성체줄기세포 치료 후 뇌 경색 쥐 모델 뇌 조직에서 기체크로마토그래피-질량분석기를 이용한 아미노산 프로파일 분석

Abstract

An efficient method for the simultaneous profiling of protein and non-protein amino acids (AAs) as their N(O,S)-ethoxycarbonyl/tert-butyldimethylsilyl (EOC/TBDMS) derivatives was developed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. In this study, the EOC reaction conducted in two-step pH shift mode (initially at pH 7.5 phosphate buffer then raised to pH 12) for the simultaneous analysis of 30 AAs including 3,4-dihydroxyphenylalanine as their N(O,S)-EOC/TBDMS derivatives. Then the aqueous solution was acidified with concentrated sulfuric acid (pH ≤ 2), followed by solvent extraction with diethyl ether and ethyl acetate in sequence. The combined extracts were evaporated to dryness under a gentle stream of nitrogen. And the remaining active hydrogen atoms were converted to TBDMS derivatives for the direct analysis by GC-SIM-MS. Under the optimal conditions, linear response in the range of 0.5-10.0 g/ml was achieved with good correlation coefficient (r≥0.996). The precision showed low relative standard deviation less than 10%, and the accuracy (% RE) varied from -10.9 to 9.8 for 30 AAs studied. When applied to rat brain tissues from normal control group, transient middle cerebral artery occlusion (MCAo) group and MCAo group following intravenous human mesenchymal stem cells (hMSC) transplantation, the levels of some AAs of the MCAo and MCAo + hMSC groups were significantly different from those of the control group. And normalized AA levels of MCAo and MCAo + hMSC groups were transformed into distorted star patterns which was different from control mean. The present method has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the transplantation effects of MSCs in stroke.