Activation coefficient, glutathione reductase

In pregnant women, there is a progressive increase in the erythrocyte glutathione reductase activation coefficient (an index of functional riboflavin nutritional status Section 7.5.2), which resolves on parturition despite the daily secretion of 200 to 400 /rg (0.5 to 1 /rmol) of riboflavin into milk. This suggests that the estrogen-induced riboflavin binding protein can sequester the vitamin for fetal uptake at the expense of causing functional deficiency in the mother. 

Like glutathione reductase, pyridoxine oxidase is sensitive to riboflavin depletion. In normal subjects and in experimental animals, the EGR and pyridoxine oxidase activation coefficients are correlated, and both reflect riboflavin nutritional status. In subjects with glucose 6-phosphate dehydrogenase deficiency, there is an apparent protection of EGR, so that even in riboflavin deficiency it does not lose its cofactor, and the EGR activation coefficient remains within the normal range. The mechanism of this protection is unknown. In such subjects, the erythrocyte pyridoxine oxidase activation coefficient gives a response that mirrors riboflavin nutritional status (Clements and Anderson, 1980). 

Riboflavin status is assessed by (1) determination of urine riboflavin excretion, (2) a functional assay using the activation coefficient of stimulation of the enzyme glutathione reductase by FAD, or (3) direct measurement of riboflavin or its metabolites in plasma or erythrocytes. The advantages and disadvantages of functional or direct methods have been discussed in the section on thiamine. 

The reference interval for eiythrocyte riboflavin using a fluorometric method is 10 to 50jig/dL (266 to 1330nmol/L). The reference interval for serum or plasma levels of riboflavin is 4 to 24 Lig/dL (106 to 638 nmol/L). Guidance reference intervals for the activation coefficient of erythrocyte glutathione reductase by FAD are 1.20 (adequacy), 1.21 to 1.40 (marginal deficiency), and 1.41 and above (deficiency)... 

The coenzyme activity of Alg-NADP was determined enzymatically by the glutathione reductase system (38). The assay mixture consisted of 250 pmol Tris-HCl buffer (pH 8.0), 2.7 [xmol EDTA, 13.2 pmol GSSG, an appropriate amount of Alg-NADP", and 20 units of GR in a total volume of 2.87 ml. The reaction was started by the addition of the enzyme and the reaction mixture was incubated at 30°C. Absorbance at 340 nm was measured with a double beam spectrophotometer. The reference contained all components except for Alg-NADP. In the calculation of the amount of Alg-NADPH produced, a molar absorption coefficient for NADPH of 6.22 X 10 l-mol -cm was used. 

A more generous estimate of requirements, and the basis of reference intakes, is the intake at which there is normalization of the activity of the red cell enzyme glutathione reductase, a flavoprotein whose activity is especially sensitive to riboflavin nutritional status. Normal values of the activation coefficient (section 11.7.4.1) are seen in subjects whose habitual intake of riboflavin is between 1.2 and 1.5 mg/day. 

Glutathione reductase is especially sensitive to riboflavin depletion, and the usual way of assessing riboflavin status is by measurement of the activation of red blood cell glutathione reductase by FAD added vitro (section 11.6.4.1). An activation coefficient > 1.7 indicates deficiency. 

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