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Riboflavin, vitamin B2, serves in its phosphorylated form as the prosthetic group for a number of tissue enzymes important in biological oxidations (1). The involved substrates include glucose, lactic acid, the d-amino acids, and other compounds of biological interest (1, 2, 3). Riboflavin deficiency has been described in man (4), and it seems possible that further clinical study of this vitamin may reveal alterations in its metabolism which will increase our knowledge of the r6le played by these enzymatic oxidation systems in health and in disease. To aid in interpreting such a study, a convenient method has been sought for following the urinary excretion of riboflavin. It is known that riboflavin occurs in urine in the form of uroflavin, a pigment almost identical with it in composition,' properties, and vitamin activity (5), but the methods usually recommended for its determination (5, 6, 7, 8, 9) were not found by the writer to be sufficiently convenient or accurate for routine use. More specific and accurate determinations are possible, and smaller and more convenient volumes of urine (I to 10 cc. instead of 100 to 500 cc.) can be used when the pigment is estimated not by its optical density but by its intense greenish-yellow fluorescence.2 In order to do this, the effect of interfering substances must be avoided. With urines of normal individuals containing 0.5 gamma or more flavin per cc., this is readily accomplished by diluting down the salts and other materials to the point where they no longer affect fluorescence (10) and by destroying the greater part of other pig-

THE URINARY EXCRETION OF RIBOFLAVIN FLUOROMETRIC METHODS FOR ITS ESTIMATION