Erythrocyte riboflavin

Although riboflavin is fundamentally involved in metabolism, and deficiencies are found in most countries, it is not fatal as there is very efficient conservation of tissue riboflavin. Riboflavin deficiency is characterized by cheilosis, lingual desquamation and a seborrheic dermatitis. Riboflavin nutritional status is assessed by measurement of the activation of erythrocyte glutathione reductase by FAD added in vitro. 

Today, biochemical deficiency of riboflavin is accepted in the absence of clinical signs of deficiency. Biochemical signs of deficiency include change in the amount of the vitamin which is excreted in the urine, or change in the level of activity of a red blood cell (erythrocyte) enzyme, which is known as the erythrocyte glutathione reductase. Requirements for the vitamin are defined as that amount which will prevent both clinical and biochemical signs of deficiency. 

Darby, W. J. (1972) Application of the erythrocyte glutathione reductase assay in evaluating riboflavin nutritional status in a high school student population. Am. J. Clin. Nutr. 

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. 

Tissue concentrations of flavin coenzymes in hypothyroid animals may be as low as in those fed a riboflavin-deficient diet, in hypothyroid patients, erythrocyte glutathione reductase (EGR) activity may be as low, and its activation by FAD added in vitro (Section 7.5.2) as high, as in riboflavin-deficient subjects. Tissue concentrations of flavin coenzymes and EGR are normalized by the administration of thyroid hormones, with no increase in riboflavin intake (Cimino et al., 1987). 

Glutathione reductase is especially sensitive to riboflavin depletion, in deficient animals, the activity of glutathione reductase responds earlier and more markedly than any other index of riboflavin stams apart from liver concentrations of flavin coenzymes and the activity of hepatic flavokinase (Prentice and Bates, 1981a, 1981b). The activity of the enzyme in erythrocytes can therefore be used as an index of riboflavin status. 

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). 

Pyridoxine phosphate oxidase is a flavoprotein, and activation of the erythrocyte apoenzyme by riboflavin 5 -phosphate in vitro can be used as an index of riboflavin nutritional status (Section 7.4.3). However, even in riboflavin deficiency, there is sufficient residual activity of pyridoxine phosphate oxidase to permit normal metabolism ofvitamin Be (Lakshmi and Bamji, 1974). Pyridoxine phosphate oxidase is inhibited by its product, pyridoxal phosphate, which binds a specific lysine residue in tbe enzyme. In tbe brain, tbe Ki of pyridoxal phosphate is of the order of 2 /xmol per L - the same as the brain concentration of free and loosely bound pyridoxal phosphate, suggesting that this inhibition may be a physiologically important mechanism in the control of tissue pyridoxal phosphate (Choi et al., 1987). 

DuttaP (1991) Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum. Journal of Protozoology 38,479-83. 

Prentice AM and Bates CJ (1981a) A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status. 1. Rate and specificity of response in acute deficiency. British Journal of Nutrition 45,37-52. 

Saubcriich, H. E., Judd, J. H., Nichoalds, C. F.., Broquist, H. P, and Darby, W. J, (1972). Application of the erythrocyte glutathione reductase assay in evaluating riboflavin status in a high school student population. Am. /. Cfjri. Nutr, 25, 756-762. 

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. 

Direct measurement of riboflavin, FMN, and FAD in plasma or erythrocytes may be made by HPLC, usually with fluorescence detection after protein precipitation or by capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). In a study of riboflavin status and FMN and FAD concentrations in plasma and erythrocytes from elderly subjects at baseline and after low-dose riboflavin supplementation, using both activation coefficient measurements and CE-LIF, it was concluded that concentrations of aU Ba vitamers except plasma FAD are potential... 

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)... 

Hustad S, McKinley MC, McNulty H, Schneede J, Strain fJ, Scott JM, Ueland PM. Riboflavin, flavin mononucleotide, and flavin adenine dinudeotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation. Clin Chem 2002 48 1571-7. 

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