The Study Of Serum Lipidomics In Patients With Non-alcoholic Fatty Liver Disease And Type 2 Diabetes Mellitus

Objective:The distribution of serum lipids in patients with type 2 diabetes mellitus combined with nonalcoholic fatty liver disease（NAFLD）was analyzed to investigate the changes in serum lipid composition in non alcoholic simple fatty liver（NAFL）,nonalcoholic steatohepatitis（NASH）,and different degrees of liver lipid degeneration,inflammatory injury and liver fibrosis.The correlation between lipid composition and inflammatory markers and liver related indexes was preliminarily analyzed to explore the relationship between the change of lipid composition and the progress of disease.And screening serum markers specific for nonalcoholic steatohepatitis（NASH）,it also opens new ideas for the target of the treatment of NASH.Methods:Object:40 cases of type 2 diabetes patients combined with nonalcoholic fatty liver,liver biopsy has been carried out,they were selected from September 2014to October 2017 in the department of Endocrinology and metabolism,Tianjin Third Hospital,including 17 male patients and 23 female patients,aged 30-71 years,the average age was（54.30±11.28 years）.Group:1.The 40 patients were divided into simple fatty liver group（NAFL:22）and steatohepatitis group（NASH:18 cases）according to the results of liver biopsy（SAF score）.2.Then the NAFL group（22cases）was divided into S1 group（1-2 points）and S2 group（3 points or more）according to the SAF liver fat score of liver biopsy.3.The NASH group（18 cases）was divided into N1 group（1-2 points）and N2 group（3 points or more）according to the liver fat content score of liver biopsy SAF.4.the steatohepatitis group（NASH:18）was divided into A1 group（2 points）and A2 group（3-4 points）according to SAF liver inflammation activity.5.All patients（40 cases）were divided into NAFL control group（no inflammation and fibrosis group,group NAFL_N）,NASH without fibrosis group（F0,NASH_N）,NASH with mild fibrosis group(F1,NASH _F1)and NASH with progressive fibrosis group(F2-F3 phase,NASH_F2-32-3 group),according to SAF liver biopsy.Index:The above groups were compared with the following indexes:collect the clinical data of each case,including:general condition,blood pressure,blood glucose,glycosylated hemoglobin（HbA1c）,white blood cell（WBC）,platelet（PLT）,blood fat,liver function,uric acid（UA）,serum iron（PE）,unsaturated iron binding capacity（UIBC）and ferritin（SF）,visceral fat Area,liver hardness,liver adipose attenuation,NLR,NASH score（z=-2.604+0.0025×ferritin（ug/L）+0.027×AST（U/L）+0.0687×BMI（kg/m²）-0.0052×platelet count（×10⁹/L）+0.7136（diabetes=1,no=0）+0.5723（hypertension=1,no=0）,p=100×exp（z）/[1+exp（z）],p value is NASH score）,NFS（NFS=-1.675+0.037×age（age）+0.094×BMI（kg/m²）+1.13×impaired glucose regulation/diabetes（yes=1,no=0）+0.99×AST/ALT ratio-0.013×platelet count（×10⁹/L）-6.6×albumin（g/L））.Interleukin-6（IL-6）and tumor necrosis factor-alpha（TNF-α）were measured by immunoenzyme adsorption.The determination of serum lipid composition and arachidonic acid（AA）were carried out by the method of liquid spectrum mass spectrometry.Application of Simca-P software in the analysis of lipidomics,the others were analyzed by SPSS24.0software to find out the difference between groups,and the relationship between different lipid components and inflammatory markers and liver indexes was analyzed.Results:1.Liver stiffness,NASH score,AST,ALP,GGT,vLDL-C,FBG,SF,IL-6 and TNF-αin group NASH were all higher than in NAFL group（p<0.05）,while AA level in NASH group was lower than in NAFL group（p<0.05）.5 phosphatidylcholine（PC）,5 phosphatidyl ethanolamine（PE）,1 phosphatidyl serine（PS）,2 triglycerides（TG）,3sphingomyelin（SM）,palmioyl phenylalanine,1 ceramide（Cer）were higher in the NASH group than in the NAFL group;6 triglyceride（TG）and 2 PC levels decreased in NASH group.The total PE level in group