Assessment of reduced glutathione: Comparison of an optimized fluorometric assay with enzymatic recycling method

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• 作者：Tomá ; &scaron ; Rou&scaron ; ar^a ; ^b ; ^{tomas.rousar@upce.cz} ; Otto Ku膷era^b ; Halka Lotková ; ^b ; Zuzana 膶ervinková ; ^b
• 关键词：Glutathione ; Glutathione assay ; Spectrofluorometric method
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：15 April, 2012
• 卷：423
• 期：2
• 页码：236-240
• 文件大小：326 K

摘要

Glutathione is an important tripeptide involved in a variety of cellular processes. Thus, precise knowledge of its levels is essential. Glutathione exists in two free forms鈥攔educed and oxidized鈥攁nd a number of methods exist to measure its levels. The aim of our work was to optimize a spectrofluorometric assay for reduced glutathione based on the reaction between glutathione and o-phthalaldehyde. We found that a change of excitation wavelength to 340 nm and modification of pH to 6.0 enhance sensitivity and specificity of the method (intraassay coefficient of variation CV <3%, interassay CV = 5.1%, recovery = 98-102%, linearity = 0-1000 渭M GSH, calibration R² = 1.00). We also anticipated possible effect of various amino acids on the fluorescence signal, but no interference was found. We compared the optimized fluorometric method with a popular enzymatic recycling glutathione assay and found very strong correlation of results (r = 0.99, n = 45). We introduce here an optimized fluorometric method possessing sufficient sensitivity and specificity that is comparable to the enzymatic glutathione assay. Because the fluorometric assay procedure is faster and lower in cost, it could be ideal for routine analysis of reduced glutathione levels in a large number of samples.