Analytical Methods Development For Disease Biomarker Discovery

Since the completion of the first whole-genome sequence of a free-living organism,we began to realize the paucity of our knowledge with respect to the existence,let alone the function,of the novel genes thereby uncovered.More recently,completion of the human genome sequence has accelerated further the demand for determining the biochemical function of orphan genes and for validating them as molecular targets for therapeutic intervention.The search for biomarkers that can serve as indicators of disease progression or response to therapeutic intervention has also increased. Functional studies have thus emphasized analyses at the level of gene expression (transcriptomics),protein translation(proteomics) including post-translational modifications,and the metabolic network(metabolomics),with a view to a 'systems biology' approach of defining the phenotype and bridging the genotype-to-phenotype gap.There is active debate in the research community over the exact definition of the 'metabolome',but it was first defined by Oliver et al.as the quantitative complement of all of the low molecular weight molecules present in cells in a particular physiological or developmental state.Metabolomics is gaining increasing interest in drug discovery and disease diagnostics and treatment.The concept was recently introduced as the global analysis of all metabolites in a sample(metabolomics) and the analysis of metabolic responses to drugs or diseases(metabonomics).Metabolomics has proven to be very rapid and superior to any other post-genomics technology for pattern-recognition analyses of biological samples.Changing steady state concentrations and fluctuations of metabolites that occur within milliseconds are a result of biochemical processes such as signaling cascades:metabolomic techniques are instrumental in measuring these changes rapidly and sensitively. Metabolite data can be complemented by protein,transcript and external (environmental) data,thereby leading to the identification of multiple physiological biomarkers embedded in correlative molecular networks that are not approachable with targeted studies.In view of the chemical and physical diversity of small biological molecules, the challenge remains of developing protocols to gather the whole 'metabolome'. No single technique is suitable for the analysis of different types of molecules, which is why a mixture of techniques has to be used.GC-MS is a combined system where volatile and thermally stable compounds are first separated by GC and then eluting compounds are detected traditionally by electron-impact mass spectrometers.In metabolomics,GC-MS has been described as the gold standard,although it is biased against non-volatile, high-MW metabolites.Volatile,low-MW metabolites can be sampled and subsequently analyzed directly,including breath and plant volatiles.However, the majority of metabolites analyses require chemical derivatisation at room or elevated temperatures to provide volatility and thermal stability prior to analysis.In this thesis,we focused on the development of series of novel techniques and methods to analyze the low-molecular weight molecules in the biological fluids and tissues and find the potential biomarkers of fatal diseases such as cancers,diabetes and inborn errors of metabolism.This dissertation is divided into four parts.In Chapter 1,advances in metabolomics research,current analytical platforms and methodologies,applications of f metabolomics research techniques in the field of biomarkers research,drug discovery,and clinical diagnostics and so on were summarized in details.The intention and meaning of this dissertation were explained.In chapter 2,GC-MS and solid-phase microextrction with on-fiber derivatization was developed for analysis of acetone in human plasma and breath.It is demonstrated that breath analysis could be applied to diagnosis of diabetes.In the study of lung cancer biomarkers,we developed headspace solid-phase microextraction for determination of volatile compounds in blood. Using this method,for the first time,we found two aldehyde biomarkers (hexanal and heptanal) in lung cancer blood.Further,headspace solid-phase microextrction with on-fiber derivatization technique was developed for determination of aldehydes in blood.The results proved that hexanal and heptanal might be biomarkers of lung cancer.In chapter 3,we developed two new approaches to the analysis of the lung cancer biomarkers,hexanal and heptanal in human blood that was based on headspace single-drop microextraction(HS-SDME) with(droplet) derivatization, followed by gas chromatography-mass spectrometry(GC-MS).Aldehydes in blood were headspace extracted,concentrated,and derivatized by a suspended microdrop solvent containing the derivatization agent O-(2,3,4,5,6-pentaXuorobenzyi)hydroxylamine hydrochloride.The aldehyde oximes formed in the microdrop solvent were analyzed by GC-MS.the proposed method was applied to the quantiWcation of hexanal and heptanal in cancer blood and normal blood.Due to sample extraction,concentration,and derivatization being performed in a single step,the method provided a simple, rapid,low-cost,and efficient approach to analysis of aldehydes in blood samples.In chapter 4,a novel technique of aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry was developed for the determination of organic acids in urine.The analytical procedure involves derivatization of organic acids to their ethyl esters with diethyl sulfate,headspace sampling,and GC/MS analysis.The proposed method was applied to the determination of methylmalonic acid and glutaric acid in urine.The experimental parameters and method validation were studied. Consequently,in-situ derivatization/HS-SPME/GC/MS is an alternative and powerful method for determination of organic acids as biomarkers in biological fluids.In summary,the main contributes of this dissertation is that we initially developed a series of novel techniques and methods to quick and quantitative measure the low-molecular weight metabolites in biological fluids and tissues. We established HS-SPME/GC/MS and HS-SDME/GC/MS to analyze the aldehydes and short chain fatty acids in blood and urine to find the biomarkers of feral diseases.We aimed at exploring and finding out new techniques in sample preparation,derivatization and enrichment of metabolome research fields,so that more breakthroughs can be obtained in the metabolome research study.