iron overload, as showed by the hepatic iron deposits found in rats treated with higher rHuEPO dose that seems to trigger the upregulation of hepcidin synthesis through the activation of the BMP6/SMAD pathway. The data suggested that liver iron overload was an important stimuli for hepcidin synthesis, stronger than the inhibitory effect of high rHuEPO doses; moreover, the findings raised the hypothesis that when high inflammation (triggering hepcidin expression) was associated with increased iron stores in hemodialysis patients, hepcidin expression was also upregulated via BMP6, enhancing hepcidin synthesis, leading, therefore, to worsening of anemia and, eventually, to a hyporesponse/resistance to rHuEPO therapy. © 2016 BioFactors, 42(3):296-306, 2016.

  5. 促红细胞生成素抑制正常及缺铁性贫血大鼠铁调素的表达%Inhibitory Effect of Erythropoietin on Hepcidin Expression in Normal and IDA Rats

    Institute of Scientific and Technical Information of China (English)

    明丽娟

    2012-01-01

    Objective: To investigate the effect of erythropoietin ( EPO ) on the serum hepcidin expression in iron deficiency anemia ( IDA ) rats. Method: IDA rat model was established by repeated bloodletting and low-iron diet feeding. The hepcidin expression in normal rats with or without EPO intervention and IDA rats with or without EPO intervention was respectively detected and compared. Result: After the intra-peritoneal injection of EPO, hepcidin expression and C/EBPα levels decreased in the IDA group. EPO also significantly inhibited hepcidin expression in the normal rats( P<0. 05 ). Conclusion: Hepcidin plays an important role in the pathogene-sis of IDA. Both in normal and IDA rats, EPO can decrease hepcidin expression levels by declining C/EBPα to hinder the activation of the hepcidin promoter.%目的:探讨促红细胞生成素(EPO)在缺铁性贫血(IDA)大鼠模型中对铁调素(hepcidin)表达的影响.方法:通过反复放血和饲喂低铁饲料,建立IDA大鼠动物模型,比较药物干预组(EPO)与非干预组及正常对照组与正常给药组(EPO)大鼠血清hepcidin表达的差异.Western blot检测肝脏内信号因子CCAAT/增强子结合蛋白α(C/EBPα)含量.结果:IDA模型组腹腔注射EPO后,血清hepcidin和肝脏C/EBPα含量显著降低.EPO对正常大鼠hepcidin表达也有显著抑制作用(P<0.05).结论:hepcidin可能参与了IDA的发生,在正常大鼠和IDA大鼠中,EPO能通过下调C/EBPα阻碍其激活hepcidin启动子最终抑制hepcidin的表达.

  6. Inflammation neither increases hepatic hepcidin nor affects intestinal (59)Fe-absorption in two murine models of bowel inflammation, hemizygous TNF(ΔARE/+) and homozygous IL-10(-/-) mice.

    Science.gov (United States)

    Buffler, M; Becker, C; Windisch, W; Schümann, K

    2015-10-01

    Hepcidin-synthesis was reported to be stimulated by inflammation. In contrast, hepcidin synthesis was inhibited by TNFα and serum hepcidin was low. To elucidate these contradictions, we compare data on hepcidin expression, on iron absorption and homoeostasis and markers of inflammation between two murine models of intestinal inflammation and corresponding wild-types as determined by standard methods. In TNF(ΔARE/+) and IL-10(-/-)-mice hepatic hepcidin expression and protein content was significantly lower than in corresponding wild-types. However, (59)Fe whole-body retention showed no difference between knock-outs and corresponding wild-types 7d after gavage, in neither strain. Compared to wild-types, body weight, hepatic non-haem iron content, hemoglobin and hematocrit were significantly decreased in TNF(ΔARE/+) mice, while erythropoiesis increased. These differences were not seen in IL-10(-/-) mice. Duodenal IL-6 and TNFα content increased significantly in TNF(ΔARE/+) mice, while ferritin-H decreased along with hepatic hepcidin expression, ferritin L, and non-haem iron. In IL-10(-/-) mice, these changes were less marked or missing for non-haem iron. Duodenal ferritin-L and ferroportin increased significantly, while HFE decreased. Our results corroborate the conflicting combination of low hepcidin with inflammation and without increased intestinal iron absorption. Speculating on underlying mechanism, decreased hepcidin may result from stimulated erythropoiesis. Unaltered intestinal iron-absorption may compromise between the stimulation by increased erythropoiesis and inhibition by local and systemic inflammation. The findings suggest intense interaction between counterproductive mechanisms and ask for further research.

  7. Expression of Hepcidin and Ferroportin in the Placenta, and Ferritin and Transferrin Receptor 1 Levels in Maternal and Umbilical Cord Blood in Pregnant Women with and without Gestational Diabetes

    Science.gov (United States)

    Yang, Anqiang; Zhao, Jun; Lu, Minhua; Gu, Ying; Zhu, Yunlong; Chen, Daozhen; Fu, Jinyan

    2016-01-01

    Background: Regulation of iron transfer from mother to fetus via the placenta is not fully understood and the relationship between stored iron status in the mothers’ serum and gestational diabetes (GDM) in case–control studies is controversial. The present study aimed to detect circulating soluble transferrin receptor (sTfR) and ferritin levels in maternal and umbilical cord blood. We also examined the expression of hepcidin (Hep), transferrin receptor (TfR1), and ferroportin (FPN) in the placenta in pregnant women with and without GDM at full term. Methods: Eighty-two women participated (42 with GDM and 40 without GDM [controls]). Maternal samples were collected at 37–39 weeks’ gestation. Umbilical cord blood was collected at birth. Ferritin and sTfR levels in maternal serum and umbilical cord blood, and Hep, TfR1, and FPN protein expression in plac enta were compared between the GDM and non-GDM groups. Serum ferritin (SF) was measured by electrochemiluminescence assay and sTfR was measured by ELISA. Hep, TfR1, and FPN expression was measured by immunohistochemistry. Results: Maternal serum sTfR levels were significantly elevated in the GDM group compared with the non-GDM group (p = 0.003). SF levels in cord blood in the GDM group were significantly higher than those in the non-GDM group (p = 0.003). However, maternal hemoglobin and SF, and umbilical cord sTfR levels were not different between the groups. In placental tissue, FPN expression was higher and hepcidin expression was lower in the GDM group compared with the non-GDM group (p = 0.000 and p = 0.044, respectively). There was no significant difference in TfR1 between the groups (p = 0.898). Conclusions: Women with GDM transport iron more actively than those without GDM at term pregnancy. Maternal iron metabolism in GDM may play a role in fetal/placental iron demand and in the overall outcome of pregnancy. PMID:27483296

  8. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  9. Ontogeny and modulation after PAMPs stimulation of β-defensin, hepcidin, and piscidin antimicrobial peptides in meagre (Argyrosomus regius).

    Science.gov (United States)

    Campoverde, Cindy; Milne, Douglas J; Estévez, Alicia; Duncan, Neil; Secombes, Christopher J; Andree, Karl B

    2017-10-01

    Antimicrobial peptides (AMPs), components of innate immunity, play an important role in protecting fish. In this study we report the molecular cloning of full open reading frames and characterization of expression of three AMP genes (β-defensin (defb), hepcidin (hep2), piscidin (pisc) in meagre (Argyrosomus regius). A phylogenetic analysis of the expressed sequences obtained shows the defensin isoform forms a clade with the other members of the beta class of this family, hepcidin corresponds to hepcidin 2, and piscidin corresponds to class I of its respective family. Gene expression profiles of AMPs was investigated, by means of quantification of mRNA in nine development stages, from 8 days post-hatching (dph) to accomplishment of juvenile form (120 dph). During development it was demonstrated defb, hep2, pisc were expressed in all stages of larval development and in juvenile tissues (kidney, spleen gut and gill). Moreover, expression patterns suggest the expression levels of theses AMPs are influenced by live prey (rotifer, Artemia) and first intake of commercial diet. Induction experiments in vivo (24 h) and in vitro (4, 12, 24 h) with PAMPs (LPS, poly (I:C), β-glucan) revealed significant changes in gene expression of the three AMP genes, in kidney, spleen, gut and gill. However, expression profiles differed in magnitude and time course response. defb expression shows a similar trend in vivo and in vitro in kidney at 24 h after LPS and β-glucan stimulation. The hep2 expression levels were up-regulated upon β-glucan challenge in vivo, more in gut and gills than kidney, while in vitro hep2 expression was up-regulated in kidney cells by LPS, poly (I:C), β-glucan (4 h). pisc expression was up-regulated in kidney cells, splenocytes by β-glucan, but in gill cells by poly (I:C) and β-glucan in vivo. However, pisc expression was upregulated in kidney cells by β-glucan and gill cells by LPS at 4 post-stimulation in vitro. These data suggest that AMPs

  10. Development and validation of a UHPLC-MS/MS assay for colistin methanesulphonate (CMS) and colistin in human plasma and urine using weak-cation exchange solid-phase extraction.

