NMR-Based Metabolic Phenotyping Techniques and Applications

  • John C. Lindon
Reference work entry


Determination of the comprehensive small metabolite constitution of biofluids and tissues through untargeted and targeted methods and the characterization of changes in abnormal situations is usually accomplished using both NMR spectroscopy and chromatography linked to mass spectrometry. The approach has been termed variously metabonomics, metabolomics, and metabolic phenotyping. Here the use of NMR spectroscopy in metabolic phenotyping is described with a brief history of the development of the approach, a summary of the techniques used and a survey of past and current applications in drug adverse effects, disease diagnosis and prognosis, personalized or stratified medicine approaches, and evaluation of population risk factors from large-scale epidemiological studies. The concept of dedicated phenome centers is introduced.


Adverse effects Chemometrics Disease Diagnosis Efficacy Epidemiology Magic-angle spinning Metabolic phenotyping Metabolomics Metabonomics MS NMR Statistics Pharmacometabonomics Personalized medicine Population Pharmaceuticals Prognosis Risk factors 


  1. 1.
    Nicholson JK, Lindon JC, Holmes E. “Metabonomics”: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 1999;29:1181–9.CrossRefGoogle Scholar
  2. 2.
    Lindon JC, Nicholson JK, Holmes E, Everett JR. Metabonomics: Metabolic processes studied by NMR spectroscopy of biofluids. Concepts Magn Reson. 2000; 12:289–320.CrossRefGoogle Scholar
  3. 3.
    Nicholson JK, Connelly J, Lindon JC, Holmes E. Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Disc. 2002;1:153–61.CrossRefGoogle Scholar
  4. 4.
    Teranishi R, Mon TR, Robinson AB, Cary P, Pauling L. Gas chromatography of volatiles from breath and urine. Anal Chem. 1972;44:1820.CrossRefGoogle Scholar
  5. 5.
    Pauling L, Robinson AB, Teranishi R, Cary P. Quantitative analysis of urine vapor and breath by gas-liquid partition chromatography. Proc Nat Acad Sci USA. 1971;68:2374–6.CrossRefGoogle Scholar
  6. 6.
    Tanaka K, Isselbacher KJ. The isolation and identification of N-isovalerylglycine from urine of patients with isovaleric acidemia. J Biol Chem. 1967;242:296672.Google Scholar
  7. 7.
    Chalmers RA, Lawson AM, Watts RW. Studies on the urinary acidic metabolites excreted by patients with beta-methylcrotonylglycinuria, propionic acidaemia and methylmalonic acidaemia, using gas-liquid chromatography and mass spectrometry. Clin Chim Acta. 1974;52:4351.Google Scholar
  8. 8.
    Iles RA, Hind AJ, Chalmers RA. Use of proton nuclear magnetic resonance spectroscopy in detection and study of organic acidurias. Clin Chem. 1985;31:1795–801.Google Scholar
  9. 9.
    Engelke UF, Liebrand-van Sambeek ML, de Jong JG, Leroy JG, Morava E, Smeitink JA, Wevers RA. N-acetylated metabolites in urine: proton nuclear magnetic resonance spectroscopic study on patients with inborn errors of metabolism. Clin Chem. 2004;50:58–66.CrossRefGoogle Scholar
  10. 10.
    Chalmers RA, Watts RW. The quantitative extraction and gas-liquid chromatographic determination of organic acids in urine. Analyst. 1972;97:958–67.CrossRefGoogle Scholar
  11. 11.
    Nicholson JK, O’Flynn MP, Sadler PJ, Macleod AF, Juul SM, Sönksen PH. Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects. Biochem J. 1984;217:365–75.CrossRefGoogle Scholar
  12. 12.
    Nicholson JK, Wilson ID. High resolution proton magnetic resonance spectroscopy of biological fluids. Prog NMR Spectrosc. 1989;21:449–501.CrossRefGoogle Scholar
  13. 13.
