Analytical Science and Technology (분석과학)

The Korean Society of Analytical Sciences (한국분석과학회)
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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

이온쌍 역상 HPLC를 이용한 인체 말초혈액단핵구에서 이노신 5'-일인산 탈수소효소 활성의 정량적 측정

  • Shin, Hye-Jin (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory) ;
  • Kwon, Soon-Ho (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory) ;
  • Park, Ji-Myeong (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory) ;
  • Kwon, Soon-Hyo (Dept. of Internal Medicine, Soonchunhyang University Hospital) ;
  • Lee, Kyoung-Ryul (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory) ;
  • Kim, Young-Jin (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory) ;
  • Lee, Sang-Hoo (Dept. of Bioanalysis, Seoul Medical Science Institute & Seoul Clinical Laboratory)
  • 신혜진 ((재)서울의과학연구소 질량분석부) ;
  • 권순호 ((재)서울의과학연구소 질량분석부) ;
  • 박지명 ((재)서울의과학연구소 질량분석부) ;
  • 권순효 (순천향대학병원 내과) ;
  • 이경률 ((재)서울의과학연구소 질량분석부) ;
  • 김영진 ((재)서울의과학연구소 질량분석부) ;
  • 이상후 ((재)서울의과학연구소 질량분석부)
  • Received : 2010.10.01
  • Accepted : 2010.11.22
  • Published : 2010.12.25
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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 ${\beta}$-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 ($r^2$=0.999999) in the range of $0.2-50.0\;{\mu}M$ and the limit of quantification (LOQ) was $0.2\;{\mu}M$. 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 = $27.70{\pm}6.28\;nmol/h/mg$ protein).

본 연구에서는 이온쌍 역상 HPLC/UV를 이용하여 건강한 한국인에서 분리된 말초혈액단핵구(PBMCs)에서 이노신 5'-일인산 탈수소효소(IMPDH)의 활성을 측정하였다. IMPDH는 이노신 5'-일인산(IMP)을 잔토신 5'-일인산(XMP)로 전환시키는 베타-니코틴아마이드 아데닌 디뉴클로티드 수화물(${\beta}-NAD^+$) 의존성 탈수소효소이며, 이것의 활성은 기질인 IMP와 조효소인 $NAD^+$의 존재 하에서 분해한 PBMCs로 부터 생성된 XMP에 해당하는 HPLC 크로마토그램을 정량적으로 분석함으로써 측정하였다. 생성된 XMP는 260 nm에서 검출하였다. 이동상으로는 7 mM tetra-n-butylammonium hydrogen sulfate가 포함된 37 mM potassium dihydrogen phosphate (pH 5.5)와 methanol의 혼합용액(85:15, v/v)을 사용하였으며, 유속은1 mL/min이었다. 정량 범위는 $0.2-50.0\;{\mu}M$이었으며, 이 때 정량 한계(LOQ)는 $0.2\;{\mu}M$이었다. 또한, 본 연구에서 확립된 시험법은 일내 정밀성(0.88-1.47%), 정확성(98.74-99.99%)과 일간 정밀성(0.85-5.24%), 정확성(99.95-101.65%)을 측정하여 검증하였다. 11명의 건강한 한국인에 대한 IMPDH 활성 측정 결과, 18.29-36.60 nmol/h/mg protein(평균값 $27.70{\pm}6.28\;nmol/h/mg$ protein)이었다.

Keywords

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