Abstract
Analytical techniques used for multivariate analysis of endogenous metabolites in biological systems (e. g., metabolomics, metabonomics) must be capable of accurately and selectively monitoring many known and unknown molecules that span a diverse chemical spectrum and over extremely large dynamic concentration ranges. Mass spectrometric (MS) and electrochemical array (EC-Array) detection have been widely used for multi-component analysis with applicability to low-level (fmol) metabolites. Described here are practical considerations and results obtained with the combined use of EC-Array and MS for HPLC-based multivariate metabolomic analysis. Data presented include the study of changes in rat urinary metabolite profiles associated with xenobiotic toxin exposure analyzed by HPLC using water:acetonitrile binary gradient conditions and post-column flow splitting between EC-Array and MS detectors. Results show complementary quantitative and qualitative analysis and the ability to differentiate sample groups consistent with xenobiotic-induced histopathological changes. The potential applicability of this hyphenated technique for biomarker elucidation through measurement of redox active compounds that are commonly associated with disease pathology and xenobiotic toxicity is discussed. The use of EC reactor cells in series with MS is also presented as a means of producing likely metabolites to facilitate structural elucidation and confirmation.
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Nobeli, I.; Ponstingl, H.; Krissinel, E. B.; Thornton, J. M. A Structure-Based Anatomy of the E. coli Metabolome. J. Mol. Biol. 2003, 334, 697–719.
Castrillo, J. I.; Oliver, S. G. Yeast as a Touchstone in Post-Genomic Research: Strategies for Integrative Analysis in Functional G Enomics. J. Biochem. Mol. Biol. 2004, 37, 93–106.
Weckwerth, W. Metabolomics in Systems Biology. Annu. Rev. Plant Biol. 2003, 54, 669–689.
Maharjan, R. P.; Ferenci, T. Global Metabolite Analysis: The Influence of Extraction Methodology on Metabolome Profiles of Escherichia coli. Anal. Biochem. 2003, 313, 145–154.
Nicholson, J. K.; Lindon, J. C.; Holmes, E. “Metabonomics”: Understanding the Metabolic Responses of Living Systems to Pathophysiological Stimuli via Multivariate Statistical Analysis of Biological NMR Spectroscopic Data. Xenobiotica 1999, 29, 1181–1189.
Raamsdonk, L. M.; Teusink, B.; Broadhurst, D.; Zhang, N.; Hayes, A.; Walsh, M. C.; Berden, J. A.; Brindle, K. M.; Kell, D. B.; Rowland, J. J.; Westerhoff, H. V.; van Dam, K.; Oliver, S. G. A Functional Genomics Strategy that Uses Metabolome Data to Reveal the Phenotype of Silent Mutations. Nat. Biotechnol. 2001, 19, 45–50.
Williams, G. M.; Iatropoulos, M. J. Alteration of Liver Cell Function and Proliferation: Differentiation Between Adaptation and Toxicity. Toxicol. Pathol. 2002, 30, 41–53.
Robertson, D. G.; Reily, M. D.; Albassam, M.; Dethloff, L. A. Metabonomic Assessment of Vasculitis in Rats. Cardiovasc. Toxicol. 2001, 1, 7–19.
Nicholson, J. K.; Connelly, J.; Lindon, J. C.; Holmes, E. Metabonomics: A Platform for Studying Drug Toxicity and Gene Function. Nat. Rev. Drug Discov. 2002, 1, 153–161.
Holmes, E.; Nicholls, A. W.; Lindon, J. C.; Connor, S. C.; Connelly, J. C.; Haselden, J. N.; Damment, S. J.; Spraul, M.; Neidig, P.; Nicholson, J. K. Chemometric Models for Toxicity Classification Based on NMR Spectra of Biofluids. Chem. Res. Toxicol. 2000, 13, 471–478.
Bailey, W. J.; Ulrich, R. Molecular Profiling Approaches for Identifying Novel Biomarkers. Expert Opin. Drug Safety 2004, 3, 137–151.
Strickland, P. T.; Routledge, M. N.; Dipple, A. Methodologies for Measuring Carcinogen Adducts in Humans. Cancer Epidemiol. Biomarkers Prev. 1993, 2, 607–619.
Hensley, K.; Williamson, K. S.; Floyd, R. A. Measurement of 3-Nitrotyrosine and 5-Nitro-γ-Tocopherol by High-Performance Liquid Chromatography with Electrochemical Detection. Free Rad. Biol. Med. 2000, 28, 520–528.
Shigenaga, M. K.; Ames, B. N. Assays for 8-Hydroxy-2′-Deoxyguanosine: A Biomarker of in Vivo Oxidative DNA Damage. Free Rad. Biol. Med. 1991, 10, 211–216.
