URIDINE DIPHOSPHOGLUCOSE CONTENT OF HUMAN ERYTHROCYTES - ASSESSMENT BY CONVERSION TO URIDINE DIPHOSPHOGLUCURONATE

To settle the ongoing controversy regarding differential uridine dipho sphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnor mality in patients with galactosemia, we derived a combined enzymatic- high-performance liquid chromatography (HPLC) assay. Uridine diphospho glucuronate (UDPGA), the unique product of UDPG dehydrogenase activity , was separated and quantified by HPLC in extracts of human erythrocyt es. The quantity of UDPGA produced in cell filtrates incubated with th e enzyme corresponds to the amount of UDPG directly determined by HPLC . The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fl uorometric measurement of the production of reduced nicotinamide adeni ne dinucleotide varied with the enzyme purity and activity. The combin ed enzymatic-HPLC method confirms the direct determinations of UDPG co ntent of normal erythrocytes. The results indicate that, under appropr iate conditions, the fluorometric-based assay will give accurate estim ates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.