Effect of extracts from high- or low-V/C FBs on the GSH levels in a cell-free system and in isolated G6PD-normal and G6PD-deficient RBCs. Fresh FBs with high-V/C (average content of V/C in raw seeds: 4.75 g/kg wet weight) and with low-V/C (Divine cultivar, average content of V/C in raw seeds: 0.16 g/kg wet weight), were grown and collected at Institut National de la Recherche Agronomique, Dijon, France. FB seeds were immediately flash-frozen after harvest and kept on dry ice until usage. Thirty minutes before use, FBs were thawed, dehulled, suspended in degassed, nitrogen-flushed isotonic phosphate-buffered saline (PBS) at 50% weight-to-volume ratio, and homogenized in the cold in a nitrogen atmosphere with a high-speed GVA2-type homogenizer (Krups GmbH, Offenbach am Main, Germany). The slurry was centrifuged for 10 minutes at 15 000g and 4°C, and V/C assayed in the resulting extracts as indicated¹⁹  with pure vicine (Serva, Heidelberg, Germany) as a standard. Before starting incubations, the FB extracts were treated with 5 mg/mL β-glucosidase from almonds (Sigma, St. Louis, MO) during 60 minutes at 37°C to enhance the generation of the active compounds D and I from the inactive V/C. (A) Effect of high-V/C and low-V/C FB extracts on GSH oxidation in a cell-free system. GSH was dissolved at 2.5 mM (final concentration) in PBS-glucose, and FB extract added. The final concentration of V/C in the test system was 2.5 mM or 0.1 mM for high- and low-V/C, respectively. In control samples, PBS-glucose was added instead of the FB extract. The test solutions were incubated at 37°C. The GSH concentration was assessed time dependently as indicated.²³  Mean values ±standard deviation (SD) of 3 independent experiments. (B) Effect of high-V/C FB extracts on GSH oxidation in isolated RBCs from G6PD-normal and G6PD-deficient subjects. Washed RBCs freshly isolated from hemizygous G6PD-deficient (open squares) and G6PD-normal (filled squares) subjects were resuspended in PBS-glucose at a hematocrit of 50%, supplemented at time 0 with extracts from high-V/C FBs at 2.5 mM V/C (final concentration) and incubated at 37°C. The GSH concentration in RBCs was assessed time dependently as indicated.²³  Mean values ± SD of 3 independent experiments from 3 different subjects. (C) Effect of low-V/C FB extracts on GSH oxidation in isolated RBCs from G6PD-normal and G6PD-deficient subjects. Washed RBCs freshly isolated from hemizygous G6PD-deficient (open circles) and G6PD-normal (filled circles) subjects were resuspended in PBS-glucose at a hematocrit of 50%, supplemented at time 0 with extracts from low-V/C FBs at 0.1 mM V/C (final concentration), and incubated at 37°C. The GSH concentration in RBCs was assessed time dependently as indicated.²³  Mean values ± SD of 3 independent experiments from 3 different subjects.