Abstract
Targeted profiling is a library-based method of using mathematically modeled reference spectra for quantification of metabolite concentrations in NMR mixture analysis. Metabolomics studies of biofluids, such as urine, represent a highly complex problem in this area, and for this reason targeted profiling of 1H NMR spectra can be hampered. A number of the issues relating to 1H NMR spectroscopy can be overcome using 13C{1H} NMR spectroscopy. In this work, a 13C{1H} NMR database was created using Chenomx NMR Suite, incorporating 120 metabolites. The 13C{1H} NMR database was standardized through the analysis of a series of metabolite solutions containing varying concentrations of 19 distinct metabolites, where the metabolite concentrations were varied across a range of values including biological ranges. Subsequently, the NMR spectra of urine samples were collected using 13C{1H} NMR spectroscopy and profiled using the 13C{1H} NMR library. In total, about 30 metabolites were conclusively identified and quantified in the urine samples using 13C{1H} NMR targeted profiling. The proton decoupling and larger spectral window provided easier identification and more accurate quantification for specific classes of metabolites, such as sugars and amino acids with overlap in the aliphatic region of the 1H NMR spectrum. We discuss potential application areas in which 13C{1H} NMR targeted profiling may be superior to 1H NMR targeted profiling.
Similar content being viewed by others
References
Aardema, M. J., & MacGregor, J. T. (2002). Toxicology and genetic toxicology in the new era of “toxicogenetics”: Impact of “-omics” technologies. Mutation Research, 499, 13–25. doi:10.1016/S0027-5107(01)00292-5.
Belton, P. S., Colquhoun, I. J., Kemsley, E. K., et al. (1998). Application of chemometrics to the 1H NMR spectra of apple juices: Discrimination between apple varieties. Food Chemistry, 61, 207–213. doi:10.1016/S0308-8146(97)00103-9.
Bermel, W., Bertini, I., Felli, I. C., Piccioli, M., & Pierattelli, R. (2006). 13C-detected protonless NMR spectroscopy of proteins in solution. Progress in Nuclear Magnetic Resonance Spectroscopy, 48, 25–45. doi:10.1016/j.pnmrs.2005.09.002.
Burchiel, S. W., Knall, C. M., Davis, J. W., Paules, R. C., Boggs, S. E., & Afshari, C. A. (2001). Analysis of genetic and epigenetic mechanisms of toxicity: Potential roles of toxicogenomics and proteomics in toxicology. Toxicological Sciences, 59, 193–195. doi:10.1093/toxsci/59.2.193.
Chatham, J. C., & Seymour, A.-M. L. (2002). Cardiac carbohydrate metabolism in Zucker diabetic fatty rats. Cardiovascular Research, 55, 104–112. doi:10.1016/S0008-6363(02)00399-1.
Consonni, R., Cagliani, L. R., Benevelli, F., Spraul, M., Humpfer, E., & Stocchero, M. (2008). NMR and chemometric methods: A powerful combination for characterization of balsamic and traditional balsamic vinegars of Modena. Analytica Chimica Acta, 611, 31–40. doi:10.1016/j.aca.2008.01.065.
Duarte, I. F., Barros, A., Almeida, C., Spraul, M., & Gil, A. M. (2004). Multivariate analysis of NMR and FTIR data as a potential tool for the quality control of beer. Journal of Agricultural and Food Chemistry, 52, 1031–1038. doi:10.1021/jf030659z.
Duus, J. Ø., Goftredsen, C. H., & Bock, K. (2000). Carbohydrate structural determination by NMR spectroscopy: Modern methods and limitations. Chemical Reviews, 100, 4589–4614. doi:10.1021/cr990302n.
Eriksson, L., Antti, H., Gottfries, J., et al. (2004). Using chemometrics for navigating in the large data sets of genomics, proteomics, and metabonomics (gpm). Analytical and Bioanalytical Chemistry, 380, 419–429. doi:10.1007/s00216-004-2783-y.
Fan, T. W.-N. (1996). Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy, 28, 161–219.
Fan, T. W. M., Lane, A. N., Shenker, M., Bartley, J. P., Crowley, D., & Higashi, R. M. (2001). Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS. Phytochemistry, 57, 209–221. doi:10.1016/S0031-9422(01)00007-3.
Fiehn, O., Kopka, J., Dörmann, P., Altmann, T., Trethewey, R. N., & Willmitzer, L. (2000). Metabolite profiling for plant functional genomics. Nature Biotechnology, 18, 1157–1161. doi:10.1038/81137.
