Tamás Lőrincz1, András Szarka2. 1. Department of Applied Biotechnology and Food Science, Laboratory of Biochemistry and Molecular Biology, Budapest University of Technology and Economics, 1111 Szent Gellért tér 4, Budapest, Hungary. 2. Department of Applied Biotechnology and Food Science, Laboratory of Biochemistry and Molecular Biology, Budapest University of Technology and Economics, 1111 Szent Gellért tér 4, Budapest, Hungary; Pathobiochemistry Research Group of Hungarian Academy of Sciences and Semmelweis University, 1444 Budapest, P.O. Box 260, Budapest, Hungary. Electronic address: szarka@mail.bme.hu.
Abstract
INTRODUCTION: Glutathione (GSH) through its important function in the antioxidant protection of cells and in the conjugation of drugs and xenobiotics has crucial importance in pharmacology and toxicology. Since GSH is most often measured in liver tissue and different cell organelles it is important to choose the method that best suits for the determination of GSH. METHODS: The GSH content of cell organelles isolated from control and BSO-treated liver tissues was determined by the GSH-NEM-HPLC-UV, monochlorobimane-GSH-HPLC-fluorescence method and DTNB-GSH recycling assay to find the most suitable method for GSH determination from cell organelles. RESULTS: The GSH level of organelles could easily be measured by the monochlorobimane-HPLC-fluorescent method. The addition of monochlorobimane to the homogenisation buffer prevented the oxidation of GSH during isolation. The formation of monochlorobimane-GSH adduct was accelerated by the intrinsic GST activity of samples, however the omission of GST from the GSH standards could cause the overestimation of GSH content of biological samples. NEM is an excellent thiol protective agent and the GSH-NEM conjugate can be directly analysed by HPLC-UV, but the relatively high limit of detection made the method unsuitable for the determination of GSH from cell organelles. Although the DTNB-GSH recycling assay is quite simple and rapid the stabilization of GSH and the efficiency of detection lag behind the monochlorobimane-HPLC-fluorescent method. DISCUSSION: The monochlorobimane-HPLC-fluorescent method can be advised for the determination of GSH from pharmacologically and toxicological relevant cell organelles and liver tissue whilst addition of monochlorobimane to the homogenisation buffer prevented the autoxidation of GSH.
INTRODUCTION:Glutathione (GSH) through its important function in the antioxidant protection of cells and in the conjugation of drugs and xenobiotics has crucial importance in pharmacology and toxicology. Since GSH is most often measured in liver tissue and different cell organelles it is important to choose the method that best suits for the determination of GSH. METHODS: The GSH content of cell organelles isolated from control and BSO-treated liver tissues was determined by the GSH-NEM-HPLC-UV, monochlorobimane-GSH-HPLC-fluorescence method and DTNB-GSH recycling assay to find the most suitable method for GSH determination from cell organelles. RESULTS: The GSH level of organelles could easily be measured by the monochlorobimane-HPLC-fluorescent method. The addition of monochlorobimane to the homogenisation buffer prevented the oxidation of GSH during isolation. The formation of monochlorobimane-GSH adduct was accelerated by the intrinsic GST activity of samples, however the omission of GST from the GSH standards could cause the overestimation of GSH content of biological samples. NEM is an excellent thiol protective agent and the GSH-NEM conjugate can be directly analysed by HPLC-UV, but the relatively high limit of detection made the method unsuitable for the determination of GSH from cell organelles. Although the DTNB-GSH recycling assay is quite simple and rapid the stabilization of GSH and the efficiency of detection lag behind the monochlorobimane-HPLC-fluorescent method. DISCUSSION: The monochlorobimane-HPLC-fluorescent method can be advised for the determination of GSH from pharmacologically and toxicological relevant cell organelles and liver tissue whilst addition of monochlorobimane to the homogenisation buffer prevented the autoxidation of GSH.
Authors: Archit Rastogi; Christopher W Clark; Sarah M Conlin; Sarah E Brown; Alicia R Timme-Laragy Journal: Redox Biol Date: 2019-06-05 Impact factor: 11.799
Authors: Mariama Salihu; Gaber El-Saber Batiha; Keneth Iceland Kasozi; George D Zouganelis; Souty M Z Sharkawi; Eman Ibrahim Ahmed; Ibe Michael Usman; Halima Nalugo; Juma J Ochieng; Ibrahim Ssengendo; Olatayo Segun Okeniran; Theophilus Pius; Kyobe Ronald Kimanje; Eric Simidi Kegoye; Ritah Kenganzi; Fred Ssempijja Journal: Toxicol Rep Date: 2022-03-29