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Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts

Abstract

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4–5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.

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Figure 1: 600 MHz 1H NMR spectrum of control rat urine, displaying hundreds of resolved peaks.
Figure 2: 600 MHz 1H NMR spectrum of blood serum sample.
Figure 3: Example of edited spectra of a plasma sample [all were acquired with solvent (water) suppression].
Figure 4: Mean score trajectories of principal component analysis of urinary NMR spectral data for each dose group (control, low-dose, high-dose) showing progression of metabolic effects of hydrazine treatment.
Figure 5: Score (t) scatter plots for PC1 versus PC2 from a principal component analysis model using the data acquired at two different sites using the same protocols for the analysis of split aliquots in a study on hydrazine toxicity (triangles and circles represent the two different datasets, respectively).
Figure 6: Metabonomics identifies different responses from the urine profiles of animals treated with different toxins.

References

  1. Fiehn, O. Metabolomics—the link between genotypes and phenotypes. Plant Mol. Biol. 48, 155–171 (2002).

    Article  CAS  Google Scholar 

  2. 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 29, 1181–1189 (1999).

    Article  CAS  Google Scholar 

  3. Bell, J.D., Sadler, P.J., Morris, V.C. & Levander, O.A. Effect of ageing and diet on proton NMR spectra of rat urine. Magn. Reson. Med. 17, 414–422 (1991).

    Article  CAS  Google Scholar 

  4. Bollard, M.E. et al. Investigations into biochemical changes due to diurnal variation and estrus cycle in female rats using high resolution 1H NMR spectroscopy of urine and pattern recognition. Anal. Biochem. 295, 194–202 (2001).

    Article  CAS  Google Scholar 

  5. Phipps, A.N., Wright, B., Stewart, J. & Wilson, I.D. Use of proton NMR for determining changes in metabolite excretion profiles induced by dietary changes in the rat. Pharm. Sci. 3, 143–146 (1997).

    CAS  Google Scholar 

  6. Gavaghan, C.L., Wilson, I.D. & Nicholson, J.K. Physiological variation in metabolic phenotyping and functional genomic studies: use of orthogonal signal correction and PLSDA. FEBS Lett. 530, 191–196 (2002).

    Article  CAS  Google Scholar 

  7. Holmes, E. et al. Chemometric models for toxicity classification based on NMR spectra of biofluids. Chem. Res. Toxicol. 13, 471–478 (2000).

    Article  CAS  Google Scholar 

  8. Gartland, K.P., Bonner, F.W. & Nicholson, J.K. Investigations into the biochemical effects of region-specific nephrotoxic lesions. Mol. Pharmacol. 35, 242–250 (1989).

    CAS  PubMed  Google Scholar 

  9. Beckwith-Hall, B.M. et al. Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins. Chem. Res. Toxicol. 11, 260–272 (1998).

    Article  CAS  Google Scholar 

  10. Robertson, D.G., Reily, M.D., Lindon, J.C., Holmes, E. & Nicholson, J.K. Metabonomic technology as a tool for rapid throughput in vivo toxicity screening. in Compr. Toxicol., Molecular and Cell. Toxicol., 14, 583–610, eds. Vanden Heuvel, J.P., Perdew, G.H., Mattes, W.B., Greenlee, W.F., Elsevier (2002).

    Google Scholar 

  11. Garrod, S. et al. High resolution magic angle spinning 1H NMR spectroscopic studies on intact rat renal cortex and medulla. Magn. Reson. Med. 41, 1108–1118 (1999).

    Article  CAS  Google Scholar 

  12. Moka, D. et al. Biochemical classification of kidney carcinoma biopsy samples using magic-angle-spinning 1H nuclear magnetic resonance spectroscopy. J. Pharm. Biomed. Anal. 17, 125–132 (1998).

    Article  CAS  Google Scholar 

  13. Cheng, L.L. & Pohl, U. The role of NMR-based metabolomics in cancer. in Handbook of Metabonomics and Metabolomics (eds. Lindon, J.C., Nicholson, J.K. & Holmes, E.) 345–374 (Elsevier, Amsterdam, 2006).

