PURINE ENZYME-ACTIVITIES IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS - COMPARISON OF A NEW NON-RADIOCHEMICAL HIGHPERFORMANCE LIQUID-CHROMATOGRAPHY PROCEDURE AND A RADIOCHEMICAL THIN-LAYER CHROMATOGRAPHY PROCEDURE

Purine enzyme activities are usually assayed by radiochemical procedur es and often TLC is part of the separation method. In screening patien ts with rheumatic diseases, these procedures have shown disadvantages like a relatively large coefficient of variation (C.V.) and time-insta bility. We describe a non-radiochemical reversed-phase HPLC micro-meth od with UV detection for measurement of activities of purine 5'-nucleo tidase (5'NT; EC 3.1.3.5), purine nucleoside phosphorylase (PNP; EC 2. 4.2.1) and hypoxanthine guanine phosphoribosyltransferase (HGPRT; EC 2 .4.2.8) in human peripheral blood mononuclear cells (PBMC). The HPLC p rocedure is compared with the radiochemical TLC procedure by testing b oth with a 5'NT and a PNP assay. Reproducibility is tested with 14 hea lthy controls in each procedure. Short-term and long-term time-stabili ty is tested by comparing enzyme activities measured immediately after preparation of the PBMC (week 0) with those found after freezing and storage at -20 degrees C for a maximum of 10 weeks. The HPLC procedure is preferable to the radiochemical TLC procedure because it shows sig nificantly better reproducibility and better time-stability and in add ition is non-radiochemical and less time-consuming.