DEVELOPMENT OF METHODIC OF TOTAL GLUTATHIONE AMOUNT DETERMINATION

conference paper

English

Glutathione plays important role in biochemical processes associated with oxidative stress in bacterial, plant and animals cells. Glutathione is occurring in the reduced (GSH) and oxidized form (GSSG) in the organism. The ratio between GSH and GSSG is considered as a marker of oxidative stress. During the sample preparation the destabilization of the GSH and GSSG forms is occurring which might be a problem for subsequent analysis. Aim of this study was the optimization and of quantitative reduction GSSG to GSH for HPLC-ED determination using tris (2-carboxyethyl) phosphin (TCEP) and ethylendiamintetracetic acid (EDTA). The efficiency of reduction of GSSG was analyzed using high pressure liquid chromatography with electrochemical detection (HPLC-ED). For GSH and GSSG determination we used gradient elution with methanol on reversed-phase column and coulometric detection. Limit of detection for GSH and GSSG was determined as 30 nM and 200 nM respectively. For qualitative reduction of GSSG to GSH we optimized the concentration of reducing agent (1, 2, 4, 6 mM), time of reaction (1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60 minutes) and pH range of tested buffers (Britton-Robinson, Borate buffer, Phosphate buffer and Acetate buffer). The influence of addition of EDTA was also tested. We determined the optimal condition of the reducing reaction as 2 mM TCEP in Borate buffer (pH 9) and reaction time 15 minutes. No influence of EDTA addition on the reducing reaction was observed. The usage of the method was estimated for concentration range 60 nM - 200 mu M of GSSG.

Total GSH determination; Oxidized glutathione (GSSG); Tris(2-carboxyethyl) phosphine (TCEP); ethylenediaminetetraacetic acid (EDTA)

KOMÍNKOVÁ, M.; ZÍTKA, O.; MERLOS RODRIGO, M.; ZEHNÁLEK, J.; HAVEL, L.; ADAM, V.; KIZEK, R.

22. 11. 2012

978-80-7375-836-3

MENDELNET 2012

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1224

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