The Research Of A Novel Method To Detect Serum Myeloperoxidase And Its Values In The Risk Stratification Of Coronary Heart Disease And Prediction Of Acute Coronary Events

Background:Clinical and epidemiological studies have shown that high serumlevels of low density lipoprotein cholesterol (LDL-C), and other metabolicdisorders are associated with increased incidence of coronary heart disease(CHD) after atherosclerotic plaque rupture. Nowdays, the study betweenatherosclerosis and CHD-related inflammatory factors has become thefocus because atherosclerosis as a chronic inflammatory disease has beenrecognized. Recent study found that as a new inflammatory marker forprediction of acute coronary syndrome (ACS), myeloperoxidase (MPO) haseffects on a range of factors that influence the atherosclerosis of arterialwall if elevated its expression and concentration, suggests that it contributeto the atherosclerosis pathogenesis and progression in CHD patients.Currently ELISA and selective repression methods were generally appliedfor determination of serum MPO in domestic and foreign most laboratories.However, the extensive clinical application of these two methods is restricted because of many influencing factors, poor precision andcomplicated operation.Objective:This research was design to establish a novel rapid quantitativemethod of particles enhanced transmission immunoassay based on thespecific antigen-antibody reaction, which is used for the measurement ofserum MPO levels. The value for the risk stratification of CHD wasexplored by combined detection of serum MPO, interleukin-6(IL-6) andhigh sensitivity C-reactive protein (hs-CRP). The possible association wasinvestigated between MPO with each of the variables, including ACS,LDL-C and other parameters of metabolic disorders, inflammatory markers,and traditional CHD risk factors as well as life style habits in CHD patients.The relationship was prospectively studied between serum MPO baselinelevel and ACS incidence in patients with stable angina pectoris (SAP).Methods:①The method of particles enhanced transmission immunoassay todetermine serum MPO levels was established by MPO antibody coatedlatex particles surface, and this method is evaluated according to Clinicaland Laboratory Standards Institute (CLSI).②The serum levels of MPO, IL-6and hs-CRP in CHD patients[acute myocardial infarction (AMI), unstable angina pectoris (UAP) andSAP] and controls [non-CHD disease patients (NCHD) and healthy subjects] were determined, respectively. Diagnostic efficiency of ACS wascalculated by matrix decision method.③The possible associations between serum MPO levels with ACS,metabolic-related risk parameters [LDL-C, high density lipoproteincholesterol (HDL-C), total cholesterol (TC), triglyceride (TG),homocysteine (Hcy), fasting blood glucose (FBG) and insulin (INS)],circulating markers of inflammation (IL-6and hs-CRP) and traditionalCHD risk factors as well as life style habits [body mass index (BMI),diabetes, hypertension, gender, age, smoking and alcohol intake) wereinvestigated by logistic regression analysis.④One hundred forty-six SAP patients was prospectively investigatedthe acute coronary events. The incidence of ACS was compared betweenelevated baseline MPO group and normal-low baseline MPO group.Results:①The self-established method of particles enhanced transmissionimmunoassay used determination of serum MPO levels was developed.This method had a good accuracy, its relatively error was≤9.95%; andhigh precision with intra-assay CV≤1.92%and inter-assay CV≤3.00%;and a wide linear detection range of0ng/mL-1300ng/mL; and a highsensitivity with5ng/mL of lower limit of detection; and stronganti-interference ability, which did not effect on the results when bilirubin≤513μmol/L, ascorbic acid≤0.40g/L as well as triglycerides≤11.3 mmol/L. There were well correlation between self-established method withELISA as well as selective repression method, the square of the correlationcoefficient were0.9865and0.9867, respectively.②ACS group had significantly higher levels of MPO compared toSAP, NCHD and healthy groups (P <0.001). The diagnostic efficiency ofcombined detection of three inflammatory markers (MPO, IL-6andhs-CRP) with serial tests for ACS were74.53%for sensitivity,79.82%forspecificity,89.64%for positive predictive value,57.23%for negativepredictive value and76.12%for accuracy, respectively.③MPO was positively correlated with LDL-C, TC, TG, Hcy, FBG,IL-6, hs-CRP, age, SBP, DBP and BMI, and negatively correlated withHDL-C levels. The areas under receiver operating characteristic (ROC)curve of MPO for diagnosing ACS was0.84(95%CI0.80-0.88, P <0.001).The optimal cut-off value (sensitivity; specificity) of MPO was164.77ng/mL (79.1%and82.1%).Stepwise multivariate logistic regression analysis revealed that LDL-C(III versus I tertile, OR:3.24,95%CI:1.67-6.29, P=0.001); HDL-C (IIIversus I tertile, OR:0.19,95%CI:0.11-0.34, P <0.001); TC (III versus Itertile, OR:2.94,95%CI:1.55-5.58, P=0.001); hs-CRP (III versus I tertile,OR:2.30,95%CI:1.32-3.99, P=0.003); age (III versus I tertile, OR:2.37,95%CI:1.36-4.13, P=0.002); BMI (III versus I tertile, OR:2.03,95%CI:1.17-3.54, P=0.012); ACS (yes versus no, OR2.74,95%CI:1.71-4.39, P <0.001) and smoking (yes versus no, OR2.62,95%CI:1.62-4.25, P <0.001) were associated with elevated serum MPO levels independent of TG,Hcy, UA, FBG, INS, IL-6, SBP, DBP, gender and alcohol intake. Amongthem, LDL-C and ACS had the highest odds ratio in quantitative andqualitative variables, respectively. There were significant increase trends inthe prevalence of ACS and high LDL-C levels from I to III MPO tertile,which were46.4%and8.9%for I,68.0%and31.5%for II,88.8%and59.8%for III tertile, respectively (P <0.001).④The prospective study of a primary6-month follow-up in146SAPpatients aged38-84years revealed that elevated baseline MPO levels had ahigher incidence of ACS (11/43) than that (9/103) of patients withnormal-low baseline MPO levels (28.9%vs8.3%, χ2=10.10, P=0.001).Conclusions:①The self-established method of particles enhanced transmissionimmunoassay used determination of serum MPO levels has a highsensitivity, strong specificity, good accuracy, wide linear range, wellanti-interference ability and ease of operation. There is a well correlationbetween self-established method with ELISA as well as selective repressionmethod. This method is suitable for use in automatic biochemicalanalyzers.②Combined detection of MPO, IL-6and hs-CRP can be used indiagnosis of CHD at different pathological stages and improve the specificity and positive predictive value for diagnosing ACS, which ishelpful for the risk stratification of CHD.③The present study provides the clinical epidemiological evidencethat elevated serum MPO levels are significantly associated with theprevalence of ACS as well as high LDL-C levels in CHD patients. Theinteractions between MPO and LDL-C contribute to the atherosclerosispathogenesis and progression of the patients with CHD. MPO may be avaluable inflammatory marker for the diagnosis of ACS.④Primary prospective study has demonstrated that MPO is avaluable inflammatory marker for the prediction of future acute coronaryevents in patients with CHD. The more elevated baseline MPO the CHDpatients have, the more higher incidence of ACS they would suffer.