The nutritional attempt to investigate progressive muscular dystrophy with Duchenne type in human.

这项研究是为了诊断DMD患者的营养状态而开始的。维生素B，即硫胺素，在血液中以四种化学形式存在：一磷酸硫胺素(TMP)、二磷酸硫胺素(TDP)、三磷酸硫胺素(TTP)和游离硫胺素。根据柱后荧光法，用高效液相色谱(HPLC)对这些物质进行定量分析。结果表明，虽然DMD患者血清总硫胺素含量与对照组无差异，但TDP和TTP的比例有一定差异。另一方面，正常人血液在不同温度下保存后，其高效液相色谱定量测定值发生变化。这可能是由于催化TDP合成TTP的TDP激酶的存在所致。以蛋白质结合的TDP为标记物，采用制备型等电聚焦装置(Rotofor)和TDP配基亲和层析对TDP激酶进行纯化。…此外，该同位素蛋白被用作TDP激酶全酶状态的重构材料，但没有观察到该激酶的作用。用平衡透析仪评估了同位素蛋白的TDP结合活性，但尝试也没有成功(平成1年)。因此，我们进一步研究了TDP-Kinase的性质，发现该酶含有一个SH基团，存在于红细胞中，需要一定的金属离子和血浆中的某些低分子组分作为辅因子。提纯过程中新增加了40%~60%饱和度的硫酸铵分级和DEAE-SephaceL柱。二硫苏糖醇的加入有效地引入了三种相对分子质量分别为21OKD、18OKD和125KD的蛋白质。为了分离这三种蛋白质，已经尝试使用TDP-配体亲和色谱柱、染料-配基亲和色谱柱以及Rotoformn和Rotofor。同时，以DMD患者的血液为研究对象，探讨TDP-Kinase与DMD的关系。结果表明，DMD患者TDP-Kinase活性明显高于正常对照组，TTP含量升高(平成3年)。目前尚不清楚患者的状态是由于疾病还是疾病的结果，这项研究应该继续下去。较少

This research has been started for diagnosis of the nutritional state of the patient with DMD. Vitamin B, (thiamin)is presence in blood as four chemical forms : thiamin monophosphate(TMP), thiamin diphosphate(TDP), thiamin triphosphate(TTP), and free thiamin. The quantitative analyses of these are performed by high-performance liquid chromatography(HPLC)according to a post column fluorescence method. In the resuits, although there is no difference of the total thiamin content between the DMD patient and control, a certain difference is observed in the proportions of TDP and TTP. On the other hand, the quantitative values by HPLC changed under the storage of normal human blood at various temperature. These facts might be due to the existence of the enzyme : TDP Kinase which catalysis the synthesis of TTPfrom TDP. The purification of TDP Kinase was performed using protein-bound TDP as a marker by a preparative isoelectric focusing apparatus(Rotofor)and TDP-ligand affinity chromatography. … More The isotated protein was used as reconstitutive material for HoLoenzyme state of the TDP Kinase, but the action of the Kinase was not observed. The TDP-binding activity of the isotated protein was assessed by an equilibrium dialysis apparatus, but the attempt was unsuccessful too(Heisei 1 year). Therefore, the nature of TDP Kinase has been further investigated and has been revealed that the enzyme possessed a SH-group, existed in erythrocytes, and required a certain metal ion and a certain low-molecular component in plasma as a cofactor'(Heisei 2 year). Fractionation with ammonium sulfate between 40 and 60% saturation and DEAE -SephaceL column were newly added in purification procedure. The addition of dithiothreitol to the sample effectively introduced three proteins having molecular weight as 21OKD, 18OKD, and 125KD. For separation of the three proteins, attempts to use TDP-ligand affinity column, Dye -ligand affinity column and Rotoformn and Rotofor have been carried out. While, the relationship between TDP Kinase and DMD was investigated by using of the blood of DMD patients. The results showed that the activity of TDP Kinase was more high in DMD patients than controls, therefore, the TTP content resulted in an increase(Heisei 3 year). It is unknown that the state of the patients is due to the disease or is a result of it, this study shoud be continued. Less