| A metabonomic method based on complementary hydrophilic interaction chromatography and reversed-phase liquid chromatography combining with tandem mass spectrometry (HILIC-MS/MS and RPLC-MS/MS) has been established using electrospray ionization (ESI) in both positive and negative ion modes. Then this method combining with time-course analysis of discriminating metabolites was implemented to the metabonomic study on Walker 256 tumor-bearing rats to find the potential biomarkers that were closely associated with tumor progression. Based on the research above, pharmaco-metabonomic study of 10-hydroxycamptothecin (HCPT) were accomplished to obtain HCPT metabolites and the alternations of endogenous metabolites resulting from the intervention of drugs under different pathological states. The metabonomic analysis of Walker 256 tumor model revealed 47 potential biomarkers that had regular change trend and were closely associated with tumor progression,15 of them were identified including 9 medium-chain acylcarnitines. These acylcarnitines owned similar change trends, and reflected the reduction ofβ-oxidation of long-chain fatty acids. In the pharmaco-metabonomic study of HCPT, by the comparison of the urine samples of healthy and Walker 256 tumor-bearing rats administrated high or low dosage HCPT, the parent drug of HCPT and HCPT glucuronide were obtained, and 14 endogenous metabolites that were closely related to the toxicity or the effectiveness of exogenous drugs were obtained. Furthermore,6 more reliable biomarkers and 3 HCPT sensitive biomarkers were obtained from the fifteen potential biomarkers acquired in metabonomics study on Walker 256 tumor-bearing rats. These studies demonstrate that this metabonomic approach can help to find potential biomarkers that are closely associated with the tumor progression, and provide valuable biochemical insights into metabolic alterations and caner progression in tumor-bearing biosystems. Using metabonomics approach, the investigation of in vivo metabolic process of anti-tumor drugs and the investigation of metabolic alteration of endogenous metabolites resulting from the intervention of drugs can obtain information of many aspects, including drug metabolites, the endogenous metabolites that were closely related to the toxicity or the effectiveness of drug and so on.1. Establishment of metabonomic analysis method based on LC-MS technologyIn order to improve the separation and detection of high polar metabolites in urine samples, a metabonomic method based on complementary HILIC-MS/MS and RPLC-MS/MS had been established. HILIC-MS and RPLC-MS in ESI positive and negative ion modes were used for metabonomics analysis of SPE water elution and methanol elution, respectively. In addition, the precision, sensitivity and recovery rate of this method were investigated in detail using some representative urinary metabolites and the results demonstrated that this method had high precision, good sensitivity and recovery rate, and could be used for the metabolomic analysis of rat urine.2. Metabonomic study on Walker 256 tumor modelBased on established metabonomic approach, with the help of time-course analysis of discriminating metabolites, the metabonomic research had been performed on Walker 256 tumor-bearing rats to find urinary biomarkers that are closely associated with tumor progression. In tumor metabonomics, the change trends of cancer biomarkers should be in accordance with tumor growth or cancer progression. To obtain these biomarkers, the change trends of discriminating metabolites during tumor progression were investigated and the results showed that the metabolites that increased throughout the cancer progression resulted from the changes of the physical state of Walker 256 tumor-bearing rats, while the metabolites that rose first and fell later during the cancer progression resulted from tumor growth. They were potential biomarkers that were closely associated with tumor progression. By this strategy, forty-seven potential biomarkers were obtained and fifteen of them were identified including twelve carnitine derivatives, two amino acid derivatives and one nucleoside. Furthermore, the biological significance of those potential biomarkers was investigated and explained. The results showed that such disruption might result from elevated carcinoma cell proliferation, reducedβ-oxidation of fatty acids and the loss of immunocompetence in Walker 256 tumor-bearing rats. These potential biomarkers had regular change trends in urine during tumor progression, so the concentrations of these metabolites might be helpful to monitor cancer progression in Walker 256 tumor-bearing rats with noninvasive techniques and might provide valuable reference for the monitoring of human cancer progression.Additionally, the metabolic correlation analysis has been introduced into metabonomics studies for biomarker identification and the explanation of biological significance of potential biomarkers. The results showed that this strategy can be useful for finding the correlation among the potential biomarkers and further provided biochemical insights into metabolic alterations in Walker 256 tumor-bearing rats.3. Study on analytical method of pharmaco-metabonomics based on Walker 256 tumor model.Based on above research, the analytical method of pharmaco-metabonomics was established using metabonomic approach on healthy and Walker 256 tumor-bearing rats administrated HCPT. The in vivo metabolic process of anti-tumor drugs on healthy and tumor-bearing animals and the investigation of metabolic alteration of endogenous metabolites resulting from the intervention of drugs were investigated in detail. By the comparison of the urine samples of healthy and Walker 256 tumor-bearing rats administrated high or low dosage HCPT, the parent drug of HCPT and HCPT glucuronide were obtained, and the metabolic alternations resulting from different administration time, dosage, and pathological state were analyzed. Furthermore,14 endogenous metabolites that were closely related to the toxicity or the effectiveness of exogenous drugs were obtained from the discriminating metabolites between the high or low dosage groups and corresponding control groups, respectively. In addition,6 more reliable biomarkers and 3 HCPT sensitive biomarkers were selected from the fifteen potential biomarkers acquired in metabonomics study on Walker 256 tumor-bearing rats. These studies demonstrated that this pharmaco-metabonomic method can acquire drugs metabolic information, drug toxicity and effectiveness, and can be used for the screening of more reliable potential biomarkers and drug sensitive biomarkers. This pharmaco-metabonomic approach maybe provides novel way for pharmaceutical research.4. Investigation of normalization method for metabonomic analysis of urine sampleIt is essential to choose one ideal normalization approach for LC-MS based metabonomics on urine samples. However, it is difficult to evaluate the normalization approaches because there is no unified criterion to evaluate the normalization results. In this study, the advantages and disadvantages of common normalization approaches were investigated on sequential diluted urine samples aiming to choose one best normalization method. Furthermore, the favorite normalization approach was verified using urine samples of rats of pre-and post-inoculation of Walker 256 carcinoma cells. The results showed that the control of injection volumes by their creatinine values could eliminate the intra-group differences caused by the variation of the concentration of urinary metabolites, and could bring better parallelism and clustering effect. In addition, peak area normalization could further eliminate the intra-class differences. Based on the study above, we proposed that the best normalization method is the combination of peak area normalization and the control of injection volumes of urine samples by their creatinine values.
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