    Commodari F, Arnold DL, Sanctuary BC, Shoubridge EA. 1H NMR characterization of normal human cerebrospinal fluid and the detection of methylmalonic acid in a vitamin B12 deficient patient. NMR Biomed. 1991;4:192–200.CrossRefGoogle Scholar
  14. 14.
    Albert K, Michele S, Günther U, Fial M, Gall H, Saal J. 13C NMR investigation of synovial fluids. Magn Reson Med. 1993;30:236–40.CrossRefGoogle Scholar
  15. 15.
    Williamson MP, Humm G, Crisp AJ. 1H nuclear magnetic resonance investigation of synovial fluid components in osteoarthritis, rheumatoid arthritis and traumatic effusions. Br J Rheumatol. 1989;28:23–7.CrossRefGoogle Scholar
  16. 16.
    Brown JC, Sadler PJ, Spalton DJ, Juul SM, Macleod AF, Sönksen PH. Analysis of human aqueous humour by high resolution 1H NMR spectroscopy. Exp Eye Res. 1986;42:357–62.CrossRefGoogle Scholar
  17. 17.
    Lynch MJ, Masters J, Pryor JP, Lindon JC, Spraul M, Foxall PJD, Nicholson JK. Ultra high field NMR spectroscopic studies on human seminal fluid, seminal vesicle and prostatic secretions. J Pharm Biomed Anal. 1994;12:5–19.CrossRefGoogle Scholar
  18. 18.
    Tomlins AM, Foxall PJ, Lynch MJ, Parkinson J, Everett JR, Nicholson JK. High resolution 1H NMR spectroscopic studies on dynamic biochemical processes in incubated human seminal fluid samples. Biochim Biophys Acta. 1998;1379:367–80.CrossRefGoogle Scholar
  19. 19.
    Oliver SG, Winson MK, Kell DB, Baganz F. Systematic functional analysis of the yeast genome. Trends Biotechnol. 1998;16:373–8. Erratum in: Trends Biotechnol. 1998; 16:447.CrossRefGoogle Scholar
  20. 20.
    van der Werf MJ, Jellema RH, Hankemeier T. Microbial metabolomics: replacing trial-and-error by the unbiased selection and ranking of targets. J Ind Microbiol Biotechnol. 2005;32:234–52.CrossRefGoogle Scholar
  21. 21.
    Bundy JG, Osborn D, Weeks JM, Lindon JC, Nicholson JK. An NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress. FEBS Lett. 2001;500:31–5.CrossRefGoogle Scholar
  22. 22.
    Phalaraksh C, Reynolds SE, Wilson ID, Lenz EM, Nicholson JK, Lindon JC. A metabonomic analysis of insect development: 1H NMR spectroscopic characterisation of changes in the composition of the haemolymph of larvae and pupae of Manduca sexta. Sci Asia. 2008;34:27986.CrossRefGoogle Scholar
  23. 23.
    Brennan L. NMR-based metabolomics: from sample preparation to applications in nutrition. Prog NMR Spectrosc. 2014;83:42–9.CrossRefGoogle Scholar
  24. 24.
    Lindon JC, Nicholson JK, Holmes E, Antti H, Bollard ME, Keun H, Beckonert O, Ebbels TM, Reily MD, Robertson D, Stevens GJ, Luke P, Breau AP, Cantor GH, Bible RH, Niederhauser U, Senn H, Schlotterbeck G, Sidelmann UG, Laursen SM, Tymiak A, Car BD, Lehman-McKeeman L, Colet JM, Loukaci A, Thomas C. Contemporary issues in toxicology – the role of metabonomics in toxicology and its evaluation by the COMET project. Toxicol Appl Pharmacol. 2003;187:13746.CrossRefGoogle Scholar
  25. 25.
    Cheng LL, Lean CL, Bogdanova A, Wright Jr SC, Ackerman JL, Brady TJ, Garrido L. Enhanced resolution of proton NMR spectra of malignant lymph nodes using magic-angle spinning. Magn Reson Med. 1996;36:653–8.CrossRefGoogle Scholar
  26. 26.
