Study On Analytical Method Of Pterins And Amino Acids In Rat Urine

According to the reports in the literature, pterins and amino acids were involved in and played an important role in the metabolism of organisms. When the living organisms suffered from cancer or other disease, the concentrations of pterins and amino acids would change significantly, so the two kinds of compound had the potential to be tumor biomarkers. In this paper, the author reviewed pterins and amino acids on analysis methods, sample preparation technologies and correlation with tumor, at last, introduced the modeling methods of bladder cancer model. On this basis, using chromatography analysis technology, we established new simultaneous determination methods of xanthopterin, isoxanthopterin, tyrosine and tryptophan in rat urine, applied the methods to the analysis of bladder cancer model rat urine samples, carried out the preliminary study on the bladder cancer biomarkers. The main research results are as follows:1. Objective:A bladder cancer model of SD rats was established using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) as inductive regent and the pathology diagnosis results were compared with the results of MRI diagnosis technology. Methods:112SD rats (6weeks of age) were randomly divided into two groups by weight,42rats were in the control group and70rats were in the model group. The model group was given0.05%BBN in water and the control group was treated with tap water. The whole experiment continued for35weeks, the changes of the bladder were studied with MRI scanning and pathology every5weeks after treatment. At the end of the experiment, all the animals were put to death for pathological examination. Results: At the25th week, the rats of model group were diagnosed as bladder cancer by MRI scanning, while the pathology examination found cancer cell early at15th week. No bladder tumor was detected by MRI scanning and pathological examination in the control group. At the35th week, all model group rats were judged as bladder cancer with pathology, the control group rats had not been found tumor tissues with MRI and pathology during the whole experiment process. Conclusion: The establishment of a rat bladder cancer model with BBN is successful and simple. The pathological diagnosis of bladder cancer is earlier than MRI diagnosis.2. A simple, rapid and sensitive method for the determination of xanthopterin, isoxanthopterin, tyrosine and tryptophan in mouse urine by UPLC-DAD has been developed. In order to correct the individual difference, creatinine was chosen as internal standard. After removed protein with methanol and purified with C18solid phase extraction column, the mouse urine samples were detected directly by UPLC-DAD. The limits of instrument detection of the five analytes were0.011,0.007,0.030,0.025and0.003mg/L, respectively. The recoveries were in the range of96.3%～106.3%except that of xanthopterin were in the range of60.8%～80.7%. The relative standard deviations were lower than9.85%.3. A simple, rapid and sensitive method for the determination of xanthopterin, isoxanthopterin, tyrosine and tryptophan in mouse urine by UPLC-FLD has been developed. After removed protein with methanol and acetonitrile, the mouse urine samples were purified with C18solid phase extraction column and then detected directly by UPLC-FLD. The limits of instrument detection of the four analytes were5、0.3、4and2μg/L, respectively. The recoveries were in the range of85.0%～110.0%except that of xanthopterin were in the range of61.1%～78.0%. The relative standard deviations were lower than10.2%.4. A simple, rapid and sensitive method for the determination of xanthopterin, isoxanthopterin, tyrosine and tryptophan in mouse urine by UPLC-DAD-FLD has been developed. After removed protein with methanol and acetonitrile, the urine samples were purified with C18solid phase extraction column and detected directly by UPLC-DAD-FLD. The limits of instrument detection of the five analytes were5、5、0.3、10and3μg/L, respectively. The recoveries were in the range of80.0%～102.8%except that of xanthopterin were in the range of61.1%～77.1%. The relative standard deviations were lower than10.9%. The method has been applied to the determination of bladder model rat’s urines, it was found that with the appearance and development of bladder cancer, the concentration of the four analytes(Xan, Isoxan, Tyr and Trp) in urine has all changed and the variation of isoxanthopterin was the most significant one.