The Glutathione Reductase Test

One rather serious drawback of the urinary excretion index for the assessment of riboflavin status is that it is relatively insensitive at low-to-moderate intakes, because the rate of excretion changes slowly and not very predictably with increasing intake in this region. Another important practical drawback is that 24-h urine samples are not easy to collect and excretion rates may fluctuate over short time periods. A more stable index was therefore sought and was identified in the degree of unsaturation of the red blood cell enzyme, glutathione reductase (NADPH: oxidized-glutathione oxidoreductase EC 1.6.4.2), with respect to its flavin cofactor, flavin adenine dinucleotide (FAD) (Figure 3).

As noted earlier, an inadequate supply of dietary riboflavin results in low circulating levels, and hence a gradual progressive loss of cofactor from this red cell flavoenzyme over a period of several weeks. Since the enzyme protein (apoenzyme) remains intact and reac-tivatable by FAD, it is possible to remove a small sample of blood, collect, wash and hemolyse the red cells, and then measure glutathione reductase activity with and without the FAD cofactor. If the individual is riboflavin replete, then the added FAD has almost no effect and the 'activation coefficient,' or ratio of FAD stimulated to unstimulated activity ('EGRAC') is between 1.0 and 1.3-1.4. If the individual is deficient, then added FAD produces a larger stimulation, and the 'activation coefficient' is higher. For people living in communities with very low intakes of riboflavin and a significant prevalence of clinically recognizable deficiency, activation coefficients as high as 2.0-3.0 are quite common. In Western countries, few values as high as 2.0 are encountered. However, recent population surveys in the UK have indicated that the proportion of values between 1.3 and 1.8 is considerable across all age ranges. Whether this apparent evidence of marginal deficiency has a technical, assay-related explanation, or is a result of decreasing intakes of riboflavin-rich foods, such as cows' milk, is uncertain.

This test has the advantage that it is highly sensitive to, and predictive of, the extent of tissue depletion in the range of severe-to-moderate deficiency; it is robust and requires only a small sample of blood, and it can be automated by modern enzyme rate reaction analyzers. Riboflavin supplements given to deficient subjects result in rapid and reproducible restoration of the saturated condition of the enzyme,

Gut Diet -Riboflavin Diet----(little or no riboflavin)

Blood

Red cell

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