    Google Scholar 

  14. Swanson, M.G. et al. Proton HR-MAS spectroscopy and quantitative pathologic analysis of MRI/3D-MRSI-targeted postsurgical prostate tissues. Magn. Reson. Med. 50, 944–954 (2003).

    Article  CAS  Google Scholar 

  15. Bollard, M.E. et al. Metabolic profiling of the effects of D-galactosamine in liver spheroids using 1H NMR and MAS NMR spectroscopy. Chem. Res. Toxicol. 15, 1351–1359 (2002).

    Article  CAS  Google Scholar 

  16. Nicholson, J.K., Foxall, P.J., Spraul, M., Farrant, R.D. & Lindon, J.C. 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. Anal. Chem. 67, 793–811 (1995).

    Article  CAS  Google Scholar 

  17. Teahan, O. et al. Impact of analytical bias in metabonomic studies of human blood serum and plasma. Anal. Chem. 78, 4307–4318 (2006).

    Article  CAS  Google Scholar 

  18. Waters, N.J., Holmes, E., Waterfield, C.J., Farrant, R.D. & Nicholson, J.K. NMR and pattern recognition studies on liver extracts and intact livers from rats treated with α-napthylisothiocyanate. Biochem. Pharmacol. 64, 67–77 (2002).

    Article  CAS  Google Scholar 

  19. Coen, M. et al. An integrated metabonomic investigation of acetaminophen toxicity in the mouse using NMR spectroscopy. Chem. Res. Toxicol. 16, 295–303 (2003).

    Article  CAS  Google Scholar 

  20. Wang, Y. et al. Spectral editing and pattern recognition methods applied to high-resolution magic-angle spinning 1H nuclear magnetic resonance spectroscopy of liver tissues. Anal. Biochem. 323, 26–32 (2003).

    Article  CAS  Google Scholar 

  21. Keun, H.C. et al. Analytical reproducibility in 1H NMR-based metabonomic urinalysis. Chem. Res. Toxicol. 15, 1380–1386 (2002).

    Article  CAS  Google Scholar 

  22. Brown, F.F., Campbell, I.D., Kuchel, P.W. & Rabenstein, D.C. Human erythrocyte metabolism studies by 1H spin echo NMR. FEBS Lett. 82, 12–16 (1977).

    Article  CAS  Google Scholar 

  23. Brindle, K.M., Brown, F.F., Campbell, I.D., Grathwohl, C. & Kuchel, P.W. Application of spin-echo nuclear magnetic resonance to whole-cell systems. Membrane transport. Biochem. J. 180, 37–44 (1979).

    Article  CAS  Google Scholar 

  24. Nicholson, J.K., Buckingham, M.J. & Sadler, P.J. High resolution 1H n.m.r. studies of vertebrate blood and plasma. Biochem. J. 211, 605–615 (1983).

    Article  CAS  Google Scholar 

  25. Nicholson, J.K. et al. Proton nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects. Biochem. J. 217, 365–375 (1984).

    Article  CAS  Google Scholar 

  26. Nicholson, J.K., Timbrell, J.A. & Sadler, P.J. Proton NMR spectra of urine as indicators of renal damage. Mercury-induced nephrotoxicity in rats. Mol. Pharmacol. 27, 644–651 (1985).

    CAS  PubMed  Google Scholar 

  27. Nicholson, J.K. & Wilson, I.D. High resolution proton magnetic resonance spectroscopy of biological fluids. Prog. Nucl. Magn. Reson. Spectrosc. 21, 449–501 (1989).

    Article  CAS  Google Scholar 

  28. Nicholson, J.K., Connelly, J., Lindon, J.C. & Holmes, E. Metabonomics: a platform for studying drug toxicity and gene function. Nat. Rev. Drug Discov. 1, 153–162 (2002).

    Article  CAS  Google Scholar 

  29. Lindon, J.C., Nicholson, J.K. & Holmes, E. (eds.) The Handbook of Metabonomics and Metabolomics (Elsevier, Amsterdam, 2006).

