Human Plasma (human + plasma)

Distribution by Scientific Domains

Terms modified by Human Plasma

  • human plasma protein
  • human plasma sample

  • Selected Abstracts


    Validation of an LC,MS Method for the Detection and Quantification of BZP and TFMPP and their Hydroxylated Metabolites in Human Plasma and its Application to the Pharmacokinetic Study of TFMPP in Humans,

    JOURNAL OF FORENSIC SCIENCES, Issue 5 2010
    Ushtana Antia M.Sc.
    Abstract:, An LC,MS method was developed for benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP), constituents of "party pills" or "legal herbal highs," and their metabolites in human blood plasma. Compounds were resolved using a mixture of ammonium formate (pH 4.5, 0.01 M) and acetonitrile (flow rate of 1.0 mL/min) with a C18 column. Calibration curves were linear from 1 to 50 ng/mL (R2 > 0.99); the lower limit of quantification (LLOQ) was 5 ng/mL; the accuracy was >90%; the intra- and interday relative standard deviations (R.S.D) were <5% and <10%, respectively. Human plasma concentrations of TFMPP were measured in blood samples taken from healthy adults (n = 6) over 24 h following a 60-mg oral dose of TFMPP: these peaked at 24.10 ng/mL (±1.8 ng/mL) (Cmax) after 90 min (Tmax). Plasma concentrations of 1-(3-trifluoromethyl-4-hydroxyphenyl) piperazine peaked at 20.2 ng/mL (±4.6 ng/mL) after 90 min. TFMPP had two disposition phases (t½ = 2.04 h (±0.19 h) and 5.95 h (±1.63 h). Apparent clearance (Cl/F) was 384 L/h (±45 L/h). [source]


    Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubes

    JOURNAL OF SEPARATION SCIENCE, JSS, Issue 6 2007
    Xin Li
    Abstract Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300,1800 range) in about 50 ,L of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis. [source]


    Simultaneous determination of ten antihistamine drugs in human plasma using pipette tip solid-phase extraction and gas chromatography/mass spectrometry

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2006
    Chika Hasegawa
    Ten antihistamine drugs, diphenhydramine, orphenadrine, chlorpheniramine, diphenylpyraline, triprolidine, promethazine, homochlorcyclizine, cyproheptadine, cloperastine and clemastine, have been found to be extractable from human plasma samples using MonoTip C18 tips, inside which C18 -bonded monolithic silica gel was fixed. Human plasma (0.1,mL) containing the ten antihistamines was mixed with 0.4,mL of distilled water and 25,µL of a 1,M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C18 phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. The eluate was injected into a gas chromatography (GC) injector without evaporation and reconstitution steps, and was detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of the ten drugs from each other and from impurities was generally satisfactory using a DB-1MS column (30,m,×,0.32,mm i.d., film thickness 0.25,µm). The recoveries of the ten antihistamines spiked into plasma were 73.8,105%. The regression equations for the ten antihistamines showed excellent linearity with detection limits of 0.02,5.0,ng/0.1,mL. The within-day and day-to-day coefficients of variation for plasma were not greater than 9.9%. The data obtained from determination of diphenhydramine and chlorpheniramine in human plasma after oral administration of the drugs are also presented. Copyright © 2006 John Wiley & Sons, Ltd. [source]


    Fluid Labor and Blood Money: The Economy of HIV/AIDS in Rural Central China

    CULTURAL ANTHROPOLOGY, Issue 4 2006
    SHAO Jing
    This ethnographically grounded "epidemiology" implicates China's liberalized economy in the HIV epidemic among commercial plasma donors in rural central China. It uncovers the pathological confluence of spheres of economic circulations that have created the conditions for value to be extracted not through labor but from human plasma harvested from agricultural producers. This critique has emerged out of, and in turn informed, efforts to forestall the secondary epidemic of AIDS among donors already infected by HIV. The specific history of the production and consumption of blood products in China shows how biotechnology broadly defined can be powerfully refracted by local configurations of economy, technology, and social relations. The ideologically sustained second-order "reality" of benevolent economic imperatives needs to be brought into the critical focus of cultural anthropology. [source]


