Purine enzyme activities in peripheral blood mononuclear cells: comparison of a new non-radiochemical high-performance liquid chromatography procedure and a radiochemical thin-layer chromatography procedure.
Purine enzyme activities are usually assayed by radiochemical procedures and often TLC is part of the separation method. In screening patients with rheumatic diseases, these procedures have shown disadvantages like a relatively large coefficient of variation (C.V.) and time-instability. We describe a non-radiochemical reversed-phase HPLC micro-method with UV detection for measurement of activities of purine 5'-nucleotidase (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 procedure is compared with the radiochemical TLC procedure by testing both with a 5'NT and a PNP assay. Reproducibility is tested with 14 healthy controls in each procedure. Short-term and long-term time-stability 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 significantly better reproducibility and better time-stability and in addition is non-radiochemical and less time-consuming.
Stolk
J N
JN
Department of Rheumatology, University Hospital Nijmegen, Netherlands.
De Abreu
R A
RA
Boerbooms
A M
AM
de Koning
D G
DG
de Graaf
R
R
Kerstens
P J
PJ
van de Putte
L B
LB
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008875:Middle Aged; D009708:Nucleotidases / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D011854:Radiochemistry; D012016:Reference Values; D015203:Reproducibility of Results
D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase; D009708:Nucleotidases; C029296:purine 5'-nucleotidase