[Determination of glutathione with the GSH-400 method: value of derivative spectrophotometry]

The GSH-400 method (Oxis International SA) performed in acid supernatant, provides an assay of reduced glutathione (L-gamma-glutamyl-L-cysteinylglycine) (GSH) in biological samples by a two step chemical reaction followed by a spectrophotometric detection. We improved the analytical performances of the assay by using derivative spectrophotometry (delta). The second order derivative (delta 2) spectrophotometry gave identical signals with either a GSH standard solution or a erythrocyte lysate containing the same amount of GSH, whereas the corresponding absorbances (Abs.) were different. The linearity range of the method was 20 to 2,000 mumol/l. Detection limit (DL) was not modified with derivative spectrophotometry (DL Abs. = 7 mumol/l; DL delta 2 = 12 mumol/l). The GSH-400 method was not suitable for plasma GSH determination. Erythrocyte GSH concentrations (M +/- SD) in 23 healthy subjects (8 men, 15 women, median age 36) obtained with absorbance measurements (2.66 +/- 0.60 mmol/l) were significantly higher than those obtained with delta 2 (2.30 +/- 0.41 mmol/l) (p < 0.001). Erythrocyte lysate storage at -20 degrees C did not lead to satisfactory conservation (65% decrease of the GSH concentration in erythrocytes after 15 days freezing). The derivative spectrophotometric detection improved analytical performances of GSH-400 method, when compared to the absorbance measurement at 400 nm.