Study On Metabolic Profiles Of Hypertension

Metabonomics is the quantitative measurement of the multiparametric time-related metabolic responses of a complex (multicellular) system to a pathophysiological intervention or genetic modification. Metabolomics apply useful modeling tools for the classification and prediction of physiological and pathological states from metabolite profiles of biofluids such as plasma and urine. Thus metabonomics seeks to assess the global system level homeostatic and pathological responses to interventions or stressors. Metabolome is the result of whole expression of proteome, transcriptome, genome which reflect the biochemical status of tissues directly. Urine and plasma sampling are non-invasive and commonly used for in vivo metabolic profiling. It is very important for the illustration of the complicated life systems because of the sensitive response of the changes of the philological and pathological status of the life. Metabonomics has become an important hotspot for the research of disease and healthy in the world now.Metabolite profiling analysis represents an extremely useful tool that finds application in many aspects of disease diagnosis, in the characterization of pathological states and disorders of cell and organisms, in physiological and taxonomic studies. By determining humoral metabolites of the same kind and similar biomarkers that associated to pathological and physiological state, metabolite profiling analysis discover the mechanism of disease for aided clinical diagnosis and treatment. In the present study, we have developed GC and HPLC approaches as determination means of metabolic profiling analysis, to differentiate healthy persons and hypertension patients. Plasma metabolite content from healthy persons and hypertension patients were acquired using GC and HPLC methods. We also established a metabolite fingerprinting analysis method for plasma and urine by HPLC/TOF-MS. Using pattern recognition, it was capable of distinguishing normal blood pressure plasma samples from hypertensive patients. It was showed that the analysis of metabolite in plasma can recognize hypertension condition. The present study was a successful research of hypertension using metabolic profiling analysis approach. The study provided theory for the early-stage diagnose and prevention of disease and pathopoiesis mechanism.1. Determination of free fatty acids in plasma by derivatization GC methodTo establish a GC method for determining 10 kinds of free fatty acids in plasma. The samples were derivatised before analysis. The free fatty acids in plasma were esterified using H2S04-methanol. The influence of temperature, derivatizing time and different derivatizing conditions on the extent of esterification reaction was investigated. The free fatty acids in plasma can be esterified in 10% H2SO4-methnol at 56℃for 1h. The standard calibration curve was obtained from the ratio value of chromatographic area of fatty acids and internal standard and the concentration of fatty acids. The calibration curves showed linearity over the studied concentration ranges(r> 0.99). The intra-day and inter-day precisions were 1.8%-3.9% and 2.3%-5.6%, respectively. The spiked recoveries were between 88.4% and 104%. The method was applied in the determination of the concentration of the plasma free fatty acids in hypertensive patients and healthy subjects. The experimental data were further analyzed by the principal component and classification method, which indicated that there was a clear differentiation between healthy people and hypertensive patients with the classification value of 85.2%.2. Determination of amino acids in plasma by derivatization HPLC methodA reversed phase high performance liquid chromatographic (RP-HPLC) method for simultaneous determination of 22 free plasma amino acids was established by using 2,4-dinitrochlorobenzene as pre-column derivatization agent. Free amino acids were derivatized in borate buffer solution (pH 9.0) at 90℃for 1.5h, the mole ratio of derivatization agent and amino acid were 4:1. The amino acid derivatives were separated on a Kromasil C18 column with gradient elution mode and detected at wavelength of 360 nm. The calibration curves showed linearity over the studied concentration range. The intra-day and inter-day precisions were 1.7%-4.1% and 2.8%-6.9%, respectively. The spiked recoveries were between 90.0% and 106%. The method was applied in the determination of the concentration of the free plasma amino acids in hypertensive patients and healthy subjects. The experimental data were further analyzed by the principal component analysis and classification method, which indicated that there was a clear differentiation between healthy people and hypertensive patients with the classification value of 94.4%.3. Metabolite fingerprinting analysis of plasma and urine by HPLC/TOF-MS methodA HPLC/TOF-MS method for metabolite fingerprinting analysis of plasma and urine was established. The samples were separated on a Kromasil C18 column with gradient elution mode and detected in positive mode. The samples were stable in 12h for the RE was±9.4 in plasma and±10.1% in urine. The equipment precision were 0.98%-3.4% for plasma and 0.84%-4.1% for urine. The method was applied in the metabolite fingerprinting analysis of samples from hypertensive patients and healthy subjects.