Establishment of a rapid diagnosis of congenital lactic acidosis using peripheral blood samples

儿童先天性乳酸酸中毒的特征是血丙酮酸和乳酸水平持续高企。在日本，报告的先天性乳酸血症病例数量正在增加。在丙酮酸脱氢酶复合体的酶、糖异生途径的关键酶和电子传递系统的酶中，已经证明了导致乳酸酸中毒的遗传性缺陷。为了建立先天性乳酸酸中毒的诊断，通常的程序是测量培养的皮肤成纤维细胞中与丙酮酸代谢有关的酶的活性。然而，皮肤成纤维细胞的培养需要很长的时间。因此，我们开发了一种利用外周血中分离的血小板和淋巴细胞来快速诊断先天性乳酸酸中毒的方法。用该方法测定了日本各地医院运送的40例先天性乳酸中毒患者外周血中(1)丙酮酸和(3)丙酮酸的脱羧率、血小板细胞色素c氧化酶活性、淋巴细胞丙酮酸羧基酶和磷酸烯醇丙酮酸羧基酶的活性。然后测定丙酮酸脱羧率和丙酮酸代谢相关酶的活性。血样运送过程中，血小板丙酮酸脱羧率、细胞色素c氧化酶活性和淋巴细胞丙酮酸羧基酶活性不稳定，呈下降趋势。因此，为了诊断酶缺陷，需要同时从对照组采集血液样本。用此法在40例先天性乳酸中毒患者中发现1例丙酮酸脱氢酶缺乏症患者。

Congenital lactic acidosis of childhood is characterized by persistently high levels of blood pyruvate and lactate. In Japan, the number of reported cases of congenital lactic acidosis is increasing. Hereditary deficiencies leading to lactic acidosis have been demonstrated in the enzymes of the pyruvate dehydrogenase complex, in the key enzymes of the pathway of gluconeogenesis and in the enzymes of the electron transport system. For establishment of a diagnosis of congenital lactic acidosis, the usual procedures are measurements of the activities of enzymes involved in pyruvate metabolism in cultured skin fibroblasts. However, it takes long time to culture skin fibroblasts. Therefore, we had developed a rapid method for diagnosis of congenital lactic acidosis using platelets and lymphocytes separated from peripheral blood samples. In this method, the rates of decarboxylation of (1-^<14>C)pyruvate and (3-^<14>C)pyruvate and the activity of cytochrome c oxidase in platelets and the activities of pyruvate carboxylase, and phosphoenol pyruvate carboxykinase in lymphocytes were measured.Platelets and lymphocytes were separated from the peripheral blood samples of 40 patients with congenital lactic acidosis which were transported from the hospitals in every part of Japan. Then, the rates of decarboxylation of pyruvate and the activities of enzymes involved in pyruvate metabolism were measured. The rates of decarboxylation of pyruvate and the activity of cytochrome c oxidase in the platelets and the activity of pyruvate carboxylase in the lymphocytes were unstable and decreased during the trans portation of blood samples. Therefore, in order to diagnose the enzyme defects, blood samples which were simultaneously obtained from control subjects were required. Using this method, we found a patient with pyruvate dehydrogenase deficiency in 40 patients with congenital lactic acidosis.