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Simple determination of erythrocyte pyrimidine 5'-nucleotidase activity in human blood by high-performance liquid chromatography.

We developed a method for the determination of erythrocyte pyrimidine 5'-nucleotidase (P5N) activity in human blood using a high-performance liquid chromatograph (HPLC). In this method, UMP (uridine 5'-monophosphate disodium salt) was used as a substrate and assay solutions were directly prepared from whole blood. Uridine formed by the enzyme reaction of P5N was determined spectrophotometrically at 260 nm after separation from the substrate and blood components by HPLC. The optimum pH of this reaction was 7.5.This HPLC method was very rapid and simple compared to the conventional method, because of eliminating the need for dialysis of erythrocyte lysate prior to the assay.The present method was applied to the examination of P51N activity in the blood of lead workers, indicating that the P5N activity is significantly inhibited in these workers.