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

儿童先天性乳酸酸中毒的特征是持续高水平的血液丙酮酸和乳酸。在日本，先天性策略性酸中毒的报告病例数正在增加。然而，在许多患有先天性乳酸性酸中毒的患者中，无法发现潜在的缺陷，并且大多数患者在婴儿期死亡或表现出生长和精神发育迟缓，因为没有足够的治疗。我们一直在努力开发一种方法来筛选先天性乳酸酸中毒患者培养的皮肤成纤维细胞中丙酮酸代谢障碍。我们开发了一种测定乳酸形成的测定方法，1- 1C<14>）培养的皮肤成纤维细胞中的丙酮酸。通过该方法，我们测量了培养的<14><14>丙酮酸脱氢酶（PDH）缺陷（1）和PDH激活缺陷（3）患者皮肤成纤维细胞中（1-^C）丙酮酸的乳酸形成率和（1-^ C）丙酮酸的脱羧率。线粒体丙酮酸转运缺陷（1），多发性羧化酶缺乏（1）。复合物I缺乏症（1）、复合物IV缺乏症（1）、原因不明的先天性战术性酸中毒（10）和16名对照受试者（细胞系的数目显示在括号中）。患者成纤维细胞的乳酸盐形成和脱羧速率低于对照成纤维细胞，因为这些患者的上限值与对照受试者的下限值重叠。与此相反，这些患者的乳酸形成和丙酮酸脱羧的比率（L/D比）没有重叠的控制，我们可以清楚地区分患者从控制subjects.Thus，L/D比的测量是有用的方法，用于筛选丙酮酸代谢障碍培养的皮肤：Bibroblasts。用这种方法，我们还发现10例不明原因的先天性策略性酸中毒患者中有2例存在丙酮酸代谢障碍。

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.