Determination of plasma activities of purine nucleoside phosphorylase by high-performance liquid chromatography: estimates of nonparenchymal cell injury after porcine liver transplantation

Abstract

An assay is described for measurement of purine nucleoside phosphorylase (PNP) in plasma by high-performance liquid chromatography (HPLC). A plasma sample was incubated with hypoxanthine and ribose-1-phosphate in phosphate-free medium at pH 7.4 to catalyse the production of inosine by plasmatic PNP. The reaction was stopped by addition of perchloric acid to inactivate the enzyme and to precipitate plasma proteins. After centrifugation and neutralization of the supernatant with NaOH the increase in the substrate inosine was determined by HPLC. Plasma activities of PNP averaged 5.0 mU/ml before and 12.3 mU/ml ( p < 0.001), 5 min after porcine liver transplantation. At the same time points, the plasma activities of the frequently used liver enzymes lactate dehydrogenase or alanine aminotransferase remained virtually unchanged. Thus, plasmatic activities of PNP may be a suitable and early indicator of ischemic alterations to the graft in vivo.