    Science.gov (United States)

    Zhao, Miao; Wu, Xiao-Jie; Fan, Ya-Xin; Guo, Bei-Ning; Zhang, Jing

    2016-05-30

    A rapid ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS and formed colistin in human plasma and urine. After extraction on a 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the eluents were mixed and injected into the UHPLC-MS/MS system directly. A Phonomenex Kinetex XB-C18 analytical column was employed with a mobile phase consisting of solution "A" (acetonitrile:methanol, 1:1, v/v) and solution "B" (0.1% formic acid in water, v/v). The flow rate was 0.4 mL/min with gradient elution over 3.5 min. Ions were detected in ESI positive ion mode and the precursor-product ion pairs were m/z 390.7/101.3 for colistin A, m/z 386.0/101.2 for colistin B, and m/z 402.3/101.2 for polymyxin B1 (IS), respectively. The lower limit of quantification (LLOQ) was 0.0130 and 0.0251 mg/L for colistin A and colistin B in both plasma and urine with accuracy (relative error, %) <± 12.6% and precision (relative standard deviation, %) <± 10.8%. Stability of CMS was demonstrated in biological samples before and during sample treatment, and in the extract. This new analytical method provides high-throughput treatment and optimized quantification of CMS and colistin, which offers a highly efficient tool for the analysis of a large number of clinical samples as well as routine therapeutic drug monitoring.

  11. Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas).

    Science.gov (United States)

    Kaplan, Amy J; Stacy, Nicole I; Jacobson, Elliott; Le-Bert, Carolina R; Nollens, Hendrik H; Origgi, Francesco C; Green, Linda G; Bootorabi, Shadi; Bolten, Alan; Hernandez, Jorge A

    2016-01-01

    The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38-0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16-2.15 g/dL) and 0.40-0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18-1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology.

  12. On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

    Science.gov (United States)

    Nicolardi, Simone; Giera, Martin; Kooijman, Pieter; Kraj, Agnieszka; Chervet, Jean-Pierre; Deelder, André M.; van der Burgt, Yuri E. M.

    2013-12-01

    Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.

  13. Prevalence of anemia and associations between neonatal iron status, hepcidin, and maternal iron status among neonates born to pregnant adolescents.

    Science.gov (United States)

    Lee, Sunmin; Guillet, Ronnie; Cooper, Elizabeth M; Westerman, Mark; Orlando, Mark; Kent, Tera; Pressman, Eva; O'Brien, Kimberly O

    2016-01-01

    Little is known about anemia and iron status in US newborns because screening for anemia is typically not undertaken until 1 y of age. This study was undertaken to characterize and identify determinants of iron status in newborns born to pregnant adolescents. Pregnant adolescents (≤ 18 y, n = 193) were followed from ≥ 12 wk gestation until delivery. Hemoglobin, ferritin, soluble transferrin receptor, serum iron, hepcidin, erythropoietin (EPO), IL-6, and C-reactive protein were assessed in maternal and cord blood. At birth, 21% of the neonates were anemic (Hb Neonates born to mothers with ferritin neonates born to mothers with longer durations of active labor. Given the importance of the iron stores at birth on maintenance of iron homeostasis over early infancy, additional screening of iron status at birth is warranted among those born to this high risk obstetric population.

  14. B cell helper assays.

    Science.gov (United States)

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  15. [The effect of exogenous antioxidants on the antioxidant status of erythrocytes and hepcidin content in blood of patients with disorders of iron metabolism regulation].

    Science.gov (United States)

    Shcherbinina, S P; Levina, A A; Lisovskaia, I L; Ataullakhanov, F I

    2013-01-01

    In many diseases associated with impairments in iron metabolism, erythrocytes exhibit an increased sensitivity to oxidative stress induced in vitro. In this study, we have examined the antioxidant status of erythrocytes from healthy donors and from 12 patients with disorders of iron homeostasis by measuring the extent of t-BHP-induced hemolysis in vitro. The extent of hemolysis observed with patient erythrocytes was significantly higher than that observed in experiment with normal cells. After therapeutic infusions of the antioxidants mexidol or emoxypin, oxidative hemolysis in patients was restored to normal values and blood hepcidin content increased significantly. A significant correlation was observed between hepcidin concentration after treatment and t-BHP-induced hemolysis before treatment. These data suggest that antioxidants may exert a favorable effect under pathological conditions associated with iron overload disease.

  16. In Vivo Effects of Pichia Pastoris-Expressed Antimicrobial Peptide Hepcidin on the Community Composition and Metabolism Gut Microbiota of Rats.

    Science.gov (United States)

    Tian, Lanfang; Chen, Siyuan; Liu, Haiyan; Guo, Mingzhang; Xu, Wentao; He, Xiaoyun; Luo, Yunbo; Qi, Xiaozhe; Luo, Hongxia; Huang, Kunlun

    2016-01-01

    Hepcidin, one kind of antimicrobial peptides, is one of the promising alternatives to antibiotics with broad spectrum of antimicrobial activity. Hepcidins cloned from different kinds of fishes have been produced using exogenous expression systems, and their in vitro antimicrobial effects have been verified. However their in vivo effects on gut microbiota and gut health of hosts remain unclear. Here we performed a safety study of hepcidin so that it can be used to reduce microbial contaminations in the food and feed. In this study, Pichia pastoris-expressed Pseudosciaena crocea hepcidin (PC-hepc) was first assessed by simulated digestion tests and then administered to male and female Sprague-Dawley (SD) rats in different concentrations. Subchronic toxicity testing, high throughput 16S rRNA sequencing of gut microbiota, and examinations on gut metabolism and permeability were conducted. The results showed PC-hepc could be digested in simulated intestinal fluid but not in simulated gastric fluid. PC-hepc had no adverse effects on general health, except causing increase of blood glucose (still in the normal value range of this index) in all trial groups of female rats and intestinal inflammation in HD group of female rats. Community composition of gut microbiota of female MD and HD groups shifted compared with control group, of which the decrease of genus Akkermansia might be related to the increase of blood glucose and intestinal inflammation. Significant increase of fecal nitroreductase activity was also observed in female MD and HD groups. Our results suggest the uses of exogenous PC-hepc in normal dosage are safe, however excess dosage of it may cause intestinal disorder of animals.

  17. A Microaffinity Assay for Plasma Fibronectin.

    Science.gov (United States)

    1983-03-25

    employed as the source of fibronectin. The purification procedure was a modification of the method of Vuento and Vaheri (15) which involves affinity...Coated Beads. The gelatin-coated Sepharose 4B beads were prepared by a minor modification of the method of Vuento and Vaheri (15). Cyanogen bromide...Soc. 23, 119-134. 8. Ruoslahti, E., Vuento , M., and Engvall, E. (1978) Biochim. Biophys. Acta 534, 210-218. 9. Todd-Kulikowski, H.D. and Parsons, R.G

  18. 快速免疫荧光法检测血浆NT-proBNP的分析性能评价%Evaluation of performance of a rapid immunofluorescence method for assaying plasma NT-proBNP

    Institute of Scientific and Technical Information of China (English)

    唐筑灵; 徐国宾

    2010-01-01

    Objective To analyze the performance of a rapid immunofluorescence assay for plasma NT-proBNP. Methods Human plasma NT-proBNP was measured by RAMP in 264 healthy cases and 78 patients with heart failure. The precision, stability, linearity and interference factors of RAMP were evaluated according to the protocold from America CLSI. Meanwhile, the results were compared with those obtained by Elecsys. Results Functional sensitivity of RAMP in 20% CV and 10% CV were 48 ng/L and 57 ng/L respectively. The linear range was 18-8 000 ng/L. NT-proBNP in plasma samples detected by RAMP were stable at room temperature for 24 hours, 4 ℃ for 3 days and - 20 ℃ for 20 days. No influences on results were observed throughout three freeze-thaw cycles. The results measured by RAMP were compared between EDTA-K2 anticoagulant plasma and heparin anticoagulant plasma, which showed that YEDTA-K2 =0. 953 9 Xheparin + 0. 365 2 ( R2 = 0. 982, P < 0. 01, n = 40 ). The results of EDTA-K2 and heparin anticoagulant plasma using RAMP and Elecsys showed no significant difference( P >0. 05). Slight hemolysis( Hb 2 g/L)had small effect on the results of PAMP and Elecsys assays and deviations of them were below 5%. However median and heavy hemolysis( Hb 3-4 g/L )obviously influenced the results with deviation were more than 15%. The values measured by RMAP fell from 390 ng/L to 82 ng/L, and those measured by Elecsys method fell from 390 ng/L to 178 ng/L when 3 different concentrations of triacylglycerol were added, the values measured by two methods fell from 7 777 ng/L to 7 741 ng/L when bilirubin ( 16-330 μmol/L) was added. The anti-interference ability of RAMP method was similar to Elecsys. Results detected with two methods in 45 EDTA-K2 anticoagulant plasma samples were analyzed with Passing and Bablok regression.The regression equation was YRAMP = 0. 972 8XElecsys - 0. 035 2 (R2 = 0. 994, P > 0. 05, n = 45 ). When heparin anticoagulant plasma samples increased to 78 samples, Passing and

  19. Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus

    2011-01-01

    OBJECTIVE: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. METHODS: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 ...

