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Optimization of spectrofluorimetric assay of glutathione in hepatocyte cultures
Authors: Roušar Tomáš | Kučera Otto | Křiváková Pavla | Lotková Halka | Červinková Zuzana
Year: 2008
Type of publication: článek ve sborníku
Name of source: Acta Medica (Hradec Králové)
Publisher name: Univerzita Karlova v Praze
Place: Praha
Page from-to: 85
Titles:
Language Name Abstract Keywords
cze Optimization of spectrofluorimetric assay of glutathione in hepatocyte cultures Glutathione, one of the most important intracellular antioxidants, exists in two forms - the reduced (GSH) and the oxidized (GSSG). Therefore, the analysis of its levels is crucial to evaluate the oxidative stress. Various types of methods have been used recently; the HPLC methods have been prefered mostly, although spectrophotometric and spectrofluorimetric methods could be used too. The aim of our work was to optimize and describe fluorimetric method based on the reaction between glutathione and o-phthaldialdehyde. Following this reaction, the isoindole compound, which possesses high fluorescence, is formed. The main disadvantage of this method described in literature has been related to low specificity of the detected fluorescence and consequently to enhancement of glutathione levels in biological material. On the other hand, our preliminary results showed no intereference, and therefore we focused on optimizing of detection and reaction conditions, especially on optimizing excitation and emission wavelength, temperature of detection, pH value. Our results: GSH assay - calibration (c = 0-500 ?M; R2= 1.00), intra-assay (c = 20 ?M;CV% = 3.67%, c = 100 ?M;CV% = 0.92%); GSSG assay - calibration (c = 0-70 ?M, R2=0.99), intra-assay (c= 3 ?M;CV% = 4.30%, c = 7 ?M;CV% = 2.80%). We found great correlation of GSH and GSSG levels, respectively, between HPLC and optimized method (r = 0.99). As the results show, we have developed fast, simple and low-cost glutathione assay comparable with HPLC methods.
eng Optimization of spectrofluorimetric assay of glutathione in hepatocyte cultures Glutathione, one of the most important intracellular antioxidants, exists in two forms - the reduced (GSH) and the oxidized (GSSG). Therefore, the analysis of its levels is crucial to evaluate the oxidative stress. Various types of methods have been used recently; the HPLC methods have been prefered mostly, although spectrophotometric and spectrofluorimetric methods could be used too. The aim of our work was to optimize and describe fluorimetric method based on the reaction between glutathione and o-phthaldialdehyde. Following this reaction, the isoindole compound, which possesses high fluorescence, is formed. The main disadvantage of this method described in literature has been related to low specificity of the detected fluorescence and consequently to enhancement of glutathione levels in biological material. On the other hand, our preliminary results showed no intereference, and therefore we focused on optimizing of detection and reaction conditions, especially on optimizing excitation and emission wavelength, temperature of detection, pH value. Our results: GSH assay - calibration (c = 0-500 ?M; R2= 1.00), intra-assay (c = 20 ?M;CV% = 3.67%, c = 100 ?M;CV% = 0.92%); GSSG assay - calibration (c = 0-70 ?M, R2=0.99), intra-assay (c= 3 ?M;CV% = 4.30%, c = 7 ?M;CV% = 2.80%). We found great correlation of GSH and GSSG levels, respectively, between HPLC and optimized method (r = 0.99). As the results show, we have developed fast, simple and low-cost glutathione assay comparable with HPLC methods. Glutathione, hepatocytes in culture, hepatotoxicity

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