基于呼出气代谢组学的肝衰竭“肝臭”中医嗅诊辨证客观化研究

Hepaticus fetor, as one of hepatic failure characteristic clinical symptoms, is due to the changes of metabolic disorder and appear breath gas.which has been found relevance to the progress of hepatic failure.Nowadays, olfactory examination on hepaticus fetor, as the basic elements of TCM diagnosis,in hepatic failure patients often have a basis on doctor’s subjective sense, which is lack of objective standard and hard to closely correspond with the syndrome differentiation.Existing hepatic failure in TCM syndrome differentiation standard, did not include the content of hepaticus fetor and the evaluation value for syndrome differentiation failed to reflect..In our previous study, there were differences existed in the feature of the hepaticus fetor between Yang huang and Yin huang.Therefore, in this project, with the breath samples from as the research object and the normal breath samples as the negative control, GC-MS-based breath metabonomics technology would be adopted to present the metabolite differentiation for screening biomarkers from different syndrome types (Yang huang and Yin huang). And a corresponding relationship would be established within different syndrome types in Chronic hepatitis B with acute hepatic failure samples. Finally, the conclusion would be tested with actual situation by the retrospective and prospective clinical samples examination based on the established metabonomics analysis methods. Aim to explain the connotation of olfactory examination, to objectify the olfactory examination on hepaticus fetor in hepatic failure patients, and to provide the reference for the prognosis judgement and curative effect evaluation of hepatic failure.

“肝臭”作为肝衰竭阶段特征性临床症候，是由于机体代谢紊乱而出现呼出气的变化所致，目前已发现肝臭与肝衰竭进展存在关联。肝臭是肝衰竭中医嗅诊辨证的基本内容，但诊察过程主观性强，未与中医辨证分型明确对应，现有的肝衰竭中医辨证标准中，未纳入肝臭中医嗅诊辨证的内容，其对辨证分型、预后判断的价值未能体现。课题组前期发现肝衰竭阳黄证、阴黄证肝臭存在差异，本项目基于气相色谱-质谱(GC-MS)联用的代谢组学方法，以慢性乙型病毒性肝炎慢加急性肝衰竭患者呼出气样本为研究对象，与正常呼出气样本对照，通过表征呼出气代谢物差异，筛选阳黄证、阴黄证呼出气代谢标志物，建立肝衰竭肝臭中医嗅诊辨证分型的明确对应，并与临床生化指标建立关联，最后对回顾性、前瞻性临床样本进行基于呼出气代谢标志物的阳黄证、阴黄证分型预判，确证肝臭中医嗅诊辨证的物质基础，实现肝衰竭肝臭中医嗅诊辨证的客观化，为肝衰竭预后判定及疗效评估提供依据。

肝臭是肝衰竭的重要临床特征，也是中医嗅诊辨证的重要内容，目前尚无对肝臭中医嗅诊辨证的客观化研究。现有《病毒性肝炎中医辨证标准》未纳入“肝臭”中医嗅诊辨证的内容，限制了肝臭中医嗅诊辨证的临床应用。本项目以乙型肝炎慢加急性肝衰竭患者为研究对象，通过代谢组学方法分析研究对象的呼出气冷凝液样本，表征呼出气代谢物及其差异，阐释肝臭中医嗅诊辨证的科学内涵。. 前期通过分析肝衰竭患者阳黄证与阴黄证的临床基线资料，建立Logistic回归方程，最终确定病程、胆碱酯酶、白细胞为判断肝衰竭阴黄证、阳黄证的独立危险因素；同时根据约登指数判定病程＞4周，WBC＜6.55×10^9/L、CHE＜1509.5U/L为辨别阴黄证的主要客观化依据；白蛋白、血小板、中性粒细胞、中性粒细胞淋巴细胞比值降低以及存在肝硬化基础可作为临床辨别阴黄证的次要客观化依据，并证实阴黄证较之阳黄证预后不佳，为临床乙型肝炎慢加急性肝衰竭阳黄证与阴黄证的诊断提供客观化依据，建立了阴黄证与临床指标的关联。之后以乙型肝炎慢加急性肝衰竭阴黄证患者呼出气样本为研究对象，发现呼出气中41种代谢物可作为区分肝衰竭患者不同预后的候选生物标志物，最终筛选四种化合物：磷脂酰肌醇、磷脂酰乙醇胺、异丙肾上腺素、苯乙烷，构建相关回归模型进行验证，模型的综合诊断价值为0.859，该呼出气代谢物组合可作为预测肝衰竭患者预后不良的重要潜在生物标志物。之后通过比较慢加急性肝衰竭与乙肝肝硬化患者的呼出气代谢物，发现十二烷醇、1-1-甲基乙氧基2-丙酮、二氧化碳、1-1-环己烯-1-基乙酮和邻环己烯这几种化合物相对丰度有显著差异，同时十二烷醇的特异性和敏感性最高，可作为预测乙肝肝硬化失代偿期进展为慢性肝衰竭的一种潜在的生物标志物。上述研究结果可作为肝衰竭无创评估方法以预测乙肝慢加急性肝衰竭的进展及预后，有利于提高中医辨证论治的精准度，为肝衰竭诊疗提供参考。