Characterization of ankylosing spondylitis and rheumatoid arthritis using 1H NMR-based metabolomics of human fecal extracts



The differences in fecal metabolome between ankylosing spondylitis (AS)/rheumatoid arthritis (RA) patients and healthy individuals could be the reason for an autoimmune disorder.


The study explored the fecal metabolome difference between AS/RA patients and healthy controls to clarify human immune disturbance.


Fecal samples from 109 individuals (healthy controls 34, AS 40, and RA 35) were analyzed by 1H NMR spectroscopy. Data were analyzed with principal component analysis (PCA) and orthogonal projection to latent structure discriminant (OPLS-DA) analysis.


Significant differences in the fecal metabolic profiles could distinguish AS/RA patients from healthy controls but could not distinguish between AS and RA patients. The significantly decreased metabolites in AS/RA patients were butyrate, propionate, methionine, and hypoxanthine. Significantly increased metabolites in AS/RA patients were taurine, methanol, fumarate, and tryptophan.


The metabolome variations in feces indicated AS and RA were two homologous diseases that could not be distinguished by 1H NMR metabolomics.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Amiot, A., Dona, A. C., Wijeyesekera, A., Tournigand, C., Baumgaertner, I., Lebaleur, Y., et al. (2015). 1H NMR spectroscopy of fecal extracts enables detection of advanced colorectal neoplasia. Journal of Proteome Research, 14(9), 3871–3881. doi:10.1021/acs.jproteome.5b00277.

    CAS  Article  PubMed  Google Scholar 

  2. Arase, T., Uchida, H., Kajitani, T., Ono, M., Tamaki, K., Oda, H., et al. (2009). The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8. Journal of Immunology, 182(11), 7074–7084. doi:10.4049/jimmunol.0900001.

    CAS  Article  Google Scholar 

  3. Azzam, M., Zou, X., Dong, X., & Xie, P. (2011). Effect of supplemental l-threonine on mucin 2 gene expression and intestine mucosal immune and digestive enzymes activities of laying hens in environments with high temperature and humidity. Poultry Science, 90(10), 2251–2256. doi:10.3382/ps.2011-01574.

    CAS  Article  PubMed  Google Scholar 

  4. Bjerrum, J. T., Wang, Y., Hao, F., Coskun, M., Ludwig, C., Gunther, U., & Nielsen, O. H. (2015). Metabonomics of human fecal extracts characterize ulcerative colitis, Crohn’s disease and healthy individuals. Metabolomics, 11, 122–133. doi:10.1007/s11306-014-0677-3.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Cani, P. D., & Delzenne, N. M. (2007). Gut microflora as a target for energy and metabolic homeostasis. Current Opinion in Clinical Nutrition & Metabolic Care, 10(6), 729–734. doi:10.1097/MCO.0b013e3282efdebb.

    Article  Google Scholar 

  6. Chen, R., Han, S., Dong, D., Wang, Y., Liu, Q., Xie, W., et al. (2015). Serum fatty acid profiles and potential biomarkers of ankylosing spondylitis determined by gas chromatography-mass spectrometry and multivariate statistical analysis. Biomedical Chromatography, 29(4), 604–611. doi:10.1002/bmc.3321.

    CAS  Article  PubMed  Google Scholar 

  7. Conterno, L., Fava, F., Viola, R., & Tuohy, K. M. (2011). Obesity and the gut microbiota: Does up-regulating colonic fermentation protect against obesity and metabolic disease? Genes and Nutrition, 6(3), 241–260. doi:10.1007/s12263-011-0230-1.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Costello, M. E., Ciccia, F., Willner, D., Warrington, N., Robinson, P. C., Gardiner, B., et al. (2015). Brief Report: Intestinal dysbiosis in ankylosing spondylitis. Arthritis & Rheumatology, 67(3), 686–691. doi:10.1002/art.38967.

    Article  Google Scholar 

  9. Fan, W. M. T. (1996). Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy, 28(2), 161–219. doi:10.1016/0079-6565(95)01017-3.

    CAS  Article  Google Scholar 

  10. Fan, T. W. M., & Lane, A. N. (2008). Structure-based profiling of metabolites and isotopomers by NMR. Progress in Nuclear Magnetic Resonance Spectroscopy, 52(2), 69–117. doi:10.1016/j.pnmrs.2007.03.002.

    CAS  Article  Google Scholar 

  11. Fischer, R., Trudgian, D.C., Wright, C., Thomas, G., Bradbury, L.A., Brown, M.A., Bowness, P., Kessler, B.M. (2012). Discovery of candidate serum proteomic and metabolomic biomarkers in ankylosing spondylitis. Molecular & Cellular Proteomics, 11 (2), M111. 013904. doi: 10.1074/mcp.M111.013904.

  12. Fukuda, S., Toh, H., Hase, K., Oshima, K., Nakanishi, Y., Yoshimura, K., et al. (2011). Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature, 469(7331), 543–547. doi:10.1038/nature09646.

