使用酶促转化的[1-(13)C]-缬氨酸标准化曲线和气相色谱-质谱法测定[1-(13)C]-α-酮异戊酸的细胞外和细胞内富集情况。
Determination of extracellular and intracellular enrichments of [1-(13)C]-alpha-ketoisovalerate using enzymatically converted [1-(13)C]-valine standardization curves and gas chromatography-mass spectrometry.
作者信息
Kulik W, Jakobs C, de Meer K
机构信息
Department of Clinical Chemistry, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
出版信息
J Chromatogr B Biomed Sci Appl. 1999 Jun 11;729(1-2):211-6. doi: 10.1016/s0378-4347(99)00158-9.
We report a validated method for the determination of extra- and intracellular [1-(13)C]-alpha-ketoisovalerate ([1-(13)C]-KIV) enrichments by gas chromatography-mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-(13)C]-valine enriched standards of known composition. Slopes of [1-(13)C]-valine standardization curves (mean+/-SD: 0.99+/-0.02, n=5) and [1-(13)C]-KIV standardization curves (mean+/-SD: 0.98+/-0.01, n=7) were not significantly different. The method was applied for the determination of [1-(13)C]-KIV enrichments in plasma and tissues during [1-(13)C]-valine infusion in a piglet. [1-(13)C]-KIV enrichment could be determined+/-0.1 MPE (C.V. 1%), and extracellular [1-(13)C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-(13)C]-KIV enrichment.
我们报告了一种通过气相色谱-质谱法测定细胞外和细胞内[1-(13)C]-α-酮异戊酸（[1-(13)C]-KIV）丰度的经过验证的方法。通过对已知组成的[1-(13)C]-缬氨酸富集标准品进行酶促氧化来制备标准曲线。[1-(13)C]-缬氨酸标准曲线的斜率（平均值±标准差：0.99±0.02，n = 5）和[1-(13)C]-KIV标准曲线的斜率（平均值±标准差：0.98±0.01，n = 7）无显著差异。该方法用于测定仔猪在输注[1-(13)C]-缬氨酸期间血浆和组织中的[1-(13)C]-KIV丰度。[1-(13)C]-KIV丰度的测定精度可达±0.1 MPE（变异系数1%），并且细胞外[1-(13)C]-KIV丰度是细胞内（骨骼肌、骨生长板）[1-(13)C]-KIV丰度的可靠估计值。
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