| Background:Hepatolenticular degeneration,also known as Wilson's disease(WD),is caused by the mutation of ATP7 B gene on the autosomal gene,which leads to the disorder of copper metabolism and the deposition of copper in multiple organs of the body.If not treated in time and reasonably,the progressive aggravation may lead to disability or even death.However,there are still some deficiencies in the current diagnosis basis.The degree of ceruloplasmin reduction is different or even normal,and it can also be reduced in other liver diseases;liver copper is a invasive test;ATP7B gene detection is relatively expensive;therefore,more sensitive and specific diagnostic markers are needed in clinical work.The pathological changes of the liver in WD patients were similar to those in non-alcoholic fatty liver,but the serum triglyceride level in WD patients was low,which was opposite to that in non-alcoholic fatty liver patients.However,the mechanism of lipid changes in WD has not been fully elucidated,and there are different opinions.Therefore,it is necessary to use the method of lipid metabonomics to study the lipid changes in WD deeply,in order to explore the markers for diagnosis and clarify the mechanism.Through the application of lipid metabonomics technology to detect lipid substances in WD serum,to explore the change rule of lipid substances,so as to explain the phenomenon of inconsistency between serum indexes and pathological changes,and explore the metabolic markers that may be valuable for disease diagnosis and explain the mechanism.Aims:The serum samples of WD patients,family members and healthy controls were detected by lipometabonomics to find out the different metabolisms,so as to find out the metabolic markers with diagnostic value,find out the affected metabolic pathway and explore the lipid metabolism mechanism of WD.Methods:According to the research method of genetic diseases,130 subjects were included in this study,which were divided into patients group(P)34,family group(F)31 and control group(C)65.Fasting blood samples were collected in the early morning of the study subjects,the serum samples were pretreated,and then positive and negative ions were detected by ultra-high performance liquid chromatographyhigh resolution mass spectrometry.Lipidsearch software was used to process the collected lipomics data,including peak detection and lipid structure identification.The data matrix values were selected according to conditions,standardized and then imported into metaboanalyst 4.0 online software and Simca-p 14.1 for pattern discrimination analysis.Through principal component analysis,partial least squares discrimination analysis and orthogonal partial least squares discrimination analysis,the model was constructed according to groups,and the main differential metabolites between groups were found.According to t-test and ANOVA,p-value(FDR correction)of significance level between groups was obtained to build volcano map,look for different metabolites between groups,and find the affected metabolic pathway based on KEGG database and human metabolite datase database.SPSS 25.0 software was used to analyze the general situation and laboratory indexes of the subjects.Logistic regression analysis and ROC curve of the subjects were used to analyze the diagnostic value of compound molecules for WD.Results:1.The sex ratio of the patients was 1:1,which was consistent with the inheritance law of euchromosome;62% of the patients were young;nearly half of the patients were liver type,35% were mixed type,and all were mixed type of liver type and brain type;79% of the patients had a significant decrease of CP < 0.1g/l,but at the same time,20% of the patients had a slight decrease of ceruloplasmin in the range of 0.1-0.2g/l;16 patients were detected The mean value of triglyceride was 0.85 mmol/L(0.28-1.8 mmol/L).43 people were detected in the control group,the mean value was 1.19 mmol/L.the level of patients was generally lower than that of the control group,and the difference between the two groups was statistically significant.2.The coefficient of variation of 98% lipid quality control samples is less than 30%,which shows good quantitative accuracy in the whole experiment process,and the correlation between quality control samples is between 0.99-1,so the reproducibility of the whole experiment is very good.3.After building the data model,it is found that there are differences between groups,and the prediction effect of this sample is good without any fitting,the experimental results are reliable and have clinical prediction significance.4.42 different metabolites were found between the patient group and the control group,mainly including triglyceride,phosphatidylcholine,phosphatidylethanolamine,lysophosphatidylcholine and ceramide,and a few of them were glycosphingolipids,cholesterol esters,phosphatidylinositol,phosphatidylserine and phytosphingosine.The two metabolic pathways were sphingolipid pathway and glycerophosphingolipid pathway.5.In WD patients,triglycerides with saturated fatty acid structure increased,acetaldehyde phosphatidylethanolamine decreased,and ultra long chain ceramide decreased.6.No significant metabolites were found between patients and their families,patients with different clinical phenotypes,patients with different ceruloplasmin levels,family members and healthy controls.7.The area under ROC curve of WD predicted by LPC(18:0),LPC(18:1p),LPC(17:0)and LPC(15:0)was 0.981,0.959,0.980 and 0.960 respectively.The area under ROC curve predicted by TG(38:0),TG(36:0)and TG(47:0)was 0.947,0.905 and 0.908,respectively.The area under ROC curve predicted by ceramide cerg1(d42:2),cer34:0 and cer41:2 was 0.915,0.909 and 0.911 respectively.The area under ROC curve predicted by DG(34:0)and DG(36:0)was 0.925 and 0.920 respectively.The area under ROC curve predicted by phosphatidylserine PS(34:0)and PS(35:0)was 0.911 and 0.919 respectively.8.LPC(17:0)and LPC(18:0),PC(32:1)and PC(37:5),PC(32:1)and PC(39:5),PC(32:1)and PC(39:6)have higher diagnostic value.The area under ROC curve is 0.989,0.967,0.972 and 0.949 respectively.Conclusions:1.There were significant differences in lipid metabolism between WD patients and healthy controls.The affected metabolic pathways are sphingolipid pathway and glycerophosphingolipid pathway.2.In WD patients,the change of saturated fatty acid and unsaturated fatty acid ratio of triglyceride molecules is the cause of low serum triglyceride and liver lipid deposition.3.The change of phospholipid in cell membrane causes the destruction of hepatocyte membrane,which leads to hepatocyte apoptosis and focal necrosis.Lysophosphatidylcholine LPC(17:0),LPC(18:0),phosphatidylcholine PC(32:1),PC(37:5),PC(39:5),PC(39:6)binding ceruloplasmin have the potential as clinical diagnostic markers of WD. |
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