Ofenstein J P, Dandurand D M, Kiechle F L
Department of Pediatrics, William Beaumont Hospital, Royal Oak, MI 48073-6769.
Ann Clin Lab Sci. 1992 Nov-Dec;22(6):406-13.
To assess mitochondrial function (pyruvate dehydrogenase [PDH] activity), cells were grown in the appropriate media to confluence, rinsed and incubated in glucose free media containing 25 microM L-lactate and [1-14C]-D,L-lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of 14CO2 generated per mg of protein per minute. Basal activity varied with cell number and the cell type studied: fibroblast 2.26 +/- 0.01; Chinese hamster ovary (CHO) 42 +/- 0.4; BC3H-1 52 +/- 2.1 nmol per mg per minute. The CHO cells screened for PDH activity decreased their dependence on lactate as a substrate in the presence of 5mM glucose by 60 percent. Increasing the cold lactate concentration diluted the labelled lactate available for pyruvate oxidation in a dose dependent manner. The mitochondrial inhibitor rotenone (25 microM) decreased assay activity by > 75 percent in CHO and BC3H-1 cells. The lactate oxidation assay was shown to be sensitive enough to measure insulin stimulation of PDH in a dose dependent manner with maximum activity occurring at concentrations between 1 microU per ml and 100 microU per ml.
为评估线粒体功能（丙酮酸脱氢酶[PDH]活性），将细胞在合适的培养基中培养至汇合，冲洗后在含有25微摩尔L-乳酸和[1-14C]-D，L-乳酸的无葡萄糖培养基中孵育。乳酸氧化以每分钟每毫克蛋白质产生的14CO2的纳摩尔数表示的乳酸氧化量来衡量。基础活性随细胞数量和所研究的细胞类型而变化：成纤维细胞为2.26±0.01；中国仓鼠卵巢（CHO）细胞为42±0.4；BC3H-1细胞为52±2.1纳摩尔每毫克每分钟。筛选出的具有PDH活性的CHO细胞在存在5毫摩尔葡萄糖的情况下，对乳酸作为底物的依赖性降低了60%。增加冷乳酸浓度以剂量依赖性方式稀释了可用于丙酮酸氧化的标记乳酸。线粒体抑制剂鱼藤酮（25微摩尔）使CHO和BC3H-1细胞中的测定活性降低了>75%。乳酸氧化测定显示出足够的敏感性，能够以剂量依赖性方式测量胰岛素对PDH的刺激，最大活性出现在每毫升1微单位至100微单位的浓度之间。
