| The glycosaminoglycan (GAG) content, composition and structure in serum havebeen suggested to play a role in chronic disease and may provide useful diagnostic orprognostic markers. The aim of this study was to assess the level ofglycosaminoglycan and their disaccharides in various chronic liver diseases and test ifsome specific compounds could be used as prognostic markers.This research optimized a protocol to separate and purify serum GAGs, includingenzymolysis extraction, anion exchange chromatography, and ultrafiltrationcentrifugal desalination. This protocol is well applicable to mass spectrometryanalysis. In addition, one studied the fragmentation of chondroitin sulfate/hyaluronicacid (CS/HA) disaccharides. The most abundant fragment ion were employed asquantitative ion for multiple reaction monitoring (MRM) in LC-MS. This study alsooptimized an Aminoacridone assisted high performance liquid chromatography(AMAC-HPLC) to analyze the composition of CS/HA disaccharides. Bothquantitative methods were used to analysis disaccharides from a wide range of serumsamples and biomarker screening was implemented to these disaccharides. Thisresearch provided insights for the establishment of next generation biomarkers andearly detection of liver diseases.This thesis was focused on the following contents:1. Four anion exchange chromatography means were compared, includingDEAE-Cellulose, DEAE-52, DEAE-Sephacel, and Vivapure Ion Exchange.Results showed that Vivapure Ion Exchange approach worked best, thanks to its high repeatability and recovery (76.6%), which would be further employed as thefollowing experiments.2. The structure of CS/HA disaccharides was analyzed by LC-ESI-QQQ,fragmentations were also proposed and illustrated. The most abundant product ionwas chosen as the MRM quantitative ion. Base the degree of sulfation ofdisaccharides, this method could safely separate disaccharides and each run wouldbe less than10min.3. Aminoacridone was employed as fluorescent derivation agent to increasedisaccharides sensitivity. AMAC-HPLC method was developed to quantifydisaccharides and analyze their composition. At the optimal conditions, baselineseparation of nine CS/HA disaccharides was achieved. The excess AMAC wouldnot influence disaccharides analysis, which simplified the procedure as a whole.The LOD and LOQ were0.36ng and1.07ng respectively, RSD were lower than4.6%. By adding disaccharide standard mixture, AMAC-HPLC and LC-ESI-QQQwere compared and evaluate their accuracy. Both methods yield similar results.4. AMAC-HPLC was used to analyze four different biological samples, includingWistar rat brain, rat serum, human serum and fetal bovine serum. One found that4SCSis the major component of the disaccharides, the contents were43.8%,29.4%,69.7%and83.0%, respectively.0SCSwere the most abundant CS disaccharide inrat serum, while human serum and fetal bovine serum were14.1%and5.5%,respectively. Moreover, there was a huge disaccharide compositional differencebetween the serum samples and the brain sample. The highly sulfated CSdisaccharides were very low in three serum samples, Tri SCS, the highest sulfatedCS disaccharide, could not be detected. Whereas the content of Tri SCSin rat brainwas16.9%, the second highest sulfated CS disaccharides, SBCS, SECS, and SDCSwere10.7%,8.8%, and6.5%, respectively.5. Quantitative analyzed CS/HA disaccharides from multiple samples, includinghealthy, hyperlipidemia, NAFLD, liver cirrhosis, hepatocellular carcinoma of human serum, and various orotic acid induced NAFLD rat serum. One found that4SCSwas elevated6%in liver cirrhosis,7%in hepatocellular carcinoma;0SHAwaselevated34.8%in liver cirrhosis,36.3%in hepatocellular carcinoma, in comparedwith healthy human serum samples.0SCSwas found significantly declining inorotic acid induced NAFLD rat group, compared with control group, while foursaponin treated group (Scc, Ag, Ds, Sfc) rising in different extent, compared withthe NAFLD rat group. Instead, the trend of4SCSwas in the opposite direction of0SCS, among the rat serum samples. According to the above results, one proposedthat4SCS,0SHAand0SCShave potentials to be prognostic markers for liver disease. |
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