The association of fecal microbiota and fecal, blood serum and urine metabolites in myalgic encephalomyelitis/chronic fatigue syndrome

Abstract

Introduction

The human gut microbiota has the ability to modulate host metabolism. Metabolic profiling of the microbiota and the host biofluids may determine associations significant of a host–microbe relationship. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a long-term disorder of fatigue that is poorly understood, but has been linked to gut problems and altered microbiota.

Objectives

Find changes in fecal microbiota and metabolites in ME/CFS and determine their association with blood serum and urine metabolites.

Methods

A workflow was developed that correlates microbial counts with fecal, blood serum and urine metabolites quantitated by high-throughput 1H NMR spectroscopy. The study consists of thirty-four females with ME/CFS (34.9 ± 1.8 SE years old) and twenty-five non-ME/CFS female (33.0 ± 1.6 SE years old).

Results

The workflow was validated using the non-ME/CFS cohort where fecal short chain fatty acids (SCFA) were associated with serum and urine metabolites indicative of host metabolism changes enacted by SCFA. In the ME/CFS cohort a decrease in fecal lactate and an increase in fecal butyrate, isovalerate and valerate were observed along with an increase in Clostridium spp. and a decrease in Bacteroides spp. These differences were consistent with an increase in microbial fermentation of fiber and amino acids to produce SCFA in the gut of ME/CFS patients. Decreased fecal amino acids positively correlated with substrates of gluconeogenesis and purine synthesis in the serum of ME/CFS patients.

Conclusion

Increased production of SCFA by microbial fermentation in the gut of ME/CFS patients may be associated with deleterious effects on the host energy metabolism.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

BCFA:

Branched-chain fatty acids

IBD:

Irritable bowel disease

ME/CFS:

Myalgic encephalomyelitis/chronic fatigue syndrome

NOESY:

Nuclear overhauser effect spectroscopy

NMR:

Nuclear magnetic resonance

PCA:

Principal component analysis

SCFA:

Short chain fatty acids

TOCSY:

Total correlated spectroscopy

References

  1. Armstrong, C. W., McGregor, N. R., Butt, H. L., & Gooley, P. R. (2014). Metabolism in chronic fatigue syndrome. Advances in Clinical Chemistry, 66, 121–172.

    CAS  Article  PubMed  Google Scholar 

  2. Armstrong, C. W., McGregor, N. R., Lewis, D. P., Butt, H. L., & Gooley, P. R. (2015). Metabolic profiling reveals anomalous energy metabolism and oxidative stress pathways in chronic fatigue syndrome patients. Metabolomics, 11, 1626–1639. doi:10.1007/s11306-015-0816-5.

    CAS  Article  Google Scholar 

  3. Armstrong, C. W., McGregor, N. R., Sheedy, J. R., Buttfield, I., Butt, H. L., & Gooley, P. R. (2012). NMR metabolic profiling of serum identifies amino acid disturbances in chronic fatigue syndrome. Clinica Chimica Acta; International Journal of Clinical Chemistry, 413, 1525–1531. doi:10.1016/j.cca.2012.06.022.

    CAS  Article  PubMed  Google Scholar 

  4. Baticz, O., Tomoskozi, S., Vida, L., & Gaal, T. (2002). Relationship between concentration of citrate and ketone bodies in cow’s milk. Acta Veterinaria Hungarica, 50, 253–261. doi:10.1556/AVet.50.2002.3.1.

    CAS  Article  PubMed  Google Scholar 

  5. Belenguer, A., Duncan, S. H., Holtrop, G., Anderson, S. E., Lobley, G. E., & Flint, H. J. (2007). Impact of pH on lactate formation and utilization by human fecal microbial communities. Applied and Environmental Microbiology, 73, 6526–6533. doi:10.1128/AEM.00508-07.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Borody, T., Leis, S., Campbell, J., Torres, M., & Nowak, A. (2011). Fecal microbiota transplantation (FMT) in multiple sclerosis (MS). American Journal of Gastroenterology, 106, S352–S352.

