血中乙醇和胆汁酸分析方法的建立及临床应用

发布时间：2018-01-19 00:19

  本文关键词： 乙醇 胆汁酸 电化学传感器 妊娠期无症状高胆汁酸血症 妊娠肝内胆汁淤积症　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：建立灵敏、简单、可用于血液中生物活性物质测定的分析方法是临床检验诊断学学科发展的重要方向之一。电化学分析法灵敏、快速,且所用仪器简单、易于微型化,在临床疾病的辅助诊断和标志物的快速检测中扮演着重要角色。而液相色谱-质谱联用技术具有灵敏度高、结果准确且重现性好等优点,可实现血液中已知和未知的多种代谢物同时检测。鉴于此,本文以氧化/还原型烟酰胺腺嘌呤二核苷酸(NAD+/NADH)为基础,建立了简单、实用的血液中乙醇和总胆汁酸(total bile acid,TBA)的电化学分析方法;并采用超高效液相色谱-四级杆飞行时间质谱(UPLC-Triple TOF-MS/MS)技术对妊娠肝内胆汁淤积症(intrahepatic cholestasis of pregnancy,ICP)和妊娠期无症状高胆汁酸血症(asymptomatic hypercholanaemia of pregnancy,AHP)患者血清胆汁酸代谢轮廓进行全面分析。主要内容包括以下四个部分:1.免修饰丝网印刷碳电极(SPCE)-电化学测定血浆乙醇方法的建立及应用。利用乙醇脱氢酶(ADH)和NAD+特异性催化乙醇生成乙醛和NADH的氧化-还原体系,采用SPCE并结合直接稀释血液的策略,实现血浆中乙醇的电化学测定。血浆样本经磷酸盐缓冲液(PBS)稀释后与ADH和NAD+室温共反应,再滴加到SPCE表面,采用示差脉冲伏安法(DPV)进行测定。该方法线性良好(0.10~3.20 mg/m L,r=0.9943),灵敏度较高(LOD,40.0mg/m L)。精密度和回收率分别为(RSD,5.1%~9.4%)和(80.1%~103%)。将建立的方法用于20例饮酒自愿者血浆乙醇的测定并与气相色谱(GC)法测定结果比较,相关性良好(r=0.9311)。该方法简单、准确,有望用于酒精中毒、酒驾和醉驾等的快速筛查。2.基于双重氧化的血清TBA超灵敏电化学传感器的构建及临床应用。含3α羟基类固醇脱氢酶(3α-HSD)、NAD+和三联吡啶钌(Ru(bpy)32+)的PBS溶液滴加到SPCE电极表面并施加恒电位,加入稀释血样,3α-HSD特异性催化血清中胆汁酸生成NADH,NADH与被氧化的Ru(bpy)33+反应生成Ru(bpy)32+,Ru(bpy)32+再被氧化为Ru(bpy)33+产生电化学信号,采用计时电流法测定,实现血清TBA超灵敏检测。该方法测定血清TBA的线性良好(5.0~150.0mmol/L,r=0.9983),灵敏度较高(LOD,0.40mmol/L)。精密度和回收率分别为(RSD,6.4%~11.8%)和(91.4%~108%)。将建立的方法用于正常孕妇(20例)、ICP患者(30例)和AHP患者(22例)血清样本测定并与酶循环法测定结果比较,相关性良好(r=0.9372)。与正常孕妇相比,ICP与AHP患者血清TBA均显著增高。该方法灵敏、快速有望用于床旁检测。3.AHP与ICP患者血清胆汁酸代谢轮廓分析。利用UPLC-Triple TOF-MS/MS对27例正常孕妇、26例AHP患者和59例ICP患者血清中57种胆汁酸的代谢轮廓进行分析。采用PLS-DA显示AHP患者的血清胆汁酸代谢轮廓与正常孕妇和ICP孕妇均具有明显差异,VIP值1的24种胆汁酸,其中甘氨脱氧胆酸(GDCA)、牛磺三羟基胆汁酸的同分异构体4(Ttri-4)、牛磺-ω-鼠胆酸(T-ω-MCA)、甘氨猪胆酸(GHCA)、甘氨三羟基胆汁酸同分异构体3(Gtri-3)这5种胆汁酸在三组间均有显著差异(P0.05)。采用Logistic回归筛选出疾病组(AHP和ICP)与正常组的联合诊断标志物甘氨胆酸(GCA)、甘氨三羟基胆汁酸同分异构体1(Gtri-1)和牛磺二羟基胆汁酸同分异构体1(Tdi-1),ROC曲线下面积(AUC)为0.989,灵敏度98.9%,特异性100%;鉴别AHP与ICP的联合诊断标志物石胆酸(LCA)、Gtri-1和鹅脱氧胆酸(CDCA),AUC为0.956,灵敏度92.1%,特异性91.7%。胆汁酸代谢轮廓分析与成分胆汁酸的检测有助于临床对AHP的认识,诊断与鉴别诊断。4.芯片-电化学法检测血清TBA的方法学研究。固相萃取(SPE)预处理的血清样本经聚二甲基硅氧烷(polydimethylsiloxane,PDMS)芯片通道电驱动进样后电泳到检测池,血清胆汁酸与检测池中的3α-HSD、Ru(bpy)32+和NAD+发生酶促偶联-双重氧化还原反应,生成的Ru(bpy)32+在SPCE电极上被氧化产生电信号,采用计时电流法进行分析。该方法测定血清TBA的线性良好(1.0~100.0mmol/L,r=0.9995),灵敏度较高(LOD,0.10mmol/L)。精密度和回收率分别为(RSD,5.9%~8.9%)和(94.9%~102%)。该方法为后续建立微流控芯片-电化学检测胆汁酸代谢轮廓奠定基础。
[Abstract]:To establish a sensitive, simple and can be used for determination of bioactive substances in the blood is a clinical test for the diagnosis of discipline development one of the important directions. The electrochemical analysis method is sensitive, rapid, and the instrument is simple, easy miniaturization, plays an important role in the rapid detection and diagnosis marker of clinical disease. The liquid chromatography-mass spectrometry technique has high sensitivity, accuracy and repeatability, can achieve a variety of known and unknown metabolites in the blood are detected simultaneously. In view of this, the oxidation of nicotinamide adenine dinucleotide / (NAD+/NADH) as the foundation, established a simple, practical and total bile acid ethanol the blood (total bile, acid, TBA) and electrochemical analysis method; using ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (four UPLC-Triple TOF-MS/MS) on pregnancy with intrahepatic cholestasis (int Rahepatic cholestasis of