Yanhua Chen

Integrated ionization approach for RRLC-MS/MS-based metabonomics: finding potential biomarkers for lung cancer.

An integrated ionization approach of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) combining with rapid resolution liquid chromatography mass spectrometry (RRLC-MS) has been developed for performing global metabonomic analysis on complex biological samples. This approach was designed to overcome the low ionization efficiencies of endogenous metabolites due to diverse physicochemical properties as well as ion suppression, and obtain comprehensive metabolite profiles in LC-MS analysis. Ionization capability and applicability were manifested by improved ionization efficiency and enlarged metabolite coverage in analysis on typical urinary metabolite standards and urine samples from healthy volunteers. The method was validated by the limit of detection and precision. When applied to the global metabonomic studies of lung cancer, more comprehensive biomarker candidates were obtained to reflect metabolic traits between healthy volunteers and lung cancer patients, including 74 potential biomarkers in positive ion mode and 59 in negative ion mode. Taking identical potential biomarkers of any two or three ionization methods into account, analysis using ESI-MS in positive (+) and negative (-) ion mode contributed to 70 and 64% of the total potential biomarkers, respectively. The biomarker discovery capability of (+/-) APCI-MS accounted for 45 and 42% of the overall; meanwhile (+/-) APPI-MS amounted for 39 and 54%. These results indicated that potential biomarkers with vital biological information could be missed if only a single ionization method was used. Furthermore, 11 potential biomarkers were identified including amino acids, nucleosides, and a metabolite of indole. They revealed elevated amino acid and nucleoside metabolism as well as protein degradation in lung cancer patients. This proposed approach provided a more comprehensive picture of the metabolic changes and further verified identical biomarkers that were obtained simultaneously using different ionization methods.

Global and Targeted Metabolomics of Esophageal Squamous Cell Carcinoma Discovers Potential Diagnostic and Therapeutic Biomarkers

Diagnostic and therapeutic biomarkers useful for esophageal squamous cell carcinoma (ESCC) have the ability to increase the long term survival of cancer patients. A metabolomics study, using plasma from four groups including ESCC patients before, during, and after chemoradiotherapy (CRT) and healthy controls, was originally carried out by LC-MS to determine global alterations in the metabolic profiles and find biomarkers potentially applicable to diagnosis and monitoring treatment effects. It is worth pointing out that a clear clustering and separation of metabolic data from the four groups was observed, which indicated that disease status and treatment intervention resulted in specific metabolic perturbations in the patients. A series of metabolites were found to be significantly altered in ESCC patients versus healthy controls and in pre- versus post-treatment patients based on multivariate statistical data analysis (MVDA). To further validate the reliability of these potential biomarkers, an independent validation was performed by using the selected reaction monitoring (SRM) based targeted approach. Finally, 18 most significantly altered plasma metabolites in ESCC patients, relative to healthy controls, were tentatively identified as lysophosphatidylcholines (lysoPCs), fatty acids, l-carnitine, acylcarnitines, organic acids, and a sterol metabolite. The classification performance of these metabolites were analyzed by receiver operating characteristic (ROC)1 analysis and a biomarker panel was generated. Together, biological significance of these metabolites was discussed. Comparison between pre- and post-treatment patients generated 11 metabolites as potential therapeutic biomarkers that were tentatively identified as amino acids, acylcarnitines, and lysoPCs. Levels of three of these (octanoylcarnitine, lysoPC(16:1), and decanoylcarnitine) were closely correlated with treatment effect. Moreover, variation of these three potential biomarkers was investigated over the treatment course. The results suggest that these biomarkers may be useful in diagnosis, as well as in monitoring therapeutic responses and predicting outcomes of the ESCC.

RRLC-MS/MS-based metabonomics combined with in-depth analysis of metabolic correlation network: finding potential biomarkers for breast cancer

A metabonomics strategy based on rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS), multivariate statistics and metabolic correlation networks has been implemented to find biologically significant metabolite biomarkers in breast cancer. RRLC-MS/MS analysis by electrospray ionization (ESI) in both positive and negative ion modes was employed to investigate human urine samples. The resulting data matrices were analyzed using multivariate analysis. Application of orthogonal projections to latent structures discriminate analysis (OPLS-DA) allowed us to extract several discriminated metabolites reflecting metabolic characteristics between healthy volunteers and breast cancer patients. Correlation network analysis between these metabolites has been further applied to select more reliable biomarkers. Finally, high resolution MS and MS/MS analyses were performed for the identification of the metabolites of interest. We identified 12 metabolites as potential biomarkers including amino acids, organic acids, and nucleosides. They revealed elevated tryptophan and nucleoside metabolism as well as protein degradation in breast cancer patients. These studies demonstrate the advantages of integrating metabolic correlation networks with metabonomics for finding significant potential biomarkers: this strategy not only helps identify potential biomarkers, it also further confirms these biomarkers and can even provide biochemical insights into changes in breast cancer.