    Google Scholar 

  7. Borody, T. J., Brandt, L. J., & Paramsothy, S. (2014). Therapeutic faecal microbiota transplantation: current status and future developments. Current Opinion in Gastroenterology, 30, 97–105. doi:10.1097/MOG.0000000000000027.

    Article  PubMed  Google Scholar 

  8. Borody, T. J., & Khoruts, A. (2012). Fecal microbiota transplantation and emerging applications. Nature Reviews. Gastroenterology and Hepatology, 9, 88–96. doi:10.1038/nrgastro.2011.244.

    CAS  Article  Google Scholar 

  9. Bourriaud, C., et al. (2005). Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident. Journal of Applied Microbiology, 99, 201–212. doi:10.1111/j.1365-2672.2005.02605.x.

    CAS  Article  PubMed  Google Scholar 

  10. Canani, R. B., Costanzo, M. D., Leone, L., Pedata, M., Meli, R., & Calignano, A. (2011). Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World Journal of Gastroenterology: WJG, 17, 1519–1528. doi:10.3748/wjg.v17.i12.1519.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Canfora, E. E., Jocken, J. W., & Blaak, E. E. (2015). Short-chain fatty acids in control of body weight and insulin sensitivity. Nature Reviews Endocrinology, 11, 577–591. doi:10.1038/nrendo.2015.128.

    CAS  Article  PubMed  Google Scholar 

  12. Cardona, M. E., Collinder, E., Stern, S., Tjellstrom, B., Norin, E., Midtvedt, T. (2005). Correlation between faecal iso-butyric and iso-valeric acids in different species. Microbial Ecology in Health and Disease, 17, 177–182.

    CAS  Article  Google Scholar 

  13. Carruthers, B. M., et al. (2003). Myalgic encephalomyelitis/chronic fatigue syndrome: clinical working case definition, diagnostic and treatment protocols. J Chronic Fatigue Syndrome, 11, 7–36.

    Article  Google Scholar 

  14. Corby, P. M., et al. (2005). Microbial risk indicators of early childhood caries. Journal of clinical microbiology, 43, 5753–5759. doi:10.1128/JCM.43.11.5753-5759.2005.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. den Besten, G., et al. (2013a). Gut-derived short-chain fatty acids are vividly assimilated into host carbohydrates and lipids. American Journal of Physiology Gastrointestinal and Liver Physiology, 305, G900–G910. doi:10.1152/ajpgi.00265.2013.

    CAS  Article  PubMed  Google Scholar 

  16. den Besten, G., van Eunen, K., Groen, A. K., Venema, K., Reijngoud, D. J., & Bakker, B. M. (2013b). The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. Journal of Lipid Research, 54, 2325–2340. doi:10.1194/jlr.R036012.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Dorrestein, P. C., Mazmanian, S. K., & Knight, R. (2014). Finding the missing links among metabolites, microbes, and the host. Immunity, 40, 824–832. doi:10.1016/j.immuni.2014.05.015.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Fremont, M., Coomans, D., Massart, S., & De Meirleir, K. (2013). High-throughput 16 S rRNA gene sequencing reveals alterations of intestinal microbiota in myalgic encephalomyelitis/chronic fatigue syndrome patients. Anaerobe, 22, 50–56. doi:10.1016/j.anaerobe.2013.06.002.

    CAS  Article  PubMed  Google Scholar 

  19. Georgiades, E., et al. (2003). Chronic fatigue syndrome: new evidence for a central fatigue disorder. Clinical Science (London, England: 1979), 105, 213–218. doi:10.1042/CS20020354.

    CAS  Article  Google Scholar 

  20. Giloteaux, L., Goodrich, J. K., Walters, W. A., Levine, S. M., Ley, R. E., Hanson, M. R. (2016). Reduced diversity and altered composition of the gut microbiome in individuals with myalgic encephalomyelitis/chronic fatigue syndrome. Microbiome, 4, 30 doi:10.1186/s40168-016-0171-4.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Guarner, F., & Malagelada, J. R. (2003). Gut flora in health and disease. Lancet, 361, 512–519. doi:10.1016/S0140-6736(03)12489-0.