pregnancy, ICP) and no symptoms of pregnancy hyperbileacidemia (asymptomatic hypercholanaemia of pregnancy, AHP) in patients with serum bile acid metabolic profile were analyzed. The main contents include the following four parts: 1. free modified screen printed carbon electrode (SPCE) - the establishment and application of plasma ethanol electrochemical determination methods. The use of alcohol dehydrogenase (ADH) - and NAD+ specific catalytic oxidation of ethanol to acetaldehyde and the reduction of NADH by SPCE system, combined with the direct blood dilution strategy, electrochemical determination of ethanol in plasma. Plasma samples were extracted with phosphate buffer solution (PBS) were diluted with ADH and NAD+ at room temperature, and then dropped to the SPCE surface. By using differential pulse voltammetry (DPV) were measured. This method has good linearity (0.10~3.20 mg/m, L, r=0.9943), high sensitivity (LOD, 40.0mg/m L). The precision and recovery rate were RSD (, 5.1%~9.4%) and (80.1%~103%). The established method for the measurement of 20 cases of drinking alcohol and plasma volunteers with gas chromatography (GC) method for the determination of results, good correlation (r=0.9311). This method is simple, accurate, is expected to be used for fast screening of.2. alcohol poisoning, drunk driving and drunk driving and other construction and clinical application of serum TBA double oxidation ultra sensitive electrochemical sensor based on containing 3 alpha hydroxysteroid dehydrogenase (3 alpha, NAD+ and -HSD) terpyridyl ruthenium (Ru (bpy) 32+) PBS solution is dripped into the SPCE electrode surface and applying a constant potential, diluted blood samples, 3 alpha -HSD specific catalytic serum bile acid to generate NADH, NADH and Ru (bpy) 33+ oxidation reaction of Ru (bpy) 32+, Ru (bpy) 32+ is oxidized to Ru (bpy) 33+ produce electrochemical signals, measured by chronoamperometry, the serum TBA ultra sensitive detection. The linear determination of serum TBA and this method is good (5.0~150.0mmol/ L, r=0.9983), high sensitivity (LOD, 0.40mmol/L). The precision and recovery rate respectively (RSD, 6.4%~11.8%) and (91.4%~108%). The established method for normal pregnant women (20 cases), ICP patients (30 cases) and AHP patients (22 cases) serum samples were measured and compared with the results of enzyme recycling determination, good correlation (r=0.9372). Compared with the normal pregnant women, ICP and AHP in sera of patients with TBA were significantly increased. The method is sensitive, rapid analysis is expected to be used for bedside detection of.3.AHP and ICP in serum of patients with bile acid metabolic profile. On 27 pregnant women with UPLC-Triple TOF-MS/MS, 26 cases of 57 kinds of metabolic profile in patients with AHP and 59 ICP patients serum bile acids were analyzed. The PLS-DA showed the serum bile acid metabolic profile in patients with AHP and normal pregnant women and pregnant women with ICP has the obvious difference, VIP value of 24 kinds of 1 bile acids, including glycine deoxycholate acid (GDCA), three hydroxy bile acid with sodium 鍒嗗紓鏋勪綋4(Ttri-4),鐗涚：-蠅-榧犺儐閰，

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