Investigation Of Volatile Organic Metabolites In Lung Cancer Pleural Effusions By SPME-GC/MS Metabolomics Method

Lung cancer is a leading cause of cancer-related death,accounted for 1.59 million deaths worldwide in 2012.Pleural effusion is usually induced directly by lung disease and closely related to the progress of the disease.Therefore,it is considered as more specific for lung disorders than other body fluids due to its close correlation with the affected organ.Pleural effusion may be a valuable sample source for observation of changes in low molecular-weight volatile organic metabolites.Volatile organic compounds(VOCs),which widely exist in human body fluids and tissues,may be absorbed as contaminants from the environment(exogenous)or may be generated in the body(endogenous).Endogenous VOCs are products of the body metabolism and often have close relationship with some diseases.Therefore,their existence and content are the important information of health status.In this study,headspace solid-phase microextraction(HS-SPME)combined with gas chromatography/mass spectrometry(GC/MS)method was applied for the investigation of low molecular-weight volatile organic metabolites(VOMs)in pleural effusion samples from lung cancer and benign inflammatory diseases.Principle component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)were built to separate benign from malignant pleural effusion samples and to find dysregulated metabolites between the two groups.In addition,a quantitative analysis method was developed for examining the concentration of the dysregulated VOMs in benign and malignant effusions,providing the basis for establishing diagnosis standards in other body fluids such as blood,urine and even saliva in the future.Meanwhile,it will be a complementary method for the differentiation between benign and malignant pleural effusions.The main work and results of this study are as follows:1.Volatile organic metabolites in the collected 54 pleural effusion samples(22 malignant effusions and 32 benign effusions)were investigated by HS-SPME-GC/MS.A total of 36 volatile metabolites belonging to nine distinct chemical classes were identified by comparison of fragmentation patterns with a standard mass chromatogram using a NIST 02 library and verified by reference compounds,when available.The peak areas of the chemical classes were firstly determined with quantified ions.Ketones,alcohols,and benzene derivatives were the main chemical classes for the metabolomic profile of malignant group,whereas in the benign group,ketones,benzene derivatives and esters are the chemical classes that contributed most to the metabolomic profile.Ketones,alcohols and phenols exhibit significant differences(p<0.05)between the two groups.Then,Principle component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)were built to separate benign from malignant pleural effusion groups.The PLS-DA was good enough for classification of the two groups.According to multi-criteria assessment(1)VIP>1;(2)|Pcorr|>0.58;(3)p<0.05,cyclohexanone and 2-ethyl-1-hexanol have been screened out with a significant influence on the classification.2.XCMS Online has been used for automated pre-processing of theGC/MS-based metabolomic data of pleural effusion samples(20 malignant effusions and 20 benign effusions).Consequently,8 feature groups exhibited statistical difference(p<0.05)in benign and malignant pleural effusions.Unsupervised principle component analysis was good enough for classification of the two groups.The obtained results exhibited similarity with quantified ions,although the different effusion samples were used.3.The concentration of the four dysregulated VOMs(2-ethyl-1-hexanol,cyclohexanone,1,2,4,5-tetramethylbenzene and naphthalene)in 40 pleural effusion samples were further determined by external standard method.The results showed the concentrations of four dysregulated VOMs in malignant effusion samples were significantly higher than those in benign ones,especially for cyclohexanone and 2-ethyl-1-hexanol.Remarkably,the median concentration of cyclohexanone in malignant effusion samples is 2.43 ?g/mL(be equivalent to 24796 nM),which is much higher than that in lung cancer urine samples reported in literature(average concentration 3360 nM).