Incubation of NAD(P)H2: glutathione oxidoreductase (EC 1.6.4.2) with flavin adenine dinucleotide for maximal stimulation in the measurement of riboflavin status.

Thurnham DI, Rathakette P.

Some modifications to the erythrocytes glutathione reductase assay for riboflavin status are described. 2. Cusum analysis of results collected on a quality-control (QC) haemolysate, analysed separately at the beginning and end of each batch of samples over a period of 20 weeks, suggested that the activation coefficient (AC) was higher at the end of a batch than at the beginning. 3. The higher AC was due to higher FAD-stimulated enzyme activities of the QC samples measured at the end of the day, by comparison with the beginning, and this suggested that the conditions of assay were not optimal. 4. The conditions required to achieve maximal coupling of FAD to glutathione reductase (NAD(P)H2: glutathione oxidoreductase; EC 1.6.4.2) were therefore examined and found to be 15 min at 35 degrees by comparison with the 5-7 min incubation used by most workers. 5. Alternatively, where samples are prepared in batches, the enzyme and FAD should be pre-incubated in the reaction mixture for 2 h at 4 degrees or 1 h at 25 degrees before the standard incubation of 5 min at 35 degrees. 6. Additionally, the use of cummulative sum (cusum) analysis on the QC results suggested that there was a slight deterioration of QC samples after 4-weeks storage. However, the QC results obtained, remained within 2 standard deviations of initial results over a 20-week period, suggesting that the deterioration was very slight.