Preliminary Study Of Inflammation On Animal Model By Hydrophilic Chromatography (HPLC-HILIC-DAD) Based Metabonomics

Objective:(1) Establishing an HPLC-HILIC-DAD based profiling of rat biological fluids with good reproducibility and,simple sample preparation. On the basis of that, metabolic phenotype of the inflammatory model and the control group will be obtained. Chromatography peaks related to the difference of metabolic phenotype of the inflammation model from the normal group can be detected and identified.(2) Development of an HPLC-HILIC-DAD method to quantify simuLtaneously hippuric acid, uric acid, allantoin, creatine and creatinine in rat urine, and understanding whether the levels of these metabolites have differences in gender and inflammation treatment.Methods:(1) The inflammation rat was induced by carrageen glue. Urine, blood, liver and kidney tissue of the inflammation group and control group were collected respectively. Samples was analyzed by developed HPLC-HILIC-DAD method. Principal components analysis (PCA) and Partial least squares-Discriminant analysis (PLS-DA) were employed to classify the inflammation group to the control group and the chromatography peaks related to the classification was obtained.(2)16rats were divided into four groups randomLy,each for4rats. There are Female normal group, inflammation of the female group,male normal group and inflammation of the male group respectively. The12-hour urine samples of rat were collected at room temperature. After pretreatment of those, the contents of hippuric acid, uric acid, allantoin, creatine and creatinine were quantified by HPLC-HILIC-DAD method as follows:ZIC-HILIC column,0.25%acetonitrile (A) and0.5%phosphoric acid+0.005mol-L-1ammonium dihydrogen phosphate (B,75:25) for isocratic elution, the column was maintained at30℃, flow rate was set at0.8mL·min-1and detection was set to205nm.ResuLts: (1) The developed HPLC-HILIC-DAD method have good reproducibility and simple sample preparation. On the basis of analysis of the profilings by PCA, we found that there was different metabolic phenotype between the inflammation group and the control group. After PLS-DA analysis some chromatography peaks in biological fluids (urine and kidney extraction) related to the classification was confirmed. These peaks were successfuLly isolated into discrete chromatographic peak after semi-preparative HPLC. The chromatographic peaks in time of26.5-26.6,33.2-33.4min and33.5-34.0min were identified as uric acid, creatine and creatinine respectively. The identification of other peaks is undergoing.(2) The HPLC-HILIC-DAD method developed was validated to be specific, good linear range from3-250μg-mL-1, good precision and high accuracy. The urinary content ratio of uric acid to creatinine was found to be significantly different between male rats and female ones; and the urinary content ratio of uric acid to creatinine and creatine to creatinine was found to be significantly different between inflammatory rats and normal ones.Conclusion:(1) A readily obtained and simple sample pretreatment HPLC-HILIC-DAD rat biological fluid profiling with good reproducibility was developed.(2) There is different HPLC-HILIC-DAD based metabolic phenotype in inflammation group and the normal group.The chromatography peaks related to the differences were obtained and some of those have been identified.(3) An HPLC-HILIC-DAD method to quantify simuLtaneously hippuric acid, uric acid, allantoin, creatine and creatinine in rat urine was successfuLly developed. The urinary content ratio of uric acid to creatinine was found to be significantly different between male rats and female ones. The levels of hippuric acid and allantoin decreased obviously in urine of inflammation rats indicating that inflammation may have affects on the nucleotide catabolism and the reguLation of intestinal flora in the body.