Jonsson, P.; Gullberg, J.; Nordstrom, A.; Kusano, M.; Kowalczyk, M.; Sjostrom, M.; Moritz, T. A Strategy for Identifying Differences in Large Series of Metabolomic Samples Analyzed by GC/MS. Anal. Chem. 2004, 76, 1738–1745.
Verdonk, J. C.; Ric de Vos, C. H.; Verhoeven, H. A.; Haring, M. A.; van Tunen, A. J.; Schuurink, R. C. Regulation of Floral Scent Production in Petunia Revealed by Targeted Metabolomics. Phytochemistry 2003, 62, 997–991008.
Gamache, P.; Ryan, E.; Svendsen, C.; Murayama, K.; Acworth, I. N. Simultaneous Measurement of Monoamines, Metabolites, and Amino Aacids in Brain Tissue and Microdialysis Perfusates. J. Chromatogr. 1993, 614, 213–220.
Shi, H.; Vigneau-Callahan, K. E.; Shestopalov, A. I.; Milbury, P. E.; Matson, W. R.; Kristal, B. S. Characterization of Diet-Dependent Metabolic Serotypes: Proof of Principle in Female and Male Rats. J. Nutr. 2002, 132, 1031–1038.
Devanesan, P.; Todorovic, R.; Zhao, J.; Gross, M. L.; Rogan, E. G.; Cavalieri, E. L. Catechol Estrogen Conjugates and DNA Adducts in the Kidney of Male Syrian Golden Hamsters Treated with 4-Hydroxyestradiol: Potential Biomarkers for Estrogen-Initiated Cancer. Carcinogenesis 2001, 22, 489–497.
Todorovic, R.; Devanesan, P.; Higginbotham, S.; Zhao, J.; Gross, M. L.; Rogan, E. G.; Cavalieri, E. L. Analysis of Potential Biomarkers of Estrogen-Initiated Cancer in the Urine of Syrian Golden Hamsters Treated with 4-Hydroxyestradiol. Carcinogenesis 2001, 22, 905–911.
Gamache, P.; Freeto, S. M.; Acworth, I. N. Coulometric Array HPLC Analysis of Lipid Soluble Vitamins and Antioxidants. Am. Clin. Lab. 1999, 18, 18–19.
Buchholz, A.; Hurlebaus, J.; Wandrey, C.; Takors, R. Metabolomics: Quantification of Intracellular Metabolite Dynamics. Biomol. Eng. 2002, 19, 5–15.
Plumb, R.; Granger, J.; Stumpf, C.; Wilson, I. D.; Evans, J. A.; Lenz, E. M. Metabonomic Analysis of Mouse Urine by Liquid-Chromatography Time of Flight Mass Spectrometry (LC-TOFMS): Detection of Strain, Diurnal, and Gender Differences. Analyst 2003, 128, 819–823.
Lafaye, A.; Junot, C.; Ramounet-Le Gall, B.; Fritsch, P.; Tabet, J. C.; Ezan, E. Metabolite Profiling in Rat Urine by Liquid Chromatography/Electrospray Ion Trap Mass Spectrometry. Application to the Study of Heavy Metal Toxicity. Rapid Commun. Mass Spectrom. 2003, 17, 2541–2549.
Shockcor, J. P.; Holmes, E. Metabonomic Applications in Toxicity Screening and Disease Diagnosis. Curr. Top. Med. Chem. 2002, 2, 35–51.
Cadenas, S.; Barja, G.; Poulsen, H. E.; Loft, S. Oxidative DNA Damage Estimated by oxo8dG in the Liver of Guinea Pigs Supplemented with Graded Dietary Doses of Ascorbic Aacid and α-Tocopherol. Carcinogenesis 1997, 18, 2373–2377.
Yanagawa, K.; Takeda, H.; Egashira, T.; Matsumiya, T.; Shibuya, T.; Takasaki, M. Changes in Antioxidative Mechanisms in Elderly Patients with Noninsulin-Dependent Diabetes Mellitus. Investigation of the Redox Dynamics of α-Tocopherol in Erythrocyte Membranes. Gerontology 2001, 47, 150–157.
Bugianesi, R.; Serafini, M.; Simone, F.; Wu, D.; Meydani, S.; Ferro-Luzzi, A.; Azzini, E.; Maiani, G. High-Performance Liquid Chromatography with Coulometric Electrode Array Detector for the Determination of Quercetin Levels in Cells of the Immune System. Anal. Biochem. 2000, 284, 296–300.
Christen, S.; Jiang, Q.; Shigenaga, M. K.; Ames, B. N. Analysis of Plasma Tocopherols α, γ, and 5-Nitro-γ in Rats with Inflammation by HPLC Coulometric Detection. J. Lipid Res. 2002, 43, 1978–1985.