    Voltammetric Sensor for Sodium Nitroprusside Determination in Biological Fluids Using Films of Poly- L -Lysine

    ELECTROANALYSIS, Issue 9 2007
    Claudece Pereira, Francisco
    Abstract Sodium nitroprusside (NP), a commercial vasodilator, can be pre-concentrated on vitreous carbon electrode modified by films of 97.5%: 2.5% poly- L -lysine (PLL): glutaraldehyde (GA). This coating gives acceptable anion exchange properties whilst giving the required improvement of adhesion to the glassy carbon electrode surface. Linear response range and detection limit on nitroprusside in B-R buffer pH,4.0, were 1×10,6 to 2×10,5 mol L,1 and 1×10,7 mol L,1, respectively. The repeatability of the proposed sensor, evaluated in term of relative standard deviation, was measured as 4.1% for 10 experiments. The voltammetric sensor was directly applied to determination of nitroprusside in human plasma and urine samples and the average recovery for these samples was around 95,97% without any pre treatment. [source]


    Simultaneous determination of memantine and amantadine in human plasma as fluorescein derivatives by micellar electrokinetic chromatography with laser-induced fluorescence detection and its clinical application

    ELECTROPHORESIS, Issue 11 2010
    Hsin-Hua Yeh
    Abstract A nonionic surfactant MEKC method with LIF detection was developed for the simultaneous determination of memantine, an anti-Alzheimer's disease agent, and amantadine, an anti-Parkinson's disease drug, in human plasma. Before analysis, the plasma samples were pretreated by liquid,liquid extraction with ethyl acetate, and derivatized with 6-carboxyfluorescein N -hydroxysuccinimide ester. The chemical derivatization is performed with 6-carboxyfluorescein N -hydroxysuccinimide ester in ACN , 5,mM pH 9.0 borate buffer (40:60, v/v) at 35°C for 3,h. After the derivatization reaction, hydrodynamic injection (0.5,psi, 8,s) was used to introduce the derivatized solution, and the separation was performed in borate buffer (30,mM, pH 9.5) with the nonionic surfactant Brij-35® (0.07%, w/v); the separation voltage was 6,kV. The linear ranges of the method for the determination of memantine and amantadine in human plasma were over a range of 2.0,60.0,ng/mL. The detection limit was 0.5,ng/mL (S/N=3). This method was applied successfully to monitor the concentration of memantine or amantadine in patients with Alzheimer's disease or Parkinson's disease. [source]


    Direct automatic determination of free and total anesthetic drugs in human plasma by use of a dual (microdialysis,microextraction by packed sorbent) sample treatment coupled at-line to NACE,MS

    ELECTROPHORESIS, Issue 10 2009
    Gabriel Morales-Cid
    Abstract This paper reports for the first time the use of microextraction by packed sorbent in combination with CE. The combined system was used to determine anesthetic drugs in human plasma. A microdialysis fiber was coupled on-line to the microextraction unit in order to distinguish between free and total concentrations of drugs. The system was automated by connecting the microextraction unit to a syringe pump and interfacing it to a computer. The ensuing method allows the determination of 10,,g/L concentrations of free drugs and 1,,g/L concentrations of total drugs from only 200,,L of sample with an RSD of less than 9%. [source]


    Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on-column field-amplified sample stacking and its application in Alzheimer's disease,

    ELECTROPHORESIS, Issue 17 2008
    Hsin-Hua Yeh
    Abstract Field-amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid,liquid extraction with isopropanol/n -hexane (v/v 3:97) and subsequent quantification by FASS-CE was used. Before sample loading, a water plug (0.5,psi, 6,s) was injected to permit FASS. Electrokinetic injection (7,kV, 90,s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60,mM, pH 4.0) with sodium octanesulfonate 40,mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28,kV and detected at 200,nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1,50,ng/mL. The limit of detection was 0.1,ng/mL (S/N=3, sampling 90,s at 7,kV). One female volunteer (54 years old) was orally administered a single dose of 10,mg donepezil (Aricept®, Eisai), and blood samples were drawn over a 60,h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas. [source]


