Elucidation of a Novel Metabolic Pathway of Morphine and Its Physiological Role

我们证实吗啡6-脱氢酶产生的吗啡酮（MO）对小鼠的毒性约为吗啡(M)的9倍（s.c注射）和4倍（i.c注射）。经MO预处理，单次滴注预处理1小时后，M的镇痛活性下降至对照组的20%，且这种作用在MO预处理后至少持续168小时。MO被进一步代谢为吗啡酮-谷胱甘肽加合物（MO- gsh），该代谢物在豚鼠胆汁中排泄。MO- gsh给药（500 mg/kg）无毒性，但给药剂量约为灌胃给药MO致死量的2倍。现将目前的调查总结如下。1. 产生MO的酶活性局限于细胞质中，在较小程度上也存在于动物（豚鼠、大鼠、家兔、小鼠和仓鼠）的每个组织（肝、肾、肺、小肠和脑）的微粒体中。2. 羟基自由基清除剂抑制了M的微粒体反应；因此，有可能由nadph -细胞色素P-450产生的羟基自由基形成MO。结果表明，MO是由Fe(II)- edta -抗坏血酸形成的。3. 痕量的M-GSH和mo -半胱氨酸加合物也被检测到，并通过与真实样品的比较进行了鉴定。4. 建立了用高效液相色谱法测定豚鼠尿液和胆汁中钼及其非共轭代谢物的方法。5. 用马来酸二乙酯（500 mg/kg）或与M同时注射石胆酸-3-硫酸酯（25 mg/kg）对豚鼠进行预处理，与未处理的动物相比，MO- gsh的胆道排泄减少，MO的胆道排泄增加。6. 经s.c注射纳洛酮（25 mg/kg）预处理的豚鼠胆道中MO和MO- gsh的排泄量均增加。7. 经二维核磁共振鉴定，MO-GSH的结构为8(S)-（谷胱甘肽-S-基）二氢吗啡酮。8. 在MO-GSH上的正极gaba - mass上观察到典型的三个钠离子阳离子峰。这些钠离子离子可用于代谢物的检测和鉴定。少

We have demonstrated that morphinone (MO), which produced by morphine 6-dehydrogenase, gave about 9 times (s.c. injection) and 4 times (i.c. injection) of the toxicity of morphine (M) in mice. By the pretreatment of MO, the analgesic activity of M was decreased to 20 % that of control after 1 hr pre-treatment by a single i.c. injection, and this effect continued for at least 168 hr after MO pretreat-ment. MO was further metabolized to from morphinone-glutathione adduct (MO-GSH), and this metabolite was excreted in the bile of guinea pig. MO-GSH gave no toxicity by s.c. injection (500 mg/kg), however, it gave about 2 timea of the lethal dose of MO in mice by i.c. injection.Summary of the present investigations are as follows. 1. The enzyme activity to produce MO was localized in the cytosol and to a lesser extent in the micro-somes of each tissue (liver, kidney, lung, small intestine and brain) of animals (guinea pig, rats, rabbits, mice and hamsters). 2. The production of MO from M by … More microsomal reaction was suppressed by hydroxyl radical scavengers; consequently, it is possible that the hydroxyl radical generated from NADPH-cytochrome P-450 func-tions to form MO. It was shown that MO was formed by Fe(II)-EDTA-ascorbate. 3. Trace amounts of M-GSH and MO-cysteine adduct were also detected and identified by comparison with the authentic samples. 4. We establish the method for determination of not only MO but also M and its unconjugated metabolites in the urine and bile of guinea pig by high-performance liquid chromatography. 5. Pretreatment of guinea pig with diethyl maleate (500 mg/kg) or simutaneous injection of lithocholic acid-3-sulfate (25 mg/kg) with M decreased the biliary excretion of MO-GSH and increased that of MO in comparison with untreated animals. 6. Pretreatment of guined pig by s.c. injection with naloxone (25 mg/kg) increased biliary excretion of both MO and MO-GSH. 7. the unambiguous structure assignment of MO-GSH was identified as 8(S)-(glutathion-S-yl)dihydromor-phinone by two dimensional NMR spectroscopy. 8. The typical three sodium cationized ion peaks are observed on the positive GAB-Mass with sodium iodide on the MO-GSH. These sodium cationized ion praks were available for the detection and iden-tification of the metabolite. Less