    Article  PubMed  Google Scholar 

  22. Guinane, C. M., & Cotter, P. D. (2013). Role of the gut microbiota in health and chronic gastrointestinal disease: understanding a hidden metabolic organ. Therapeutic Advances in Gastroenterology, 6, 295–308 doi:10.1177/1756283X13482996.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Hornig, M., et al. (2015). Distinct plasma immune signatures in ME/CFS are present early in the course of illness. Science Advances, 1, e1400121. doi:10.1126/sciadv.1400121.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Huda-Faujan, N., et al. (2010). The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects. The Open Biochemical Journal, 4, 53–58. doi:10.2174/1874091X01004010053.

    CAS  Article  Google Scholar 

  25. Human Microbiome Project, C (2012). Structure, function and diversity of the healthy human microbiome. Nature, 486, 207–214. doi:10.1038/nature11234.

    Article  Google Scholar 

  26. Jacobson, W., Saich, T., Borysiewicz, L. K., Behan, W. M., Behan, P. O., & Wreghitt, T. G. (1993). Serum folate and chronic fatigue syndrome. Neurology, 43, 2645–2647.

    CAS  Article  PubMed  Google Scholar 

  27. Li, M., et al. (2008). Symbiotic gut microbes modulate human metabolic phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 105, 2117–2122. doi:10.1073/pnas.0712038105.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Macfabe, D. F. (2012). Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders. Microbial Ecology in Health and Disease, 23, 19260. doi:10.3402/mehd.v23i0.19260.

    Article  Google Scholar 

  29. Maes, M., Kubera, M., Uytterhoeven, M., Vrydags, N., Bosmans, E. (2011). Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Medical Science Monitor, 17, SC11–SC15.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. McGregor, N. R., Dunstan, R. H., Zerbes, M., Butt, H. L., Roberts, T. K., & Klineberg, I. J. (1996). Preliminary determination of a molecular basis of chronic fatigue syndrome. Biochemical and Molecular Medicine, 57, 73–80.

    CAS  Article  PubMed  Google Scholar 

  31. Moayyedi, P., et al. (2015). Fecal microbiota transplantation induces remission in patients with active ulcerative colitis in a randomized controlled trial. Gastroenterology, 149, 102–109.e6. doi:10.1053/j.gastro.2015.04.001.

    Google Scholar 

  32. Naviaux, R. K., et al. (2016). Metabolic features of chronic fatigue syndrome. Proceedings of the National Academy of Sciences of the United States of America, 113, E5472–E5480. doi:10.1073/pnas.1607571113.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Niblett, S. H., et al. (2007). Hematologic and urinary excretion anomalies in patients with chronic fatigue syndrome. Experimental Biology and Medicine (Maywood, N. J.), 232, 1041–1049. doi:10.3181/0702-RM-44.

    CAS  Article  Google Scholar 

  34. Nicholson, J. K., et al. (2012). Host-gut microbiota metabolic interactions. Science, 336, 1262–1267. doi:10.1126/science.1223813.

    CAS  Article  PubMed  Google Scholar 

  35. Pall, M. L. (2002). Levels of nitric oxide synthase product citrulline are elevated in sera of chronic fatigue syndrome patients. Journal of Chronic Fatigue Syndrome, 10, 37–41. doi:10.1300/J092v10n03_04.

    Article  Google Scholar 

  36. Rasmussen, H. S., Holtug, K., & Mortensen, P. B. (1988). Degradation of amino-acids to short-chain fatty-acids in humans: an invitro study. Scandinavian Journal of Gastroenterology, 23, 178–182. doi:10.3109/00365528809103964.

    CAS  Article  PubMed  Google Scholar 

  37. Rigottier-Gois, L., Rochet, V., Garrec, N., Suau, A., & Dore, J. (2003). Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16 S rRNA probes. Systematic and Applied Microbiology, 26, 110–118. doi:10.1078/072320203322337399.

    Article  PubMed  Google Scholar 

  38. Schaubeck, M., et al. (2016). Dysbiotic gut microbiota causes transmissible Crohn’s disease-like ileitis independent of failure in antimicrobial defence. Gut, 65, 225–237. doi:10.1136/gutjnl-2015-309333.