    Moka D, Vorreuther R, Schicha H, Spraul M, Humpfer E, Lipinski M, Foxall PJD, Nicholson JK, Lindon JC. Biochemical classification of kidney carcinoma biopsy samples using magic-angle-spinning 1H nuclear magnetic resonance spectroscopy. J Pharm Biomed Anal. 1998;17:125–32.CrossRefGoogle Scholar
  27. 27.
    Garrod S, Humpfer E, Spraul M, Connor SC, Polley S, Connelly J, Lindon JC, Nicholson JK, Holmes E. High resolution magic angle spinning 1H NMR spectroscopic studies on intact rat renal cortex and medulla. Magn Reson Med. 1999;41:1108–18.CrossRefGoogle Scholar
  28. 28.
    Dunn WB, Lin W, Broadhurst D, Begley P, Brown M, Zelena E, Vaughan AA, Halsall A, Harding N, Knowles JD, Francis-McIntyre S, Tseng A, Ellis DI, O'Hagan S, Aarons G, Benjamin B, Chew-Graham S, Moseley C, Potter P, Winder CL, Potts C, Thornton P, McWhirter C, Zubair M, Pan M, Burns A, Cruickshank JK, Jayson GC, Purandare N, Wu FC, Finn JD, Haselden JN, Nicholls AW, Wilson ID, Goodacre R, Kell DB. Molecular phenotyping of a UK population: defining the human serum metabolome. Metabolomics 2015; 11: 9–26.CrossRefGoogle Scholar
  29. 29.
  30. 30.
    Psychogios N, Hau DD, Peng J, Guo AC, Mandal R, Bouatra S, Sinelnikov I, Krishnamurthy R, Eisner R, Gautam B, Young N, Xia J, Knox C, Dong E, Huang P, Hollander Z, Pedersen TL, Smith SR, Bamforth F, Greiner R, McManus B, Newman JW, Goodfriend T, Wishart DS. The human serum metabolome. PLoS One. 2011;6:e16957.CrossRefGoogle Scholar
  31. 31.
    Bouatra S, Aziat F, Mandal R, Guo AC, Wilson MR, Knox C, Bjorndahl TC, Krishnamurthy R, Saleem F, Liu P, Dame ZT, Poelzer J, Huynh J, Yallou FS, Psychogios N, Dong E, Bogumil R, Roehring C, Wishart DS. The human urine metabolome. PLoS One. 2013;8:e73076.CrossRefGoogle Scholar
  32. 32.
    Lindon JC, Nicholson JK. Spectroscopic and statistical techniques for information recovery in metabonomics and metabolomics. Ann Rev Anal Chem. 2008;1:45–69.CrossRefGoogle Scholar
  33. 33.
    Khandelwal P, Beyer CE, Lin Q, McGonicle P, Schechter LE, Bach C. Nanoprobe NMR spectroscopy and in vivo microdialysis: new analytical methods to study brain neurochemistry. J Neurosci Mehtods. 2004;133:181–9.CrossRefGoogle Scholar
  34. 34.
    Griffin JL, Nicholls AW, Keun HC, Mortishire-Smith RJ, Nicholson JK, Keuhn T. Metabolic profiling of rodent biological fluids via 1H NMR spectroscopy using a 1 mm microlitre probe. Analyst. 2002;127:582–4.CrossRefGoogle Scholar
  35. 35.
    Keun HC, Beckonert O, Griffin JL, Richter C, Moskau D, Lindon JC, Nicholson JK. Cryogenic probe 13C NMR spectroscopy of urine for metabonomic studies. Anal Chem. 2002;74:4588–93.CrossRefGoogle Scholar
  36. 36.
    Lindon JC, Nicholson JK, Wilson ID. Directly-coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development. J Chromatogr B. 2000;748:233–58.CrossRefGoogle Scholar
  37. 37.
    Waters NJ, Holmes E, Williams A, Waterfield CJ, Farrant RD, Nicholson JK. NMR and pattern recognition studies on the time-related metabolic effects of alpha-naphthylisothiocyanate on liver, urine, and plasma in the rat: an integrative metabonomic approach. Chem Res Toxicol. 2001;14:1401–12.CrossRefGoogle Scholar
  38. 38.
