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KMID : 0385520100230060531
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Analytical Science & Technology
2010 Volume.23 No. 6 p.531 ~ p.536
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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
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Shin Hye-Jin
Kwon Soon-Ho
Park Ji-Myeong
Kwon Soon-Hyo
Lee Kyoung-Ryul
Kim Young-Jin
Lee Sang-Hoo
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Abstract
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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 ㎚. 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 n㏖/h/㎎ protein (mean = 27.70 ± 6.28 n㏖/h/㎎ protein).
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KEYWORD
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IMP, XMP, IMPDH activity, PBMCs, HPLC
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