Establishment Of The Electrochemical Method For Detecting Plasma Indoxyl Sulfate And Its Application

Objective:1.To establish a simple and rapid electrochemical method for the detection of plasma indoxyl sulfate（IS）.2.The plasma IS in patients with gestational diabetes mellitus（GDM）and healthy pregnant women were detected by the established electrochemical method.Moreover,the levels of IS were investigated and compared in both physiological and pathological states of pregnancy.3.To establish a new electrochemical method for the simultaneous detection of IS and indole（IND）.Methods:1.Based on the properties of the oxidation of IS at a certain potential,cyclic voltammetry（CV）and differential pulse voltammetry（DPV）were used for qualitative and quantitative analysis of IS,respectively.The plasma samples were treated by protein precipitation with acetonitrile.And the supernatant was blown to dry by nitrogen,and then the residue was redissolved in buffer solution.Finally,the solution was added onto the surface of screen printed carbon electrodes（SPCE）for detecting IS by DPV method.After the methodology evaluation,the established electrochemical method was used to determine IS in plasma as well as the method of high performance liquid chromatography fluorescence detection（HPLC-FLD）.2.The concentrations of plasma IS in 20 GDM patients and 20 healthy pregnant women were detected by the established electrochemical method.Clinical basic information and relevant laboratory indicators of the subjects were collected,and the data were statistically analyzed.3.Based on the different electrochemical properties of IS and IND on SPCE,DPV was used to analyze IS and IND simultaneously.Results:1.At the optimal experimental conditions,the linear range of IS was from 1.0 to 160.0μmol/L,and the regression equation was y=0.0369 x+0.3460（R²=0.9942）,and the limit of detection（LOD）was 0.2μmol/L（S/N=3）.The relative standard deviations of intra-day and inter-day precisions were less than 5.1%and 6.9%,respectively.Moreover,the recoveries were from 96.1%to 106.9%.The established electrochemical method had a good correlation with HPLC-FLD method（r=0.8956,p=0.000）.2.The concentrations of plasma IS in GDM group（13.70μmol/L）was significantly higher than that in control group（6.22μmol/L）,and the difference was statistically significant（p<0.05）.In addition,the levels of diastolic blood pressure,1 h postprandial blood glucose（1 h PG）,2 h postprandial blood glucose（2 h PG）and insulin of pregnant women were statistically different between GDM group and the control group（p<0.05）.The correlation analysis showed that the concentrations of IS were positively correlated with the levels of 1 h PG and 2 h PG（p<0.05）,and negatively correlated with the levels of direct bilirubin and lactic dehydrogenase（p<0.05）.As odds ratio was 1.554（p<0.05）,the regression analysis showed that IS was a risk factor for patients with GDM.The receiver operating characteristic（ROC）analysis showed that IS had a good diagnostic efficiency for GDM.The area under the curve（AUC）was 0.873 with the sensitivity of 70.0%and the specificity of 100.0%.3.At the optimal experimental conditions,the linear ranges of the electrochemical method were from 2.0 to 47.9μmol/L for IS and from 0.9 to34.1μmol/L for IND,respectively.The regression equations were y_（IS）=0.0215 x+0.0044（R²=0.9966）for IS and y_（IND）=0.0297 x+0.0156（R²=0.9922）for IND,respectively.LODs were 0.4μmol/L for IS and 0.2μmol/L for IND（S/N=3）,respectively.The relative standard deviations of intra-day precisions were less than 1.8%for IS and 4.5%for IND,and the relative standard deviations of inter-day precisions were less than 4.0%for IS and8.1%for IND,respectively.The recoveries were from 97.7%to 105.9%for IS and from 96.4%to 107.0%for IND,respectively.Conclusions:1.A simple,sensitive and cost-effective electrochemical method was established with unmodified SPCE for direct detection of plasma IS.As the SPCE could be mass-produced and be easily miniaturized,the established method has potential application in both laboratory detection and clinical researches.2.IS was a risk factor for patients with GDM and it had a good efficiency in GDM diagnosis with a high specificity,indicating IS could be used as a new index for GDM diagnosis.The metabolic disorders of IS were discovered in patients with GDM,suggesting that IS may play an important role in the occurrence and development of GDM.3.A new,simple and rapid electrochemical method was established to detect IS and IND simultaneously with a good repeatability.This method provided a new idea for the construction of the sensitive and specific electrochemical sensor of IND and IS,and the study of the metabolism of IND and IS in vivo.