An automated flavin adenine dinucleotide-dependent glutathione reductase assay for assessing riboflavin nutriture

Abstract

An automated AutoAnalyzer method using 5:5′dithiobis-2-nitrobenzoic acid is described for determining whole blood gluthathione reductase (BGR) activity and for measuring in vitro activation of BGR with flavin adenine dinucleotide (FAD). BGR activity is expressed as µmoles glutathione regenerated from oxidized glutathione per ml of whole blood (WB) or per g of hemoglobin. The stimulatory effect of FAD on BGR activity divided by the activity without FAD determined the activity coefficient (AC). We found that NADPH and oxidized glutathione assay concentrations of 0.100 mmole/liter and 0.250 mmole/liter, respectively, in 0.1 mole/liter phosphate buffer, pH 7.4, gave consistent results when WB, before assay, was diluted 20-fold. WB samples to be stored are initially diluted 10-fold with distilled water and frozen. Prior to assay, two aliquots of the sample are diluted 2-fold, one aliquot with distilled water and another with 46 µmole/liter FAD. With sample and manifold dilutions the assay FAD concentration is 1.0 µmole/liter: assay concentrations greater than 5.0 µmole FAD/liter were shown to be inhibitory. We examined blood samples from 617 children in the age range 6 to 60 months and determined the normal AC range to be between 1.00 and 1.35. Six weaned rats (23 days of age), maintained on a riboflavin-deficient diet, showed a mean AC of 1.23, 1.54, 2.02, and 2.41 at 23, 26, 30, and 36 days of age, respectively. Six control rats maintained an AC of 1.23 ± 0.05 (SD) during the same period.