Effect Of Perchlorate On Lipid Metabolism Based LC-MS

BakgroundPerchlorate,an endocrine disruptor,enters the body through water and food,and interferes with the regulatory function of the hypothalamus-pituitary-thyroid endocrine axis(HPT),causing lipid metabolism disorders.Previous studies have shown that perchlorate affects the pathological characteristics and routine indexes of tissue slices related to lipid metabolism,but few deeper researches on the mechanism.Lipidomics is widely used to comprehensively characterize the changing trends of lipid metabolites in the body.Therefore,the study was designed to screen the differential metabolites of perchlorate affecting lipid metabolism,identify potential biomarkers based on LC-MS,and the metabolic pathways were analyzed to explore the mechanism.Method(1)Hep G2 cells were poisoned with perchlorate,which were blank control group(C group),low-dose group(L group,5?M),medium-dose group(M group,50?M),and high-dose group(H group,100?M).Cell samples were collected 24 hours after exposure,and cell lipids were extracted.C57BL/6J mice were exposed to perchlorate,which were blank control group(C group),low-dose group(L group,0.1 mg·kg^-1·d^-1),and medium-dose group(M group,1 mg·kg^-1·d^-1)and high-dose group(H group,10 mg·kg^-1·d^-1).The serum and liver samples of mice were obtained,lipids were extracted.The MRM and Scheduled MRM scanning modes were used to screen 2131 lipid compounds in mixed samples of cells,mouse serum and liver respectively.The chromatography and mass spectrometry conditions were optimized,and the stability and reproducibility of the method were verified.(2)The oil red O sections,TC,TG,ACC and MDH enzyme content of Hep G2 cells were detected.The different metabolites of perchlorate affecting cell lipid metabolism were screened,and the potential biomarkers were identified.Finally,the effect of perchlorate on cell lipid metabolism pathways were analyzed.(3)The HE and Oil Red O sections of the liver of the mice were observed.The different metabolites of perchlorate affecting serum and liver were screened,and the potential biomarkers were identified.The metabolic pathways of perchlorate affecting serum and liver lipids were analyzed.Result(1)There were 800 kinds of lipid compounds in a single time for the positive ion mode,including 8 kinds of monoglycerides(MAG),28 kinds of diglycerides(DAG),and 313 kinds of triglycerides(TAG),73 kinds of phosphatidylcholine(PC),63 kinds of phosphatidylethanolamine(PE),18 kinds of phosphatidylserine(PS),42 kinds of lysophosphatidylcholine(LPC),25 kinds of lysophosphatidylethanolamine(LPE),68 kinds of sphingomyelin(SM),157 kinds of ceramide(Cer)and 5 kinds of cholesterol lipids(CE).And 147 kinds of lipid compounds for the negative ion mode,including 4 kinds of lysophosphatidylinositol(LPI),19 kinds of phosphatidylserine(LPS),77 kinds of phosphatidylglycerol(PG)and 47 kinds of phosphatidylcholines(PI).The method had the characteristics of high throughput,high response,good reproducibility,and strong stability.