Willems H L, de Kort T F, Trijbels F J, Monnens L A, Veerkamp J H
Clin Chem. 1978 Feb;24(2):200-3.
We measured pyruvate oxidation in intact leukocytes and fibroblasts by measuring 14C02 production. The optimal pyruvate concentration appeared to be higher than that usually applied. Activities remained constant during the incubation and were proportional to the amount of tissue protein added. Man values (+/-SD) were 2.8 +/- 0.9 nmol/h per 10(6) cells and 37 +/- 14 nmol/h per mg of protein for leukocytes and fibroblasts, respectively, for [1-14C]pyruvate oxidation; and 2.1 +/- 0.8 nmol/h per 10(6) cells and 18 +/- 7 nmol/h per mg of protein, respectively for [2-14C]pyruvate oxidation. We compared oxidation rates of pyruvate and 2-oxoglutarate by intact cells with those of isolated mitochondria. The ratio of 14CO2 production vs. activity of mitochondrial marker enzyme demonstrated that the rate of pyruvate oxidation can adequately be assayed in intact cells, but that the permeability of the cell membrane is rate-limiting in the oxidation of 2-oxoglutarate. No significant oxidation of other intermediates of the citric acid cycle was found, presumably owing to a low rate of transport of these substances across the cell membrane.
我们通过测量¹⁴CO₂的生成来测定完整白细胞和成纤维细胞中的丙酮酸氧化。最佳丙酮酸浓度似乎高于通常使用的浓度。在孵育过程中活性保持恒定，并且与添加的组织蛋白量成正比。对于[1-¹⁴C]丙酮酸氧化，白细胞和成纤维细胞的平均值（±标准差）分别为每10⁶个细胞2.8±0.9 nmol/h和每毫克蛋白37±14 nmol/h；对于[2-¹⁴C]丙酮酸氧化，分别为每10⁶个细胞2.1±0.8 nmol/h和每毫克蛋白18±7 nmol/h。我们将完整细胞中丙酮酸和2-氧代戊二酸的氧化速率与分离线粒体的氧化速率进行了比较。¹⁴CO₂生成与线粒体标记酶活性的比率表明，丙酮酸氧化速率可以在完整细胞中得到充分测定，但细胞膜的通透性是2-氧代戊二酸氧化的限速因素。未发现柠檬酸循环其他中间产物有明显氧化，推测是由于这些物质跨细胞膜的转运速率较低。
