RAPID LIQUID-CHROMATOGRAPHIC ASSAY OF GLUTATHIONE IN CULTURED-CELLS

被引:31
作者
LEROY, P
NICOLAS, A
THIOUDELLET, C
OSTER, T
WELLMAN, M
SIEST, G
机构
[1] FAC SCI PHARMACEUT & BIOL NANCY,CHIM ANALYT & BROMATOL LAB,CNRS,URA 597,BP 403,F-54001 NANCY,FRANCE
[2] FAC SCI PHARMACEUT & BIOL NANCY,CTR MEDICAMENT,CNRS,URA 597,F-54001 NANCY,FRANCE
关键词
D O I
10.1002/bmc.1130070208
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion-pair reversed phase rapid high performance liquid chromatography. The use of a 4 mum particle, 5 cm long column (Superspher 100 RP 18 end-capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o-phthalaldehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 mug/ml, with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.
引用
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页码:86 / 89
页数:4
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