title: 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.
abstract: 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.
authors.affiliation: Department of Rheumatology, University Hospital Nijmegen, Netherlands.
authors.forename: J N
authors.initials: JN
authors.lastname: Stolk
authors.forename: R A
authors.initials: RA
authors.lastname: De Abreu
authors.forename: A M
authors.initials: AM
authors.lastname: Boerbooms
authors.forename: D G
authors.initials: DG
authors.lastname: de Koning
authors.forename: R
authors.initials: R
authors.lastname: de Graaf
authors.forename: P J
authors.initials: PJ
authors.lastname: Kerstens
authors.forename: L B
authors.initials: LB
authors.lastname: van de Putte
mesh_terms: 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
chemical_list: D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase; D009708:Nucleotidases; C029296:purine 5'-nucleotidase