THE FLUOROMETRIC MEASUREMENT OF THE NUCLEOTIDES OF RIBOFLAVIN AND THEIR CONCENTRATION IN TISSUES

Abstract

Riboflavin, flavin adenine dinucleotide and flavi mononucleotide were estimated fluorimetrically by methods based on the knowledge that the dinucleotide is less fluorescent than riboflavin and that riboflavin can be separated from its nucleotides by partition between benzyl alcohol and water. The fluorescent properties of the 3 substances were studied. Fluorescence of the dinucleotide was found to increase as the pH of the aqueous solution decreased. In tests with various solvents, maximum fluorescence was obtained in a mixture of acetate buffer, ethanol and benzyl alcohol. On mild hydrolysis of the dinucleotide, the fluorescence increased, possibly owing to the liberation of the mononucleotide. The fluorescence of riboflavin and both its nucleotides was quenched in the presence of adenosine or adenylic acid. The fluorimetric method of estimation was compared with the enzymic method and found to be .satisfactory. By means of the fluorimetric method it was demonstrated that almost all the riboflavin in rat kidney was present as riboflavin monophosphate and dinucleotide. In liver, kidney, heart, brain and skeletal muscle of rats, the dinucleotide accounted for 70 to 90 per cent, of the riboflavin, and almost no free riboflavin was found. A similar destruction was found in normal rats and in rats deprived of riboflavin. Values for riboflavin derivatives are reported for 16 other rat tissues.-A. M. Copping.