Gorin, P. A. J. (1981). Carbon–13 nuclear magnetic resonance spectroscopy of polysaccharides. Advances in Carbohydrate Chemistry and Biochemistry, 38, 13–104. doi:10.1016/S0065-2318(08)60309-1.
Hatada, K., & Kitayama, T. (2004). NMR spectroscopy of polymers. Berlin: Springer.
Hidalgo, F. J., & Zamora, R. (2003). Edible oil analysis by high-resolution nuclear magnetic resonance: Recent advances and future perspectives. Trends in Food Science & Technology, 14, 499–506.
Holmes, E., & Antti, H. (2002). Chemometric contributions to the evolution of metabonomics: Mathematical solutions to characterising and interpreting complex biological NMR spectra. Analyst (London), 127, 1549–1557. doi:10.1039/b208254n.
Holmes, E., Loo, R. L., Stamler, J., et al. (2008). Human metabolic phenotype diversity and its association with diet and blood pressure. Nature, 453, 396–400. doi:10.1038/nature06882.
Hyberts, S. G., Heffron, G. J., Tarragona, N. G., et al. (2007). Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction. Journal of the American Chemical Society, 129, 5108–5116. doi:10.1021/ja068541x.
Jeener, J., Meier, B. H., Bachmann, P., & Ernst, R. R. (1979). Investigation of exchange processes by two-dimensional NMR spectroscopy. The Journal of Chemical Physics, 71, 4546–4553. doi:10.1063/1.438208.
Keun, H. C., Beckonert, O., Griffin, J. L., et al. (2002). Cryogenic probe 13C NMR spectroscopy of urine for metabonomic studies. Analytical Chemistry, 74, 4588–4593. doi:10.1021/ac025691r.
Kikuchi, J., Shinozaki, K., & Hirayama, T. (2004). Stable isotope labeling of Arabidopsis thaliana for an NMR-based metabolomics approach. Plant and Cell Physiology, 45, 1099–1104. doi:10.1093/pcp/pch117.
Kovacs, H., Moskau, D., & Spraul, M. (2005). Cryogenically cooled probes—a leap in NMR technology. Progress in Nuclear Magnetic Resonance Spectroscopy, 46, 131–155. doi:10.1016/j.pnmrs.2005.03.001.
Krawczyk, H., Gryff-Keller, A., Gradowska, W., Duran, M., & Pronicka, E. (2001). 13C NMR spectroscopy: A convenient tool for detection of argininosuccinic aciduria. Journal of Pharmaceutical and Biomedical Analysis, 26, 401–408. doi:10.1016/S0731-7085(01)00420-4.
Lewis, I. A., Schommer, S. C., Hodis, B., et al. (2007). Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H–13C NMR spectra. Analytical Chemistry, 79, 9385–9390. doi:10.1021/ac071583z.
Lindon, J. C., Holmes, E., & Nicholson, J. K. (2001). Pattern recognition methods and applications in biomedical magnetic resonance. Progress in Nuclear Magnetic Resonance Spectroscopy, 39, 1–40. doi:10.1016/S0079-6565(00)00036-4.
Lindon, J. C., Holmes, E., & Nicholson, J. K. (2004). Toxicological applications of magnetic resonance. Progress in Nuclear Magnetic Resonance Spectroscopy, 45, 109–143. doi:10.1016/j.pnmrs.2004.05.001.
Lindon, J. C., Nicholson, J. K., & Everett, J. R. (1999). In Webb, G. A. (Ed.), NMR spectroscopy of biofluids. Annual reports on NMR spectroscopy (Vol. 38, pp. 1–88). London: Academic Press.
Lindon, J. C., Nicholson, J. K., Holmes, S., & Everett, J. R. (2000). Metabonomics: Metabolic processes studied by NMR spectroscopy of biofluids. Concepts in Magnetic Resonance, 12, 289–320. doi:10.1002/1099-0534(2000)12:5<289::AID-CMR3>3.0.CO;2-W.
Lindon, J. C., Nicholson, J. K., Holmes, E., et al. (2003). Contemporary issues in toxicology the role of metabonomics in toxicology and its evaluation by the COMET project. Toxicology and Applied Pharmacology, 187, 137–146. doi:10.1016/S0041-008X(02)00079-0.
