Whole Blood Metabolomics By~1H NMR Spectroscopy In The Diagnosis Of Papillary Thyroid Carcinoma

Foreword:In recent years,the incidence of thyroid cancer has increased significantly,and among all types of them the incidence of papillary thyroid carcinoma(PTC)is the highest.There are rarely no metastasis in PTC,and the lymph node metastasis is the first important metastasis pathway.Although many studies have focused on the metabolic changes of PTC,few studies focused on the analysis of the whole blood metabolism in patients with PTC.At the same time,there are many studies on the risk factors of lymph node metastasis of PTC,but the metabolic characteristics of patients with positive or negative lymph node metastasis are less studied.The purpose of this study is to find differential metabolites between patients with PTC and normal people,with or without lymph node metastasis,and to analyze effective metabolites and metabolic pathways that can assist in the diagnosis of PTC.We want to find out the potential role of metabolites as a predictor of prognosis in patients with PTC.Methods:In this study,whole blood samples of 80 patients who underwent modified radical thyroidectomy in the Department of Breast and Thyroid Surgery of Shandong Provincial Hospital from March 2018 to October 2018 were collected as samples in the PTC group,while whole blood samples of 47 normal adults were collected which were used as healthy controls.According to the presence or absence of lymph node metastasis,the PTC group samples were further divided into PTC group with lymph node metastasis and PTC group without lymph node metastasis.All samples were pretreated,mixed with methanol,centrifuged,evaporated to dryness and added with phosphate buffered saline to fully extract metabolites in whole blood.All samples were collected by proton nuclear magnetic resonance(1H NMR)spectral data.The collected spectral data export by manual phasing,normalized saving and other steps.The data of PTC patient group and healthy control group,lymph node metastasis group and no lymph node metastasis group were compared,including principal components analysis(PCA)to exclude abnormal samples,and partial least squares discriminant analysis(PLS-DA),and orthogonal partial least squares discriminant analysis(OPLS-DA)to generate score maps,S-Plots maps,and variable importance in the projection(VIP)maps to determine possible differential metabolites.The MetaboAnalyst4.0 website is used to analyze the possible differential metabolites obtained from this,further narrow the range of differential metabolites,draw ROC curves to detect the credibility of the differential metabolites,and find possible metabolic pathways based on the differential metabolites.Results:Of all the 80 patients,44 had lymphatic metastases,divided into two groups:PTC group and healthy control group,PTC group with lymph node metastasis and PTC group without lymph node metastasis.The abnormal samples were deleted during PCA analysis;When the PLS-DA analysis was performed separately,it was found that the comparison between the two groups was good for disturbance test.According to OPLS-DA analysis,the PTC group and the healthy control group was significantly different from the score map,but there was no significant difference between the PTC group with lymph node metastasis and the PTC group without lymph node metastasis.It was found from the S-Plots chart the former comparison has obvious differences in metabolites,the possible metabolic differences are known from the VIP diagram,and the latter comparison has no significant difference in contrast.Through the MetaboAnalyst4.0 website,based on the analysis of possible differential metabolites obtained from the S-Plots plot and VIP plot of the PTC group and the healthy control group,a total of 20 differential metabolites were screened out.PTC patients were relatively decreased in the concentrations of arginine,glutamic acid,methionine,hydroxybutyric acid,aspartic acid,tyrosine,threonine,histidine,phenylalanine and formic acid,while lactic acid,proline,1-Methylhistidine,creatine,glucose,ornithine,isopropanol,and hypoxanthine increased.The differential metabolite results were verified by the area under the ROC curve(AUC).All AUC scores exceeded 0.9,which means the result has high reliability.According to the differential metabolites,MetaboAnalyst4.0 website was used to analyze the metabolic pathways,and 7 potential metabolic pathways were obtained,including ? histidine metabolic pathway;?arginine biosynthetic pathway;? phenylalanine,tyrosine and Tryptophan biosynthetic pathway;? arginine and proline metabolism pathway;? phenylalanine metabolism pathway;? pyruvate metabolism pathway;?alanine,aspartic acid and glutamic acid metabolism pathway.Conclusion:This study was the first research to use whole blood to determine the differences in metabolic components between PTC patients and healthy adults.Studies have found that the metabolic information carried by whole blood is sufficient to separate PTC patients from healthy adults,providing theoretical support for the use of whole blood,a more convenient sample,for the differential diagnosis of PTC patients.This study showed that compared with healthy adults,PTC patients had lower lysine levels,increased lactic acid levels,and abnormal histidine metabolism pathways,which fully explained the metabolic differences between PTC patients and healthy adults,but there was no highly specific differential metabolism.At the same time,in the comparison of the PTC group with and without lymph node metastasis,no significantly different metabolites were found,which need to be studied with a larger number of experiments in the future.