    Determination of vigabatrin in human plasma by means of CE with LIF detection

    ELECTROPHORESIS, Issue 19 2007
    Alessandro Musenga
    Abstract A method has been developed for the quantitation of the antiepileptic drug vigabatrin (VGB) in human plasma. It is based on CE with LIF detection. The effect of the pH of the buffer and of N -methylglucamine (GLC) as BGE constituent was investigated. The final BGE consisted of 50,mM borate buffer, pH,9.0, with 100,mM GLC and enabled separation within 12,min at 20,kV voltage. An SPE procedure was used for the pretreatment of biological samples, based on mixed-mode lipophilic-cation exchange cartridges, followed by a derivatization step with 6-carboxyfluorescein- N -succinimidyl ester (CFSE). Fluorescence was excited by an Ar-ion laser (,exc,=,488,nm). Linearity was observed in the 10,120,,g/mL plasma concentration range. Extraction yield was >96%, precision (expressed as RSD) <6.7% and accuracy (recovery) was between 97.0 and 101.6%. The method has been successfully applied to the analysis of VGB in plasma of epileptic patients undergoing therapy with the drug. [source]


    Determination of sertraline and N -desmethylsertraline in human plasma by CE with LIF detection

    ELECTROPHORESIS, Issue 11 2007
    Alessandro Musenga
    Abstract A method has been developed for the analysis of the antidepressant drug sertraline together with its main metabolite N -desmethylsertraline (DMS) in human plasma. It is based on CE with LIF detection (,,=,488,nm). A SPE procedure is employed for biological sample pretreatment, followed by a derivatization step with FITC; reboxetine was the internal standard. The effect of CD, acetone and N -methyl- D -glucamine (GLC) as constituents of the BGE for analyte separation was investigated. The final BGE consisted of 20,mM carbonate buffer, pH,9.0, with 2.5,mM heptakis(2,6-di- O -methyl)-,-CD, 50,mM GLC and 20% v/v acetone. With 30,kV applied voltage, the electrophoretic run is completed in 7.5,min. Linearity was observed in the plasma concentration range from 3.0 to 500,ng/mL for sertraline and 4.0 to 500,ng/mL for DMS. Extraction yield was >97.1%, precision , expressed as RSD% , was <3.7, accuracy (recovery) was >95.6%. Due to its sensitivity and selectivity, the method was suited for the analysis of plasma samples from patients undergoing therapy with sertraline. [source]


    Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

    ELECTROPHORESIS, Issue 4-5 2005
    Vincenzo Pucci
    Abstract A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1,20 ,g/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 ,g/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.* [source]


    Direct electrochemical detection of glucose in human plasma on capillary electrophoresis microchips

    ELECTROPHORESIS, Issue 21-22 2004
    Yan Du
    Abstract We developed an electrochemical detector on a hybrid chip for the determination of glucose in human plasma. The microchip system described in this paper consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate. The copper microelectrode is fabricated by selective electroless deposition. The fabrication of the decoupler is performed by platinum electrochemical deposition on the metal film formed by electroless deposition. Factors influencing the performance, including detection potential, separation field strength, and buffer concentration, were studied. The electrodes exhibited good stability and durability in the analytical procedures. Under optimized detection conditions, glucose responded linearly from 10 ,M to 1 mM. Finally, glucose in human plasma from three healthy individuals and two diabetics was successfully determined, giving a good prospect for a new clinical diagnostic instrument. [source]


    Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

    ELECTROPHORESIS, Issue 13 2004
    Jack Zheng
    Abstract A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1,5.0 ,g/mL with a limit of detection as low as 0.05 ,g/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection. [source]


    S -Adenosyl methionine/S -adenosyl- L -homocysteine ratio determination by capillary electrophoresis employed as a monitoring tool for the antiviral effectiveness of adenosine analogs

