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

酮症酸中毒的发作是相当常见的儿童，但我们有时会遇到严重的病人。有可能在这些患者中隐藏了遗传性代谢紊乱，如有机酸血症。GC/MS可以很容易地检测出许多有机酸。近年来，与有机酸中毒密切相关的酮体分解代谢紊乱症也得到了认识。酮体疾病包括3-酮硫酶缺乏症、胞质乙酰乙酰辅酶a硫酶（CT）缺乏症和琥珀酰辅酶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，目前很难准确评估培养细胞的CT活性。我们开发了一个系统来精确诊断与严重酮症酸中毒相关的疾病。1)自动GC/MS数据分析和疾病检测系统的开发：该自动化系统能够分析尿有机酸和化学诊断有机酸血症，这通常与严重的酮症酸中毒有关。我们通过检测以前已知疾病患者的尿液样本证实了该系统的实用性。2)建立简单实用的CT缺乏症检测方法：我们利用培养的淋巴细胞，开发了一种实用的CT检测方法，分离线粒体硫酶或SCOT，两者都与酮体分解代谢有关，使用快速分离细胞质和细胞器部分的地黄碱。通过免疫印迹和免疫滴定实验验证了分离效果。

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.