NASH increased and the PC/PE value decreased,the difference was statistically significant（p<0.05）.2.The level of SF in group S2 was higher than that in group S1,the difference was statistically significant（p<0.05）,but there was no significant difference in other clinical indexes and IL-6,TNF-αand AA levels.The levels of TG（54:4）and TG（54:5）in group S2 were significantly lower than those in S1 group（p<0.005）.3.There was no significant difference in clinical data between N1 and N2 groups.The level of TNF-αin group N2 was higher than that in group N1,and the difference was statistically significant（p<0.05）.N2 group PC（43:2）,PC（36:5）^e[PC（20:5/16:0）],PC（36:5）^f[PC（22:5/14:0）]were lower than N1 group,the difference was statistically significant（p<0.05）.4.There was no significant difference in clinical data between A1 and A2 groups.The level of IL-6 in group A2 was significantly higher than that in group A1,and the difference was statistically significant（p<0.005）.The levels of PC（36:1）^a[PC（19:1/17:0）],PE（35:0）,PE（38:1）,PS（37:0）,TG（58:1）and TG（29:0）were higher in group A2 than those in group A1（p<0.05）,the levels of PC（34:1）,PE（34:1）^c[PE-NMe2（18:1/16:0）]and PS（33:0）were significantly higher than those in A1group（p<0.005）.5.The liver hardness and SF in group NASH_F2-32-3 were significantly higher than those in group NAFL_N（p<0.005）,higher than those in group NASH_N（p<0.05）.The AST in group NASH_F2-32-3 was significantly higher than that in group NAFL_N（p<0.005）,higher than that in group NASH_N and NASH_F1（p<0.05）.The GGT in group NASH_F2-32-3 was higher than that in group NAFL_N and group NASH_N（p<0.05）.The vLDL-C in group NASH_F11 was significantly higher than that in group NAFL_N（p<0.005）,higher than that in group NASH_N（p<0.05）.The NASH score in group NASH_F2-32-3 was higher than that in group NAFL_N（p<0.05）.The level of IL-6 in group NASH_F2-32-3 was higher than that in group NASH_N（p<0.05）,which was significantly higher than that in group NAFL_N（p<0.005）.The level of TNF-αin group NASH_N and group NASH_F2-32-3 was higher than that in group NAFL_N（p<0.05）.Lipidomics analysis revealed that 2 PC,2PE,1 PS,1 SM and 1 TG are differences in four groups.Among them,the PC（36:1）^a and PE（38:1）in group NASH_F2-32-3 and group NASH_F11 were higher than those in NAFL_N group（p<0.05）.PC（34:1）in group NASH_F2-32-3 was higher than that in group NAFL_N and NASH_N（p<0.05）.PE（39:1）in group NASH_F2-32-3 was higher than that in group NAFL_N（p<0.05）.The PS（33:0）in group NASH_F2-32-3 was significantly higher than that in group NAFL_N（p<0.005）,higher than that in NASH_N group（p<0.05）.In group NASH_N and group NASH_F1,SM（39:1）was higher than that in group NAFL_N（p<0.05）.The TG（29:0）in group NASH_F2-32-3 and group NASH_F11 was significantly higher than that in group NAFL_N（p<0.005）.6.The differential lipids were analyzed by single factor and multi factor correlation analysis showed that IL-6 was positively correlated with PS（33:0）,and the difference was statistically significant（p<0.05）.Liver stiffness was positively correlated with PE（34:1）^c,and ALT was negatively correlated with TG（60:6）,and the difference was statistically significant（p<0.05）.AST was positively correlated with PS（33:0）,GGT was positively correlated with PE（34:1）^c,and the difference was statistically significant（p<0.005）.Conclusion:1.The main lipid components in the non alcoholic simple fatty liver are the triglycerides with different saturation.With the increase of liver fat content,the low triglyceride decreases.2.The changes of lipids in nonalcoholic steatohepatitis are mainly glycerol phospholipids,sphingolipids and triglycerides.3.PE（34:1）^c and PS（33:0）may become serum markers of nonalcoholic steatohepatitis.4.It seems that in patients with nonalcoholic steatohepatitis,the degree of liver injury with low triglyceride saturation is relatively mild.