  20. An LC-MS/MS assay for the quantitative determination of 2-pyridyl acetic acid, a major metabolite and key surrogate for betahistine, using low-volume human K2 EDTA plasma.

    Science.gov (United States)

    Soni, Krunal; Bhatt, Chandrakant; Singh, Kanchan; Bhuvaneshwari, P C; Jha, Anil; Patel, Palak; Patel, Harilal; Srinivas, Nuggehally R

    2017-02-01

    Betahistine is widely used for the treatment of vertigo. Owing to first-pass metabolism, 2-pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC-MS/MS method was developed and validated for quantitation of 2PAA using turbo-ion spray in a positive ion mode. A solid-phase extraction was employed for the extraction of 2PAA and 2PAA d6 (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 μm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile-methanol (90:10% v/v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m/z 138.1 to m/z 92.0 and m/z 142.1 to m/z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter- and intra-batch assays were <10%. The developed method was used to support clinical sample analysis.

  1. Radioreceptor assay method for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Mori, K.F.; Wood, R.J. (Bureau of Drug Research, Health and Welfare Canada, Ottawa, Ontario. Health Protection Branch)

    1984-01-01

    A sensitive practical radioreceptor assay method for pharmaceutical insulin products has been developed with partially purified rat liver plasma membranes and the optimal conditions under which the best overall assay performance is obtainable have been defined. Intra- and inter-assay variations of the method averaged 7.3 and 12.2% of the man, respectively, when expressed as the coefficient of variation. Potency estimates of an insulin product obtained with the proposed method correlated well with those determined by the mouse convulsion bioassay method. Liver membranes prepared according to the method could be stored for up to ten weeks at 4/sup 0/C and for 6 months or more at -18/sup 0/C without losing insulin-binding ability.

  2. Anemia, ineffective erythropoiesis, and hepcidin: interacting factors in abnormal iron metabolism leading to iron overload in β-thalassemia.

    Science.gov (United States)

    Gardenghi, Sara; Grady, Robert W; Rivella, Stefano

    2010-12-01

    β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

  3. Isolation, Purification and Identification of Recombinant Human Hepcidin%重组hepcidin的分离纯化及鉴定

    Institute of Scientific and Technical Information of China (English)

    朱亚平; 袁其朋; 张怀

    2006-01-01

    建立了重组hepcidin的分离纯化方法,并鉴定其抗菌活性.经金属螯合初步纯化的重组蛋白在cysteine/cystine氧化还原体系中氧化形成二硫键,用变性条件下的凝胶过滤除去多聚体,稀释复性后用于肠激酶酶切反应,得到重组hepcidin.融合蛋白His-hepcidin经氧化、复性后的总收率为50%,纯度大于95%.酶切后所得重组hepcidin经抑菌圈试验检验,对枯草芽孢杆菌具有抗菌活性.LC-ESI-MS与园二色光谱检测显示重组hepcidin与天然hepcidin相对分子质量相同、二级结构相似.

  4. DO HIGH BLOOD HEPCIDIN CONCENTRATIONS CONTRIBUTE TO LOW FERRITIN LEVELS IN YOUNG TENNIS PLAYERS AT THE END OF TOURNAMENT SEASON?

    Directory of Open Access Journals (Sweden)

    Ewa Ziemann

    2013-06-01

    Full Text Available The purpose of the present study was to verify whether impaired iron metabolism in young athletes is a consequence of an excessive workload during the tournament season. Low levels of ferritin (under 25 µg·L-1 have been frequently observed in young tennis players. We considered this finding to be related to the high-intensity workload or to insufficient rest, which both trigger a strong immune response. Groups of male, well-trained young tennis players (16 ± 0.9 years old, average of 10-year training experience and a control peer group participated in this study. The research consisted of two examination sessions (March and September 2010. Blood samples were collected to determine haematological and immunological parameters. Additionally, body composition and physical capacity were assessed. In both periods of the study, the trained groups were characterised by low levels of ferritin, but also elevated levels of pro- inflammatory cytokine IL-1β. Moreover, an inverse correlation between IL-1β and blood ferritin was observed. Additionally, an increased concentration of the iron homeostasis regulator hepcidin was found in blood samples (mean 71 ng·ml-1; range from 48 to 100 ng·ml-1. We concluded that the pro- inflammatory cytokine IL-1β, most likely induced by an extensive workload during the tournament season, was responsible for the low level of ferritin in young, professional athletes

  5. Effects of Endurance Exercise on Iron Metabolism and Hepcidin in Female Athletes%耐力运动对女运动员铁代谢及铁调素的影响

    Institute of Scientific and Technical Information of China (English)

    刘君雯; 聂集林

    2015-01-01

    This study aims to reveal the changes of iron metabolism and provide reference for the preven-tion and treatment of sports anemia by exploring the effects of endurance exercise on iron metabolism and hepcidin in female athletes.5 mL venous blood was taken from 12 collegiate female athletes before, immediately after,1 day after and 3 days after the 6-km round-the-city race of Hengyang in Jan.1,2015. Tests of serum iron,serum ferritin,hepcidin and IL-6 in the blood samples showed that immediately after the race,the levels of serum iron and IL-6 rose significantly (P <0.05)compared with those at the other time points;one day after the race,the serum hepcidin levels in the athletes were significantly greater (P<0.01)than those at the other time points.It can be concluded that the endurance exercise produced transient effects on serum iron,hepcidin and IL-6 in female athletes.Therefore the iron metabolism rela-tive indexes and hepcidin levels in female athletes should be frequently monitored.%为探索耐力运动对女运动员铁代谢及铁调素的影响,揭示运动时铁代谢的变化规律,并为防治运动性贫血提供依据和参考,对12名参加2015年衡阳市元旦环城赛跑(全长约6000 m)的大学女运动员,分别于赛前、赛后即刻、赛后1 d、赛后3 d 各取静脉血5 mL,测试血清铁、铁蛋白、Hepcidin 和白细胞介素6(IL 6).结果显示,赛后即刻运动员血清铁和 IL 6比其他各时间点显著上升(P <0.05),赛后1 d 运动员血清 Hepcidin 比其他各时间点显著上升(P <0.01).这说明耐力运动对女运动员血清铁、Hepcidin 和 IL 6产生了即时性影响,建议经常监控女运动员铁代谢相关指标及铁调素水平.

  6. Development and application of assays for serotonin

    Energy Technology Data Exchange (ETDEWEB)

    Gow, I.F.

    1987-01-01

    In this thesis, two assays for serotonin were developed, validated, and used to investigate the relationship between platelet aggregation, serotonin levels and sodium status and serotonin levels and platelet function in patients with cardiovascular disease. A radioimmunoassay (RIA) using an (/sup 125/I)-labelled tracer was developed and validated for the measurement of serotonin in human platelet-rich plasma (PRP) and rat serum. Antisera were raised against N-succinamylserotonin conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. An assay for serotonin using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) was developed, and used to validate the RIA. The RIA can be used to assay up to 100 samples/day compared with 10-20/day by the HPLC-ECD assay.

  7. Establishment of an assay for active von Willebrand factor in plasma and its application%血浆活化血管性vWF测定方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    韩纪举; 潘少东; 吴亚平; de GROOT Evelyn; de GROOT Philip G; 王云; 蔡洪信; 赵晓民; 夏作理

    2010-01-01

    机制提供重要依据.%Objective To establish an double-antibody sandwich method to detect the amount of active vWF in the plasma of healthy people and diabetic patients.Methods A double-antibody sandwich method has been established using llama-derived anti-active vWF antibody and horseradish peroxidaselabelled rabbit anti-human vWF polyclonal antibodies.The study randomly selected 66 healthy people,30 diabetic patients,10 cases of von Willebrand disease 2B(vWD-2B),5 cases of congenital thrombotic thrombocytopenic purpura(TIP),13 cases of acquired TIPs and 13 cases of malaria.The activated vWF level were described as the ratio of absorbance value(A)representing activated vWF value using AU/vWFa-11 antibody-based ELISA method to the concentrations of vWF antigen.At the same time,the absorbance values representing the concentrations of active vWF were measured when the concentrations of vWF antigen were 62,125,250μg/L respectively.The data were analyzed with GraphPad Prism 4 software.Under the premise of reaching a good linear regression,plasma active vWF values were calculated as the slope ratio of the sample plasma to the standard plasma.Accordingly to the ratio evaluated the levels of active vWF in plasma.Results The coefficients of variation(CVs)for intra assays were 4.21%,4.98% and 6.32% respectively.The CVs for interassays were 6.34%,7.02% and 7.69% respectively.In healthy adults,the level of active vWF in plasma was 0.74±0.35.There was no significant difference of the vWF levels[male (7.29±5.15)×10~3μg/L,female(9.42±4.67)×10~3μg/L]and active vWF levels(male 0.74±0.34,female 0.73±0.35)between male and female in healthy people(t=1.35,0.04,P>0.05).The vWF levels of the group over 40 years old[(10.64±5.39)×10~3μg/L]were higher than these below 40 years old[(6.11 ±2.84)×10~3 μL)(t=8.24,P < 0.01)],but there was no significant difference of the active vWF levels difference between these groups(over 40 years old 0.70±0.38,under 40 years old 0.78 ± 0.29,t=1.02,P>0.05).The plasma

