Metabolic Profiling Of Urinary Estrogen Metabolites And Metabonomics Analysis Of Serum For Breast Cancer On Basis Of LC-MS/MS Method

Metabolic profiling of16distinct endogenous estrogen metabolites in urine on basis of ultra-fast liquid chromatography mass spectrometry/tandem mass spectrometry (UFLC-MS/MS) and metabonomics analysis in serum on basis of rapid resolution liquid chromatography mass spectrometry/tandem mass spectrometry (RRLC-MS/MS) integrating with multivariate statistics were explored and established for breast cancer. By the metabolic profiling of estrogen metabolites, the relative imbalances in estrogen metabolic pathway associated with breast cancer were observed,2-and4-hydroxylation pathways rather than16a-hydroxylation pathway account for the predominant contribution to the initiation of oncogenic mutations, which coincides with the acknowledged quinone-mediated carcinogenesis mechanism. Meanwhile, the results indicted that estrone (E1) and estradiol (E2) as well as their hydroxylated metabolites2-hydroxyestradiol (2-OHE2) and4-hydroxyestradiol (4-OHE2) might be the potential carcinogenic biomarkers while2-methoxyestradiol (2-MeOE2) was presumed as a potential anticancer biomarker. On the other hand, the metabonomics analysis of breast cancer in serum revealed that37endogenous metabolites, which were found with significant differences between breast cancer patients and healthy controls, namely were potential biomarkers. Therefore, combining metabolic profiling of endogenous estrogen metabolites and metabonimics analysis for breast cancer in biological samples could help discover more specific potential biomarkers for more comprehensive, entire metabolism information. It could also help get insight into the association of metabolic pathway with carcinogenesis mechanism for breast cancer to provide vaulabale evidence to early diagnosis and prediction evaluation.1. Analytical method of multiplex urinary estrogen metabolites by UFLC-MS/MSLC-MS analysis applicable for16representative endogenous estrogen metabolites in urine was investigated by a UFLC system coupled with a QTRAP system in positive ion mode. Due to the similar structures, complex sample matrices and trace concentrations, sample preparation by conlusion of enzymatic hydrolysis, SPE extraction and dansyl chloride derivatization was studied in detail, and also MRM scan mode based on LC-MS/MS was systematically optimized, finally a rapid UFLC-MS/MS method was developed for simultaneous quantitative analysis of16urinary estrogen metabolites. In addition, the specificity, sensitivity, matrix effect, recovery, accuracy, precision and stability of this method were inverstigated and validated, and the result demonstrated that this method was reliable and effective with high sensitivity, good precision and could be competitive for the simultaneous quantification of16estrogen metabolites in a large number of urine samples in16min.2. Metabolic profiling of urinary estrogen metabolites for breast cancerBased on the developed and validated UFLC-MS/MS method, metabolic profling of16estrogen metabolites in169breast cancer patients and76health controls was performed. According to the health status and menopausal status, all urine sample were separated into four groups during statistical analysis as follows:40premenopausal healthy female (H-pre),36postmenopausal healthy female (H-post),83premenopausal breast cancer patients (BC-pre),86postmenopausal breast cancer patients (BC-post). Typical MRM chromatographic profiles of16estrogen metabolites on urine samples were firstly adjusted in accordance with creatinine. And then statistical analysis was carried out by average comparison and paired t-test. Since the total exposure to estrogen is strongly affected by a variety of factors especially menstrual cycle in premenopausal female, and relative lower level in estrogen excretion and a more stable profiles in postmenopausal female, we focus on the assay of the concentration difference between breast cancer patients and health controls to explore perturbations in the metabolic pathway of estrogen metabolites and its association with carcinogenesis mechanism for breast cancer. As a result, E1and E2, as well as2-OHE2and4-OHE2were produced in higher abundance, whereas16α-hydroxyestrone (16a-OHE1) and2-MeOE2were decreased in the breast cancer group. In view of the metabolic pathway and carcinogenesis mechanism, imbalances among the three hydroxylation pathways were believed to relate with the initiation and development of breast cancer. El and E2with increased level in breast cancer individuals synergistically lead to the apprearance of spontaneous mutations which eventually increases breast cancer risk. The results provide key evidence supporting that2-and4-hydroxylation of parent estrogens, rather than the16a-hydroxylation pathway, strongly contributed to the initiation of breast cancer through a quinone-mediated mechanism. Especially2-OHE2and4-OHE2showed as key metabolites. The lower excretion of2-MeOE2in breast cancer cases hinted that methylation might play an important role in protection again rapid tumor growth, and therefore2-MeOE2was speculated of antiproliferative ability. In summary, E1, E2,2-OHE2and4-OHE2were considered as potential carcinogenic biomarkers while2-MeOE2as a potential anticancer biomarker, suggesting that these five estrogen metabolites paly significant role in the initiation and development of breast cancer. The results yield valuable application in the fields of early diagnosis, screening, prevention.3. RRLC-MS/MS based Metabonomics analysis in serum for breast cancerMetabonomics analysis in serum was carried out on basis of a RRLC system coupled with a QTOF system integrating with multivariate statistics in both positive and negative ion mode for breast cancer. As a complementary for metabolic profiling of estrogen metabolites for breast cancer, metabonomics analysis of72breast cancer patients and81health controls in serum were aiming at discovery of more specific endogenous potential biomarkers for more global, complete metabolism information, thereby in support of revealing the mechanism of breast cancer risk. As a result,37low-molecular-weight discriminated metabolites were detected to have significant differences between breast cancer patients and healthy controls, whose abnormal changes might be highly associated with the perturbations in metabolism, which were believed as potential biomarkers. Then High resolution MS and MS/MS spectra analysis were performed for for the identification of the metabolites of interest,12of which were eventually confirmed with further validation by metabolite database searching as well as standard samples comparison., including6amino acids and their derivatives,3carnitine and its derivatives, Hypoxanthine, N-methy-2-pyrrolidone (NMP) and Allantoin.