    ELECTROPHORESIS, Issue 10-11 2004
    Elena Sbrana
    Abstract S -Adenosyl- L -homocysteine hydrolase (SAHh) inhibitors have long been used as broad-range antivirals and have been recently evaluated as an experimental therapy of filovirus infections. In response to the need for a rapid laboratory testing method that could assess antiviral potency in vivo, our group developed a capillary electrophoresis (CE) method for the determination of the S -adenosyl- L -homocysteine (SAH) to S -adenosyl- L -methionine (SAM) ratio. After chloroacetaldehyde derivatization, SAH and SAM were detected using laser-induced fluorescence detection with a HeCd laser. Separation and quantitation of both SAH and SAM in human plasma were achieved in less than 1 min. The proposed method is rapid and reliable, and could be easily applied to routine monitoring of clinical and preclinical trials subjects. [source]


    Determination of ribavirin in human serum and plasma by capillary electrophoresis

    ELECTROPHORESIS, Issue 10-11 2004
    Michael C. Breadmore
    Abstract The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 ,L sample and reconstitution of the dried extract into 100 ,L of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 ,g/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-,-2b for a hepatitis C virus infection. [source]


    Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin

    ELECTROPHORESIS, Issue 14 2003
    Klaus Altland
    Abstract Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70,80°C in neutral to mild alkaline buffers or at 37°C and slightly acidic pH (6,7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis. [source]


    IgG2 containing IgM,IgG immune complexes predominate in normal human plasma, but not in plasma of patients with warm autoimmune haemolytic anaemia

    EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006
    Dorothea Stahl
    Abstract:, The different physicochemical and sterical properties of IgG subclasses may favour a selective enrichment of defined IgG subclasses in IgM,IgG immune complexes (IC) of human plasma under physiological conditions. Such enrichment of IgG subclasses in IgM,IgG IC of plasma may differ from the normal IgG subclass distribution in plasma itself, and contribute to the physiological functions of IgM,IgG IC. Systematic studies on the IgG subclass distribution in IgM,IgG IC in humans are lacking. Using specific analytical techniques to characterise IgM,IgG IC in human plasma (i.e. fast protein liquid chromatography, enzyme-linked immunosorbent assay, affinity biosensor technology), and taking warm autoimmune haemolytic anaemia (WAIHA) of humans as a disease model, we here demonstrate that: (i) IgG2 is the predominant IgG subclass in IgM,IgG IC under physiological conditions, (ii) the predominance of IgG2 within IgM,IgG IC may get lost in polyclonal IgG-mediated autoimmune disease and (iii) the IgG subclass distribution in IgM,IgG IC influences the interaction between IC and blood cells involved in antigen presentation. The data presented here therefore extend the physiological function of IgG2, which is the protective immune response towards carbohydrate antigens in bacterial infections, and suggest IgG2-dependent regulation of immune responses to self-immunoglobulin in humans. The disturbed IgG subclass distribution in IgM,IgG IC of patients with WAIHA might influence activity of self-reactive B cells involved in the pathophysiology of the disease. [source]


    Synthesis and Characterization of Thrombin Conjugated ,-Fe2O3 Magnetic Nanoparticles for Hemostasis

    ADVANCED ENGINEERING MATERIALS, Issue 12 2009
    Ofra Ziv
    Abstract Thrombin is the final protease produced in the clotting pathways. Thrombin has been used in the clinic more than six decades for topical hemostasis and wound management. In human plasma the half-life of thrombin is shorter than 15 seconds due to close control by inhibitors. In order to stabilize thrombin, this enzyme was conjugated covalently and physically to ,-Fe2O3 magnetic nanoparticles. The physical conjugation was accomplished through adsorption of thrombin to BSA coating on the nanoparticles. The coagulant activity of the covalently bound thrombin was significantly lower than that of the physically adsorbed thrombin. Leakage of the physically bound thrombin into PBS containing 4% HSA was negligible. The physical conjugation of thrombin onto the nanoparticles stabilized the thrombin against its major inhibitor antithrombin III and improved its storage stability. At optimal CaCl2 concentration, the clotting time by the bound thrombin is shorter than that of the free enzyme. This novel conjugated thrombin may be an efficient candidate for topical hemostasis and wound healing. [source]