  8. Hepcidin与疾病关系和其临床意义的研究进展%Recent progress in diagnosis applications of hepcidin in human disorders

    Institute of Scientific and Technical Information of China (English)

    牛诗雯; 宁波; 刘罡; 魏景艳; 聂广军

    2012-01-01

    铁是人体必需的微量元素,是血红蛋白、肌红蛋白及多种酶的重要组成成分,广泛地参与氧气输运、氧化还原反应、细胞增殖与分化、基因表达调控等基本生命过程.机体铁稳态对生命体新陈代谢的平衡起着至关重要的作用.铁稳态依赖铁吸收、转运和储存、再循环利用等代谢过程共同调节.铁调素(Hepcidin)是铁代谢调节中最关键的调节分子,成熟的铁调素是一个由25个氨基酸组成的功能性小肽类激素,可以通过调节小肠上皮细胞和巨噬细胞表面的相关铁转运蛋白来调控机体内铁的储存和利用.铁调素同时受到机体铁水平的反馈,免疫应答和红细胞生成等因素的共同调节.许多铁代谢疾病、炎症和各种原因引起的贫血与铁调素的异常表达相关.因此,对于铁调素的检测不但可以反映机体的铁代谢状况,结合其他临床指标还能够辅助诊断和有针对性地检测相关疾病的治疗效果.%Iron is an essential trace element with the capacity to accept and donate electrons in some fundamental biochemical reactions. It is an indispensable component of cytochromes, oxygen-binding molecules (i.e., hemoglobin and myoglobin), and many enzymes. Human iron homeostasis largely depends on the regulation of physiological processes of iron absorption, transportation and storage. Hepcidin, a 25-amino acid peptide hormone in mature form is produced by hepatocytes and the key regulator of iron metabolism. Hepcidin binds to the cellular iron exporter ferroportin located on the membrane of intestinal enterocytes and macrophages and triggers internalization and degradation of ferroportin, which consequently suppresses iron uptake and recycling. Meanwhile, hepcidin can be regulated by the feedback of iron levels, immune response, erythropoiesis and other factors. Abnormal expression of hepcidin is involved in iron metabolism disorders, inflammation and anemia. Therefore

  9. Cross-sectional study of expression of divalent metal transporter-1, transferrin, and hepcidin in blood of smelters who are occupationally exposed to manganese

    Directory of Open Access Journals (Sweden)

    Qiyuan Fan

    2016-09-01

    Full Text Available Background Manganese (Mn is widely used in industries including the manufacture of Mn-iron (Fe alloy. Occupational Mn overexposure causes manganism. Mn is known to affect Fe metabolism; this study was designed to test the hypothesis that workers exposed to Mn may have an altered expression of mRNAs encoding proteins in Fe metabolism. Methods Workers occupationally exposed to Mn (n = 71 from a Mn–Fe alloy factory and control workers without Mn-exposure (n = 48 from a pig-iron plant from Zunyi, China, were recruited for this study. Blood samples were collected into Trizol-containing tubes. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Metal concentrations were quantified by atomic absorption spectrophotometry. Results Working environment and genetic background of both groups were similar except for marked differences in airborne Mn concentrations (0.18 mg/m3 in Mn–Fe alloy factory vs. 0.0022 mg/m3 in pig-Fe plant, and in blood Mn levels (34.3 µg/L vs. 10.4 µg/L. Mn exposure caused a significant decrease in the expression of divalent metal transporter-1 (DMT1, transferrin (Tf and hepcidin by 58.2%, 68.5% and 61.5%, respectively, as compared to controls, while the expression of transferrin receptor (TfR was unaltered. Linear regression analysis revealed that expressions of DMT1, Tf and hepcidin were inversely correlated with the accumulative Mn exposure; the correlation coefficients (r are −0.47, −0.54, and −0.49, respectively (p < 0.01. Conclusion The data suggest that occupational Mn exposure causes decreased expressions of DMT1, Tf and hepcidin in blood cells; the finding will help understand the mechanism underlying Mn exposure-associated alteration in Fe homeostasis among workers.

  10. Hiperpigmentación cutánea y homeostasis del hierro: rol de la hepcidina Cutaneous hyperpigmentation and homeostasis of iron: role of the hepcidin

    Directory of Open Access Journals (Sweden)

    C. Wolf

    2007-06-01

    Full Text Available La hiperpigmentación cutánea por melanina en zonas expuestas al sol puede estar asociada a un desequilibrio en la homeostasis del hierro. La hepcidina es un péptido responsable de la regulación negativa de la absorción del hierro en el intestino delgado y de su liberación por los macrófagos. Posee capacidad antimicrobiana. Es sintetizada en el hígado, secretada al torrente circulatorio y excretada por la orina. La sobreexpresión causa anemia y su déficit, sobrecarga de hierro (acumulación en diferentes órganos y hemocromatosis hereditaria. Los antagonistas de la hepcidina podrían utilizarse en el tratamiento de la anemia resistente a eritropoyetina, asociada a procesos crónicos. Por su parte, los agonistas o sustancias que estimulen la producción de hepcidina, podrían constituir un tratamiento en enfermedades con sobrecarga de hierro (siderosis y por consiguiente, corregir la hiperpigmentación asociada.The cutaneous hyperpigmentation by melanin in zones of the skin exposed to the sun can be associated to an imbalance in the homeostasis of the iron. The hepcidin is a peptide responsible for the negative regulation of the absorption of the iron in the small intestine and of its liberation by the macrophages. It has, in addition, antimicrobial capacity. It is synthesized in the liver, secreted to the circulatory torrent and excreted by the urine. Its overexpression causes anemia and its deficit iron overload (accumulation in different organs and hereditary hemochromatosis, The antagonists of the hepcidin, could be used in the treatment of anemia resistant to erythropoyetin associated to chronic processes. On the other hand, the agonists or substances that stimulate the hepcidin production, could constitute a treatment in diseases with overload of iron (siderosis and therefore, to correct the associate.hyperpigmentation.

  11. Studies of Antimicrobial Peptides Hepcidin and Scygonadin in Chinese Marine-cultured Fishes and Mud Crab%海洋鱼类和青蟹抗菌肽hepcidin和scygonadin的研究

    Institute of Scientific and Technical Information of China (English)

    王克坚

    2011-01-01

    nilotica , Pseudosciaena crocea . Meanwhile, a novel anionic antimicrobial peptide named scygonadin was in detailed described, which was first isolated by HPLC from the seminal plasma of Scylla serrata and was characterized by mass spectral analysis in his group. lts cDNA sequence was revealed and its expression patterns in tissues and in the developmental stages from embryo to maturation in crabs were investigated. Furthermore, the synthetic and recombinant peptides of hepcidin and scygonadin are confirmed to be potent active against Gram-positive bacteria, Gram-negative bacteria and fungi. Based on the developed appropriate methods for producing large quantities of AMPs using gene engineering techniques and combining with the strong antimicrobial activity these AMPs are potential to be used in veterinary medicine and aquaculture.

  12. Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant.

    Science.gov (United States)

    Yuki, Kyoko E; Eva, Megan M; Richer, Etienne; Chung, Dudley; Paquet, Marilène; Cellier, Mathieu; Canonne-Hergaux, François; Vaulont, Sophie; Vidal, Silvia M; Malo, Danielle

    2013-01-01

    Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial

  13. Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant.

    Directory of Open Access Journals (Sweden)

    Kyoko E Yuki

    Full Text Available Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16, a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1 gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15, erythropoietin (Epo and heme oxygenase 1 (Hmox1 exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are

  14. The Regulatory Mechanism of Hepcidin Expression by Interleukin 6(IL-6) in Exercise%运动引起的白细胞介素6的改变与铁调素的调控

    Institute of Scientific and Technical Information of China (English)

    闫芬; 李颖; 刘树欣; 刘玉倩

    2011-01-01

    运动可改变机体铁状态,过度运动会引起机体铁缺乏,甚至导致机体出现运动性贫血,影响运动员成绩.近几年研究表明铁调素(Hepcidin)是调控机体铁代谢的关键因素,而运动中产生的白细胞介素6(IL-6)是影响Hepcidin表达变化的重要原因之一.介绍了IL-6对Hepcidin表达的影响、运动对IL-6表达调控的研究进展,综合分析了运动引起的IL-6的改变及其对Hepcidin的调控机制.%The iron status can be changed during exercise. Excessive exercise can lead to iron deficiency, even sports anemia. Accordingly athletes performance is greatly affected by iron status. The researches on the regulatory mechanism of iron metabolism show that hepcidin plays an important role in iron metabolism,and IL -6 is one of the important reasons for the expression of hepcidin in exercise. This review reveals the regulaory mechanism of IL-6 on hepcidin expression in exercise.

