Fluorimetric HPLC detection of endogenous riboflavin 4′,5′-cyclic phosphate in rat liver at nanomolar concentrations

We have investigated the biological occurrence of riboflavin 4′,5′-cyclic phosphate (cyclic FMN or cFMN), the flavin product known to be formed in vitro from FAD by the rat liver enzyme FAD-AMP lyase (cyclizing) or FMN cyclase (EC 4.6.1.15). The expected difficulties were the low concentration of the compound, the tendency of the more abundant FAD to decompose chemically to cFMN, and the acid lability of cFMN itself. A protocol was devised to prepare deproteinized rat liver extracts, avoiding conditions prone to the chemical formation of cFMN and making exposure to conditions of cFMN degradation as short as possible. In these extracts, cFMN was assayed by HPLC with fluorimetric detection. The identity of liver cFMN was confirmed by its HPLC separation from other known flavins, its coelution with authentic cFMN, and its susceptibility to acid degradation, yielding a mixture of 5′-FMN and 4′-FMN. The amount of total cFMN recovered in the liver extracts was 22 ± 11 pmol/g fresh tissue. Careful control experiments were performed to rule out the possibility that this could be a chemical product of FAD degradation during extract preparation. These controls indicated that, on average, 97% of the measured extract concentration of cFMN, i.e., about 21 ± 10 pmol/g, was already present in the liver at the beginning of the process and was extracted from the tissue. This figure is likely to be an underestimation of the hepatic content, as indicated by control experiments.