Human Hair Targeting Lipidomics Analysis Technology And Its Application In Cushing's Disease

Hair has unique advantages in long-term disease monitoring,disease process research and pathogenesis research since it is noninvasive,time sequential and easy storing compared to other bio-specimens.Thus researchers have paid more and more attention on it.There are a little reports of hair metabolomics on diagnosis and pathogenesis of the disease.The sensitive and reliable hair metabolomics analysis has significant importance for the expandence of hair application in disease research.This thesis focus on hair metabolomics.Hair pre-treatment method were carefully optimized to achieve extraction of different types of endogenous compounds.The hair target lipidomics covering core metabolites of lipid and steroid metabolism network were established based on previous study.The established method was applied to profile the hair metabolites of rare Cushing disease for exploring the potential biomarkers associated with the disease,providing noninvasive monitoring indicators for clinical diagnosis and prognosis evaluation.The washing solvent,extraction method and the length of the hair fragment were optimized for the lipid compounds in human hair.A simple hair pretreatment method was established as following:the hair was washed with the shampoo-n-hexane method,then cut into 5 mm pieces.Sphingolipid,glyceride and phospholipid compounds were extracted using chloroform-methanol extraction method from 30mg hair.The high performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to profile hair sphingolipids and quantify the detected sphingolipids in hair.High performance liquid chromatography coupled with high resolution mass spectrometry was used to profile hair triglycerides and phospholipids and quantify the detected triglycerides and phospholipids in hair.Method validation was carried out.For sphingolipids,the linear range of standard substances were 0.50-160 pmol/mg and 0.05-16 pmol/mgwith r>0.99.LOQ were 0.50 pmol/mg and LOD were 0.125-0.25 pmol/mg.The intra-precision was less than 14.9%and inter-precision was less than 16.7%.The accuracy was between 80.2-119%.The extraction recovery of each standard substance at each concentration level was between 79.6-104%.The matrix effect was between 80.5-119%.For glyceride and phospholipid,the linear range of standard substances were 0.50-100 pmol/mg with r>0.99.LOQ were 0.50 pmol/mg and LOD for these compounds were 0.125-0.25 pmol/mg.The intra-precision was less than 20.1%and inter-precision was less than 17.3%.The accuracy is between 80.0-120%.The extraction recovery of each standard substance at each concentration level was between 80.7-99.9%.The matrix effect was between 86.9-118%.The results showed that the method meet the requirements of characterization and determination of lipid compounds in human hair.The established method was successfully applied to hair samples of male and female.32 sphingolipids,72 glycerides and 2 phospholipids in hair were successfully profiled and quatified and 29 compounds showed sex-related significant differences.In addition,the lipid concentration trends were explored from the proximal to more distal hair segments.The washing solvent,extraction method and the length of the hair fragment were optimized for the steroid compounds in human hair.A simple hair pretreatment method was established as following:he hair was washed with the shampoo-n-hexane method,then cut into 5 mm pieces.Steroid compounds from 30mg hair were extracted using methanol(18h).The ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to profile hair steroids and quantify the detected steroids in hair.The previous established ultra-high performance liquid chromatography triple quadrupole mass spectrometry method for plasma steroids was optimized,increasing the throughput of the steroids compounds from 20 to 28.Method validation was performed.The linear range of standard substances were 0.25-100 pg/mg and 0.05-20 pg/mgwith r>0.99.LOQ were 0.05-0.25 pg/mg and LOD for these compounds were 0.05-0.1 pg/mg.The intra-precision was less than 17.9%and inter-precision was less than 16.5%.The accuracy is between 79.5-115%.The extraction recovery of each standard substance at each concentration level was between 77.1-106%.The matrix effect was between 81.5%and 119%except for corticosterone(127-134%)and 17-OHPROG(67.6-72.5%).The results showed that the method meet the requirements of characterization and determination of steroids in human hair.The established method was successfully applied to hair samples of male and female.17 steroids in hair were successfully profiled and quatified and 5 compounds showed sex-related significant differences.In addition,the steroid concentration trends were analyzed from the proximal to more distal hair segments.In the thesis,the established method was applied to hair samples of Cushing disease patients group and control group.17 steroids,33 sphingolipids,100 glycerides and 4 phospholipids were detected.According to the standard of variable importance value(VIP)>1 and significant difference in T test,40 potential biomarkers were found to present different status of Cushing disease group and control group,including 6 steroids,3 sphingolipids and 31 glycerides.The result of hair metabolomics of Cushing disease provided valuable data for the future study in the new indicators of clinical diagnosis of Cushing disease and it may be complementary to the clinical diagnosis mode and provide noninvasive monitoring indicators for cinical effective treatment and prognosis.