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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

Published online by Cambridge University Press:  09 March 2007

D. I. Thurnham  and
Prapimporn Rathakette
D. I. Thurnham
Affiliation:
Department of Human Nutrition, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
Prapimporn Rathakette
Affiliation:
Department of Human Nutrition, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
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Abstract

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1. Some modifications to the erythrocyte glutathione reductase assay for riboflavin status are described.

2. Cusum analysis of results collected on a quality-control (QC) haemolysate, analysedseparately 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° 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° or 1 h at 25° before the standard incubation of 5 min at 35°.

6. Additionally, the use of cummulative sum (cusum) analysis on the QC results suggested that there was a slight deterioration of QC sample after 4-weeks storage. However, theQC results obtained, remained within 2 standard deviations of initial results over a 20-week period, suggesting that the deterioration was very slight.

Type
Papers of direct relevance to Clinical and Human Nutrition
Information
British Journal of Nutrition , Volume 48 , Issue 3 , November 1982 , pp. 459 - 466
Copyright
Copyright © The Nutrition Society 1982

References

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