    TNF-, suppresses dendritic cell death and the production of reactive oxygen intermediates induced by plasma withdrawal

    EXPERIMENTAL DERMATOLOGY, Issue 5 2004
    Hong-Duck Um
    Abstract:, Mature dendritic cells (DCs) were generated by culturing human peripheral blood monocytes for 7 days and, then, treating them with a cytokine cocktail for 2 days. The viability of the mature DCs (Day 9) obtained was approximately 60,70%, and this gradually declined when they were recultured in X-VIVO 15 media containing 2% human plasma (40% viability after 3 days of reculture). DC death accelerated on withdrawing plasma from the culture (20% viability after 3 days). However, the addition of tumor necrosis factor-, (TNF-,) to the medium completely restored DC viability in the absence of plasma. Such a protective effect was not afforded by other cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1, (IL-1,), IL-4, IL-6 and prostaglandin E2 which are used for the maturation of DCs. These results indicate that TNF-, is specifically required to maintain the viability of mature DCs. The withdrawal of plasma rapidly (within 15 min) elevated cellular levels of reactive oxygen intermediates (ROIs), which have been proposed to regulate the ability of DCs to control inflammatory reactions. The possibility that ROIs act as mediators of DC death was eliminated by the observation that scavengers of ROIs, such as catalase, N -acetylcysteine, glutathione, failed to prolong DC life span in the absence of plasma. Interestingly, TNF-, was found to almost completely abolish the production of ROIs induced by plasma withdrawal. To summarize, our results suggest that TNF-, controls not only the inflammatory functions of DCs but also their survival. [source]


    Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma

    FEBS JOURNAL, Issue 22 2009
    Michael J. Krisinger
    Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. [source]


    Complete high-density lipoproteins in nanoparticle corona

    FEBS JOURNAL, Issue 12 2009
    Erik Hellstrand
    In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull-down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high-density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A-I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions. [source]


    Hydrolysis of acetylthiocoline, o -nitroacetanilide and o- nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesterase

    FEBS JOURNAL, Issue 7 2009
    Marķa F. Montenegro
    Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o -nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o -nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species () of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The kcat/Km values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N -(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 × 106 m,1·min,1, 0.8 × 106 m,1·min,1, and 17.5 × 106 m,1·min,1, respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o -nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained. [source]


    Unmasking a hyaluronan-binding site of the BX7B type in the H3 heavy chain of the inter-,-inhibitor family

    FEBS JOURNAL, Issue 3 2001
    Laetitia Jean
    The inter-,-inhibitor (I,I) family gathers together several plasma protease inhibitors such as I,I and pre-,-inhibitor (P,I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I,I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I,I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P,I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P,I may vary with the folding and/or fragmentation of this protein. [source]


    Safety and efficacy of a plasma-derived monoclonal purified factor VIII concentrate during 10 years of follow-up

    HAEMOPHILIA, Issue 6 2007
    E. P. MAUSER-BUNSCHOTEN
    Summary., In 1995, AAFACT®, a new monoclonal purified factor VIII concentrate (FVIII), derived from human plasma, was introduced in the Netherlands. The monoclonal purification based production process includes a viral inactivation step by solvent/detergent treatment. Products manufactured according to this procedure, for example Hemofil M® are used worldwide. The aim of the present study was to assess inhibitor development in a large cohort of previously treated patients (PTPs) who were followed up for 10 years. In addition, efficacy, HIV and hepatitis C virus (HCV) transmission, and allergic reactions were monitored. All 165 patients with severe haemophilia A (FVIII < 1%) known at the van Creveldkliniek who ever used AAFACT® during the period from October 1995 to September 2005 were included. Two of them were previously untreated patients (PUPs) and two others had <50 exposure days. Data on FVIII consumption, number of exposures, bleedings and hospitalization days were collected from start of AAFACT® until last clinical and laboratory evaluation while on this product. At the end of follow-up, 91 patients were still using this plasma-derived FVIII. Median age at start of follow-up was 26 years (range 1,52). None of the patients reported lack of efficacy. Median FVIII consumption per patient during follow-up was 2058 IU kg,1 bodyweight per year, and median number of exposures was 148 per year. During 1029 patient-years of follow-up, one inhibitor was diagnosed in a previously treated patient PTP. This patient developed high titre inhibitor following surgery for which he, during 1 week, had been treated with continuous infusion with recombinant FVIII. No inhibitor occurred during 68 cases of surgery using continuous infusion with AAFACT®. No viral transmissions or other adverse events occurred during 10 years of follow-up; AAFACT® appeared to be an effective and safe FVIII product. [source]


