Screening And Identification Of Serum Biomarkers Of Type 2 Diabetes Mellitus With Peripheral Arterial Disease

Objective:Peripheral arterial disease was one of the most common categories of the macrovascular disease,which was the main cause of death and disability in type 2 diabetic patients.By far,there were no special biomarkers for the early diagnosis of it.So,finding the biomarkers of type 2 diabetes mellitus(T2DM)with peripheral arterial disease was extremely important.By using relative quantitative proteomics technology to compare the serum of type 2 diabetic patients with or without peripheral arterial disease,we could find the expression of the protein spectrum.And then,applying the enzyme linked immunosorbent assay(ELISA)technique to further detect the differentially expressed proteins which we choose,we could find the biomarkers of T2DM with peripheral arterial disease that was designed to provide help for diagnosis and treatment.Methods:1.The patients and healthy individuals were chosen in the first affiliated hospital of Dalian Medical University from Jan.to Dec.in 2014.According to with or without T2DM and peripheral arterial disease,they were divided into four groups and every group had thirty cases,including non-diabetes and non-macrovascular disease group(NDNM),T2DM and non-macrovascular disease group(NDM),T2DM with subclinical macrovascular disease group(DSM)and T2DM with peripheral arterial disease group(DPA).We collected their serum and analyzed the clinical data.2.Isobaric tags for relative and absolute quantitation(iTRAQ)and Uniprot were used to analyze the expression of protein spectrum in order to find the differentially expressed proteins.Then kyoto encyclopedia of gene and genomes pathway was used to screen and analyze the main metabolism/signal transduction pathways of those proteins.3.ELISA technique was applied to detect five kinds of the differentially expressed proteins that we chose from the pathways above,four of them were diagnostic indicators for T2DM with subclinical macrovascular disease named as insulin-like growth factor 1(IGF-1),alpha-enolase(ENO1),alpha 2 macroglobulin 2(A2M)and complement 7(C7).Two of them were diagnostic indicators for T2DM with peripheral arterial disease named as IGF-1 and insulin-like growth binding protein 3(IGFBP-3).Results:1.The risk factor of T2DM with subclinical macrovascular disease was age,the OR value was 1.111.The risk factors of T2DM with peripheral arterial disease were age,duration of T2DM and sex,the OR values were 1.225,1.575 and 0.014 respectively.2.By using iTRAQ,we found eight hundred and eighty-eight proteins finally.There were thirty-four relative differentially expressed proteins in the comparison of DSM vs.DNM(eighteen proteins increased and sixteen proteins decreased).Nine of them participated in four metabolism/signal transduction pathways(IGF-1 receptor mediated signal pathway,complement and coagulation cascades pathway,salivary secretion pathway and biosynthesis of amino acids pathway).Meanwhile,there were fifty-two relative differentially expressed proteins in the comparison of DPA vs.DNM(thirty-four proteins increased and eighteen proteins decreased).Three of them participated in one metabolism/signal transduction pathway(IGF-1 receptor mediated signal pathway).3.The results of ELISA indicated that,DSM vs.DNM,IGF-1 level was downregulated(61.80±16.53ng/ml vs.73.90±26.65ng/ml,P=0.044),ENO1 level was downregulated(147.07±69.48 U/ml vs.201.59±95.76 U/ml,P=0.017),A2M level was upregulated(1281.51±344.39μg/ml vs.1097.57±231.70μg/ml,P=0.019),C7 level was upregulated(80.98±13.11±g/ml vs.74.75±10.80μg/ml,P=0.056),IGF-1,ENO1 and A2M all had the same regulated trend with iTRAQ,and had statistical difference between the two groups;C7 had the same regulated trend with iTRAQ,though no statistical difference between the two groups.DPA vs.DNM,IGF-1 level was downregulated(58.12±28.11ng/ml vs.73.90±26.65ng/ml,P=0.032),IGFBP-3 level was downregulated(1937.53±621.94ng/ml vs.2499.72±746.61ng/ml,P=0.020),IGF-1 and IGFBP-3 both had the same regulated trend with iTRAQ,and also had statistical difference between the two groups.4.The results of receiver operating characteristic curve indicated that ENO1 had statistical significance in diagnosing T2DM with subclinical macrovascular disease;IGF-1 and IGFBP-3 had statistical significance in diagnosing T2DM with peripheral arterial disease,moreover,IGFBP-3 was better than IGF-1 in diagnosis.Conclusions:1.Age,duration of T2DM and male are the risk factors of T2DM with peripheral arterial disease.2.Thirty-four differentially expressed proteins are the biomarkers of T2DM with subclinical macrovascular disease which nine of them participate in four metabolism/signal transduction pathways,including IGF-1 receptor mediated signal pathway,complement and coagulation cascades pathway,salivary secretion pathway and biosynthesis of amino acids pathway;fifty-two differentially expressed proteins are the biomarkers of T2DM with peripheral arterial disease which three of them participate in one metabolism/signal transduction pathway,which is the IGF-1 receptor mediated signal pathway.3.ENO1 may be recognized as a diagnostic indicator of T2DM with subclinical macrovascular disease;IGF-1 and IGFBP-3 may be recognized as diagnostic indicators of T2DM with peripheral arterial disease.