A new and versatile method for determination of thiolamines of biological importance

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Abstract

A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C18 reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, Nϵ-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.

Introduction

The tripeptide glutathione (GSH) is the major nonprotein thiol in mammalian cells where it is present in millimolar concentrations. GSH serves many diverse physiological functions including redox homeostasis, amino acid transport, and protection against reactive oxygen species. Another major function of GSH is the detoxication of reactive metabolites generated during xenobiotic metabolism, which can lead to GSH depletion [1], [2], [3]. GSH biosynthesis is limited by the amount of available cysteine. The administration of cysteine in various forms has been shown to maintain GSH levels and protect against xenobiotic toxicity [3], [4]. We are currently interested in the development of new cysteine prodrugs directed at elevating depleted GSH levels resultant of toxic insults. These studies would be facilitated by a suitable detection method for the measurement of biologically relevant thiolamines.

A variety of high-perfromance liquid chromatography (HPLC) methods are available for the detection and quantitation of thiol and disulfide containing compounds [5], [6]. Many of these methods utilize thiol-specific reagents that attach a chromophore to the thiol group thereby assisting in separation and detection. The more commonly used HPLC methods for the determination of thiols and disulfides are based on the formation of fluorescent derivatives of thiols using reagents such as monobromobimane, o-phthalaldehyde, or N-substituted maleimides. Fluorometric detection provides good sensitivity for thiol measurement but lacks the ability to simultaneously measure thiols and disulfides [5]. Disulfides are detected only as their free thiol form following a separate reduction step. Another commonly used HPLC method in which thiols and disulfides can be measured in the same sample uses electrochemical detection. However, this method suffers from a severe problem related to the sensitivity of the detector to interference from oxidizable impurities [5]. Another widely used HPLC method developed by Reed and coworkers is dependent on trapping free thiol groups with iodoacetic acid followed by the formation of N-dinitrophenylated (DNP) derivatives by reaction with Sanger’s reagent [7], [8]. The derivatives are then separated on an ion-exchange column and monitored at 365 nm. This procedure has been successful in the simultaneous measurement of GSH and GSH derivatives, as well as other thiols and disulfides, in the same sample [9], [10], [11]. Although UV–VIS detection is not as sensitive as other detection methods, the sensitivity of the Reed method has been reported in the nanomole range [7]. The primary disadvantage of this procedure is the inability to analyze thiol compounds of neutral charge (i.e., no free carboxyl group). Also, the high salt concentration necessary for the elution of the derivatives is damaging to the HPLC instrumentation, and the column itself becomes derivatized by Sanger’s reagent leading to reduced performance over time.

We have developed a new method for determining concentrations of GSH, oxidized GSH (GSSG), cysteine, and cystine in a single analysis. In the method described here, both free thiols and amino groups are dinitrophenylated with Sanger’s reagent and the derivatives are separated on a C18 reversed-phase column. This method allows the simultaneous determination of both reduced and oxidized thiol compounds. The development and validation of this method is presented.

Section snippets

Chemicals

GSH, GSSG, l-cysteine, l-cystine, Nϵ-methyl-l-lysine, bathophenanthrolinedisulfonic acid (BPDS), trifluoroacetic acid (TFA), 70% perchloric acid (PCA), 2,4-dinitrofluorobenzene (DNFB), Eagle’s minimum essential medium (EMEM), antibiotic antimycotic solution (100×), Hank’s balanced salt solution (without Ca2+ or Mg2+), trypsin 1:250, EDTA, and phosphate-buffered saline (PBS), pH 7, were purchased from Sigma–Aldrich Chemical Company (St Louis, MO, USA). Fetal bovine serum (Fetal Clone I) was

Sample preparation

During sample preparation, cell and tissue samples were homogenized in 10% PCA in the presence of the metal ion chelator, BPDS, to prevent auto-oxidation of thiols and thiol–disulfide exchange. PCA was chosen for sample homogenization in this procedure in order to facilitate subsequent precipitation of the acid as the potassium salt. This prevented the separation of the reaction mixture into aqueous and organic phases. Previously, salicylic acid, which did not precipitate as a salt, was used,

Discussion

We report a versatile method for the simultaneous measurement of biological thiols and disulfides including GSH, GSSG, cysteine, and cystine. This method is a modification of the techniques described by Mertens et al. [10] and Reed et al. [7], [8]. We exploited the reactivity of DNFB with both thiol and amino groups [9] to produce N,S-di-DNP derivatives of GSH and cysteine. Nϵ-Methyllysine, GSSG, and cystine were treated with DNFB to give N,N′-di-DNP derivatives. The derivatives were separated

Acknowledgements

This study was supported by the University of Utah College of Pharmacy, a University of Utah Technology Innovation Grant, and NIH 5R29 GM 44785 and 1R01 GM 58913. The Finnegan MAT 95 mass spectrometer and the Micromass Quattro II Triple Quadrupole mass spectrometer were supported by the University of Utah Institutional Funds Committee and the National Science Foundation Grants CHE-9002690 and CHE-9708413, respectively.

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Present address: Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.

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