  30. Bollard, M.E., Stanley, E.G., Lindon, J.C., Nicholson, J.K. & Holmes, E. NMR-based metabonomics approaches for evaluating physiological influences on biofluid composition. NMR Biomed. 18, 143–162 (2005).

    Article  CAS  Google Scholar 

  31. Bales, J.R., Sadler, P.J., Nicholson, J.K. & Timbrell, J.A. Urinary excretion of acetaminophen and its metabolites as studied by proton NMR spectroscopy. Clin. Chem. 30, 1631–1636 (1984).

    CAS  PubMed  Google Scholar 

  32. Lenz, E.M. & Wilson, I.D. Analytical strategies in metabonomics. J. Proteome Res. 6, 443–458 (2007).

    Article  CAS  Google Scholar 

  33. van der Greef, J. et al. Evaluation of field desorption and fast atom-bombardment mass spectrometric profiles by pattern recognition techniques. Anal. Chim. Acta 150, 45–52 (1983).

    Article  CAS  Google Scholar 

  34. Wilson, I.D. et al. High resolution “ultra performance” liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies. J. Proteome Res. 4, 591–598 (2005).

    Article  CAS  Google Scholar 

  35. Gartland, K.P. et al. Pattern recognition analysis of high resolution 1H NMR spectra of urine. A nonlinear mapping approach to the classification of toxicological data. NMR Biomed. 3, 166–172 (1990).

    Article  CAS  Google Scholar 

  36. Gartland, K.P., Beddell, C.R., Lindon, J.C. & Nicholson, J.K. The application of pattern recognition methods to the analysis and classification of toxicological data derived from proton NMR spectroscopy of urine. Mol. Pharmacol. 39, 629–642 (1991).

    CAS  PubMed  Google Scholar 

  37. Ebbels, T. et al. Toxicity classification from metabonomic data using a density superposition approach: 'CLOUDS'. Anal. Chim. Acta 490, 109–122 (2003).

    Article  CAS  Google Scholar 

  38. Antti, H. et al. Statistical experimental design and partial least squares regression analysis of biofluid metabonomic NMR and clinical chemistry data for screening of adverse drug effects. Chemom. Intell. Lab. Syst. 73, 139–149 (2004).

    Article  CAS  Google Scholar 

  39. Cloarec, O. et al. Statistical total correlation spectroscopy: an exploratory approach for latent biomarker identification from metabolic 1H NMR data sets. Anal. Chem. 77, 1282–1289 (2005).

    Article  CAS  Google Scholar 

  40. Crockford, D.J. et al. Statistical HeterospectroscopY (SHY), a new approach to the integrated analysis of NMR and UPLC-MS datasets: application in metabonomic toxicology studies. Anal. Chem. 78, 363–371 (2006).

    Article  CAS  Google Scholar 

  41. Lindon, J.C. et al. Contemporary issues in toxicology—the role of metabonomics in toxicology and its evaluation by the COMET project. Toxicol. Appl. Pharmacol. 187, 137–146 (2003).

    Article  CAS  Google Scholar 

  42. Lindon, J.C. et al. The Consortium for Metabonomic Toxicology (COMET): aims, activities and achievements. Pharmacogenomics 6, 691–699 (2005).

    Article  CAS  Google Scholar 

  43. Ebbels, T.M.D. et al. Prediction and classification of drug toxicity using probabilistic modeling of temporal metabolic data: The Consortium of Metabonomic Toxicology Screening approach. J. Proteome Res. 6, 3944–3951 (2007).

    Article  Google Scholar 

  44. Lindon, J.C. & Holmes, E. A survey of metabonomics approaches for disease characterization. In Handbook of Metabonomics and Metabolomics (eds. Lindon, J.C., Nicholson, J.K. & Holmes, E.) 413–442 (Elsevier, Amsterdam, 2006).

    Google Scholar 

  45. Moolenaar, S.H., Engelke, U.F. & Wevers, R.A. Proton nuclear magnetic resonance spectroscopy of body fluids in the field of inborn errors of metabolism. Ann. Clin. Biochem. 40, 16–24 (2003).

    Article  CAS  Google Scholar 

  46. Brindle, J.T. et al. Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H NMR-based metabonomics. Nat. Med. 8, 1439–1445 (2002).

