Study of early detection of patients with inherited metabolic disorders characterized clinically by severe ketoacidosis.

酮症酸中毒的发作在儿童时期相当常见，但我们有时会遇到严重的患者。有一种可能性，遗传代谢紊乱，如有机酸中毒是隐藏在这样的病人。许多有机酸类化合物都可以用GC/MS快速检测出来，与有机酸类化合物密切相关的酮体分解代谢障碍物也被发现。酮体疾病包括3种类型的疾病，3-酮硫解酶缺乏症、细胞溶质乙酰乙酰辅酶A硫解酶（CT）缺乏症或琥珀酰辅酶A：3-酮酸辅酶A转移酶（SCOT）缺乏症。后两种疾病可能难以单独通过GC/MS诊断，因为其代谢谱通常是非特异性的。此外，目前难以准确评估培养细胞中的CT活性。我们开发了一种系统来精确诊断与严重酮症酸中毒相关的疾病。1）开发自动GC/MS数据分析和疾病检测系统：该自动系统能够分析尿有机酸并化学诊断有机酸中毒，这通常与严重酮症酸中毒相关。我们通过检测患有先前已知疾病的患者的尿液样本证实了该系统的有用性。2）开发用于检测CT缺乏的简单实用的测定方法：我们使用培养的淋巴母细胞并开发了CT的实用测定方法，从线粒体硫解酶或SCOT分离，两者都与酮体catalysts相关，使用细胞溶质和细胞器组分的快速digitaxin分离。通过免疫印迹和免疫滴定实验证实了分离的有效性。

Episodes of ketoacidosis are rather common in childhood, but we sometimes come across severe patients. There is a possibility that inherited metabolic disorders like organic acidemias are concealed in such patients. Many organic acidemias can be readily detected by GC/MS.Recently, ketone body catabolic disoredrs, which are closely related to organic acidemias, have also been recognized. The ketone body disorders include 3 types of diseases, 3-ketothiolase deficiency, cytosolic acetoacetyl-CoA thiolase (CT) deficiency or succinyl-CoA : 3-ketoacid CoA transferase (SCOT) deficiency. The latter two diseases may be difficult in diagnosis by GC/MS alone, because its metabolic profile is often nonspecific. Further, accurate assessment of CT activity in cultured cells is difficult at the present time. We developed a system to precisely diagnose the diseases associated with severe ketoacidosis.1) Development of the system of automated GC/MS data profiling and disease detection : This automated system enabled to profile urinary organic acids and chemically diagnose organic acidemias, which are often associated with severe ketoacidosis. We confirmed the usefulness of this system by testing urine samples from patients with previously known diseases.2) Development of simple and practical assay method for the detection of CT deficiency : We used cultured lymphoblasts and developed a practical assay method of CT,separating from mitochondrial thiolases or SCOT,both related to ketone body catabolism, using rapid digitonine separation of cytosol and organelle fractions. The efficiency of separation were confirmed by immunoblotting and immunotitration experiments.