Determination of intracellular glutathione and thiols by high performance liquid chromatography with a gold electrode at the femtomole level: comparison with a spectroscopic assay.
Glutathione (GSH) is an important thiol, which has multiple functions in human metabolism, including the detoxification of xenobiotics, radioprotection and antioxidant defense. Here we provide a sensitive and specific method to quantify intracellular GSH and other thiols using an electrochemical detector coupled to a high performance liquid chromatograph (HPLC-ECD). This HPLC-ECD system includes a specially devised gold electrode with a large surface area and a thin gasket to provide an extremely high sensitivity to thiols. The standard curve for GSH showed a good linear relationship at low femtomole levels (r=0.970). We could simultaneously detect GSH, cysteine, N-acetylcysteine, gamma-glutamyl-cysteine and cysteinyl-glycine by this method. We compared the specificity and sensitivity of this method with those of the conventional spectroscopic method by measuring the amounts of GSH in HL-60 cell extracts. Although the values obtained from these methods were closely correlated (r=0.984), the electrochemical method was much more specific for GSH. This method could detect 2 fmol of GSH and was 6 orders and 2-3 orders of magnitude more sensitive than the spectroscopic method and previous methods using HPLC, respectively. As an example of the application of this method, we demonstrated that the time-dependent alteration in intracellular GSH and cysteine levels could be easily measured using buthionine sulfoximine, an inhibitor of GSH synthesis. On the basis of these results, the advantage of this electrochemical method is extremely sensitive and specific to detect femtomole levels of GSH and other various thiols.
Hiraku
Yusuke
Y
Department of Hygiene, Mie University, School of Medicine, Tsu, 514-8507, Mie, Japan.
Murata
Mariko
M
Kawanishi
Shosuke
S
D000111:Acetylcysteine / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D004151:Dipeptides / Q000032:analysis; D004563:Electrochemistry; D004566:Electrodes; D005978:Glutathione / Q000032:analysis*; D006046:Gold; D018922:HL-60 Cells; D006801:Humans; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis*
D004151:Dipeptides; D013438:Sulfhydryl Compounds; C028505:cysteinylglycine; D006046:Gold; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine; D000111:Acetylcysteine