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Optimized fluorimetric assay of glutathione in hepatocyte tissue cultures.
Authors: Roušar Tomáš | Kučera Otto | Křiváková Pavla | Mužáková Vladimíra | Červinková Zuzana
Year: 2007
Type of publication: článek ve sborníku
Name of source: Clinical Chemistry and Laboratory Medicine Special Supplement
Publisher name: Walter de Gruyter GmbH & Co. KG
Place: Berlin
Page from-to: S206
Titles:
Language Name Abstract Keywords
cze Optimalizovaná fluorimetrická metoda pro stanovení glutathionu v hepatocytárních kulturách Optimalizovaná fluorimetrická metoda pro stanovení glutathionu v hepatocytárních kulturách
eng Optimized fluorimetric assay of glutathione in hepatocyte tissue cultures. Background: Glutathione is one of the most important antioxidants. It plays crucial role in xenobiotic detoxification, cysteine metabolism and in regulation of redox state in the cell generally. Due to the importance of that compound, the proper information about its levels is essencial for better understanding of cellular redox state, especially in liver. Methods: Hepatocytes were isolated from male Wistar rats by two step collagenase perfusion. Isolated hepatocytes were cultivated in collagen coated Petri dishes. After lysis, the samples were deproteinized by metaphosphoric acid and centrifuged. The samples were frozen in -80°C until analyzed. Fluorimetric method is based on the reaction between reduced form of glutathione (GSH) and o-phthaldialdehyde. Consequently, the fluorescent product is detected spectrofluorimetrically. Results: We optimized well-known fluorimetric GSH-assay described by Hissin in 1976. We found optimal wavelength of excitation (335 nm) and emission (420 nm), respectively. After modification of reaction and detection, we proved that optimal pH value is much lower than published. After optimalization of pH value, temperature and detection conditions, we determined analytical assay parameters as follows (in comparison with results using the published method before): linearity, 1-500 ?M (calibration curve, R2=1.00); intra-assay 6 ?M and 100 ?M GSH (n=10), CV 0.9% and 2.7% (non-optimized method CV 4.1 and 16.0%), respectively. Conclusion: The optimized fluorimetric method is due to changes in detection and reaction parameters much more accurate and sensitive and therefore we can recommend it in analysis of different biological material. Glutathione; fluorescence; oxidative stress

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