    FEIBA® safety profile in multiple modes of clinical and home-therapy application

    HAEMOPHILIA, Issue 2004
    H. Luu
    Summary., The development of neutralizing antibodies to factor VIII or IX therapeutic concentrates remains the most serious and challenging complication in the management of patients with haemophilia A and B. FEIBA®, Anti-Inhibitor Coagulant Complex, is an activated prothrombin complex concentrate that has been used to treat patients with such complications for almost 30 years. The mechanism of action of FEIBA® has been proposed to involve simultaneous FVIII/FIX inhibitor bypassing action in the common, intrinsic and extrinsic coagulation pathways. FEIBA® is derived from human plasma that undergoes stringent viral screening followed by significant viral inactivation and removal. To date, there have been no confirmed reports of transmission of hepatitis A, B or C, or of human immunodeficiency viruses associated with the use of the current, vapour-heat-treated FEIBA® concentrate. The incidence of thrombotic adverse events recorded in the Baxter pharmacovigilance database for the 10-year postmarket period (1990,99) was approximately 4 : 100 000 infusions of FEIBA®. Almost all documented thrombotic events with FEIBA® occurred with doses that exceeded dosing recommendations, and known risk factors for cardiovascular disease were evident in more than 80% of the patients involved. Overall, clinical data have shown FEIBA® to be safe and well-tolerated for use in a wide variety of clinical settings, including treatment of bleeding episodes, management of surgical procedures, home therapy, long-term prophylaxis, and prophylaxis during immune tolerance induction, when used according to dosing guidelines. [source]


    Hepatitis G virus in clotting factor concentrates

    HAEMOPHILIA, Issue 1 2003
    E. Alonso-Rubiano
    Summary. Blood-borne hepatitis is a well-known complication in patients with bleeding disorders. A recently discovered parentally transmitted virus, hepatitis G [GB virus C (GBV-C)] has an increased prevalence in patients with haemophilia. Clotting factor concentrates derived from pools of human plasma currently undergo viral inactivation techniques known to be effective against hepatitis B, C and HIV; however, the effectiveness of current purification and viral inactivation techniques against newly discovered viruses such as GBV-C is unknown. A total of 37 vials of clotting factor concentrates manufactured in the USA from 1981 to 1995 were tested for the presence of GBV-C virus. All samples that did not undergo a specific viral inactivation step were positive for GBV-C. Viral inactivation techniques that did not uniformly remove GBV-C included vapour heat treatment and dry heat treatments for less than 144 h. All samples treated by pasteurization, solvent detergent or dry heat for 144 h, were negative for the presence of GBV-C. [source]


    Simultaneous determination of maraviroc and raltegravir in human plasma by HPLC-UV

