Publikace detail

Optimization of spectrofluorimetric assay of glutathione in hepatocyte cultures

Autoři: Roušar Tomáš | Kučera Otto | Křiváková Pavla | Lotková Halka | Červinková Zuzana

Rok: 2008

Druh publikace: článek ve sborníku

Název zdroje: Acta Medica (Hradec Králové)

Název nakladatele: Univerzita Karlova v Praze

Místo vydání: Praha

Strana od-do: 85

Tituly:

Jazyk	Název	Abstrakt	Klíčová slova
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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