    Mirnezami R, Jinenez B, Li JV, Kinross J, Veselkov K, Goldin RD, Holmes E, Nicholson JK, Darzi A. Rapid diagnosis and staging of colorectal cancer via high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy of intact tissue biopsies. Ann Surg. 2014;259:1138–49.CrossRefGoogle Scholar
  39. 39.
    Lamers RJAN, Wessels ECHH, van der Sandt JJM, Venema K, Schaafsma G, van der Greef J, van Nesselrooij JHJ. A pilot study to investigate effects of inulin on Caco-2 cells through in vitro metabolic fingerprinting. J Nutr. 2003;133:3080–4.CrossRefGoogle Scholar
  40. 40.
    Veselkov KA, Lindon JC, Ebbels TMD, Crockford D, Volynkin VV, Holmes E, Davies DB, Nicholson JK. Recursive segment-wise peak alignment of biological H-1 NMR spectra for improved metabolic biomarker recovery. Anal Chem. 2009;81:56–66.CrossRefGoogle Scholar
  41. 41.
    Farrant RD, Lindon JC, Rahr E, Sweatman BC. An automatic data reduction and transfer method to aid pattern recognition analysis and classification of NMR spectra. J Pharm Biomed Anal. 1992;10:141–4.CrossRefGoogle Scholar
  42. 42.
    Lindon JC, Nicholson JK, Holmes E. Summary recommendations for standardization and reporting of metabolic analyses. Nature Biotech. 2005;23:833–8.CrossRefGoogle Scholar
  43. 43.
    Sansone S-A, Nikolau B, van Ommen B, Kristal BS, Taylor C, Robertson D, Lindon JC, Griffin JL, Sumner LW, van der Werf M, Hardy NW, Morrison N, Mendes P, Kaddurah-Daouk R, Goodacre R, Fan T, Fiehn O. The metabolomics standards initiative. Nature Biotech. 2007;25:846–8.Google Scholar
  44. 44.
    Beckonert O, Keun HC, Ebbels TMD, Bundy J, Holmes E, Lindon JC, Nicholson JK. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat Protoc. 2007;2:2692–703.CrossRefGoogle Scholar
  45. 45.
    Dona AC, Jimenez B, Schaefer H, Humpfer E, Spraul M, Lewis MR, Pearce JTM, Holmes E, Lindon JC, Nicholson JK. Precision high-throughput proton NMR spectroscopy of human urine, serum, and plasma for large-scale metabolic phenotyping. Anal Chem. 2014;8:9887–94.CrossRefGoogle Scholar
  46. 46.
  47. 47.
  48. 48.
  49. 49.
  50. 50.
    Lindon JC, Holmes E, Nicholson JK. Toxicological applications of magnetic resonance. Prog NMR Spectrosc. 2004;45:109–43.CrossRefGoogle Scholar
  51. 51.
    Keun HC, Ebbels TMD, Antti H, Bollard M, Beckonert O, Schlotterbeck G, Senn H, Niederhauser U, Holmes E, Lindon JC, Nicholson JK. Analytical reproducibility in 1H NMR-based metabonomic urinalysis. Chem Res Toxicol. 2002;15:1380–6.CrossRefGoogle Scholar
  52. 52.
    Shipkova P, Vassallo J, Aranibar N, Hnatyshyn S, Zhang H, Clayton TA, Cantor GH, Sanders M, Coen M, Campbell A, Lindon JC, Nicholson JK, Lehman-McKeeman L. Urinary metabolites of 2-bromoethanamine in the rat identified by stable isotope labeling: evidence for carbamoylation and glutathione conjugation. Xenobiotica. 2011;41:144–54.CrossRefGoogle Scholar
  53. 53.
