Fluorometric measurements of riboflavin and its natural derivatives in small quantities of blood serum and cells.

The concentrations of many of the vitamins or their natural derivatives in blood serum and cells reflect the relatively recent intake of the particular dietary essential (e.g., ascorbic acid). Such a relationship between blood level and intake has led to the usefulness of chemical analytical methods for the evaluation of nutritional status. However, there is, at present, insufficient information available to indicate whether or not measurements of the blood levels of riboflavin in any of its forms would be helpful in this way. This paucity of dat.a on riboflavin blood levels is primarily attributable to the lack of satisfactory analytical methods. Results with microbiological methods (l-3) have been somewhat discordant, apparently because of the difficulty of measuring the low concentrations of the vitamin in the presence of persistent substances in blood extracts which may enhance or inhibit bacterial growth. The microbiological methods also fail to differentiate between riboflavin and its derivatives. By a manometric technique (4, 5), the flavin-adenine-dinucleotide has been measured in red cells and plasma. This procedure requires so much material and labor as to appear impracticable for wide usage. The recent development of a very sensitive fluorometer (6) now makes possible methods which are simple, rapid, and reproducible to 3 to 5 per cent, and by which riboflavin can be differentiated from its natural derivatives. With this instrument the fluorescence of as little as 0.2 my of riboflavin in volumes of 0.5 ml. is readily measurable. Methods will be described for determining (1) both free and total riboflavin in 50 cmm. of blood serum (25 c.mm. for total alone), (2) the free riboflavin, flavin-monoand dinucleotide as separate fractions in 0.2 ml. of serum, (3) the total riboflavin in 20 c.mm. of red cells or whole blood, and (4) the total riboflavin in white cells and platelets from 0.1 ml.