Liquid chromatography-tandem mass spectrometry-based plasma metabonomics delineate the effect of metabolites' stability on reliability of potential biomarkers.

Metabonomics is an important platform for investigating the metabolites of integrated living systems and their dynamic responses to changes caused by both endogenous and exogenous factors. A metabonomics strategy based on liquid chromatography-mass spectrometry/mass spectrometry in both positive and negative ion modes was applied to investigate the short-term and long-term stability of metabolites in plasma. Principal components analysis and ten types of identified metabolites were used to summarize the time-dependent change rules in metabolites systematically at different temperatures. The long-term stability of metabolites in plasma specimens stored at -80 °C for five years was also studied. Analysis of these subjects identified 36 metabolites with statistically significant changes in expression (p < 0.05) and found a kind of metabolite with a hundred-fold change. The stability of metabolites in blood at 4 °C for 24 h was also investigated. These studies show that a thorough understanding of the effects of metabolite stability are necessary for improving the reliability of potential biomarkers.

Human Metabolic Responses to Chronic Environmental Polycyclic Aromatic Hydrocarbon Exposure by a Metabolomic Approach

The toxicities of polycyclic aromatic hydrocarbons (PAHs) have been extensively explored due to their carcinogenic and mutagenic potency; however, little is known about the metabolic responses to chronic environmental PAH exposure among the general population. In the present study, 566 healthy volunteers were dichotomized into exposed and control groups to investigate PAH-induced perturbations in the metabolic profiles. Nine urine PAH metabolites were measured by a sensitive LC-MS/MS method to comprehensively evaluate the PAH exposure level of each individual, and the metabolic profiles were characterized via a LC-MS-based metabolomic approach. PAH exposure was correlated to its metabolic outcomes by linear and logistic regression analyses. Metabolites related to amino acid, purine, lipid, and glucuronic acid metabolism were significantly changed in the exposed group. 1-Hydroxyphenanthrene and dodecadienylcarnitine have potential as sensitive and reliable biomarkers for PAH exposure and its metabolic outcomes, respectively, in the general population. These findings generally support the hypothesis that environmental PAH exposure causes oxidative stress-related effects in humans. The current study provides new insight into the early molecular events induced by PAH exposure in the actual environment.

Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography-tandem mass spectrometry: a case study for breast cancer.

A rapid, sensitive, specific and accurate analytical method of ultra-fast liquid chromatography combined with tandem mass spectrometry (UFLC-MS/MS) was established for simultaneous quantitative analysis of 16 distinct endogenous estrogens and their metabolites (EMs) in postmenopausal female urine. The quantitative method utilized a hydrolysis/extraction/derivatization step and a UFLC system to achieve separation in 16 min. The lower limit of quantitation for each estrogen metabolite was 2 pg mL(-1) with the percent recovery of a known added amount of estrogen at 93.2-109.3%. The intra-batch accuracy and precision for all analytes were 87.5-107.7% and 0.6-11.7%, respectively, while inter-batch accuracy and precision were 87.0-105.8% and 1.2-10.2%, respectively. Using this developed and validated method, the comprehensive metabolic profiling of 16 EMs in urine samples of 86 postmenopausal female breast cancer patients and 36 healthy controls was investigated by systematic statistical analysis. As a result, the circulating levels of 6 EMs were found to be different by a comparison of patients and healthy controls. The parent estrogens, estrone (E1) and 17β-estradiol (E2), as well as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) were produced in higher abundance, whereas 16α-hydroxyestrone (16α-OHE1) and 2-methoxyestradiol (2-MeOE2) were decreased in the breast cancer group. 2-OHE2 and 4-OHE2 in particular showed significant elevation in patients, which are consistent with the carcinogenic mechanism hypothesis that catechol estrogens can react with DNA via quinones, resulting in mutations to induce breast cancer. Thus, 2,4-hydroxylation may be the dominant metabolic pathway for parent estrogens rather than 16α-hydroxylation. The lower level of 2-MeOE2 in the breast cancer group was believed to correlate with its protective effect against tumor formation. This study could provide valuable information on the association of the EM metabolic pathway with carcinogenesis as well as identify potential biomarkers for estrogen-induced breast cancer risk.

Assessment of data pre-processing methods for LC-MS/MS-based metabolomics of uterine cervix cancer.

