Glutathione reductase, riboflavin status

Determination of the effective functioning of particular enzymes or metabolic pathways potentially may be useful in demonstrating adequacy of provision. Enzymes in plasma that may be helpful in this regard are glutathione peroxidase as an index of selenium status, and red cell enzymes, such as transketolase (thiamine), glutathione reductase (riboflavin) or transaminase (pyridoxine), or glutathione peroxidase (selenium) are all widely used. Methyltetrahydrofolate reductase is involved in metabolism of homocysteine, hence assessment of plasma homocysteine is a useful measure of... 

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. 

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. 

In addition to the role of flavoproteins in iron metabolism, it is possible that the anemia associated with riboflavin deficiency is a consequence of the impairment of vitamin Be metabolism in riboflavin deficiency. Pyridoxine oxidase is a flavoprotein and, like glutathione reductase, is very sensitive to riboflavin depletion (McCormick, 1989). Vitamin Be deficiency can result in hypochromic anemia as a result of impaired porphyrin synthesis. Although riboflavin depletion decreases the oxidation of dietary vitamin Be to pyridoxal (Section 9.2), it is not clear to what extent there is secondary vitamin Be deficiency in riboflavin deficiency This is partly because vitamin Be nutritional status is commonly... 

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). 

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. 

Flavins are lost from the body as intael riboflavin, rather than as a breakdown product of riboflavin. Hence, vitamin status may be assessed by measuring the level of urinary riboflavin. Generally, the loss of 30 ig of riboflavin/g creatinine or less per day indicates a deficiency. This metht>d of assessment is not preferred because it is influenced by a number of factors unrelated to vitamin status. Another problem with this method is its great sensitivity to a short-term deficiency thus, it does not necessarily reflect the true concentrations of FAD and FMN in tissues. The most reliable way to assess riboflavin status is by a functional test. The test involves the assay of glutathione reductase, using red blood cells as the source of... 

Glutathione is discussed further in the section on selenium and glutathione in Chapter 10. The enzyme assay is conducted using glutathione reductase extracted from red blood cells with and without added FAD. Chmnic consumption of a diet deficient in riboflavin allows the continued synthesis of a variety of flavoproteins, but results in the accumulation of apoenzyme without its conversion to holoen-zyme. Addition of chemically pure FAD to a biological fluid containing apoenzyme results In the stimulation of enzyme activity because of the formation of the holoenzyme. It is this stimulation of enzyme activity that is used to determine vitamin status in humans. 

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. 

Use of oral contraceptives may increase the dietary requirement for riboflavin. Riboflavin status can be evaluated from the activity of erythrocyte glutathione reductase, an FAD-requiring enzyme, before and after addition of exogenous FAD. A low initial activity or a marked stimulation by FAD (or both) is indicative of ariboflavi-nosis. 

Glutathione reductase has selenium at the catalytic site, as a selenocysteine residue (section 11.15.2.5) this explains the role of selenium as an antioxidant nutrient. Glutathione reductase is a flavoprotein, and is especially sensitive to riboflavin (vitamin B ) depletion as discussed in section 11.7.4.1, measurement of glutathione reductase is used as a means of assessing riboflavin status. 

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. 

Riboflavin can be measured by microbiological or fluorimetric methods. The erythrocyte enzyme, glutathione reductase, requires FAD as a cofactor and its measurement in the presence and absence of added FAD is therefore considered a good indicator of riboflavin status. 

Figure 3 Basis of the glutathione reductase for riboflavin status (A) riboflavin sufficient (B) riboflavin deficient. Reaction of oxidized glutathione with reduced nicotinamide adenine dinucleotide phosphate.

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