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Warburg and Christian (I), in describing the preparation of flavinadenine dinucleotide (FAD), mentioned that whenever dinucleotide solutions developed a greenish fluorescence they were found to have lost their coenzyme activity.On further investigation of this phenomenon it has been found that there occurs a lo-fold increase in fluorescence when FAD is split to riboflavin phosphate (flavin mononucleotide) or to free riboflavin.This change in fluorescence has proved to be a practical means of measuring FAD.Since flavin mononucleotide (FMN) can be distinguished from riboflavin on the basis of its distribution coefficient between benzyl alcohol and aqueous solutions (2), ready analytical means are available for measuring separately FAD, FMN, and riboflavin, the three forms of riboflavin encountered in biological material.A description is given below for the fluorometric measurement of these three forms of riboflavin.The measurements are considered to be much simpler than the enzymatic procedures which were the available methods hitherto.Data are given for the fluorescent behavior of FAD, FMN, and riboflavin, the stability of FAD, and the concentration of each of these three forms of riboflavin in normal and riboflavin-deficient tissues of the rat. Measurement of Ribojlavin, FMN, and FAD in Tissues
Bessey et al. (1949) studied this question.