Elsevier

Clinica Chimica Acta

Volume 275, Issue 2, 28 July 1998, Pages 175-184
Clinica Chimica Acta

Glutathione measurement in human plasma: Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC

https://doi.org/10.1016/S0009-8981(98)00089-8Get rights and content

Abstract

Literature values for human plasma GSH vary over 10-fold despite the use of apparently valid analytical procedures for GSH measurement. The purpose of this study was to develop a procedure to minimize error in sample collection, processing and storage that could contribute to such differences. HPLC with fluorescence detection of dansyl derivatives was used for quantification. The results show that collection of blood with a butterfly needle and syringe reduces overestimation due to limited hemolysis and that use of a preservation solution designed to inhibit autooxidation and enzymatic degradation allows quantitative recovery of both GSH and GSSG. Stability tests showed that non-derivatized samples were stable for at least 2 months at −80° while dansyl derivatives were stable in the dark at 0–4° for 12 months. Results from 59 healthy individuals (20–43 years) provided a mean (±1 SD) GSH value of 2.09±1.14 micromolar.

Introduction

Plasma GSH in humans has been reported to be altered in several pathophysiologic states, including alcoholic liver disease, human immunodeficiency viral infection and ascorbate deficiency [1], yet the findings in different laboratories are difficult to compare in terms of absolute contents of GSH. For instance, the measurement of plasma GSH with monobromobimane has provided values of approximately 3–6 μM in one laboratory [2]while a variation of this method gave values under 1 μM [3]. An orthophthalaldehyde method gave values of 2–4 μM 4, 5, 6while enzymatic recycling methods have produced values from 1 to 11 μM 7, 8, 9. Because the different analytical methods for GSH have been validated and are calibrated against GSH standards, at least some of this variability is likely to be due to differences in sampling, processing and/or storage.

Three types of sample collection and processing errors have been discussed in the literature. Red blood cells contain approximately 500-times higher GSH concentration than plasma so that minor hemolysis (0.1% to 1%) can result in erroneously high plasma values. Alternatively, GSH can be lost by oxidation which occurs with a half-time of about 5 min in plasma at room temperature 10, 11, 12. Human plasma also contains γ-glutamyltranspeptidase, an enzyme that degrades GSH [13]and can be present at high activities in human plasma in association with liver disease.

In the present report, we describe a procedure that we have optimized to collect blood with minimal hemolysis, prevent GSH oxidation and degradation during processing and provide a specific and sensitive measure of GSH and GSSG under clinical conditions. In this procedure, blood is collected with a butterfly needle to minimize hemolysis and transferred immediately into a preservation solution. The preservation solution includes heparin to inhibit coagulation, serine·borate to inhibit degradation of GSH by γ-glutamyltranspeptidase [13], bathophenanthroline disulfonate to inhibit GSH oxidation [10]and iodoacetic acid to alkylate GSH [14]. The use of this preservation solution allows for the blood sample to stand at room temperature for up to 30 min without significant changes in the absolute contents or redox state of the GSH and GSSG. With the greater flexibility in timing that this approach allows, a phlebotomist can prepare the plasma after attending to the subject so that a second investigator is not needed to immediately process samples. In the protocol as described, an internal standard (γ-glutamylglutamate) is included to facilitate quantitation, and derivatization with dansyl chloride [15]has been optimized to allow quantitation of GSSG as well as GSH. Conditions for HPLC separation on an amine column have been optimized to allow simultaneous measurement of cystine (Cys2) and cysteine (Cys) with GSH and GSSG.

Section snippets

Materials

Sodium heparin, bathophenanthroline disulfonate sodium salt (BPDS), iodoacetic acid, dansyl chloride, l-serine, γ-glutamylglutamate (γ-Glu-Glu), GSH, GSSG, Cys, Cys2, γ-Glu-Cys, and sodium acetate trihydrate were from Sigma Chemical (St. Louis). The disulfide of Cys and GSH, CySH-GSH, was from Toronto Research Chemicals (Toronto). Boric acid, sodium tetraborate, potassium tetraborate, perchloric acid, acetic acid, acetone and chloroform were reagent grade and purchased locally. Methanol was

Results

Biologically important compounds separated by the method include Cys2, Cys, CySH-GSH, GSH and GSSG (Fig. 1). Studies performed to optimize conditions for derivatization showed that reaction of thiols with iodoacetic acid was complete within 5 min at pH 9.0. The rate of derivatization with dansylchloride was the same for GSH as for the internal standard (complete by 8 h at room temperature); however, disulfides, which have two amino groups, required 16 h for complete derivatization at room

Discussion

Experimental evidence suggests that plasma GSH concentration varies in response to disease and may therefore be a useful clinical measure [1]. Thus, there is a need for a standardized procedure for determination of plasma GSH. Several analytical techniques are available for measurement of GSH at concentrations that are present in human plasma, and these can be calibrated with relevant standards to provide reliable measurement. The present study has focused on aspects of sample collection and

Acknowledgements

This research was supported by NIH grant EY07892 and Research to Prevent Blindness Inc..

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