    Coen M, Want EJ, Clayton TA, Rhode CM, Hong YS, Keun HC, Cantor GH, Metz AL, Robertson DG, Reily MD, Holmes E, Lindon JC, Nicholson JK. Mechanistic aspects and novel biomarkers of responder and nonresponder phenotypes in galactosamine-induced hepatitis. J Proteome Res. 2009;8:5175–87.CrossRefGoogle Scholar
  54. 54.
    Gonzalez FJ, Fang ZZ, Ma X. Transgenic mice and metabolomics for study of hepatic xenobiotic metabolism and toxicity. Expert Opin Drug Metab Toxicol. 2015;11:869–81.CrossRefGoogle Scholar
  55. 55.
    Swann J, Wang Y, Abecia L, Costabile A, Tuohy K, Gibson G, Roberts D, Sidaway J, Jones H, Wilson ID, Nicholson JK, Holmes E. Gut microbiome modulates the toxicity of hydrazine: a metabonomic study. Mol Biosyst. 2009;5:351–5.CrossRefGoogle Scholar
  56. 56.
    Elliott P, Posma JM, Chan Q, Garcia-Perez I, Wijeyesekera A, Bictash M, Ebbels TM, Ueshima H, Zhao L, van Horn L, Daviglus M, Stamler J, Holmes E, Nicholson JK. Urinary metabolic signatures of human adiposity. Sci Trans Med. 2015; 7: 285ra62.CrossRefGoogle Scholar
  57. 57.
    Garcia-Perez I, Posma JM, Chambers ES, Nicholson JK, Mathers JC, Beckmann M, Draper J, Holmes E, Frost G. An analytical pipeline for quantitative characterization of dietary intake: application to assess grape intake. J Agric Food Chem. 2016;64:2423–31.CrossRefGoogle Scholar
  58. 58.
    Duarte IF, Diaz SO, Gil AM. NMR metabolomics of human blood and urine in disease research. J Pharmaceut Biomed Anal. 2014;93:17–26.CrossRefGoogle Scholar
  59. 59.
    Clayton TA, Lindon JC, Cloarec O, Antti H, Charuel C, Hanton G, Provost J-P, Le Net J-L, Baker D, Walley RJ, Everett JR, Nicholson JK. Pharmaco-metabonomic phenotyping and personalised drug treatment. Nature. 2006;440:1073–7.CrossRefGoogle Scholar
  60. 60.
    Clayton TA, Baker D, Lindon JC, Everett JR, Nicholson JK. Pharmacometabonomic identification of a significant host-microbiome metabolic interaction affecting human drug metabolism. Proc Nat Acad Sci USA. 2009;106:14728–33.CrossRefGoogle Scholar
  61. 61.
    Backshall A, Sharma R, Clarke SJ, Keun HC. Pharmacometabonomic profiling as a predictor of toxicity in patients with inoperable colorectal cancer treated with capecitabine. Clin Cancer Res. 2011;17:3019–28.CrossRefGoogle Scholar
  62. 62.
    McPhail MJ, Shawcross DL, Lewis MR, Coltart I, Want EJ, Antoniades CG, Veselkov K, Triantafyllou E, Patel V, Pop O, Gomez-Romero M, Kyriakides M, Zia R, Abeles RD, Crossey MM, Jassem W, O'Grady J, Heaton N, Auzinger G, Bernal W, Quaglia A, Coen M, Nicholson JK, Wendon JA, Holmes E, Taylor-Robinson SD. Multivariate metabotyping of plasma predicts survival in patients with decompensated cirrhosis. J Hepatol. 2016;64:1058–67.CrossRefGoogle Scholar
  63. 63.
    Nicholson JK, Holmes E, Kinross JM, Darzi AW, Takats Z, Lindon JC. Metabolic phenotyping in clinical and surgical environments. Nature. 2012;491:384–92.CrossRefGoogle Scholar
  64. 64.
