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

这项研究是为了诊断DMD患者的营养状况而开始的。维生素B（硫胺素）以四种化学形式存在于血液中：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，采用高效液相色谱（HPLC）进行定量分析。结果显示，虽然DMD患者与对照组之间的总硫胺素含量没有差异，但TDP和TTP的比例有一定差异。另一方面，人体正常血液在不同温度下的储存，HPLC的定量值也会发生变化。这些事实可能是由于酶的存在：TDP激酶催化由TDP合成ttp。采用等电聚焦仪（Rotofor）和TDP-配体亲和层析技术，以蛋白结合的TDP为标记物进行TDP激酶的纯化。将分离得到的蛋白作为TDP激酶全酶状态的重构材料，但未观察到该激酶的作用。通过平衡透析仪评估分离蛋白的tdp结合活性，但尝试也不成功（平成1年）。因此，对TDP激酶的性质进行了进一步的研究，发现该酶具有sh基团，存在于红细胞中，需要某种金属离子和血浆中某种低分子成分作为辅因子（Heisei 2年）。新增加了饱和度为40 ~ 60%的硫酸铵分馏和DEAE -SephaceL色谱柱。将二硫苏糖醇加入到样品中，有效地引入了三种分子量分别为21OKD、18OKD和125KD的蛋白质。为了分离这三种蛋白，分别尝试使用了tdp -配体亲和柱、Dye -配体亲和柱以及rotoform和Rotofor。同时，利用DMD患者的血液研究了TDP激酶与DMD的关系。结果显示，DMD患者的TDP激酶活性高于对照组，因此TTP含量增加（Heisei 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，这项研究应继续进行。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