Profiling the Concentration of Reduced and Oxidized Glutathione in Rat Brain Using HPLC/DAD Chromatographic System.

George Jîtcă,Mircea Dumitru Croitoru,Carmen Maria Rusz,Ibolya Fülöp,Bianca-Eugenia Ősz,Camil Eugen Vari,Maria Titica Dogaru,Erzsébet Fogarasi

doi:10.3390/molecules26216590

George Jîtcă, Mircea Dumitru Croitoru + Show 6 more

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https://doi.org/10.3390/molecules26216590

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Abstract

This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman′s reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.

Highlights

Accepted: 29 October 2021Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) that exhibits many biological roles, including the protection against reactive oxygen and nitrogen species (ROS and RNS)
It has been observed that the inherited enzyme defects related to GSH are very rare; in contrast, the inbalances in GSH homeostasis associated with the increase of the oxidative stress levels in central nervous system (CNS) are common in neurodegenerative diseases [7]
Measurement of GSH in biological samples is of high importance in the study of oxidative stress and exogenous substances or pathological conditions associated with oxidative state [21,22]

Summary

Introduction

Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) that exhibits many biological roles, including the protection against reactive oxygen and nitrogen species (ROS and RNS). GSH is involved in many processes, such as defense and preservation of the organism, prevention or delay of age-related diseases onset, given the proportional increase of free radicals with aging [4]. It is found mainly in its reduced form (GSH) because the enzyme responsible for converting the oxidized form (GSSG) is induced by oxidative stress. It has been observed that the inherited enzyme defects related to GSH are very rare; in contrast, the inbalances in GSH homeostasis associated with the increase of the oxidative stress levels in central nervous system (CNS) are common in neurodegenerative diseases [7]

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