  15. Early kinetics of plasma cytomegalovirus DNA load in allogeneic stem cell transplant recipients in the era of highly sensitive real-time PCR assays: does it have any clinical value?

    Science.gov (United States)

    Giménez, Estela; Muñoz-Cobo, Beatriz; Solano, Carlos; Amat, Paula; Navarro, David

    2014-02-01

    We report that in a population of allogeneic stem cell transplant recipients, determination of the viral doubling time (dt) of the cytomegalovirus (CMV) DNA plasma load predicted the eventual need for inception of preemptive antiviral therapy, whereas the level of the initial plasma CMV DNA load did not. The data thus indicated that determination of the dt of CMV DNA may be useful in the therapeutic management of CMV infection in this clinical setting.

  16. EDTA interference in electrochemiluminescence ACTH assay.

    Science.gov (United States)

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer

    2016-11-01

    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  17. 重组人红细胞生成素对多发性骨髓瘤小鼠及患者Hepcidin-25水平的影响%Effect of recombinant erythropoietin on Hepcidin-25 in mice and patients with multiple myeloma

    Institute of Scientific and Technical Information of China (English)

    陈姣; 王晓冬; 贾永前

    2015-01-01

    目的:探讨重组人红细胞生成素对多发性骨髓瘤(MM)小鼠及患者Hepcidin-25水平的影响.方法:①建立MM小鼠模型,随机分为对照组和观察组,每组15只.观察组给予尾静脉注射重组鼠源红细胞生成素100 μl(5 mg·kg1-·只-1),对照组给予尾静脉注射等体积的生理盐水.观察2组疗效,分析小鼠血红蛋白变化,并在治疗后3、6、9天处死小鼠,取肝脏,提取总RNA,采用实时荧光定量PCR分析肝脏中Hepcidin-25 mRNA水平表达情况.②选取30例MM贫血患者作为研究对象,其中16例接受重组人红细胞生成素治疗(观察组),14例未接受治疗(对照组).采用ELISA分析检测治疗前和治疗后2、4、6周患者血清Hepcidin-25及血红蛋白水平的变化.结果:①与对照组比较,观察组小鼠肿瘤生长明显延缓,生存率显著提高(P<0.05).MM小鼠模型在建模后均表现出贫血症状,观察组小鼠治疗后3、6、9天血红蛋白水平较治疗前和对照组明显增加(P<0.05),并随着治疗时间延长逐渐升高.观察组小鼠治疗后3、6、9天肝脏中Hepcidin-25 mRNA水平较治疗前和对照组明显下降(P<0.05).②30例MM贫血患者中,观察组治疗有效率明显高于对照组(87.5%∶0,P<0.05).治疗2、4、6周后,观察组血红蛋白水平较治疗前和对照组明显增加(P<0.05),血清Hepcidin-25水平较治疗前和对照组显著下降(P<0.05).结论:红细胞生成素可显著改善MM小鼠及患者贫血状态,进而抑制肝脏组织Hepcidin-25 mRNA表达水平,导致血清Hepcidin-25水平下调.

  18. Effect of N-acetylcysteine on the accuracy of the prothrombin time assay of plasma coagulation factor II plus VII plus X activity in subjects infused with the drug. Influence of time and temperature

    DEFF Research Database (Denmark)

    Thorsen, S.; Teisner, A.; Jensen, S.A.;

    2009-01-01

    Objectives: The prothrombin time (PT) assay of factor II+VII+X activity is an important predictor of liver damage in paracetamol poisoned patients. It complicates interpretation of results that the antidote, acetylcysteine (NAC) depresses this activity. The aim was to investigate if NAC influences...

  19. Effect of N-acetylcysteine on the accuracy of the prothrombin time assay of plasma coagulation factor II+VII+X activity in subjects infused with the drug. Influence of time and temperature

    DEFF Research Database (Denmark)

    Thorsen, Sixtus; Teisner, Ane; Jensen, Søren Astrup;

    2009-01-01

    OBJECTIVES: The prothrombin time (PT) assay of factor II+VII+X activity is an important predictor of liver damage in paracetamol poisoned patients. It complicates interpretation of results that the antidote, acetylcysteine (NAC) depresses this activity. The aim was to investigate if NAC influences...

  20. Hepcidin蛋白与前列腺癌骨转移的相关性研究%Role of Hepcidin in prostate cancer patients with bone metastasis

    Institute of Scientific and Technical Information of China (English)

    薛冬; 何小舟; 许贤林; 周萃星; 徐宁

    2013-01-01

    Objective To detect the expression and correlation of serum Hepcidin,IL-6,sTfR,BMP6 diversity and explore the role of serum Hepcidin in prostate cancer with bone metastasis.Methods From January 2012 to March 2012,serum Hepcidin,IL-6,sTfR,BMP6 diversity were tested by ELISA in 25 prostate cancer patients with bone metastasis,30 prostate cancer patients without bone metastasis and 30 patients with BPH were used as the control group.The mean patient's age was 67 years (range from 55 to 75 years).In prostate cancers with bone metastasis group,the mean PSA base line was 138.0 μg/L (ranged from 20.0-1500 μg/L),the prostate cancers without bone metastasis group,mean PSA was 10.2 μg/L(ranged from 3.5-28.2 μg/L),and in BPH group,the mean PSA was 3.7 μg/L (ranged from 0.3-14.2 μg/L).Venous blood samples were taken in fasting mornings,then stored 3 ml in EDTA anticoagulant vacuum tube and centrifuged at4 ℃ for 10 min,the isolated serum were then preserved in-80 ℃ refrigerator.The competitive in-phase enzyme-linked immunoassay (ELISA) was used to detect serum Hepcidin,IL-6 and sTfR and BMP6 levels.Results Serum Hepcidin expressions in three groups were 67.7 ± 40.6 μg/L,37.5 ± 15.3 μg/L and 34.3 ± 10.7 μg/L,respectively.For prostate cancers with bone metastasis group,serum Hepcidin expression were higher than control group (P < 0.05),and associated with IL-6(22.5 ±22.1 μg/L),sTfR (5.7 ± 2.6 μg/L),BMP6 (429.3 ± 188.4 μg/L),correlation coefficients were 0.972,-0.987,0.971 (P < 0.05).Conclusions Increased serum Hepcidin level might be a sensitive index for diagnosis and prognosis of prostate cancers with bone metastasis.%目的 观察前列腺癌骨转移患者血清中IL-6、sTfR、BMP6等指标的变化,分析3个指标之间的相关性,探讨Hepcidin蛋白在前列腺癌骨转移中的作用.方法 选取2012年1月至2012年3月前列腺癌骨转移患者25例、前列腺癌无骨转移患者30例和BPH患者30例,年龄55~75岁,平均67

  1. 铁调素的表达与调节机制%Expression and regulatory mechanism of hepcidin

    Institute of Scientific and Technical Information of China (English)

    刘树欣; 卢红梅; 刘玉倩; 王海涛; 康红哲; 李文惠; 陈军

    2010-01-01

    背景:细胞铁的外向转运是由铁调素调节的.铁调素的表达受机体信号因子的影响,如血清铁水平、促红细胞生成素、炎症、低氧、氧化应激等.这些刺激通过肝实质细胞表面的组织相容性Ⅰ型跨膜糖蛋白HFE,转铁蛋白受体1,2,铁调素调节蛋白激活各种信号通路,包括BMP-SMAD、JAK-STAT和HIF1通路,进而改变铁调素基因的转录,调节铁调素的表达水平,但调节铁调素表达的分子机制还不明确.目的:从影响铁调素表达的信号因子及信号通路两方面入手,总结并分析铁调素表达的调节机制.方法:由第一作者通过计算机检索NCBI和SpringerLink(2000/2010)英文数据库,检索词为"Hepcidin,Hemochromatosis,Hemoiuvelin,BMP signaling pathway,HFE,TfR1,TfR2".分别从影响铁调素表达的信号因子及参与其表达调控的信号通路两方面进行总结,介绍近年来在铁调素表达调控机制方面的最新研究进展.结果与结论:共检索到210篇文章,按纳入和排除标准对文献进行筛选,共纳入37篇文章.结果表明铁调素的表达受机体信号因子的影响,如肝实质细胞表面的遗传性血色素沉着症侯选基因、转铁蛋白受体1,2等,可通过激活各种信号通路,包括BMP-SMAD,JAK-STAT和HIF1通路,进而改变铁调素基因的转录,并调节其表达.

