STUDIES ON IN VIVO METABOLISM OF TRYPTOPHAN AND ITS METABOLITES USING COMBINED GAS CHROMATOGRAPHY / MASS SPECTROMETRY WITH USE OF STABLE ISOTOPE LABELED COMPOUNDS

本文研究了色氨酸代谢物的GC/MS分析方法，并将氘标记物应用于色氨酸代谢物的GC/MS分析，得到了如下结果：建立了5-羟基吲哚-3-乙酸的高灵敏GC/MS分析方法，吲哚-3-乙酸和犬尿氨酸，并证实该方法可用于使用氘代色氨酸的代谢研究。L-色氨酸-3，3-d_2成功地用作色氨酸代谢为色胺、5-羟色胺、吲哚-3-乙酸和5-羟基吲哚-3-乙酸的示踪剂。然而，由于D-H交换反应，氘代色氨酸不能用作色氨酸到犬尿氨酸代谢研究的示踪剂。因此，合成了L-色氨酸-2，3-^C_2，用于色氨酸到其上述代谢物的同时代谢研究。<13>用稳定同位素标记的GC/MS方法，对尿中5-羟色胺和色胺的合成部位进行了研究。在5-羟色胺合成方面，除脑外，没有其他器官的5-羟色胺-d_2与内源性5-羟色胺的摩尔比高于尿。因此，尿中的5-羟色胺-d_2可能反映了脑内5-羟色胺的合成。尿中色胺-d_2可能来源于肝脏或其它外周器官。据信，血清素不能通过血脑屏障。然而，支持这一建议的数据尚未得到明确证实。因此，研究了5-羟色胺的渗透。并证实血清素能透过血脑屏障。本文对四名孤独症儿童进行了阿糖胞苷d_2的静注实验，发现阿糖胞苷d_2的排泄模式异常。

Investigations for development of sensitive GC/MS methods for the determination of tryptophan metabolites and application of the GC/MS methods with use of some deuterium labeled compounds to metabolic investigation were carried out and following results were obtained.Highly sensitive GC/MS methods for the determination of 5-hydroxyindole-3- acetic acid, indole-3-acetic acid and kynurenine were developed and the methods were confirmed to be useful for metabolic investigations using deuterated tryptophan. L Tryptophan-3,3-d_2 was successfully used as a tracer for metabolic investigation of tryptophan to tryptamine, serotonin, indole-3-acetic acid and 5-hydroxyindole-3- acetic acid. However, the deuterated tryptophan was not usable as a tracer for metabolic investigation of tryptophan to kynurenine because of D-H exchange reaction. L-Tryptophan-2,3-^<13>C_2 was therefore synthesized for simultaneous metabolic studies of tryptophan to its metabolites described above. By GC/MS methods with use of stable isotope labeled compounds, some experiments were carried out to investigate where urinary serotonin and tryptamine were synthesized. Concerning serotonin synthesis, there were no organs except the brain which have high molar ratio of serotonin-d_2 to endogenous one as that in urine. Therefore, it could be postulated that serotonin-d_2 in urine might reflect the serotonin synthesis in the brain. In the case of tryptamine, tryptamine-d_2 in urine could be derived form liver or other peripheral oragans. It is believed that serotonin could not pass through the blood brain barrier. However, the data supporting the suggestion have not been clearly confirmed. Therefore, the penetration of serotonin was investigated. And it was confirmed that serotonin could penetrate through blood brain barrier. Tacer experiments with use of tryptophan-d_2 were carried out in four autistic children and abnormal excretion pattern of serotonin-d_2 was found.