    IUBMB LIFE, Issue 4 2009
    Stefania Notari
    Abstract Therapeutic drug monitoring is pivotal to improve the management of HIV infection. Here, a new HPLC,UV method to quantify simultaneously maraviroc and raltegravir levels in human plasma is reported. Remarkably, this is the first method for maraviroc determination in human plasma. The volume of the plasma sample was 600 ,L. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (30 mg divinylbenzene and N -vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 200 ,L 50/50 of mobile-phase solution (0.01 M KH2PO4 and acetonitrile). Twenty microliters of these samples were injected into a HPLC,UV system, the analytes were eluted on an analytical dC18 Atlantis column (150 mm × 4.6 mm I.D.) with a particle size of 5 ,m. The mobile phase (0.01 M KH2PO4 and acetonitrile) was delivered at 1.0 mL/min with isocratic elution. The total run time for a single analysis was 10 min; maraviroc and raltegravir were detected by UV at 197 and 300 nm. The calibration curves were linear up to 2,500 ng/mL. The absolute recovery ranged between 93 and 100%. The HPLC,UV method reported here has been validated and is currently applied to monitor plasma levels of maraviroc and raltegravir in HIV-infected patients. © 2009 IUBMB IUBMB Life, 61(4):470,475, 2009 [source]


    Hereditary angioedema: an update on available therapeutic options

    JOURNAL DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT, Issue 9 2010
    Marcus Maurer
    Summary There is no cure for hereditary angioedema (HAE). Therapeutic approaches consist of symptomatic therapy for acute attacks, short-term prophylaxis before surgery, and long-term prophylaxis for those with frequent and severe attacks. In Germany, C1-INH concentrate and icatibant are licensed for acute therapy. C1-INH concentrate, which is obtained from human plasma, is administered intravenously to restore the deficient C1-INH activity. This therapy, which has been available for decades, is effective and well-tolerated. Batch documentation is required by German law. The synthetic decapeptide icatibant is administered subcutaneously. It competes with bradykinin, the responsible inducer of edema formation, for binding to the bradykinin B2 receptor. Icatibant is also effective and well-tolerated, even on repeated administration. An additional human C1-inhibitor, a recombinant human C1-inhibitor and the recombinant inhibitor of kallikrein ecallantide are currently under development. There are no licensed treatment options available in Germany for long- and short-term prophylaxis. Androgen derivatives are established in long-term prophylaxis. However, they are associated with many adverse effects, some of which are severe. Many drug interactions also limit their use. They are contraindicated in pregnancy, lactation, for children and in cases of prostate cancer. Antifibrinolytics have fewer adverse effects but are also less effective than androgens. They are contraindicated in thromboembolic disease and impaired vision. If androgen therapy has too negative an effect on quality of life, it may be worth reducing the dose or discontinuing therapy entirely and treating attacks with acute therapy. [source]


    Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose

    JOURNAL OF APPLIED TOXICOLOGY, Issue 6 2010
    Bin Li
    Abstract The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60,84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase. Copyright © 2010 John Wiley & Sons, Ltd. [source]


    Weak inhibitors protect cholinesterases from strong inhibitors (paraoxon): in vitro effect of tiapride

    JOURNAL OF APPLIED TOXICOLOGY, Issue 6 2005
    G. A. Petroianu
    Abstract Weak and reversible inhibitors of cholinesterases, when administered before potent organophosphorus inhibitors (pretreatment), have the ability, to a certain extent, to protect enzymes from inhibition. Such a protective effect was demonstrated in vitro for metoclopramide and ranitidine. The putative mode of protective action of these substances is, when administered in excess, competition for the active site of the enzyme with the more potent organophosphate. The present paper presents results using another benzamide with weak cholinesterase inhibitory properties: tiapride (TIA). The purpose of the study was to quantify in vitro the extent that TIA conferred protection, using paraoxon (POX) as an inhibitor, and to compare the results with existing data obtained using TIA as a protective agent against dichlorvos (DDVP). POX is a highly toxic non-neuropathic organophosphate. While the use of parathion (the inactive prodrug which is metabolically converted to POX) has been restricted in most countries, the organophosphate is still responsible for a large number of accidental or suicidal exposures. DDVP is a moderately toxic, non-neuropathic organophosphate. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different POX and TIA concentrations and the IC50 was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC50 of POX increases with the TIA concentration in a linear manner. The protective effect of tiapride on cholinesterase could be of practical relevance in the pretreatment of organophosphate poisoning. It is concluded that in vivo testing of TIA as an organophosphate protective agent is warranted. Copyright © 2005 John Wiley & Sons, Ltd. [source]