Screening for disorders of pyruvate metabolism by measurement of lactate production in cultured skin fibroblasts

儿童先天性乳酸性酸中毒的特点是持续高水平的血液丙酮酸和乳酸。在日本，报告的先天性战术酸中毒病例正在增加。然而，在许多先天性乳酸酸中毒患者中，由于缺乏足够的治疗，无法发现潜在的缺陷，大多数患者在婴儿期死亡或表现出生长和精神运动迟缓。因此，我们一直在尝试开发一种筛选先天性乳酸酸中毒患者培养皮肤成纤维细胞中丙酮酸代谢紊乱的方法。我们开发了一种测定培养皮肤成纤维细胞中（1-^<14>C）丙酮酸生成乳酸的方法。通过该方法，我们测量了丙酮酸脱氢酶（PDH）缺乏症(1)、PDH激活缺陷(3)、线粒体丙酮酸转运缺陷(1)、多种羧化酶缺乏症(1)、复合物I缺乏症(1)、复合物IV缺乏症(1)、（1-^<14>C）丙酮酸形成乳酸的速率和（1-^<14>C）丙酮酸脱羧的速率。原因不明的先天性战术酸中毒（10例）和对照组（16例）（细胞系数量在括号中显示）。患者的成纤维细胞乳酸形成和脱羧率低于对照组成纤维细胞，因为这些患者的高值与对照组的低值重叠。相比之下，这些患者的乳酸形成率和丙酮酸脱羧率（L/D）与对照组没有重叠，我们可以清楚地将患者与对照组区分开来。因此，测定L/D比值可作为筛选体外培养皮肤成细胞中丙酮酸代谢紊乱的一种方法。使用这种方法，我们也发现了丙酮酸代谢紊乱的十个患者中的两个先天性战术酸中毒的原因不明。

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 tactic acidosis is increasing. However, in many patients with congenital lactic acidosis, the underlying deficiency cannot be found and most patients die in infancy or show growth and psychomotor retardation because no adequate treatment is available.Therefore, we have been trying to develop a method for screening the disorders of pyruvate metabolism in cultured skin fibroblasts from patients with congenital lactic acidosis.We developed an assay method for measuring lactate formation from (1-^<14>C)pyruvate in cultured skin fibroblasts. By the method, we measured the rates of lactate formation from (1-^<14>C) pyruvate and decarboxylktion of (1-^<14>C) pyruvate in cultured skin fibroblasts from patients with pyruvate dehydrogenase (PDH) deficiency (1), defect of PDH activation (3). defect of mitochondrial pyruvate transport (1), multiple carboxylase deficiency (1). complex I deficiency (1), complex IV deficiency (1), congenital tactic acidosis of unknown cause (10) and 16 control subjects (Numbers of cell lines are shown in parentheses). The rates of lactate formation and decarboxylation in fibroblasts from the patients were lower than those in control fibroblasts, by the upper values for the these patients overlapped the lower values for control subjects. In contrast, the ratio of lactate formation and pyruvate decarboxylation (L/D ratio) of these patients did not overlap those of controls and we could clearly distinguish the patients from control subjects.Thus, measurement of L/D ratio is useful as a method for screening disorders of pyruvate metabolism in cultured skin :Bibroblasts. Using this mi-Bthod, we also found disorders of pyruvate metabolism in two of ten patients with congenital tactic acidosis of an unknown cause.