  2. 鲢抗菌肽 Hepcidin 的基因克隆和表达及抑菌活性分析

    Institute of Scientific and Technical Information of China (English)

    周卫军; 刘振兴; 柯浩; 马艳平; 郝乐; 徐明芳

    2014-01-01

    利用同源克隆的方法从鲢(Hypophthalmichthys molitrix)的肝脏中克隆出抗菌肽 Hepcidin cDNA(GenBank 登入号 KF312213)后,通过 pET32a(+)构建含抗菌肽 Hepcidin 的重组质粒的 pET-Hep/ Rosetta 菌株,在37℃、28℃和16℃下分别用0.5 mmol·L -1和1.0 mmol·L -1 IPTG 诱导表达,产物用 Ni SepharoseTM 亲和层析柱纯化并进行体外抑菌试验。鲢 Hepcidin cDNA 总长度755 bp,ORF 为282 bp,5′UTR 为108 bp,3′UTR 为365 bp,编码93氨基酸,信号肽24个氨基酸,前域42个氨基酸和成熟肽27个氨基酸。pET-Hep/ Rosetta 在28℃和16℃主要是可溶性表达,37℃主要是包涵体表达。纯化的产物经15% SDS-PAGE 电泳验证为目的产物。目的产物、pET32/Rosetta 产物以及氟苯尼考的体外抑菌试验表明,目的表达产物对无乳链球菌( Streptococcus agalactiae)、金黄色葡萄球菌(Staphylococcus aureus)、嗜水气单胞菌(Aeromonas hydrophila)等具有很好的抑菌效果。

  3. Relative Quantification of Hepcidin Gene Transcripts in Pseudosciaena crocea%大黄鱼Hepcidin基因转录物的相对定量分析

    Institute of Scientific and Technical Information of China (English)

    蔡灵; 吴曼丽; 范丹青; 杨明

    2012-01-01

    Two house-keeping genes, 18S rRNA and β-actin, were cloned as endogenous genes for relative quantification of an antimicrobial peptide hepcidin in large yellow croakers ( Pseudosciaena crocea) using the real-time RT-PCR. In comparision of the slopes of the relative standard curves between the two endogenous genes and the target gene, a relative quantification method for hepcidin gene using 18S rRNA as the calibrator gene was established. Results obtained with this method were consistent with the rusults from Northern-blot, suggesting feasibility and effectiveness of the method. This paper provides an effective method to investigate expression patterns and induction profiles of the hepcidin gene in large yellow croakers, and other immunity-related genes in fish bodies. The cloned fragments of 18S rRNA and P-actin genes from the large yellow croaker have been submitted to the GenBank, which have provided accession numbers.%以大黄鱼(Pseudosciaena crocea)管家基因18S rRNA和β-actin作为内参基因,分别比较2个内参基因建立的相对定量曲线,最终确立以18S rRNA为参比基因,定量分析大黄鱼的Hepcidin抗菌肽基因.该相对定量分析方法所得结果与Northern-blot方法一致.应用建立的Hepcidin基因的实时荧光相对定量研究方法,对大黄鱼头肾中的Hepcidin基因转录物进行相对定量,为今后开展鱼体内免疫相关基因的表达特性、诱导机制等工作奠定研究基础.同时,克隆得到的大黄鱼β-actin基因和18S rRNA基因片段已提交基因库,并获得登录号.

  4. Calorie restriction down-regulates expression of the iron regulatory hormone hepcidin in normal and D-galactose-induced aging mouse brain.

    Science.gov (United States)

    Wei, Shougang; Shi, Wenli; Li, Man; Gao, Qian

    2014-02-01

    It has been shown that iron progressively accumulates in the brain with age. Calorie restriction (CR) may allay many of the adverse effects of aging on the brain, yet the underlying mechanisms, in particular in relation to brain iron metabolism, remain unclear. This study aimed to investigate the role of CR in the regulation of cerebral cellular iron homeostasis. C57BL/6 mice were randomly divided into four groups of eight. The control group was fed a conventional diet ad libitum; the CR group received 70% of the calories of the control mouse intake per day; the D-galactose (D-gal) group received subcutaneous injection of D-gal at a dose of 100 mg/kg once daily to produce mouse model of aging; the D-gal plus CR group received both of the two interventions for 14 weeks. The Morris water maze (MWM) was employed to test the cognitive performance of all animals, and the expression of iron regulatory genes, ferroportin and hepcidin, in the cortex and hippocampus were detected by quantitative real-time PCR. Compared to the controls, the D-gal group mice showed significant spatial reference memory deficits in the MWM test, whereas the D-gal-CR group mice exhibited almost normal cognitive function, indicating that CR protects against D-gal-induced learning and memory impairment. Hepcidin mRNA expression was increased in the D-gal group, decreased in the CR group, and was basically unchanged in the D-gal-CR group. There was no statistical difference in the transmembrane iron exporter ferroportin expression between control and any of the experimental groups. The results suggest that the anti-aging effects of CR might partially lie in its capacity to reduce or avoid age-related iron accumulation in the brain through down-regulating expression of brain hepcidin--the key negative regulator for intracellular iron efflux--and that facilitating the balance of brain iron metabolism may be a promising anti-aging measure.

  5. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  6. Colorimetric protein assay techniques.

    Science.gov (United States)

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  7. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  8. Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate dimethyl benzoylphenylurea (BPU) and its five metabolites in human plasma and urine for clinical pharmacology studies.

    Science.gov (United States)

    Rudek, Michelle A; Zhao, Ming; He, Ping; Zabelina, Yelena; Jin, Runyan; Messersmith, Wells A; Wolff, Antonio C; Baker, Sharyn D

    2005-12-15

    A method has been developed for the quantitation of N-[4-(5-bromo-2-pyrimidinyloxy)-3-methylphenyl]-N'-(2-dimethylamino-benzoyl)urea (BPU) and its metabolites in human plasma and urine. BPU and metabolites were separated on a C18 column with acetonitrile-water mobile phase containing 0.1% formic acid using isocratic flow for 5 min. The analytes were monitored by tandem mass spectrometry. Calibration curves were generated over the range of 2.5-500 ng/mL for BPU, mmBPU, and aminoBPU in plasma; and 0.1-20, 0.1-20, 0.5-100, 10-2000, 1-200, and 3-600 ng/mL for BPU, mmBPU, aminoBPU, G280, G308, and G322 in urine, respectively. The method has been successfully applied to study the pharmacokinetics of BPU.

  9. Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats.

    Science.gov (United States)

    Saini, R K; Manoj, P; Shetty, N P; Srinivasan, K; Giridhar, P

    2014-07-01

    In this study, the effects of iron depletion and repletion on biochemical and molecular indices of iron status were investigated in growing male Wistar rats. We hypothesized that iron from Moringa leaves could overcome the effects of iron deficiency and modulate the expression of iron-responsive genes better than conventional iron supplements. Iron deficiency was induced by feeding rats an iron-deficient diet for 10 weeks, whereas control rats were maintained on an iron-sufficient diet (35.0-mg Fe/kg diet). After the depletion period, animals were repleted with different source of iron, in combination with ascorbic acid. Iron deficiency caused a significant (P Moringa leaf was found to be superior compared with ferric citrate in overcoming the effects of iron deficiency in rats. These results suggest that changes in the relative expression of liver hepcidin messenger RNA can be used as a sensitive molecular marker for iron deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. 白癜风患者血浆中氧化与抗氧化相关指标的检测%Assay of Oxidative Stress in Plasma of Patients with Vitiligo

    Institute of Scientific and Technical Information of China (English)

    刘志军; 刘晶; 唐显华

    2011-01-01

    目的 通过对白癜风患者及健康对照者血浆中氧化与抗氧化相关指标--超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、维生素E(VitE)及一氧化氮(NO)表达水平的测定,探讨氧化应激在白癜风发病机制及疾病发展中的作用和意义.方法 选择60例白癜风患者(患者组)和40名健康志愿者(健康对照组)为研究对象,化学法检测血浆中SOD,CAT,GSH-Px的活性及MDA,VE和NO的含量.结果 患者组血浆中MDA含量及SOD活性明显高于健康对照组(P0.05).结论 白癜风患者血浆中存在氧化-抗氧化失衡,白癜风的发病及病情活动与氧化应激可能相关.%Objective To evaluate the oxidative stress in the pathogenesis of vitiligo by examining indicators of oxidative stress in plasma, which include superoxide of dismutase (SOD), catalase( CAT), glutathione peroxidase( GSH-Px), malonaldehyde( MDA), VitE( Vitamin E) and NO( nitric oxide) . Methods Sixty patients with vitiligo and 40 healthy volunteers were included in the study. Plasma levels of SOD, CAT,GSH-Px, MDA, VitE and NO were determined by chemical method. Results Our results revealed a significantly higher level of plasma MDA and SOD, a lower level of GSH-Px in 60 patients with vitiligo compared with the control (P <0. 0l ); Compared with the control, the MDA levels and SOD activities in plasma in the active vitiligo group were significantly increased( P < 0. 0l ), whlile the GSH-Px activities, levels in the active vitiligo group were significantly decreased (r = -0. 337 ,P < 0.01 ); With the increase of the MDA, there was a linear decrease of activities about GSH-Px in plasma and a linear increase of acitivities about SOD in plasma( r =0. 347 ,P <0.01 ). Compared with the control, the CAT, VE and NO levels in plasma in the active and stable vitiligo groups were not increased significantly ( P > 0.05 ). Conclusion Our study shows that oxidative stress is

  11. A simple assay for the simultaneous determination of human plasma albendazole and albendazole sulfoxide levels by high performance liquid chromatography in tandem mass spectrometry with solid-phase extraction.