A metabolomics strategy based on rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) and multivariate statistics has been implemented to identify potential biomarkers in uterine cervix cancer. Due to the importance of the data pre-processing method, three popular software packages have been compared. Then they have been used to acquire respective data matrices from the same LC-MS/MS data. Multivariate statistics was subsequently used to identify significantly changed biomarkers for uterine cervix cancer from the resulting data matrices. The reliabilities of the identified discriminated metabolites have been further validated on the basis of manually extracted data and ROC curves. Nine potential biomarkers have been identified as having a close relationship with uterine cervix cancer. Considering these in combination as a biomarker group, the AUC amounted to 0.997, with a sensitivity of 92.9% and a specificity of 95.6%. The prediction accuracy was 96.6%. Among these potential biomarkers, the amounts of four purine derivatives were greatly decreased, which might be related to a P2 receptor that might lead to a decrease in cell number through apoptosis. Moreover, only two of them were identified simultaneously by all of the pre-processing tools. The results have demonstrated that the data pre-processing method could seriously bias the metabolomics results. Therefore, application of two or more data pre-processing methods would reveal a more comprehensive set of potential biomarkers in non-targeted metabolomics, before a further validation with LC-MS/MS based targeted metabolomics in MRM mode could be conducted.

Time-course changes in potential biomarkers detected using a metabonomic approach in Walker 256 tumor-bearing rats.

A metabonomic approach based on complementary hydrophilic interaction chromatography and reversed-phase liquid chromatography combined with tandem mass spectrometry and time-course analysis of metabolites was implemented to find more reliable potential biomarkers in urine of Walker 256 tumor-bearing rats. A major challenge in metabonomics is distinguishing reliable biomarkers that are closely associated with the genesis and progression of diseases from those that are unrelated but altered significantly. In this study, these biomarkers were selected according to the change trends of discriminating metabolites during the genesis and progression of cancer. Seven consecutive batches of urine samples from preinoculation to 16 days after were collected and analyzed. Multivariate analysis revealed 87 discriminating metabolites. Time-course analysis of discriminating metabolites was used to select more reliable biomarkers with regular and reasonable change trends. Finally, 47 were found and 15 were identified including 12 carnitine derivatives, 2 amino acid derivatives, 1 nucleoside. On the basis of time-course behaviors of these potential biomarkers, we hypothesize such disruption might result from elevated cell proliferation, reduced β-oxidation of fatty acids, and poor renal tubular reabsorption. These studies demonstrate that this method can help to find more reliable potential biomarkers and provide valuable biochemical insights into metabolic alterations in tumor-bearing biosystems.

Plasma metabolome analysis by integrated ionization rapid‐resolution liquid chromatography/tandem mass spectrometry

RATIONALE Acquiring global information on plasma-endogenous metabolites challenges metabolomics. This study has been designed to investigate the suitability of integrated ionization rapid-resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) for different kinds of metabolites in complex plasma, and provides an approach for plasma metabolomics in acquiring more comprehensive data of metabolites. METHODS Integrated ionization of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) combined with RRLC/MS/MS has been carried out to perform analysis on the global plasma metabolome of healthy volunteers. The contributions to the total numbers of ion features by RRLC/MS with ESI, APCI, and APPI in positive and negative ion modes were calculated. Representative unique and identical ions were identified. The intensities of identical ions were compared. RESULTS Each of ESI, APCI, and APPI coupled with RRLC/MS has its own advantage over the other two techniques for certain types of metabolites in plasma. LC/ESI-MS is very sensitive for detecting glycerophosphocholines, glycerophosphoethanolamines, acyl carnitines, bile acids, sulfate, etc. LC/APCI-MS is suitable for analyzing cyclic alcohols, fatty acids, and linoleic acids. LC/APPI-MS proves to be appropriate in detecting steroids, sphingolipids, some amino acids, nucleosides, and purines in plasma. CONCLUSIONS It is suggested that the integrated ionization LC/MS approach should be applied for global plasma metabolomics. Moreover, the results obtained demonstrate that it is preferable to choose certain techniques from LC/ESI-MS, LC/APCI-MS, and LC/APPI-MS for metabolite target analysis.

Methods used to increase the comprehensive coverage of urinary and plasma metabolomes by MS.

Metabolomics, focusing on comprehensive analysis of all the metabolites in a biological system, provides a direct signature of biochemical activity. Using emerging technologies in MS, it is possible to simultaneously and rapidly analyze thousands of metabolites. However, due to the chemical and physical diversity of metabolites, it is difficult to acquire a comprehensive and reliable profiling of the whole metabolome. Here, we summarize the state of the art in metabolomics research, focusing on efforts to provide a more comprehensive metabolome coverage via improvements in two fundamental processes: sample preparation and MS analysis. Additionally, the reliable analysis is also highlighted via the combinations of multiple methods (e.g., targeted and untargeted approaches), and analytical quality control and calibration methods.