Serum Lipidomics Profiling In Patients With Cyclosporin A Induced Liver Injury

Objectives:1. To establish gas chromatography-mass spectrometer (GC-MS) and electro-spray ionization high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods for the lipidomics study of human serum.2. To study the serum lipidomics in patients of CILI and non-CILI group by using the GC-MS and HPLC-MS/MS methods.3. To identify serum lipids using the lipid peak area/internal standard peak area ratio as an indicator of relative quantification by NIST 11 and HMDB databases.4. To establish discriminant models between two groups and look for the differences lipids between the two groups by using the multidimensional statistical analysis methods. To explore the potential biomarkers and lipid metabolic pathway associated CILI by using the MetaboAnalyst and KEGG online software. To have a better understanding of the pathogenesis of CILI and to get the potential biomarkers of CILI for the early diagnosis and treatment.Methods:1. Serum lipids like fatty acids were detected by GC-MS. To extract the serum lipids with n-hexane. The chromatographic separation was conducted on a VF-WAXms capillary column (0.25 mm×30 m×0.25 μm). Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The solvent delay time was set to 7.5 min. The initial oven temperature was held at 70℃ for 1 min, ramped to 180℃ at a rate of 20℃/min, then increased to 230℃ at a rate of 6℃/min, and finally held at 230℃ for 20 min. The injection volume was 1 μL with split ratio of 50:1. The temperature of the injector, transfer line and ion source (electron impact) were set at 250,270 and 230℃, respectively. The Mass data was acquired with collision energy of 70 eV by full-scan mode in the m/z range of 30-450.2. Serum lipids like phospholipids and sphingolipids were detected by HPLC-MS/MS. To extract the lipids by using methylene chloride-methanol-water (v/v/v=1:2:1).The chromatographic separation was achieved on a JADE-PAK DIOL column (250 mm×4.6mm×5μm). Mobile phase A consisted of 20 mM ammonium formate in 20% isopropanol. Mobile phase B was a mixture of acetonitrile-isopropanol (2:8, v/v). Separation was performed with an increasing gradient of 92% B (0-2 min),86% B (2-6 min) gradually,80% B (6-12 min) gradually,75% B (12-18 min) gradually,65% B (18-24min) gradually,92% B (24-30min) gradually, finally kept at 92% B (31-45 min). Twenty microliters of samples were analyzed in sequence with a flow rate of 0.3 mL/min. The analytes were detected in the positive mode and MS data were acquired over a scan range of 200-1300.3. Analysis of GC-MS spectra was automatically matching mass spectrum data with NIST 11 MS database of MSD ChemStation. To define the lipids with retention time drift<0.5min as the meaningful lipid metabolites. To accomplish the mass match of the HPLC-MS/MS spectra using the HMDB databases. And to defined the lipids with retention time drift<0.5min as the meaningful lipid metabolites.4. After using the GC-MS and HPLC-MS/MS method for the detection of the subjects serum, data statistical analysis methods including PCA, PLS-DA and OPLS-DA were used to complete pattern recognition multivariate analysis, to compare the serum lipid profiles of CILI with non-CILI group. Then the pathway analysis was conducted by MetaboAnalyst to find out the related lipid metabolism pathways. Finally, the different lipids and related pathway (impact value>0.01) were combined analyze to further understanding the biological function of lipids in the development of CILI.Results:In the present study,103 subjects were divided into CILI group (n=43) and non-CILI group (n=60) according to the results of liver function test. Fourteen lipids from GC-MS and 61 from HPLC-MS/MS were identified. After the multivariate statistical analysis with principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA),23 lipids were significantly different (VIP>1 and P<0.05) between two groups, including 5 fatty acids (FA),8 phosphatidylcholines (PC),3 phosphatidylethanolamines (PE),6 sphingomyelins (SM) and 1 cholesterol esters (CE). Lower PC/PE ratio and higher AA and FA levels were found in CILI group compared with that in non-CILI group, indicating that lipid accumulation and membrane integrity reducing in hepatocytes increase the susceptibility to liver injury. Finally, the integrative analysis of lipids indicated four metabolism pathways including glycerophospholipid metabolism, arachidonic acid metabolism, fatty acid metabolism and sphingolipid metabolism are involved in the development of CILI, the impact values for each metabolism pathway were 0.23,0.22,0.05 and 0.03.Conclusions:The present lipidomics study based on GC-MS and HPLC-MS/MS coupled with pattern recognition and metabolic pathway analysis provided a powerful mean of revealing differences between the CILI and non-CILI group. Twenty-three lipids were significantly different between two groups and four metabolism pathways, including glycerophospholipid, arachidonic acid, fatty acid and sphingolipid pathway were believed to involve in the development of CILI. Lower PC/PE ratio and higher AA and FA levels were found in CILI group compared with that in non-CILI group, indicating that lipid accumulation and membrane integrity reducing in hepatocytes increase the susceptibility to liver injury.