    CAS  Article  PubMed  Google Scholar 

  13. Gold, R., Linker, R. A., & Stangel, M. (2012). Fumaric acid and its esters: An emerging treatment for multiple sclerosis with antioxidative mechanism of action. Clinical Immunology, 142(1), 44–48. doi:10.1016/j.clim.2011.02.017.

    CAS  Article  PubMed  Google Scholar 

  14. Gregory, K. E., Bird, S. S., Gross, V. S., Marur, V. R., Lazarev, A. V., Walker, W. A., & Kristal, B. S. (2012). Method development for fecal lipidomics profiling. Analytical Chemistry, 85(2), 1114–1123. doi:10.1021/ac303011k.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Hamard, A., Mazurais, D., Boudry, G., Le Huërou-Luron, I., Sève, B., & Le Floc’h, N. (2010). A moderate threonine deficiency affects gene expression profile, paracellular permeability and glucose absorption capacity in the ileum of piglets. Journal of Nutritional Biochemistry, 21(10), 914–921. doi:10.1016/j.jnutbio.2009.07.004.

    CAS  Article  PubMed  Google Scholar 

  16. Hamer, H. M., Jonkers, D., Venema, K., Vanhoutvin, S., Troost, F., & Brummer, R. J. (2008). Review article: The role of butyrate on colonic function. Alimentary Pharmacology & Therapeutics, 27(2), 104–119. doi:10.1111/j.1365-2036.2007.03562.x.

    CAS  Article  Google Scholar 

  17. Huda-Faujan, N., Abdulamir, A. S., Fatimah, A. B., Anas, O. M., Shuhaimi, M., Yazid, A. M., & Loong, Y. Y. (2010). The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects. The Open Biochemistry Journal, 4, 53–58. doi:10.2174/1874091X01004010053.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Jacobs, S. R., Herman, C. E., MacIver, N. J., Wofford, J. A., Wieman, H. L., Hammen, J. J., & Rathmell, J. C. (2008). Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways. The Journal of Immunology, 180(7), 4476–4486. doi:10.4049/jimmunol.180.7.4476.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Lamichhane, S., Yde, C. C., Schmedes, M. S., Jensen, H. M., Meier, S., & Bertram, H. C. (2015). Strategy for nuclear-magnetic-resonance-based metabolomics of human feces. Analytical Chemistry, 87(12), 5930–5937. doi:10.1021/acs.analchem.5b00977.

    CAS  Article  PubMed  Google Scholar 

  20. Law, G. K., Bertolo, R. F., Adjiri-Awere, A., Pencharz, P. B., & Ball, R. O. (2007). Adequate oral threonine is critical for mucin production and gut function in neonatal piglets. American Journal of Physiology-Gastrointestinal and Liver Physiology, 292(5), 1293–1301. doi:10.1152/ajpgi.00221.2006.

    Article  Google Scholar 

  21. Le Gall, G., Noor, S. O., Ridgway, K., Scovell, L., Jamieson, C., Johnson, I. T., et al. (2011). Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome. Journal of Proteome Research, 10(9), 4208–4218. doi:10.1021/pr2003598.

    Article  PubMed  Google Scholar 

  22. Marcinkiewicz, J., & Kontny, E. (2014). Taurine and inflammatory diseases. Amino Acids, 46(1), 7–20. doi:10.1007/s00726-012-1361-4.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Maslowski, K. M., & Mackay, C. R. (2011). Diet, gut microbiota and immune responses. Nature Immunology, 12(1), 5–9. doi:10.1038/ni0111-5.

    CAS  Article  PubMed  Google Scholar 

  24. Munn D, Mellor A. (2012) Regulation of T cell-mediated immunity by tryptophan: U.S. Patent 8, 198, 265. 2012-6-12.

  25. Natarajan, N., & Pluznick, J. L. (2014). From microbe to man: The role of microbial short chain fatty acid metabolites in host cell biology. American Journal of Physiology-Cell Physiology, 307(11), 979–985. doi:10.1152/ajpcell.00228.2014.

    Article  Google Scholar 

  26. Nicholson, J. K., Holmes, E., Kinross, J., Burcelin, R., Gibson, G., Jia, W., & Pettersson, S. (2012). Host-gut microbiota metabolic interactions. Science, 336(6086), 1262–1267. doi:10.1126/science.1223813.

    CAS  Article  PubMed  Google Scholar 

  27. Nuenen, M. H., Ligt, R. A., Doornbos, R. P., Woude, J. C., Kuipers, E. J., & Venema, K. (2005). The influence of microbial metabolites on human intestinal epithelial cells and macrophages in vitro. FEMS Immunology and Medical Microbiology, 45(2), 183–189. doi:10.1016/j.femsim.2005.03.010.

    Article  PubMed  Google Scholar 

  28. Ohno, H. (2015). Gut microbial short-chain fatty acids in host defense and immune regulation. Inflammation and Regeneration, 35(3), 114–121. doi:10.2492/inflammregen.35.114.