    Article  CAS  Google Scholar 

  47. Nicholls, A.W., Mortishire-Smith, R.J. & Nicholson, J.K. NMR spectroscopic based metabonomic studies of urinary metabolite variation in acclimatizing germ-free rats. Chem. Res. Toxicol. 16, 1395–1404 (2003).

    Article  CAS  Google Scholar 

  48. Nicholson, J.K., Holmes, E. & Wilson, I.D. Gut microorganisms, mammalian metabolism and personalized health care. Nat. Rev. Microbiol. 3, 431–438 (2005).

    Article  CAS  Google Scholar 

  49. Clayton, T.A. et al. Pharmaco-metabonomic phenotyping and personalised drug treatment. Nature 440, 1073–1077 (2006).

    Article  CAS  Google Scholar 

  50. Dumas, M.E. et al. Assessment of analytical reproducibilty of 1H nmr spectroscopy based metabonomics for large-scale epidemiological research: the INTERMAP study. Anal. Chem. 78, 2199–2208 (2006).

    Article  CAS  Google Scholar 

  51. Liu, M., Nicholson, J.K. & Lindon, J.C. High resolution diffusion and relaxation edited one- and two-dimensional 1H NMR spectroscopy of biological fluids. Anal. Chem. 68, 3370–3376 (1996).

    Article  CAS  Google Scholar 

  52. Jenkins, H. et al. A proposed framework for the description of plant metabolomics experiments and their results. Nat. Biotechnol. 22, 1601–1606 (2004).

    Article  CAS  Google Scholar 

  53. Bino, R.J. et al. Potential of metabolomics as a functional genomics tool. Trends Plant Sci. 9, 418–425 (2004).

    Article  CAS  Google Scholar 

  54. Lindon, J.C. et al. Summary recommendations for standardization and reporting of metabolic analyses. Nat. Biotechnol. 23, 833–838 (2005).

    Article  CAS  Google Scholar 

  55. Le Belle, J.E., Harris, N.G., Williams, S.R. & Bhakoo, K.K. A comparison of cell and tissue extraction techniques using high-resolution 1H-NMR spectroscopy. NMR Biomed. 15, 37–44 (2002).

    Article  CAS  Google Scholar 

  56. Ching, Y.L., Wu, H., Tjeerdema, R.S. & Viant, M.R. Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics. Metabolomics 3, 55–67 (2007).

    Article  Google Scholar 

  57. Villas-Boas, S.G., Hojer-Pedersen, J., Akesson, M., Smedsgaard, J. & Nielsen, J. Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 22, 1155–1169 (2005).

    Article  CAS  Google Scholar 

  58. Ippolito, J.E. et al. An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers. Proc. Natl. Acad. Sci. USA 102, 9901–9906 (2005).

    Article  CAS  Google Scholar 

  59. Spraul, M. et al. Flow injection proton nuclear magnetic resonance spectroscopy combined with pattern recognition methods: implications for rapid structural studies and high throughput biochemical screening. Anal. Comm. 34, 339–341 (1997).

    Article  CAS  Google Scholar 

  60. Akoka, S., Barantin, L. & Trierweiler, M. Concentration measurement by proton NMR using the ERETIC method. Anal. Chem. 71, 2554–2557 (1999).

    Article  CAS  Google Scholar 

  61. Aue, W.P., Karhan, J. & Ernst, R.R. Homonuclear broad band decoupling and two-dimensional J-resolved NMR spectroscopy. J. Chem. Phys. 64, 4226–4227 (1976).

    Article  CAS  Google Scholar 

  62. Aue, W.P., Bartholdi, E. & Ernst, R.R. Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229–2246 (1976).

    Article  CAS  Google Scholar 

  63. Braunschweiler, L. & Ernst, R.R. Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J. Magn. Reson. 53, 521–528 (1983).

    CAS  Google Scholar 

  64. Keun, H.C. et al. Cryogenic probe 13C NMR spectroscopy of urine for metabonomic studies. Anal. Chem. 74, 4588–4593 (2002).

