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- E-ISSN 2288-8985
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Quantitative determination of inosine 5’-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells by ion-pair reversed-phase high-performance liquid chromatography
Analytical Science and Technology / Analytical Science and Technology, (P)1225-0163; (E)2288-8985
2010, v.23 no.6, pp.531-536
https://doi.org/10.5806/AST.2010.23.6.531
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(2010). Quantitative determination of inosine 5’-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells by ion-pair reversed-phase high-performance liquid chromatography. Analytical Science and Technology, 23(6), 531-536, https://doi.org/10.5806/AST.2010.23.6.531
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Abstract
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 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 (r2=0.999999) in the range of 0.2-50.0 μM and the limit of quantification (LOQ) was 0.2 μM. The intra- and inter-day precisions were between 0.88-1.47% and 0.85-5.24%,respectively. The intra- and 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).
- keywords
- IMP, XMP, IMPDH activity, PBMCs, HPLC
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