Establishment Of The Methods For Ethanol And Bile Acids In Blood And Their Clinical Applications

The establishment of sensitive,simple and useful analytic assays for the measurement of bioactive analytes in blood is one of the most important trends in clinical laboratory diagnostics.Electrochemical assay is sensitive and fast.The instruments could be integrated and miniaturized,which makes it possible to be applied in diseases diagnosis and rapid detection of biomarkers for diseases.Liquid chromatography coupled with mass spectrometry technique has many advantages,such as high sensitivity,acceptable accuracy and good repeatability for the results.Therefore,it could be used for the simultaneous detection of known and unknown metabolites in blood.As a result,in this study several simple and practical electrochemical methods based on the nicotinamide adenine dinucleotide-oxidized and icotinamide adenine dinucleotide-reduced(NAD+/NADH)oxidation-reduction system were established and applied to detect ethanol and total bile acid(TBA)quantitatively in blood.Serum metabolic profiling of bile acids in patients with intrahepatic cholestasis of pregnancy(ICP)and asymptomatic hypercholanaemia of pregnancy(AHP)were analyzed by ultrahigh performance liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry(UPLC-TripleTOF-MS/MS)technique.This dissertation includes four parts:1.The development and application of the electrochemical method for the determination of plasma ethanol with screen-printed carbon electrode(SPCE).The alcohol dehydrogenase(ADH)and NAD+ catalyze ethanol to acetaldehyde and NADH specifically.Direct dilution of blood without any other pretreatments of samples was used and the detection was performed by SPCE electrochemical analysis.The plasma samples were diluted by phosphate buffer saline(PBS)solution and ethanol in plasma reacted with ADH and NAD+ at room temperature.The mixed solution was added onto the surface of the SPCE and detected by the differential pulse voltage(DPV)technique.The linear range of the established method is from 0.10 to 3.20 mg/mL,and the regression coefficient is 0.9943.The sensitivity is satisfactory with 40.0 ?g/mL of the limits of detection(LOD).The relative standard deviations(RSD)of the precisions range from 5.1% to 9.4%,and the recoveries range from 80.1% to 103%.Twenty plasma samples were measured both by the established method and gas chromatography(GC)method.And the correlation coefficient of the two methods is satisfactory(r=0.9311).The established method is simple and accurate with the potentiality to be used for the rapid screening of alcoholism,drink driving and drunk driving.2.The development of the electrochemical sensor based on enzymatic coupling double oxidation for the measurement of serum TBA.In the mixture of 3α-HSD,NAD+,Ru(bpy)32+ and PBS solution,3α-HSD specifically catalyzed the bile acids to produce NADH,and the NADH reacts with the oxidized Ru(bpy)33+ to produce Ru(bpy)32+,and the Ru(bpy)32+ is further oxidized to Ru(bpy)33+ at the surface of the SPCE electrode to produce electrochemical signals.An ultra-sensitive electrochemical method for the determination of serum TBA by enzymatic coupling double oxidation electrochemical assay was established.Serum samples were diluted with the PBS solution and quantitatively determined by the chronoamperometry method.The linear is ranging from 5.0 μmol/L to 150.0 μmol/L,and the regression coefficient is 0.9983.The sensitivity is high(LOD,0.40 μmol/L).The RSD of the precisions range from 6.4% to 11.8% and the recoveries range from 91.4% to 108%.Serum TBA in twenty normal pregnant women,thirty ICP and twenty-two AHP were measured by the established method and the enzyme circulation method,and the correlation coefficient is satisfied(r= 0.9311).The method is helpful for rapid and sensitive detection of serum TBA for point-care-of-testing(POCT).3.UPLC-TripleTOF-MS/MS method was used to analyze serum metabolic profiling of bile acids.Profiling of 57 serum bile acids of 27 normal pregnancies,59 patients with ICP and 26 patients with AHP were analyzed by the technique mentioned above.PLS-DA model revealed that the metabolic profiling of bile acids had obvious differences among AHP,ICP patients and normal pregnant women.Twenty-four bile acids were found based on the VIP value >1,in which glycodeoxycholic acid(GDCA),the fourth isomeric component of taurine-conjugated triol-bile acid(Ttri-4),tauro-ω-muricholic acid(T-ω-MCA),glyhyocholic acid(GHCA)and the third isomeric component of glycine-conjugated triol-bile acid(Gtri-3)were significantly different among the three groups(P<0.05).The potential biomarkers of glycocholic acid(GCA),the first isomeric component of glycine-conjugated triol-bile acid(Gtri-1)and the first isomeric component of taurine-conjugated diol-bile acid(Tdi-1)were selected for the diagnosis of the disease groups and the control group.The area under the ROC(AUC)is 0.989 with a sensitivity of 98.9% and a specificity of 100%.Lithocholic acid(LCA),Gtri-1 and chenodexycholic acid(CDCA)were selected as the differential diagnosis of AHP and ICP by the logistic regression analysis.The AUC is 0.956 with a sensitivity of 92.1% and a specificity of 91.7%.The metabolic profiling of bile acids and some individual bile acids are helpful for the differential diagnosis of AHP.4.The method of the microfluidic chip combined with the electrochemical detection was established for the serum TBA.The serum samples were pretreated by the solid phase extraction(SPE)technique and then driven by electronics in the PDMS chip channel from the sample cell to the test cell.With the existence of 3α-HSD,the bile acids in serum reacted with NAD+,and the reaction production of NADH reacted with the oxidized Ru(bpy)33+ to produce Ru(bpy)32+,and the Ru(bpy)32+ is furthered oxidized at the surface of the SPCE to produce electrical signals.Therefore,TBA is quantitatively detected indirectly by the chronoamperometry method.The linearity is from 1.0 to 100.0 μmol/L,and the regression coefficient is 0.9995.The sensitivity is satisfactory with the LOD of 0.10 μmol/L.The RSD of the precisions range from 5.9% to 8.9% and the recoveries range from 94.9% to 102%.The method could be further used to establish a method that combines microfluidic chip separation with electrochemical detection for the serum bile acids metabolic profile analysis.