Malloy, C. R., Sherry, A. D., & Jeffrey, F. M. H. (1990). Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers. The American Journal of Physiology, 259, H987–H995.
Moolenaar, S. H., Poggi-Bach, J., Engelke, U. F. H., et al. (1999). Defect in dimethylglycine dehydrogenase, a new inborn error of metabolism: NMR spectroscopy study. Clinical Chemistry, 45, 459–464.
Nicholson, J. K., Connelly, J., Lindon, J. C., & Holmes, E. (2002). Metabonomics: A platform for studying drug toxicity and gene function. Nature Reviews. Drug Discovery, 1, 153–161. doi:10.1038/nrd728.
Norton, R. S., Zwick, J., & Béress, L. (1980). Natural-abundance 13C nuclear-magnetic-resonance study of toxin II from Anemonia sulcata. European Journal of Biochemistry, 113, 75–83.
Plumb, R., Granger, J., Stumpf, C., Wilson, I. D., Evans, J. A., & Lenz, E. M. (2003). Metabonomic analysis of mouse urine by liquid-chromatography-time of flight mass spectrometry (LC-TOFMS): Detection of strain, diurnal and gender differences. Analyst (London), 128, 819–823. doi:10.1039/b304296k.
Schleucher, J., Schwendinger, M., Sattler, M., et al. (1994). A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. Journal of Biomolecular NMR, 4, 301–306. doi:10.1007/BF00175254.
Shanaiah, N., Desilva, M. A., Gowda, G. A. N., Raftery, M. A., Hainline, B. E., & Raftery, D. (2007). Class selection of amino acid metabolites in body fluids using chemical derivatization and their enhanced 13C NMR. Proceedings of the National Academy of Sciences of the United States of America, 104, 11540–11544. doi:10.1073/pnas.0704449104.
Shearer, J., Duggan, G., Weljie, A., Hittel, D. S., Wasserman, D. H., & Vogel, H. J. (2008). Metabolomic profiling of dietary-induced insulin resistance in the high fat-fed C57BL/6 J mouse. Diabetes, Obesity & Metabolism, 10, 950–958.
Tennant, R. W. (2002). The National Center for Toxicogenomics: Using new technologies to inform mechanistic toxicology. Environmental Health Perspectives, 110, A8–A10.
Viant, M. R. (2003). Improved methods for the acquisition and interpretation of NMR metabolomic data. Biochemical and Biophysical Research Communications, 310, 943–948. doi:10.1016/j.bbrc.2003.09.092.
Voehler, M. W., Collier, G., Young, J. K., Stone, M. P., & Germann, M. W. (2006). Performance of cryogenic probes as a function of ionic strength and sample tube geometry. Journal of Magnetic Resonance (San Diego, Calif.), 183, 102–109. doi:10.1016/j.jmr.2006.08.002.
Wehrli, S. L., Berry, G. T., Palmieri, M., Mazur, A., Elsas, L., I. I. I., & Segal, S. (1997). Urinary galactonate in patients with galactosemia: Quantitation by nuclear magnetic resonance spectroscopy. Pediatric Research, 42, 855–861. doi:10.1203/00006450-199712000-00022.
Weljie, A. M., Dowlatabadi, R., Miller, B. J., Vogel, H. J., & Jirik, F. R. (2007). An inflammatory arthritis-associated metabolite biomarker pattern revealed by 1H NMR spectroscopy. Journal of Proteome Research, 6, 3456–3464. doi:10.1021/pr070123j.
Weljie, A., Newton, J., Jirik, F. R., & Vogel, H. J. (2008). Evaluating low-intensity unknown signals in quantitative proton NMR mixture analysis. Analytical Chemistry, 80, 8956–8965.
Weljie, A., Newton, J., Mercier, P., Carlson, E., & Slupsky, C. (2006). Targeted profiling: Quantitative analysis of 1H NMR metabolomics data. Analytical Chemistry, 78, 4430–4442. doi:10.1021/ac060209g.
Acknowledgements
This work was supported by the Human Metabolome Database Project, a research endeavor supported by Genome Canada. H. J. Vogel is a Scientist of the Alberta Heritage Foundation for Medical Research. We thank Dr Deane McIntyre for technical assistance and the maintenance of the NMR instruments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shaykhutdinov, R.A., MacInnis, G.D., Dowlatabadi, R. et al. Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy. Metabolomics 5, 307–317 (2009). https://doi.org/10.1007/s11306-009-0155-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11306-009-0155-5