Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma

The measurement of isoprostanes is a promising assay that is specific and sensitive enough to detect in vivo lipid peroxidation. We present here a gas chromatography-tandem mass spectrometry (GC/MS/MS)method that enables determination of 8-iso-prostaglandin F2α (8-iso-PGF2α)in human plasma and urine. After the addition of [2H4]-PGF2α as the internal standard to acidified plasma or urine, the samples are purified on C18 and silica cartridges, derivatised as pentafluorobenzyl esters, extracted with diethyl ether, purified on silica gel TLC plates and finally silylated. Then, 8-iso-PGF2α and its internal standard are measured by GC/MS/MS in selective-reaction monitoring mode using the transition [M −181]− to [M −181 – (3 × 90)]−. The detection limit of this method is 5 pg mL−1. Its application is presented in two situations of oxidative stress: in vitro low-density lipoprotein oxidation and in smokers. Measurement of urinary 8-iso-PGF2α levels provides a non-invasive in vivo index of free radical generation that appears not to be confounded by changes in diet.

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Liquid Chromatographic Method for the Simultaneous Determination of Eprosartan and Hydrochlorothiazide in Tablets and Human Plasma

Journal of AOAC International ◽

10.1093/jaoac/94.3.823 ◽

2011 ◽

Vol 94 (3) ◽

pp. 823-832 ◽

Cited By ~ 1

Author(s):

Fathalla Belal ◽

Amina M El-Brashy ◽

Nahed El-Enany ◽

Manar M Tolba

Keyword(s):

Simultaneous Determination ◽

Human Plasma ◽

Mobile Phase ◽

Hplc Method ◽

Rp Hplc ◽

Liquid Chromatographic Method ◽

Liquid Chromatographic

Abstract A new, specific, and sensitive RP-HPLC method was developed for the simultaneous determination of eprosartan (EPR) and hydrochlorothiazide (HCT). Good chromatographic separation was achieved using a 250 × 4.6 mm id, 5 μm particle size Symmetry® C18 column. The mobile phase acetonitrile–0.1 M phosphate buffer (35 + 65, v/v), pH 4.5, was pumped at a flow rate of 1 mL/min, with UV detection at 275 nm. The method showed good linearity in the ranges of 0.5–50 and 0.1–10 μg/mL, with LOD of 0.06 and 0.02 μg/mL and LOQ of 0.20 and 0.08 μg/mL for EPR and HCT, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixture and co-formulated tablets. The method was further extended to the in vitro and in vivo determination of the two drugs in spiked and real human plasma. Interference likely to be encountered from the co-administered drugs was studied.

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Direct determination of the redox status of cysteine residues in proteins in vivo

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2014.11.082 ◽

2015 ◽

Vol 456 (1) ◽

pp. 339-343 ◽

Cited By ~ 6

Author(s):

Satoshi Hara ◽

Yuki Tatenaka ◽

Yuya Ohuchi ◽

Toru Hisabori

Keyword(s):

Direct Determination ◽

Redox Status ◽

Cysteine Residues

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A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

Nuklearmedizin ◽

10.1055/s-0038-1624353 ◽

1987 ◽

Vol 26 (01) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

S. Selvaraj ◽

M. R. Suresh ◽

G. McLean ◽

D. Willans ◽

C. Turner ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Tumor Cell ◽

T Antigen ◽

Human Carcinomas ◽

Animal Tumor ◽

Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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Storage of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647709 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 405-416 ◽

Cited By ~ 3

Author(s):

M. R Hardeman ◽

Carina J L. Heynens

Keyword(s):

Storage Temperature ◽

Platelet Rich Plasma ◽

Blood Platelets ◽

Storage Period ◽

Serotonin Uptake ◽

Hypotonic Shock ◽

Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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Studies on the Fibrinolytic System in Human Plasma: Quantitative Determination of Plasminogen Activators and Proactivators

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646786 ◽

1979 ◽

Vol 41 (02) ◽

pp. 365-383 ◽

Cited By ~ 80

Author(s):

C Kluft

Keyword(s):

Pilot Study ◽

Plasma Level ◽

Human Plasma ◽

Plasminogen Activators ◽

Venous Occlusion ◽

Quantitative Information ◽

Optimal Recovery ◽

Dextran Sulphate ◽

Baseline Levels

SummaryEffects due to plasma plasminogen activators and proactivators are usually studied in assay systems where inhibitors influence the activity and where the degree of activation of proactivators is unknown. Quantitative information on activator and proactivator levels in plasma is therefore not availableStudies on the precipitating and activating properties of dextran sulphate in euglobulin fractionation presented in this paper resulted in the preparation of a fraction in which there was optimal recovery and optimal activation of a number of plasminogen activators and proactivators from human plasma. The quantitative assay of these activators on plasminogen-rich fibrin plates required the addition of flufenamate to eliminate inhibitors. The response on the fibrin plates (lysed zones) could be coverted to arbitrary blood activator units (BAU). Consequently, a new activator assay which enables one to quantitatively determine the plasma level of plasminogen activators and proactivators together is introduced.Two different contributions could be distinguished: an activity originating from extrinsic activator and one originating from intrinsic proactivators. The former could be assayed separately by means of its resistance to inhibition by Cl-inactivator. Considering the relative concentrations of extrinsic and intrinsic activators, an impression of the pattern of activator content in plasma was gained. In morning plasma with baseline levels of fibrinolysis, the amount of extrinsic activator was negligible as compared to the level of potentially active intrinsic activators. Consequently, the new assay nearly exclusively determines the level of intrinsic activators in morning plasma. A pilot study gave a fairly stable level of 100 ± 15 BAU/ml (n = 50). When fibrinolysis was stimulated by venous occlusion (15 min), the amount of extrinsic activator was greatly increased, reaching a total activator level of 249 ± 27 BAU/ml (n = 7).

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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