Hensley, K.; Maidt, M. L.; Pye, Q. N.; Stewart, C. A.; Wack, M.; Tabatabaie, T.; Floyd, R. A. Quantitation of Protein-Bound 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine by High-Performance Liquid Chromatography with Electrochemical Array Detection. Anal. Biochem. 1997, 251, 187–195.
Collins, A. R.; Gedik, C. M.; Olmedilla, B.; Southon, S.; Bellizzi, M. Oxidative DNA Damage Measured in Human Lymphocytes: Large Differences Between Sexes and Between Countries, and Correlations with Heart Disease Mortality Rates. FASEB J. 1998, 12, 1397–1400.
Sofic, E.; Lange, K. W.; Jellinger, K.; Riederer, P. Reduced and Oxidized Glutathione in the Substantia nigra of Patients with Parkinson’s Disease. Neurosci. Lett. 1992, 142, 128–130.
Russell, I. J.; Vaeroy, H.; Javors, M.; Nyberg, F. Cerebrospinal Fluid Biogenic Amine Metabolites in Fibromyalgia/Fibrositis Syndrome and Rheumatoid Arthritis. Arthritis Rheum. 1992, 35, 550–556.
Beal, M. F.; Matson, W. R.; Storey, E.; Milbury, P.; Ryan, E. A.; Ogawa, T.; Bird, E. D. Kynurenic Acid Concentrations are Reduced in Huntington’s Disease Cerebral Cortex. J. Neurol. Sci. 1992, 108, 80–87.
Zhou, F.; Van Berkel, G. J. Electrochemistry Combined On-Line with Electrospray Mass Spectrometry. Anal. Chem. 1995, 67, 3643–3649.
Jurva, U.; Wikstrom, H. V.; Bruins, A. P. In Vitro Mimicry of Metabolic Oxidation Reactions by Electrochemistry/Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 529–533.
Deng, H.; Van Berkel, G. J. A Thin-Layer Electrochemical Flow Cell Coupled On-Line with Electrospray-Mass Spectrometry for the Study of Biological Redox Reactions. Electroanalysis 1999, 11, 857–865.
Nicholls, A. W.; Holmes, E.; Lindon, J. C.; Shockcor, J. P.; Farrant, R. D.; Haselden, J. N.; Damment, S. J.; Waterfield, C. J.; Nicholson, J. K. Metabonomic Investigations into Hydrazine toxicity in the Rat. Chem. Res. Toxicol. 2001, 14, 975–987.
Liu, S.; Griffiths, W. J.; Sjovall, J. On-Column Electrochemical Reactions Accompanying the Electrospray Process. Anal. Chem. 2003, 75, 1022–1030.
Ferruzzi, M. G.; Sander, L. C.; Rock, C. L.; Schwartz, S. J. Carotenoid Determination in Biological Microsamples Using Liquid Chromatography with a Coulometric Electrochemical Array Detector. Anal. Biochem. 1998, 256, 74–81.
Nelson, S. D. Molecular Mechanisms of the Hepatotoxicity Caused by Acetaminophen. Semin. Liver Dis. 1990, 10, 267–278.
Jurva, U.; Wikstrom, H. V.; Weidolf, L.; Bruins, A. P. Comparison Between Electrochemistry/Mass Spectrometry and Cytochrome P450 Catalyzed Oxidation Reactions. Rapid Commun. Mass Spectrom. 2003, 17, 800–810.
Gamache, P.; Smith, R.; McCarthy, R.; Waraska, J.; Acworth, I. N. ADME/Tox Profiling Using Coulometric Electrochemistry and Electrospray Ionization Mass Spectrometry. Spectroscopy 2003, 18, 14–21.
Devanesan, P.; Santen, R. J.; Bocchinfuso, W. P.; Korach, K. S.; Rogan, E. G.; Cavalieri, E. Catechol Estrogen Metabolites and Conjugates in Mammary Tumors and Hyperplastic Tissue from Estrogen Receptor-α Knock-Out (ERKO)/Wnt-1 Mice: Implications for Initiation of Mammary Tumors. Carcinogenesis 2001, 22, 1573–1576.
Gamache, P.; Solomon, M.; Acworth, I. N.; Cole, R. Rapid On-Line Electrochemical Synthesis of Pharmaceutical Degradants and Metabolites for Profiling, Identification, and Quantitation. Pittcon 2004.
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Published online October 26, 2004
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Gamache, P.H., Meyer, D.F., Granger, M.C. et al. Metabolomic applications of electrochemistry/Mass spectrometry. J Am Soc Mass Spectrom 15, 1717–1726 (2004). https://doi.org/10.1016/j.jasms.2004.08.016
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DOI: https://doi.org/10.1016/j.jasms.2004.08.016