    Science.gov (United States)

    Wojnicz, Aneta; Cabaleiro-Ocampo, Teresa; Román-Martínez, Manuel; Ochoa-Mazarro, Dolores; Abad-Santos, Francisco; Ruiz-Nuño, Ana

    2013-11-15

    A simple, reproducible and fast (4 min chromatogram) method of liquid chromatography in tandem with mass spectrometry (LC/MS-MS) was developed to determine simultaneously the plasma levels of albendazole (ABZ) and its metabolite albendazole sulfoxide (ABZOX) for pharmacokinetic and clinical analysis. Each plasma sample was extracted by solid phase extraction (SPE) using phenacetin as internal standard (IS). The extracted sample was eluted with a Zorbax XDB-CN column using an isocratic method. The mobile phase consisting of water with 1% acetic acid (40%, A) and MeOH (60%, B), was used at a flow rate of 1 mL/min. ABZ and ABZOX were detected and identified by mass spectrometry with electrospray ionization (ESI) in the positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 5-1000 ng/mL for ABZ and 10-1500 ng/mL (full validation) or 10-5000 ng/mL (partial validation) for ABZOX, with 5 and 10 ng/mL lower limit of quantification (LLOQ) for ABZ and ABZOX, respectively. The tests of accuracy and precision, matrix effect, extraction recovery and stability of the samples for both ABZ and ABZOX did not deviate more than 20% for the LLOQ and no more than 15% for other quality controls (QCs), according to regulatory agencies.

  12. Concurrent liquid-chromatographic assay of retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin in plasma, with tocopherol acetate as internal standard.

    Science.gov (United States)

    Thurnham, D I; Smith, E; Flora, P S

    1988-02-01

    A method is described for simultaneously determining retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin in 0.25 mL of plasma. Plasma mixed with sodium dodecyl sulfate is deproteinized with ethanol containing tocopherol acetate, then extracted with heptane. The evaporated organic layer is reconstituted with mobile phase (methanol/acetonitrile/chloroform, 47/47/6 by vol) and injected onto a 100 x 4.6 mm 3-micron column of Spherisorb ODS-2 (LKB) at 1.5 mL/min. The alpha- and beta-carotenes are well resolved during the 6.5-min run. Retinol is monitored at 325 nm, the tocopherols at 292 nm, and the carotenoids at 450 nm. Extraction of concentrations as great as 135 mumol/L is complete. Intrabatch CVs were 1.7%, 2.3%, 4.1%, 10.4%, 6.4%, and 3.6% for retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin, respectively. Interbatch CVs for measurements on 30 occasions over 11 weeks were about 10% for all components except alpha-tocopherol (5.3%). Results agree well with those for retinol, alpha-tocopherol, and beta-carotene in quality-control samples.

  13. Association between ferritin, high sensitivity c-reactive protein (hsCRP and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects

    Directory of Open Access Journals (Sweden)

    Mónica Andrews Guzmán

    Full Text Available Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM; 30 obese subjects without diabetes (OB and 30 healthy subjects (Cn. We isolated peripheral mononuclear cells (PMCs and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-αIL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL-6, and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

  14. Association between ferritin, high sensitivity C-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects.

    Science.gov (United States)

    Andrews Guzmán, Mónica; Arredondo Olguín, Miguel

    2014-09-01

    Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-, IL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL- 6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

  15. 铁调素与慢性心力衰竭合并贫血关系的研究进展%Research progress in the relationship between hepcidin and chronic heart failure complicated with anemia

    Institute of Scientific and Technical Information of China (English)

    刘晶; 李英梅

    2016-01-01

    铁调素是近年来发现的一种负性调节血清铁浓度的激素,有助于维持机体内铁代谢的动态平衡。铁调素代谢紊乱可以导致血红蛋白降低等多种与铁代谢异常相关的病证。贫血是慢性心力衰竭患者的常见并发症,其与铁代谢异常紧密相关。近年来,随着铁调素在铁代谢中的重要作用被发现,铁调素与慢性心力衰竭合并贫血发生发展的紧密联系也逐渐受到重视。%Hepcidin,a hormone discovered in recent years that can negatively regulate serum iron concentration, helps to maintain the dynamic balance of iron metabolism in the body.Hepcidin metabolic disorder can lead to the reduction of hemoglobin and other diseases associated with abnormal iron metabolism.Anemia is a common complication in patients with chronic heart failure,which is closely related to abnormal iron metabolism.As the important role of hepcidin in iron metabolism is found in recent years,the close relationship between hepcidin and chronic heart failure complicated with anemia has also been found gradually.

  16. Plasma turbulence

    Energy Technology Data Exchange (ETDEWEB)

    Horton, W. [Univ. of Texas, Austin, TX (United States). Inst. for Fusion Studies; Hu, G. [Globalstar LP, San Jose, CA (United States)

    1998-07-01

    The origin of plasma turbulence from currents and spatial gradients in plasmas is described and shown to lead to the dominant transport mechanism in many plasma regimes. A wide variety of turbulent transport mechanism exists in plasmas. In this survey the authors summarize some of the universally observed plasma transport rates.

  17. Ultra-performance liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma: application to a pharmacokinetic study.

    Science.gov (United States)

    Gu, Wei; Wang, Dongyue; Pan, Zihao; Liu, Xiao; Cai, Baochang; Chen, Jun

    2016-07-01

    A rapid, simple and sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP18 UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N-oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1-500 ng/mL for brucine and strychnine and 0.2-50 ng/mL for brucine N-oxide. The intra- and inter-day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N-oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Development and validation of a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry assay for the determination of neostigmine in small-volume beagle dog plasma and its application to a pharmacokinetic study.

    Science.gov (United States)

    Cao, Di; Li, Wenxue; Zhao, Xin; Ye, Xiaolan; Sun, Fanlu; Li, Jinying; Song, Fenyun; Fan, Guorong

    2014-03-01

    A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 μL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs.

  19. Cell viability assays: introduction.

    Science.gov (United States)

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  20. Tube-Forming Assays.

    Science.gov (United States)

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  1. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  2. Assays for thrombopoietin

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, T.P.

    1977-01-01

    In summary, thrombopoietin levels have been determined indirectly by measuring thrombocytopoiesis in assay animals (platelet counting, measurement of isotope incorporation into newly formed platelets, changes in platelet sizes, or alterations in number and size of megakaryocytes) and by use of an immunoassay. Although much work remains, it seems clear at the present time that isotopic uptake into platelets of specially prepared assay mice (rebound-thrombocytosis) is superior to the other techniques now available for the measurement of thrombopoietin. However, the ideal assay for TSF which is specific, rapid, and inexpensive is yet to be developed. An immunoassay is in the development stage, but will require additional work before it can be utilized for the routine assay of TSF.

  3. New Rapid Spore Assay

    Science.gov (United States)

    Kminek, Gerhard; Conley, Catharine

    2012-07-01

    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  4. Effect of Gymnema inodorum on postprandial peak plasma glucose ...

    African Journals Online (AJOL)

    USER

    2010-02-15

    Feb 15, 2010 ... on peak plasma glucose concentrations in healthy subjects was investigated. ... fasting plasma glucose and liver function test (AST, ALT, GGT and ALP) .... culture medium was measured using an immunoradiometric assay.

