Estimates of riboflavin requirements are based on depletion/repletion studies to determine the minimum intake at which there is significant excretion of the vitamin. In deficiency there is virtually no excretion of the vitamin; as requirements are met, so any excess is excreted in the urine. On this basis the minimum adult requirement for riboflavin is 0.5–0.8 mg/day.
At intakes of 1.1–1.6 mg/day urinary excretion rises sharply, suggesting that tissue reserves are saturated.
A more generous estimate of requirements, and the basis of reference intakes, is the level of intake at which there is normalization of the activity of the red cell enzyme glutathione reductase; the activity of this flavoprotein is especially sensitive to riboflavin nutri-tional status. Normal values of the activation coeffi-cient are seen in subjects whose habitual intake of riboflavin is between 1.2 mg/day and 1.5 mg/day.
The urinary excretion of riboflavin and its metabo-lites (either basal excretion or after a test dose) can be used as an index of status. However, riboflavin excre-tion is only correlated with intake in subjects who are in nitrogen balance. In subjects in negative nitrogen balance there may be more urinary excretion than would be expected, as a result of the catabolism of tissue flavoproteins, and loss of their prosthetic groups. Higher intakes of protein than are required to maintain nitrogen balance do not affect the require-ment for riboflavin or indices of riboflavin nutri-tional status.
Glutathione reductase is especially sensitive to riboflavin depletion. The activity of the enzyme in erythrocytes can therefore be used as an index of riboflavin status. Interpretation of the results can be complicated by anemia, and it is more usual to use the activation of erythrocyte glutathione reductase (EGR) by FAD added in vitro. An activation coeffi-cient of 1.0–1.4 reflects adequate nutritional status, whereas >1.7 indicates deficiency.