(2)The lipid droplets in the cell oil red O section in the M group and H group were significantly more than in the C group.Simultaneously,the TC(P<0.01)and TG(P<0.05)contents of cells increased significantly in the H group,and ACC enzyme and MDH enzyme contents had no significant difference among each group(P>0.05).The different metabolites of Hep G2 cells were screened through multivariate statistical analysis and structural identification.They were PC(15:0/14:1),PC(16:1/14:1),PE(10:0/26:1),PE(15:0/24:0),PE(18:0/16:1),Lyso PC(22:6/0:0),Lyso PC(20:3/0:0),SM(d18:0/24:0),Cer(d18:1/24:0),PG(18:0/18:1),PG(18:1/20:3),PG(18:2/16:1),PG(18:2/20:4),PI(18:1/18:1),PI(18:1/16:1),PI(18:0/20:4),PI(18:0/18:2)and PI(16:0/20:3),respectively.The up-regulation potential biomarkers in Hep G2 cells were PI(18:1/16:1)and PI(18:0/20:4),(P<0.05),and the down-regulation potential biomarkers were PE(15:0/24:0),PE(10:0/26:1),PG(18:0/18:1),SM(d18:0/24:0)and Cer(d18:1/24:0),(P<0.05).The results indicated that perchlorate affected the glycerophospholipid metabolism and sphingolipid metabolism pathway of Hep G2 cells.(3)HE sections showed that the livers of C57BL/6J mice occurred different degrees of lesions.The number of lipid droplets in the livers of mice accumulated densely in the M and H group.The results showed that the different metabolites of perchlorate affecting serum lipid metabolism in C57BL/6J mice were PC(14:0/14:0),PC(16:1/15:1),and PC(22:1/14:0),PC(22:2/15:0),PC(3:0/16:2),Lyso PC(20:0/0:0),Lyso PC(20:2/0:0),Lyso PC(22:5/0:0),SM(d18:1/23:0),SM(d18:2/24:2),PG(16:0/18:1),PG(16:0/18:1),PG(16:0/18:0),PG(18:1/18:2),PG(18:1/18:1),PI(18:0/18:2)and PI(18:1/20:4).The up-regulation potential biomarkers in the serum of C57BL/6J mice were SM(d18:2/24:2)and Lyso PC(22:5/0:0),(P<0.05),and the down-regulation potential biomarkers were PC(14:0/14:0),PC(16:1/15:0),SM(d18:1/23:0),PC(22:2/15:0),PC(22:1/14:0),PC(3:0/16:2),Lyso PC(20:0/0:0)and Lyso PC(20:2/0:0),(P<0.05).The different metabolites of perchlorate affecting liver lipid metabolism in C57BL/6J mice were PC(18:0/14:0),PC(20:4/20:2),PC(22:1/14:0),PC(2:0/15:1),PE(18:0/18:2),Lyso PC(20:4/0:0),Lyso PC(22:6/0:0),SM(d18:1/22:0),SM(d18:1/22:1),TAG(50:2/FA16:0),TAG(52:2/FA18:1),TAG(52:3/FA16:0),TAG(52:3/FA18:1),TAG(52:4/FA16:0),TAG(52:4/FA16:1),TAG(54:5/FA18:2),PG(16:0/18:0),PG(16:0/16:0),PG(16:0/18:2),PG(18:0/18:1),PG(18:1/18:2),PG(18:1/20:4),PI(18:0/20:3)and PI(18:1/20:4).The up-regulation potential biomarkers in the livers of C57BL/6J mice were Lyso PC(20:4/0:0),Lyso PC(22:6/0:0),TAG(52:2/FA18:1),TAG(52:3/FA16:0),TAG(52:3/FA18:1),TAG(52:4/FA16:1),PG(16:0/16:0)and PI(18:1/20:4),(P<0.05).In addition,the down-regulated potential biomarkers were PC(20:4/20:2),PC(22:1/14:0),PE(18:0/18:2)and SM(d18:1/22:0),(P<0.05).The pathway analysis showed that perchlorate affected the glycerophospholipid metabolism pathway both in mouse serum and livers.Conclusion(1)The results showed that perchlorate increased the lipid droplets,TC and TG in Hep G2 cells.The potential biomarkers were PI(18:1/16:1),PI(18:0/20:4),PE(15:0/24:0),PE(10:0/26:1),PG(18:0/18:1),SM(d18:0/24:0)and Cer(d18:1/24:0).And perchlorate could cause disorders of glycerophospholipid and sphingolipid metabolism in Hep G2 cells.(2)Perchlorate caused liver lesions and lipid droplets accumulation in C57BL/6J mice,the potential biomarkers in serum were SM(d18:2/24:2),Lyso PC(22:5/0:0),PC(14:0/14:0),PC(16:1/15:0),SM(d18:1/23:0),PC(22:2/15:0),PC(22:1/14:0),PC(3:0/16:2),Lyso PC(20:0/0:0),Lyso PC(20:2/0:0).Moreover,the potential biomarkers in the liver were Lyso PC(20:4/0:0),Lyso PC(22:6/0:0),TAG(52:2/FA18:1),TAG(52:3/FA16:0),TAG(52:3/FA18:1),TAG(52:4/FA16:1),PG(16:0/16:0),PI(18:1/20:4),PC(20:4/20:2),PC(22:1/14:0),PE(18:0/18:2)and SM(d18:1/22:0).In addition,perchlorate could result in the disorder of the glycerophospholipid metabolism pathway in the serum and liver of C57BL/6J mice.(3)There were the same different metabolites in Hep G2 cells,the serum and liver of C57BL/6J mice.Perchlorate affected the glycerophospholipid metabolism pathway of Hep G2 cells and C57BL/6J mice.