| Objective: Laryngeal Cancer(LaC)is not only the second most common respiratory cancer with high incidence after lung cancer,but also the most common malignant tumor in head and neck.Because the early symptoms of laryngeal cancer are not obvious and there are no characteristic diagnostic markers in screening,it is easy to delay the opportunity of early diagnosis and treatment,which seriously threatens the life,health and quality of life of patients.Ultra-high performance liquid chromatography and high resolution mass spectrometry(UHPLC-HRMS)were used to detect and analyze the clinical samples(including serum,tissue and urine)of LaC patients,so as to identify the differential metabolites of lipid metabolism in serum,cancer and para-carcinoma tissues and urine of LaC patients and control group.The best model of lipid internal standard compounds was constructed by screening out lipid metabolic differences.The differential metabolites of urine before and after LaC operation were compared with those in control group,and the metabolic pathways that may exist differences in urine metabolism were enriched.The ideal combination of biomarkers that can be used to distinguish LaC patients from normal people was explored,so as to provide basis for screening tumor markers of LaC and theoretical basis for early detection and pathogenesis research of LaC.Methods: Serum samples from 29 patients with LaC and 36 controls were collected from the Department of Otolaryngology.Urine samples were collected from 18 LaC tissues and 16 adjacent non-cancerous tissues after pathological diagnosis.Urine samples were collected from 13 LaC patients in preoperative group,22 LaC patients in postoperative group and 19 LaC patients in control group.Using ultra-high performance liquid chromatography(UHPLC)and high resolution mass spectrometry(HRMS),nontargeted metabonomics analysis of lipids in serum and tissue samples and metabolites in urine of LaC group and control group was carried out.Supervised partial least squares discriminant analysis(PLS-DA)was performed,and the square root of standard deviation was divided by each variable to suppress noise interference.Wilcoxon and MannWhitney tests were used to compare the metabolites in blood,tissues and urine.Nonparametric tests were used to identify the metabolites with significant changes(P <0.01 and FDR < 0.05).The receiver operating characteristic(ROC)curve of binary Logistic regression was plotted with SPSS(19.0)software.Bar charts of subclasses with significant differences in Graph Pad Prism version 6.0 were drawn.The PLS-DA model was permutated 200 times to evaluate whether the model is over-fitted and whether the model is stable.Systematic data analysis of lipid metabolites in serum and tissue samples was carried out by univariate and multivariate data analysis methods,and differential metabolites were screened out.SIMCA-P(14.1.0)software was used to analyze the urine metabolites of the three groups by principal component analysis and compare them in pairs.The fold change(FC)of differential metabolites was calculated by Matlab R2016 a,and the metabolic pathways were enriched by Metabo Analyst website.Results:1.There was no significant difference in gender,age,BMI,blood glucose,hemoglobin,total bilirubin,erythrocyte and leukocyte between LaC group and control group in serum and tissue sample study.BMI and erythrocyte number in urine in control group were higher than those of LaC patients before and after operation(P < 0.05),but there was no statistical difference in other indexes.2.The relative levels of ceramide(Cer,Cer G1),sphingomyelin(SM),phosphatidyl choline(PC,PC-O),phosphatidylethanolamine(PE),phosphatidylinositol(PI),phosphatidylserine(PS)and cholesteryl ester(Ch E)in serum of LaC group were significantly higher than those in control group(P < 0.05).3.The serum levels of lysophosphatidylcholine(LPC,LPC-O),lyso Phosphatidylethanolamine(LPE,LPE-P)and diacylglycerol(DG)in LaC group were significantly lower than those in control group(P < 0.05).4.Levels of phosphatidyls(PLs)with arachidonic acid acyl chain and/or palmitoyl chain were significantly increased in LaC group,while lysophosphatidyls(LPLs)with arachidonic acid acyl chain and/or palmitoyl chain were significantly lower in LaC group than in control group(P < 0.01).5.The distribution of lipids in LaC group and para-carcinoma group mainly concentrated in triacylglycerol(TG),FA(fatty acid)and PC(phosphatidylcholine)lipids.6.There were no significant statistical differences in the distribution of ceramide(Cer,Cer G1,Cer G2),sphingomyelin(SM),lysophosphatidylcholine(LPC,LPC-o),lyso Phosphatidylethanolamine(LPE-p),phosphatidylinositol(PI),phosphatidylserine(PS),diacylglycerol(DG),cholesteryl ester(Ch E)and triacylglycerol(TG)between LaC group and para-carcinoma group.7.The content of glutamine in LaC tissue was higher than that in control group,while the content of free fatty acid decanoic acid(FFA 10: 0)and citric acid was lower than that in control group(P < 0.05).8.Pathway enrichment analysis was carried out between the urine metabolites of LaC patients after operation and the control group.There were significant differences in Aminoacyl-t RNA biosynthesis pathway and Tryptophan metabolism pathway(p < 0.05).9.The pathway enrichment of the metabolites in urine of LaC patients after operation and before operation was analyzed,and there was significant difference in Riboflavin metabolism pathway(p < 0.05).10.The levels of 5-Hydroxyindole-3-acetic acid(5-HIAA)and paraxanthine in urine of LaC patients before operation were lower than those in control group,while the levels of Glycocholic acid were higher than those in control group.The levels of 5-hydroxyindole-3-acetic acid and paraxanthine in urine of LaC patients after operation were higher than those in control group,but the levels of glycocholic acid were lower than those in control group,and the difference was statistically significant(p < 0.05).Conclusion:1.Serum study: LaC may result in increased synthesis of sphingolipids and some glycerophospholipids.PLS rich in AA residues in serum,such as PC(16: 0＿20: 4),PE(16: 0＿20: 4),PE(16: 0p＿20: 4),PE(18: 0p＿20: 4)and PI(16: 0＿20: 4),increases significantly,while AA(FA(20: 4))decreased significantly,which may result from the possible activation of long chain acyl coenzyme A synthetase 4,which promotes the accumulation of PLS,especially in LaC tumor cells characterized by rich mitochondria.Using the non-targeted metabonomics method of ultra-performance liquid chromatography and high resolution mass spectrometry technology platform,the best model containing 43 potential lipid internal standard compounds was constructed by using binary logistic regression analysis and stepwise regression(Wald)optimization algorithm.An ideal biomarker lipid panel(including LPC(16: 0)and PE(18: 0P＿20: 4))was identified,which can effectively distinguish LaC patients from healthy control cohorts.Similarly,this serum lipid plate shows ultra-high performance in predicting early LaC.This study provides evidence of systemic changes in serum lipid composition between LaC group and control group.PLs rich in ceramide,sphingomyelin and AA,which are closely related to LaC,may be potential biomarkers of LaC.2.Tissue study: The relative content of L-Glutamine in LaC group was higher than that in control group,while the content of FFA decanoic acid 10: 0 and citric acid in LaC group was lower than that in control group.Further clarification of related proteins and metabolic pathways is expected to become a potential biomarker for early diagnosis of LaC.3.Urine study: In urine study,non-targeted metabolomics analysis is carried out in LaC preoperative group,postoperative group and control group,and it is found that urine metabolites in the three groups change significantly.Among them,the contents of 5-Hydroxyindole-3-acetic acid,paraxanthine and glycocholic acid are significantly different before and after operation and in control group.Pathway enrichment analysis shows that there are statistical differences in aminoacyl t RNA synthesis pathway,tryptophan metabolism and riboflavin metabolism.A deep understanding of metabolic pathways and differential metabolites can provide theoretical basis for screening LaC related biomarkers in urine and studying the pathogenesis of LaC. |
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