线粒体乙醛脱氢酶基因多态E487K对硝酸甘油疗效和代谢的影响

【作者】 李一峰；

【导师】 金力；

【作者基本信息】 复旦大学 ， 遗传学， 2004， 博士

【摘要】 硝酸甘油是一种已经应用百余年的扩血管的药物，具有重要的临床应用价值。硝酸甘油是治疗心绞痛的一线首选药物，其中舌下含服硝酸甘油是治疗心绞痛的常规治疗方法，一般来说在含服后5-10分钟之内症状缓解。近来，有研究表明线粒体乙醛脱氢酶是体内催化硝酸甘油生物转化的主要途径，线粒体乙醛脱氢酶同时具有脱氢酶和酯酶的催化活性，其酯酶活性可以通过将硝酸甘油脱硝形成一氧化氮，进而扩张血管。线粒体乙醛脱氢酶是尼克酰胺腺嘌呤二核苷酸依赖性酶超基因家族中的一员，在线粒体乙醛脱氢酶第12个外显子存在一个G／A单核苷酸多态，相应在其编码的蛋白质第487位氨基酸残基由赖氨酸替代谷氨酸。同时这个单核苷酸多态特异性地存在于东亚人群中，并且有较高的发生频率（大约30％）。突变型酶（E487K）对其脱氢酶底物一乙醛的催化活性的严重下降（活性为正常酶活性的6％）；同时，这个单核苷酸多态也会明显的影响线粒体乙醛脱氢酶酯酶的催化活性。硝酸甘油和乙醛互为竞争性抑制剂的事实也暗示线粒体乙醛脱氢酶对硝酸甘油和乙醛的催化活性中心重叠。这让我们推测这个多态是否同时会影响硝酸甘油扩张血管效应呢?我们收集了586名临床冠脉造影确诊的冠心病患者血样，筛选单纯应用硝酸甘油治疗心绞痛的患者80名；通过舌下含服硝酸甘油治疗心绞痛临床疗效与个体线粒体乙醛脱氢酶基因型的关联分析，证明了线粒体乙醛脱氢酶E487K基因多态显著影响硝酸甘油的疗效（X²=7.59，P＜0.01）。然后，从胎儿肝脏组织中提纯了两种基因型人线粒体乙醛脱氢酶（基因型为野生型和杂合型）；同时利用昆虫细胞表达系统表达两种不同基因型人线粒体乙醛脱氢酶（基因型为野生型和异常纯合型），然后利用¹⁴C标记的硝酸甘油作为底物检测人类不同基因型线粒体乙醛脱氢酶催化硝酸甘油生物转化的酶学指标（米曼常数Km和最大速度Vmax），结果显示不同基因型线粒体乙醛脱氢酶催化硝酸甘油生物转化效率差别显著（分别相差11倍和9倍）。同时通过InsightⅡ软件的力场模拟模块模拟硝酸甘油与不同基因型线粒体乙醛脱氢酶反应的动力学过程，进一步说明了酶学性质差别的分子机制。另外，经过基因分型入选20名不同基因型线粒体乙醛脱氢酶自愿者（其中基因型为野生型10人；基因型为杂合型5人；基因型为异常纯合型5人），并由气相色谱检测他们使用相同剂量的硝酸甘油后体内各项药物代谢动力学参数，结果显示不同基因型的个体药物代谢半衰期，曲线下面积等各项参数差别不显著。通过上述系列实验充分说明了E487K单核苷酸多态明显影响线粒体乙醛脱氢酶催化硝酸甘油生物转化的效率，并且可以部分解释中国人群中心绞痛患者对硝酸甘油的药物疗效差异的机制，但是对硝酸甘油清除性代谢的影响并不显著。更多还原

【Abstract】 Nitroglycerin （NTG） has been very widely used for the management of coronary heart disease, since its first clinical application in 1876. The sublingual administration of nitroglycerin is the standard treatment for acute attacks of angina pectoris, and the symptom is usually relieved within 5 to 10 minutes.Recently, it has been shown that the Mitochondrial aldehyde dehydrogenase is responsible for the bioactivation of nitroglycerin prior to the release of NO, which is a strong vasodilator. Mitochondrial aldehyde dehydrogenase, a member of a superfamily of NAD（P）（+）-dependent enzymes has both dehydrogenase activity and eaterase activity, which can catalyze the biotransformation of nitroglycerin. A functional single nucleotide polymorphism （SNP） in exon 12, which leads to a replacement of glutamate by lysine at position 487 virtually eliminating its ability to clear acetaldehyde in the carriers, has high prevalence in Asian populations including Chinese with the allelic frequency around 30%. The fact that acetaldehyde and nitroglycerin can be the compitive inhibitor reciprocally and the mutant enzyme also lose esterase activity makes us speculate that this olymorphism can affect the biotransformation of nitroglycerin in vivo.80 stable angina pectoris patients were recruited from total 576 angiographically defined coronary heart disease patients according to the inclusion criteria, and interindividual variation of the efficacy of sublingual nitroglycerin is significantly associated with this mitochondrial aldehyde dehydrogenase polymorphism in Chinese （X² = 7.59, P < 0.01）. Further, all three genotype enzymes were obtained by either purification from human fetus livers or expression in the Bac-to-Bac baculovirus system. The kinetic properties of each genotype was characterized by the thin layer chromatography and liquid scintillation spectrometry with the [2-¹⁴C] nitroglycerin as the substrate, The catalytic efficiency of mitochondrial aldehyde dehydrogenase （Vmax/Km） of different genotype differs significantly （9 and 11 times respectively）. Furthermore, the molecular mechanism underlying the different catalytic efficiency was discussed with the use of simulation software-insight II. Finally, we enrolled 20volunteers of three genotypes （wild-type 10 individuals; heterozygote 5 individuals; mutant homozygotes 5 individuals）, After taking nitroglycerin, the pharmacokinetics curve was obtained with the use of gas chromatography. No difference was found with respect to half-life and AUC etc. These observation suggest that the presence of the A allele is at least partially but significantly responsible for the failure or reduced response to nitroglycerin; therefore, genetic factor should be involved in the consideration of the administration of nitroglycerin especially in oriental populations.更多还原