Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection

doi:10.1016/j.jpba.2006.11.028

Journal of Pharmaceutical and Biomedical Analysis

Volume 43, Issue 4, 12 March 2007, Pages 1382-1387

https://doi.org/10.1016/j.jpba.2006.11.028 Get rights and content

Abstract

A HPLC method for determination of both reduced (GSH) and oxidized (GSSG) glutathione in plasma, whole blood and rat hepatocytes has been developed and evaluated. Reduced glutathione reacts with orthophthaldehyde (OPA) to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA at pH 12. At measurement of GSSG, GSH was complexed to N-ethylmaleimide. For the separation, reverse phase column Discovery C₁₈, 150 mm × 4 mm, 5 μm, was used. The mixture of methanol and 25 mM sodium hydrogenphosphate (15:85, v/v), pH 6.0, was used as mobile phase. The analytical performance of this method is satisfactory for both GSH and GSSG. The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes, 4.3 and 5.2% for plasma. The inter-assay coefficients of variation were 5.8 and 6.2% for whole blood, 6.6 and 7.1% for rat hepatocytes, 6.9 and 7.8% for plasma. The recoveries were as follows: 98.2% (CV 3.5%) and 101.5% (CV 4.2%) for whole blood, 99.1% (2.5%) and 102.3 (4.4%) for rat hepatocytes, 94.1% (CV 7.5%) and 103.5 (CV 8.5%) for plasma. The calibration curve was linear in the whole range tested. The limit of detection was 14.0 and 5.6 fmol, respectively. The preliminary reference ranges of reduced and oxidized glutathione in a group of blood donors are (4.69 ± 0.93) and (0.28 ± 0.12) μmol/g Hb for whole blood, (1.82 ± 0.55) and (0.154 ± 0.044) μM for plasma.

Introduction

The tripeptide, glutathione (γ-glutamyl-cysteinyl-glycine) can be obtained from the diet or can be synthesized de novo in the liver [1], [2], [3], [4]. It plays important roles in biological systems. Glutathione (GSH) can readily be oxidized to its disulfide (GSSG) and the ratio of both forms is crucial for the characterization of the oxidative stress in cells. It also plays a role in the regeneration of other antioxidants. It can regenerate ascorbic acid from dehydroascorbic acid and membrane bound α-tocopherol from the α-tocopheryl radical formed during inhibition of lipid peroxidation [5], [6]. Glutathione is important in the detoxification of potentially harmful endogenous compounds and xenobiotics (e.g. α-oxoaldehydes, monoamines, polyphenoles and some drugs). It acts as cofactor for enzymes including glutathione peroxidase, and other peroxidases, dehydrochlorinase, formaldehyde dehydrogenase, glyoxylase, maleyl-acetonase isomerase, and prostaglandin endoperoxidase isomerase [7], [8]. The multifunctional properties of glutathione are reflected by the growing interest in this tripeptide.

Several methods are available for the determination glutathione in biological samples. Glutathione is measured after protein precipitation by spectrophotometry, fluorometry or by HPLC. Oxidation of GSH during sample preparation represents a major problem. The blocking of thiol group with various agents such as N-ethylmaleimide (NEM) iodoacetic acid and 2-vinyl pyridine is often used to prevent this phenomenon [9].

The most widely used technique, enzyme recycling, measures total glutathione (GSH and GSSG) in a reaction involving NADPH, 5,5′-dithiobis-(2-nitrobenzoic acid) and glutathione reductase [10].

A variety of HPLC techniques have also been developed. HPLC with ultraviolet detection requires derivatization [11], [12], [13], [14], [15], [16]. With regard to poor limit of detection it may not be sensitive enough for some biological samples. HPLC with fluorescence detection requires derivatization with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) [17], [18], [19], [20], ortophthalaldehyde (OPA) [21], [22], [23], [24], [25], monobromobimane [26], [27], [28], [29], [30], dansyl chloride [31] or 5-methyl-(2-(m-iodoacetylaminophenyl)benzoxazole) (MIPBO) [32]. HPLC with electrochemical detection, using of either amperometric or coulometric electrodes, can measure GSH and GSSG directly. These techniques avoid typical problems associated with derivatization procedures. Coulometric detection offers superior sensitivity and selectivity to the dual-amperometric approach. However, the measurement of GSSG requires a relatively high applied oxidation potential, shortening the life of the operating electrochemical cell [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46]. Quite recently liquid chromatography/mass spectrometry assays were developed for the determination of glutathione [47], [48], [49] or glutathione conjugates [50].

The aim of this study was to develop rapid, simple HPLC method for measurement both GSH and GSSG in different biological samples, suitable for clinical trials.

Section snippets

Reagents and chemicals

Reduced glutathione, oxidized glutathione, N-ethylmale-imide, orthophthalaldehyde (OPA), sodium hydroxide, hydrochloric acid, sodium hydrogenphosphate, metaphosphoric acid, EDTA, type I collagen and trypan blue were obtained from Sigma–Aldrich, medium William's E without phenol red, fetal bovine serum, penicillin, streptomycin and glutamine from Pan Biotech GmbH, HPLC-gradient grade methanol from Merck KgaA. Collagenase crude was obtained from SEVAC (Prague, Czech Republic) and insulin

HPLC analysis of GSH and GSSG

HPLC chromatogram of glutathione-OPA adduct in whole blood is shown in Fig. 2. The analytical parameters of GSH and GSSG analysis have been sufficient and are shown in Table 1. The calibration curve was linear in the whole range tested: (20.0–2000.0) μM of GSH and (2.0–200.0) μM of GSSG for whole blood and rat hepatocytes, (0.2–20.0) μM of GSH and (0.02–2.0) μM of GSSG for plasma. The limit of detection (14.0 fmol for GSH and 5.6 fmol for GSSG) was estimated as the lowest GSH and GSSG concentration,

Discussion

The research on the function of glutathione has led to the development of numerous methods for its determination. Among spectrophotometric determinations, the most considerable assay is based on the enzymatic recycling reaction discovered by Owens and Belcher [54] and developed by Tietze [10]. Cohn and Lyle [55] described a fluorimetric assay for GSH based on derivatization with OPA to form a highly fluorescent tricyclic derivative. Hissin and Hilf [53] modified this method for the

Conclusion

We developed a rapid, simple and very selective HPLC method with fluorescence detection for the determination of both reduced and oxidized glutathione in different biological samples. This method provides excellent sensitivity, precision, and accuracy in follow-up samples and is suitable for research whereas can be easily adapted for clinical testing purposes. By virtue of this method it is possible to determine both GSH and GSSG from filter paper spots in newborns.

Acknowledgement

This work was supported by Grant MSM0021627502.

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