  5. Plasma harmonics

    CERN Document Server

    Ganeev, Rashid A

    2014-01-01

    Preface; Why plasma harmonics? A very brief introduction Early stage of plasma harmonic studies - hopes and frustrations New developments in plasma harmonics studies: first successes Improvements of plasma harmonics; Theoretical basics of plasma harmonics; Basics of HHG Harmonic generation in fullerenes using few-cycle pulsesVarious approaches for description of observed peculiarities of resonant enhancement of a single harmonic in laser plasmaTwo-colour pump resonance-induced enhancement of odd and even harmonics from a tin plasmaCalculations of single harmonic generation from Mn plasma;Low-o

  6. Hepcidin measurement in the differential diagnosis of iron deficiency anemia and anemia of chronic disease%铁调素检测在缺铁性贫血与慢性贫血鉴别诊断中的价值

    Institute of Scientific and Technical Information of China (English)

    束婷婷

    2014-01-01

    目的:探讨铁调素(hepcidin)对缺铁性贫血(IDA )及慢性贫血(ACD)的鉴别诊断价值。方法采用 ELISA法检测78例体检健康人群(对照组)、40例ACD患者(ACD组)及49例IDA患者(IDA组)的血清铁调素水平。亚铁嗪比色法检测血清铁、总铁结合力水平(TIBC)。结果 IDA组血清铁调素水平低于体检组(P<0.05),ACD组铁调素水平高于对照组(P<0.05)。铁调素、血清铁、T IBC用于ACD与IDA患者鉴别诊断的ROC曲线下面积分别为0.97、0.66、0.85。结论铁调素为鉴别诊断IDA和ACD简单易行的方法,其诊断准确度优于血清铁和T IBC。%Objective To explore the value of hepcidin measurement in differential diagnosis of iron deficiency anemia(IDA) and anemia of chronic diseases(ACD) .Methods Serum hepcidin was measured by using ELISA .Serum iron(SI) and total iron binding capacity(TIBC) were determined by using Ferro zine colorimetric method .Results Hepcidin expression increased in ACD group (P<0 .05) ,but decreased in IDA group(P<0 .05) .The area under the curve of ROC for hepcidin ,SI and TIBC were 0 .97 ,0 .66 and 0 .85 respectively .Conclusion Hepcidin is a simple and safe indicator in differential diagnosis of IDA and ACD ,and its diagnos-tic accuracy is better than SI and TIBC .

  7. Effects of Different Exercise on the Serum Hepcidin of Athletes:A Meta-Analysis%不同运动方式对运动员血清抗菌多肽(Hepcidin)的影响

    Institute of Scientific and Technical Information of China (English)

    王海涛; 刘玉倩; 郑宁; 杨雯茜

    2016-01-01

    抗菌多肽(Hepcidin )是机体铁稳态的负调节子,在运动性铁缺乏的发生中发挥重要作用。目的:采用Meta分析定量评价不同运动方式对运动员血清 Hepcidin浓度的影响。方法:分别以急性运动和耐力运动的运动员在运动前后的血清 H epcidin浓度为统计量,检索截至2016年4月的相关文献,制定文献筛选标准,对纳入的研究进行 Meta分析、异质性检验和发表偏倚检验。结果:涉及急性运动的研究11项(135名运动员)和耐力运动研究7项(88名运动员)纳入 Meta分析。急性运动后运动员血清 Hepcidin增加幅度为51.94%(SMD=1.640,95% CI [1.357,1.923],z=11.37,P<0.01)。耐力运动 Hepcidin增加幅度为25.13%(SMD=0.863,95% CI[0.550,1.177],z=7.24,P<0.01)。检验均未发现显著发表偏倚。结论:急性运动和耐力运动均使运动员 Hepcidin增加,急性运动增加幅度高于耐力运动,为防止运动性贫血发生,运动员应在训练期间增加铁补充。%Hepcidin is a main negative regulator of iron metabolism ,which plays an important role in iron deficiency in athletes .Objective :The present study investigated the changes of Hepcidin concentrations in different exercise using Meta analysis .Methods :Searched related literature before April .2016 and identify including and excluding criteria .The serum Hepcidin concentrations in athletes before and after the endurance and acute exercise were selected ,and then heterogeneity test ,regression analysis for heterogeneity and publication bias were made . Results :11 studies were related to acute exercise including 135 athletes and 7 studies were re‐lated to endurance exercise including 88 athletes .Hepcidin concentrations were increased by 51 .94% in acute exercise :(SMD= 1 .640 ,95% CI [1 .357 ,1 .923] ,z=11 .37 ,P< 0 .01) . Hepcidin was increased by 25 .13% in endurance exercise :SMD

  8. Against vaccine assay secrecy.

    Science.gov (United States)

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors.

  9. Against vaccine assay secrecy

    Science.gov (United States)

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  10. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  11. Dusty plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Fortov, Vladimir E; Khrapak, Aleksei G; Molotkov, Vladimir I; Petrov, Oleg F [Institute for High Energy Densities, Associated Institute for High Temperatures, Russian Academy of Sciences, Moscow (Russian Federation); Khrapak, Sergei A [Max-Planck-Institut fur Extraterrestrische Physik, Garching (Germany)

    2004-05-31

    The properties of dusty plasmas - low-temperature plasmas containing charged macroparticles - are considered. The most important elementary processes in dusty plasmas and the forces acting on dust particles are investigated. The results of experimental and theoretical investigations of different states of strongly nonideal dusty plasmas - crystal-like, liquid-like, gas-like - are summarized. Waves and oscillations in dusty plasmas, as well as their damping and instability mechanisms, are studied. Some results on dusty plasma investigated under microgravity conditions are presented. New directions of experimental research and potential applications of dusty plasmas are discussed. (reviews of topical problems)

  12. CTL ELISPOT assay.

    Science.gov (United States)

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  13. Usefulness of human coagulation and fibrinolysis assays in domestic pigs

    DEFF Research Database (Denmark)

    Münster, Anna-Marie Bloch; Olsen, Aage Kristian; Bladbjerg, Else-Marie

    2002-01-01

    Pigs are often used as animal models in research on blood coagulation and fibrinolysis. The usefulness of the assays applied within this field, and the knowledge of reference intervals are therefore essential and of utmost importance. In the study reported here, we investigated the applicability...... time, tissue factor, tissue factor pathway inhibitor, factor VII, protein C, protein S, prothrombin fragment 1+2, antithrombin, thrombin-antithrombin complexes, fibrinogen, soluble fibrin, urokinase-type plasminogen activator, plasmin inhibitor, plasminogen activator inhibitor 1, and D-dimer. We found...... that 11 of 12 functional assays, but only 3 of 10 immunoassays, were applicable to porcine plasma, and we determined the normal range of these variables. We conclude that human functional assays are useful in porcine plasma, whereas only a few immunologic assays can be used. However, precautions must...

  14. New oligosaccharyltransferase assay method.

    Science.gov (United States)

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  15. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  16. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  17. Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin.

    Science.gov (United States)

    Gardenghi, Sara; Marongiu, Maria F; Ramos, Pedro; Guy, Ella; Breda, Laura; Chadburn, Amy; Liu, YiFang; Amariglio, Ninette; Rechavi, Gideon; Rachmilewitz, Eliezer A; Breuer, William; Cabantchik, Z Ioav; Wrighting, Diedra M; Andrews, Nancy C; de Sousa, Maria; Giardina, Patricia J; Grady, Robert W; Rivella, Stefano

    2007-06-01

    Progressive iron overload is the most salient and ultimately fatal complication of beta-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both beta-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by beta-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of beta-thalassemia.

  18. EFFECTS OF EXERCISE ON IRON METABOLISM AND HEPCIDIN%运动对铁代谢及铁调素的影响研究

    Institute of Scientific and Technical Information of China (English)

    聂集林; 刘君雯

    2011-01-01

    Iron metabolism includes such processes as iron absorption, transfer, utilization, storage and circulation.At present it is generally regarded that iron metabolism regulates hormones, the main biochemical function of which is interfering with iron absorption of the intestinal tract and iron release in the mononuclear phagocyte system.Studies have shown that prolonged high-intensity exercise leads to iron loss and disordered distribution.It can also result in an increase in hepatic expression , which may be the reason for sports anemia.%铁代谢包括铁吸收、转运、利用、储存和循环等一系列过程.铁调素(Hepcidin)是目前人们公认的铁代谢调节激素,它在铁代谢方面主要的生物学功能为抑制肠道铁吸收和抑制单核巨噬细胞系统铁释放.研究证明,长时间大强度运动导致铁丢失增多及铁分布紊乱,还可导致肝hepcidin表达增加,这些可能是运动性贫血的原因.

  19. Plasma waves

    CERN Document Server

    Swanson, DG

    1989-01-01

    Plasma Waves discusses the basic development and equations for the many aspects of plasma waves. The book is organized into two major parts, examining both linear and nonlinear plasma waves in the eight chapters it encompasses. After briefly discussing the properties and applications of plasma wave, the book goes on examining the wave types in a cold, magnetized plasma and the general forms of the dispersion relation that characterize the waves and label the various types of solutions. Chapters 3 and 4 analyze the acoustic phenomena through the fluid model of plasma and the kinetic effects. Th

  20. Plasma astrophysics

    CERN Document Server

    Kaplan, S A; ter Haar, D

    2013-01-01

    Plasma Astrophysics is a translation from the Russian language; the topics discussed are based on lectures given by V.N. Tsytovich at several universities. The book describes the physics of the various phenomena and their mathematical formulation connected with plasma astrophysics. This book also explains the theory of the interaction of fast particles plasma, their radiation activities, as well as the plasma behavior when exposed to a very strong magnetic field. The text describes the nature of collective plasma processes and of plasma turbulence. One author explains the method of elementary

  1. The corneal pocket assay.

    Science.gov (United States)

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  2. Kinetic Tetrazolium Microtiter Assay

    Science.gov (United States)

    Pierson, Duane L.; Stowe, Ray