    Article  CAS  Google Scholar 

  65. Lindon, J.C., Nicholson, J.K. & Wilson, I.D. Directly-coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development. J. Chromatogr. B 748, 233–258 (2000).

    Article  CAS  Google Scholar 

  66. Cloarec, O. et al. Virtual chromatographic resolution enhancement in cryoflow LC-NMR experiments via statistical total correlation spectroscopy. Anal. Chem. 79, 3304–3311 (2007).

    Article  CAS  Google Scholar 

  67. Smith, L.M. et al. Statistical correlation and projection methods for improved information recovery from diffusion-edited NMR spectra of biological samples. Anal. Chem. 79, 5682–5689 (2007).

    Article  CAS  Google Scholar 

  68. Deprez, S., Sweatman, B.C., Connor, S.C., Haselden, J.N. & Waterfield, C.J. Optimisation of collection, storage and preparation of rat plasma for 1H NMR spectroscopic analysis in toxicology studies to determine inherent variation in biochemical profiles. J. Pharm. Biomed. Anal. 30, 1297–1310 (2002).

    Article  CAS  Google Scholar 

  69. Maher, A.D., Zirah, S.F., Holmes, E. & Nicholson, J.K. Experimental and analytical variation in human urine in 1H NMR spectroscopy-based metabolic phenotyping studies. Anal. Chem. 79, 5204–5211 (2007).

    Article  CAS  Google Scholar 

  70. Hwang, T.L. & Shaka, A.J. Water suppression that works: excitation sculpting using arbitrary wave-forms and pulsed-field gradients. J. Magn. Reson. A 112, 275–279 (1995).

    Article  CAS  Google Scholar 

  71. Aranibar, N., Ott, K.H., Roongta, V. & Mueller, L. Metabolomic analysis using optimised NMR and statistical methods. Anal. Biochem. 355, 62–70 (2006).

    Article  CAS  Google Scholar 

  72. Sonnewald, U., Isern, E., Gribbestad, I.S. & Unsgard, G. UDP-N-acetylhexosamines and hypotaurine in human glioblastoma, normal brain tissue and cell cultures: 1H/NMR spectroscopy study. Anticancer Res. 14, 793–798 (1994).

    CAS  PubMed  Google Scholar 

  73. Henke, J., Willker, W., Engelmann, J. & Leibfritz, D. Combined extraction techniques of tumour cells and lipid/phospholipid assignment by two dimensional NMR spectroscopy. Anticancer Res. 16, 1417–1427 (1996).

    CAS  PubMed  Google Scholar 

  74. Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).

    Article  CAS  Google Scholar 

  75. Tyagi, R.K., Azrad, A., Degani, H. & Salomon, Y. Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy. Magn. Reson. Med. 35, 194–200 (1996).

    Article  CAS  Google Scholar 

  76. Beckonert, O., Monnerjahn, J., Bonk, U. & Leibfritz, D. Visualizing metabolic changes in breast-cancer tissue using 1H-NMR spectroscopy and self-organizing maps. NMR Biomed. 16, 1–11 (2003).

    Article  CAS  Google Scholar 

  77. Meiboom, S. & Gill, D. Modified spin-echo method for measuring nuclear relaxation time. Rev. Sci. Instrum. 20, 688–691 (1958).

    Article  Google Scholar 

  78. Wu, D., Chen, A. & Johnson, C.S. An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses. J. Magn. Reson. A 115, 260–264 (1995).

    Article  CAS  Google Scholar 

  79. Ernst, R.R. & Anderson, W.A. Application of Fourier transform spectroscopy to magnetic resonance. Rev. Sci. Instr. 37, 93–102 (1966).

    Article  CAS  Google Scholar 

  80. Jones, D.E. & Sternlicht, H. Fourier transform nuclear magnetic resonance I. Repetitive pulses. J. Magn. Reson. 6, 167–182 (1972).

    CAS  Google Scholar 

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Acknowledgements

We thank our academic and industrial collaborators for helpful discussions in the formulation of this paper, including those participating in the COMET project.

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Beckonert, O., Keun, H., Ebbels, T. et al. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat Protoc 2, 2692–2703 (2007). https://doi.org/10.1038/nprot.2007.376

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