Department of Biochemistry, Jagiellonian University Medical College, Cracow, Poland.
J Physiol Pharmacol. 2012 Dec;63(6):613-21.
Melatonin is a major biosynthetic product of pineal gland exerting a potent antioxidant and the reactive oxygen metabolites scavenging activities but the mechanism of formation of this indole at extrapineal sources has not been fully elucidated. It is known that the gastrointestinal (GI)-tract plays an important role as a source of melatonin synthesis but the conversion of L-tryptophan into melatonin in the GI-tract of experimental animals and humans should be further examined. In this study, the conversion of L-tryptophan to melatonin was determined in the serum collected from rats administered intragastrically with this amino acid acting as melatonin precursor. For this purpose, a simple, sensitive and reliable method was developed for simultaneous determination of six L-tryptophan metabolites in rat serum, namely, 5-hydroxytryptamnie (5-HT), 5-hydroksytryptophan (5-HTR), kynurenin (KYN), antranilic acid (AA), indole-3-acetic acid (IAA) and melatonin that were analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with UV and native fluorimetric detection with multiple wavelengths. We used nucleosil Supelco C18 5 μm 4.6 mm x 250 nm column with the standard mobile phase consisting of solvent A (water/0.1% trifluoroacetic acid (TFA) and solvent B (methanol/0.1% TFA) in gradient elution. Fifty five rats received vehicle (saline) of L-tryptophan (50 mg/kg) or melatonin (50 mg/kg) by means of intragastric gavage and they were anesthetized and sacrificed at 0, 10, 20, 30, 60, 120 or 240 min upon L-tryptophan or melatonin administration for the venous blood withdrawal. The serum collected samples were kept on ice for the HPLC determination. The average recovery of 5-HT, 5-HRT, KYN, AA, TRP, IAA, and melatonin were 99±3%, 97±1.5%, 94±2.5%, 99±2.46, 98±1.5 and 98±2%, respectively. We conclude that 1) L-tryptophan is converted to melatonin in the GI-tract during the day when the pineal gland synthesis is inhibited, and 2) the reverse phase high performance liquid chromatography (RP-HPLC) is a new sensitive and reliable method that could be successfully applied to the study of kinetics and metabolism of L-tryptophan in GI-tract.
褪黑素是松果腺产生的一种主要生物合成产物，具有很强的抗氧化和清除活性氧代谢物的活性，但 extrapineal 来源形成这种吲哚的机制尚未完全阐明。已知胃肠道 (GI) 道作为合成褪黑素的重要来源发挥作用，但应进一步检查实验动物和人类 GI 道中 L-色氨酸转化为褪黑素的情况。在这项研究中，通过测定从给予胃内 L-色氨酸的大鼠血清中测定 L-色氨酸向褪黑素的转化，L-色氨酸作为褪黑素前体。为此，开发了一种简单、灵敏和可靠的方法，用于同时测定大鼠血清中的六种 L-色氨酸代谢物，即 5-羟色胺 (5-HT)、5-羟色氨酸 (5-HTR)、犬尿氨酸 (KYN)、邻氨基苯甲酸 (AA)、吲哚-3-乙酸 (IAA) 和褪黑素，通过高效液相色谱法 (HPLC) 与紫外和原生荧光检测并用多波长进行一次色谱运行即可分析。我们使用 Nucleosil Supelco C18 5 μm 4.6 mm x 250 nm 柱，标准流动相由溶剂 A（水/0.1%三氟乙酸 (TFA)）和溶剂 B（甲醇/0.1%TFA）组成，在梯度洗脱中。55 只大鼠分别给予载体（生理盐水）或 L-色氨酸（50 mg/kg）或褪黑素（50 mg/kg），通过胃内灌胃，在给予 L-色氨酸或褪黑素后 0、10、20、30、60、120 或 240 分钟时麻醉并处死，用于静脉采血。收集的血清样品保持在冰上用于 HPLC 测定。5-HT、5-HTR、KYN、AA、TRP、IAA 和褪黑素的平均回收率分别为 99±3%、97±1.5%、94±2.5%、99±2.46%、98±1.5%和 98±2%。我们得出结论：1）白天当松果腺合成被抑制时，L-色氨酸在胃肠道 (GI) 道中转化为褪黑素，2）反相高效液相色谱法 (RP-HPLC) 是一种新的灵敏可靠的方法，可成功应用于 GI 道中 L-色氨酸的动力学和代谢研究。