    CAS  Article  Google Scholar 

  29. Peng, L., Li, Z.-R., Green, R. S., Holzman, I. R., & Lin, J. (2009). Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. The Journal of Nutrition, 139(9), 1619–1625. doi:10.3945/jn.109.104638.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Round, J. L., & Mazmanian, S. K. (2009). The gut microbiota shapes intestinal immune responses during health and disease. Nature Reviews Immunology, 9(5), 313–323. doi:10.1038/nri2515.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Ruth, M. R., & Field, C. J. (2013). The immune modifying effects of amino acids on gut-associated lymphoid tissue. Journal of Animal Science and Biotechnology, 4(1), 27. doi:10.1186/2049-1891-4-27.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Salazar, N., Dewulf, E. M., Neyrinck, A. M., Bindels, L. B., Cani, P. D., Mahillon, J., et al. (2015). Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal short-chain fatty acids in obese women. Clinical Nutrition, 34(3), 501–507. doi:10.1016/j.clnu.2014.06.001.

    CAS  Article  PubMed  Google Scholar 

  33. Samuel, B. S., Shaito, A., Motoike, T., Rey, F. E., Backhed, F., Manchester, J. K., et al. (2008). Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. Proceedings of the National Academy of Sciences, 105(43), 16767–16772. doi:10.1073/pnas.0808567105.

    CAS  Article  Google Scholar 

  34. Segain, J., De La Blétiere, D. R., Bourreille, A., Leray, V., Gervois, N., Rosales, C., et al. (2000). Butyrate inhibits inflammatory responses through NFκB inhibition: Implications for Crohn’s disease. Gut, 47(3), 397–403. doi:10.1136/gut.47.3.397.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Smith, P. M., Howitt, M. R., Panikov, N., Michaud, M., Gallini, C. A., Bohlooly-Y, M., et al. (2013). The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science, 341(6145), 569–573. doi:10.1126/science.1241165.

    CAS  Article  PubMed  Google Scholar 

  36. Tjellstrom, B., Stenhammar, L., Hogberg, L., Falth-Magnusson, K., Magnusson, K. E., Midtvedt, T., et al. (2007). Gut microflora associated characteristics in first-degree relatives of children with celiac disease. Scandinavian Journal of Gastroenterology, 42(10), 1204–1208. doi:10.1080/00365520701320687.

    Article  PubMed  Google Scholar 

  37. Wang, Z., Chen, Z., Yang, S., Wang, Y., Yu, L., Zhang, B., et al. (2012). 1H NMR-based metabolomic analysis for identifying serum biomarkers to evaluate methotrexate treatment in patients with early rheumatoid arthritis. Experimental & Therapeutic Medicine, 4(1), 165–171. doi:10.3892/etm.2012.567.

    CAS  Google Scholar 

  38. Weisbart, R. H., Min, Y., Wong, A. L., Kang, J., Kwunyeun, S., & Lin, A. (2005). Selective IgA immune unresponsiveness to Proteus mirabilis fumarate reductase A-chain in rheumatoid arthritis. The Journal of Rheumatology, 32(7), 1208–1212.

    CAS  PubMed  Google Scholar 

  39. Ye, Y., Wang, X., Zhang, L., Lu, Z., & Yan, X. (2012). Unraveling the concentration-dependent metabolic response of Pseudomonas sp. HF-1 to nicotine stress by 1H NMR-based metabolomics. Ecotoxicology, 21(5), 1314–1324. doi:10.1007/s10646-012-0885-4.

    CAS  Article  PubMed  Google Scholar 

  40. Zelante, T., Iannitti, R. G., Cunha, C., De Luca, A., Giovannini, G., Pieraccini, G., et al. (2013). Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity, 39(2), 372–385. doi:10.1016/j.immuni.2013.08.003.

    CAS  Article  PubMed  Google Scholar 

  41. Zhang, X., Zhang, D., Jia, H., Feng, Q., Wang, D., Liang, D., et al. (2015). The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nature Medicine, 21, 895–905. doi:10.1038/nm.3914.

    CAS  Article  PubMed  Google Scholar 

Download references


This study was financially supported by the National Natural Science Foundation of China (No. 81302936) and the Natural Science Foundation of Zhejiang Province (No. Q13H270006).

Author information



Corresponding author

Correspondence to Cheng-ping Wen.

Ethics declarations

Research involving human rights

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional, national research committee and with the 1964 Helsinki Declaration and its later amendments.

Conflict of interest

The authors declare no conflicts of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Tie-juan Shao and Zhi-xing He have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 457 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shao, T., He, Z., Xie, Z. et al. Characterization of ankylosing spondylitis and rheumatoid arthritis using 1H NMR-based metabolomics of human fecal extracts. Metabolomics 12, 70 (2016).

Download citation


  • Metabolomics
  • Faces
  • Ankylosing spondylitis
  • Rheumatoid arthritis
  • NMR
  • Autoimmune disorder