Quantitative determination of inosine 5'-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells by ion-pair reversed-phase high-performance liquid chromatography

A quantitative analytical method has been established for the measurement of inosine 5’monophosphate dehydrogenase (IMPDH) activity in human peripheral blood mononuclear cells (PBMCs) by ion-pair reversed-phase high performance liquid chromatography equipped with ultraviolet detection (HPLC/ UV). IMPDH is a β-nicotinamide adenine dinucleotide hydrate (NAD)-dependent dehydrogenase in which the enzyme converts inosine 5’-monophosphate (IMP) into xanthosine 5’-monophosphate (XMP). Its activity was measured by quantifying a HPLC chromatogram corresponding to XMP produced during the incubation of lysed ★ Corresponding author Phone : +82-(0)70-7115-8697 Fax : +82-(0)2-858-2814 E-mail : sprout30@scllab.co.kr 532 신혜진·권순호·박지명·권순효·이경률·김영진·이상후 Analytical Science & Technology PBMCs with IMP as a substrate and NAD as a coenzyme. XMP produced was detected at a wavelength of 260 nm. The mobile phase was composed of a mixture of 37 mM potassium dihydrogen phosphate containing 7 mM tetra-n-butylammonium hydrogen sulfate adjusted to pH 5.5 and methanol (85:15, v/v) with a flow rate of 1 mL/min. The calibration curve was linear (r=0.999999) in the range of 0.2-50.0 μM and the limit of quantification (LOQ) was 0.2 μM. The intraand inter-day precisions were between 0.88-1.47% and 0.85-5.24%, respectively. The intraand inter-day accuracies were between 98.74-99.99% and 99.95-101.65%, respectively. IMPDH activity in 11 Korean healthy volunteers ranged from 18.29 to 36.60 nmol/h/mg protein (mean = 27.70 ± 6.28 nmol/h/mg protein).