Abstract
A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 μM and 1.25 μM for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains.
Highlights
GSH is the prevalent nonprotein thiol in animal cells and the most abundant antioxidant in aerobic cells
Our goal was to validate a simple and rapid method, which allowed the simultaneous determination of the main thiol species, such as GSH and Cys, in different mouse organs by using reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet detection
Sensitive, and validated analytical methods for the quantitative evaluation of GSH and other thiol species are critical for determination of redox state in experimental models and the successful conduct of preclinical and/or clinical pharmacology studies employing molecules to restore GSH levels that can be altered in pathological conditions [2, 7]
Summary
GSH is the prevalent nonprotein thiol in animal cells and the most abundant antioxidant in aerobic cells. It is implicated in many cellular functions, such as degradation and synthesis of proteins and DNA or detoxification of toxins and carcinogens [1]. Development and validation of simple analytical methods to measure GSH and other thiols in biological samples are a prerequisite to obtain an accurate assessment of the Oxidative Medicine and Cellular Longevity degree of oxidative damage as well as indication of disease progression and evaluation of the effectiveness of antioxidant therapy [5]. Determining thiol status is important to understand the basic biochemical response of cells during a pathological condition as well as during ageing and the capacity of drugs to restore cellular glutathione homeostasis
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