    Yap IK, Brown IJ, Chan Q, Wijeyesekera A, Garcia-Perez I, Bictash M, Loo RL, Chadeau-Hyam M, Ebbels T, De Iorio M, Maibaum E, Zhao L, Kesteloot H, Daviglus ML, Stamler J, Nicholson JK, Elliott P, Holmes E. Metabolome-wide association study identifies multiple biomarkers that discriminate north and south Chinese populations at differing risks of cardiovascular disease: INTERMAP study. J Proteome Res. 2010;3:6647–54.CrossRefGoogle Scholar
  65. 65.
    Holmes E, Loo RL, Stamler J, Bictash M, Yap IK, Chan Q, Ebbels T, De Iorio M, Brown IJ, Veselkov KA, Daviglus ML, Kesteloot H, Ueshima H, Zhao L, Nicholson JK, Elliott P. Human metabolic phenotype diversity and its association with diet and blood pressure. Nature. 2008;453:396–400.CrossRefGoogle Scholar
  66. 66.
    Würtz P, Havulinna AS, Soininen P, Tynkkynen T, Prieto-Merino D, Tillin T, Ghorbani A, Artati A, Wang Q, Tiainen M, Kangas AJ, Kettunen J, Kaikkonen J, Mikkilä V, Jula A, Kähönen M, Lehtimäki T, Lawlor DA, Gaunt TR, Hughes AD, Sattar N, Illig T, Adamski J, Wang TJ, Perola M, Ripatti S, Vasan RS, Raitakari OT, Gerszten RE, Casas JP, Chaturvedi N, Ala-Korpela M, Salomaa V. Metabolite profiling and cardiovascular event risk: a prospective study of 3 population-based cohorts. Circulation. 2015;131:774–85.CrossRefGoogle Scholar
  67. 67.
    Soininen P, Kangas AJ, Wurtz P, Suna T, Ala-Korpela M. Quantitative serum nuclear magnetic resonance metabolomics in cardiovascular epidemiology and genetics. Circ Cardiovasc Genet. 2015;8:192–206.CrossRefGoogle Scholar
  68. 68.
    Crockford DJ, Holmes E, Lindon JC, Plumb RS, Zirah S, Bruce SJ, Rainville P, Stumpf CL, Nicholson JK. Statistical heterospectroscopy, an approach to the integrated analysis of NMR and UPLC-MS data sets: application in metabonomic toxicology studies. Anal Chem. 2006;78:363–71.CrossRefGoogle Scholar
  69. 69.
    Klenø TG, Kiehr B, Baunsgaard D, Sidelmann UG. Combination of ‘omics’ data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in rats and to identify potential biomarkers. Biomarkers. 2004;9:116–38.CrossRefGoogle Scholar
  70. 70.
    Rantalainen M, Cloarec O, Beckonert O, Wilson ID, Jackson D, Tonge R, Rowlinson R, Rayner S, Nickson J, Wilkinson RW, Mills JD, Trygg J, Nicholson JK, Holmes E. Statistically integrated metabonomic-proteomic studies on a human prostate cancer xenograft model in mice. J Proteome Res. 2006;5:2642–55.CrossRefGoogle Scholar
  71. 71.
    Kimhofer T, Fye H, Taylor-Robinson S, Thursz M, Hlmes E. Proteomic and metabonomics biomarkers for hepatocellular carcinoma: a comprehensive review. Br J Cancer. 2015;112:1141–56.CrossRefGoogle Scholar
  72. 72.
    Rantalainen M, Bjerrum JT, Olsen J, Nielsen OH, Wang Y. Integrative transcriptomic and metabonomic molecular profiling of colonic mucosal biopsies indicates a unique molecular phenotype for ulcerative colitis. J Proeteome Res. 2015;14:479–90.CrossRefGoogle Scholar
  73. 73.
    Gieger C, Geistlinger L, Altmaier E, Hrabé de Angelis M, Kronenberg F, Meitinger T, Mewes HW, Wichmann HE, Weinberger KM, Adamski J, Illig T, Suhre K. Genetics meets metabolomics: a genome-wide association Study of metabolite profiles in human serum. PLos Genet. 2008;4:e1000282.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Computational and Systems Medicine, Department of Surgery and CancerImperial College LondonSouth KensingtonUK

Personalised recommendations