Study On Abnormal Lipid Metabolism And Potential Biomarkers Of AD Based On Non-targeted And Targeted Lipidomics

Lipids play vital roles in the maintenance of cell membrane microenvironment,energy homeostasis,signal transduction and other biological functions.The elucidation of lipidome profiles under physiological and pathological conditions is extremely important for the development of effective drug targets for many relevant diseases.Emerging lipidomic technology will allow for discovery of a variety of lipidome and their biological functions.Alzheimer’s disease(AD)is the most dominant dementia among aging people,and its underlying mechanism and effective therapy remain to be studied.As brain is composed of the most abundant lipids and key inflammatory factors of AD pathogensis are regulated by various lipid molecules,the study on relationship between AD and lipids draws more and more attention in bioscience and pharmaceucical fields.The purpose of the current study is to establish a comprehensive and robust lipidomic methodology and utilize the AD biosamples and APP/PS1 transgenic mice models to explore abnormal lipid metabolism and potential lipid biomarker of AD.Firstly,non-targeted and targeted lipidomic analytical method was developed and validated in this study.In the non-targeted lipidomic method,methyl tertiary butyl ether(MTBE)was used to extract the lipidome from the plasma.The extracted samples were subjected to liquid chromatography time of flight mass spectrometry(HPLC-TOF-MS)to obtain the total ion chromatogram(TIC)and mass spectrometry of parent and daughter ions.The peak intensity of extracted ion chromatogram(EIC),which were extracted from TIC based on the molecular weight(MW)of lipid species,were used quantitate their amount.The exact MW of parent and daughter ions,isotope distribution pattern and peak retention time were used to identify the lipid species.Based on our untargeted lipidomic approach,we identified and analyzed about 200 plasma lipid species,which covered most abundant lipid classes in plasma,e.g.phosphatidylcholine(PC),phosphatidylethanolamine(PE),sphingomyelin(SM),ceramide(Cer),triglyceride(TG)and free fatty acid(FFA).Furthermore,we tested the lipidome ex vivo stability in plasma for the first time and found that certain lipids underwent extensive degradation.To resolve this issue,PMSF(phenylmethylsulfonyl fluoride)were selected to prevent lipid degradation,which ensured our lipidomic data more reliable and repeatable.In the targeted lipidomic method,cholesterol,cholesterol precursors(desmosterol and lanosterol),peripheral metabolite 27-hydroxy cholesterol(27-OHC)and brain specific metabolite 24-hydroxy cholesterol(24-OHC)were simultaneously determined using liquid chromatography triplequadrupole mass spectrometry(HPLC-QQQ-MS).The method validation results suggested that both non-targeted and targeted methods showed good performance in sensitivity,specificity,accuracy and precision and could be utilized in following biomarker discovery.Secondly,the plasma and brain samples from APP/PS1 transgenic mice were analyzed on basis of validated lipidomic method.The results indicated that plasma levels of very long chain fatty acid(VLCFA)and plasmenylethanolamine(PlsEtn)in APP/PS1 mice significantly increased and decreased respectively compared to the controls.Furthermore,the difference between APP/PS1 transgenic mice and controls became more and more significant as aging,and the change in cortex and hippocampus of APP/PS1 was consistent with that in plasma.It was known that both VLCFA catabolism and PlsEtn anabolism exclusively occurred in the peroxisome,so synchronic changes of VLCFA and PlsEtn might indicate that the function or number of peroxisome were altered in AD pathology progress.Further studies showed that mRNA and protein level of PPAR-γ(peroxisome proliferator activation receptor gama)significantly down regulated in cortex and hippocampus of APP/PS1 transgenic mice compared to the controls,while that change did not occur in other brain region.Based on these evidences,we believed that the down regulation of PPAR-γ resulted in the insufficient amount of peroxisome and following decrease of VLCFA catabolism and PlsEtn anabolism.Notably,the insufficiency of peroxisome also related to the decrease of reactive oxygen species(ROS),which is a well-known etiology of AD.Finally,the lipidome profiles were examined in fasted plasma samples of AD patients and cognitive normal controls.The concentrations of VLCFA and PlsEtn in plasma of AD patient significantly increased and decreased respectively,which was similar to the changes in APP/PS1 transgenic mice.The areas under ROC curve of VLCFA and PlsEtn were over 0.9,indicating that both classes of lipid could discriminate two groups of people with good accuracy.Also,poly-unsaturated fatty acid significantly decreased in plasma of AD patients compared to controls.Furthermore,the intervention of rHDL(reconstituted high density lipoprotein)in SAMP8 mice could significantly improve the cognitive function with corresponding plasma VLCFA and PlsEtn concentration changes.In summary,our lipidomic method based on LC-MS platform was versatile,robust,and quite useful for the biomarker discovery.The study indicated that VLCFA and PlsEtn may serve as the potential AD lipid biomarkers and activation of PPAR-γ might be a useful appraoch for AD disease modified therapy.The finding will provide new information for the insight into new biomarkers and targets for AD treatment and diagnosis.