    CAS  Article  PubMed  Google Scholar 

  39. Scheppach, W. (1994). Effects of short chain fatty acids on gut morphology and function. Gut, 35, S35–S38.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Sheedy, J. R., et al. (2009). Increased d-lactic Acid intestinal bacteria in patients with chronic fatigue syndrome. In Vivo (Athens, Greece), 23, 621–628.

    CAS  Google Scholar 

  41. Sheedy, J. R., Ebeling, P. R., Gooley, P. R., & McConville, M. J. (2010). A sample preparation protocol for 1 H nuclear magnetic resonance studies of water-soluble metabolites in blood and urine. Analytical Biochemistry, 398, 263–265. doi:10.1016/j.ab.2009.11.027.

    CAS  Article  PubMed  Google Scholar 

  42. Smith, E. A., & Macfarlane, G. T. (1998). Enumeration of amino acid fermenting bacteria in the human large intestine: effects of pH and starch on peptide metabolism and dissimilation of amino acids. FEMS Microbiology Ecology, 25, 355–368. doi:10.1016/S0168-6496(98)00004-X.

    CAS  Article  Google Scholar 

  43. Suarez, A., et al. (2010). Nitric oxide metabolite production during exercise in chronic fatigue syndrome: a case-control study. Journal of Women’s Health (Larchmt), 19, 1073–1077. doi:10.1089/jwh.2008.1255.

    Article  Google Scholar 

  44. Tretter, L., & Adam-Vizi, V. (2000). Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 20, 8972–8979.

    CAS  Google Scholar 

  45. Van den Abbeele, P., et al. (2013). Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. ISME Journal, 7, 949–961. doi:10.1038/ismej.2012.158.

    Article  PubMed  Google Scholar 

  46. Vasquez-Vivar, J., Kalyanaraman, B., & Kennedy, M. C. (2000). Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation. The Journal of Biological Chemistry, 275, 14064–14069.

    CAS  Article  PubMed  Google Scholar 

  47. Vrieze, A., et al. (2012). Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology, 143, 913-916.e7. doi:10.1053/j.gastro.2012.06.031.

    Article  Google Scholar 

  48. Walker, A. W., Duncan, S. H., McWilliam Leitch, E. C., Child, M. W., & Flint, H. J. (2005). pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon. Applied and Environmental Microbiology, 71, 3692–3700. doi:10.1128/AEM.71.7.3692-3700.2005.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Wang, L., Christophersen, C. T., Sorich, M. J., Gerber, J. P., Angley, M. T., & Conlon, M. A. (2012). Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder. Digestive Diseases and Sciences, 57, 2096–2102. doi:10.1007/s10620-012-2167-7.

    CAS  Article  PubMed  Google Scholar 

  50. White, P. D. (2004). What causes chronic fatigue syndrome? BMJ (Clinical Research Ed.), 329, 928–929. doi:10.1136/bmj.329.7472.928.

    Article  Google Scholar 

  51. Willis, A. T. (1991). Anaerobic culture methods. In P. N. Lovett. (Ed.), Anaerobic microbiology a practical approach the practical approach series. (pp. 1–12). New York: IRL Press at Oxford University Press.

    Google Scholar 

  52. Wong, J. M., de Souza, R., Kendall, C. W., Emam, A., & Jenkins, D. J. (2006). Colonic health: fermentation and short chain fatty acids. Journal of Clinical Gastroenterology, 40, 235–243.

    CAS  Article  PubMed  Google Scholar 

  53. Yamano, E., et al. (2016). Index markers of chronic fatigue syndrome with dysfunction of TCA and urea cycles. Scientific Reports, 6, 34990. doi:10.1038/srep34990.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Zarling, E. J., & Ruchim, M. A. (1987). Protein origin of the volatile fatty acids isobutyrate and isovalerate in human stool. Journal of Laboratory and Clinical Medicine, 109, 566–570.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors of this work would like to thank the nursing and administrative staff at the CFS Discovery clinic for their important help throughout this study.

Funding

This work was supported by Grants from the Judith Jane Mason and Harold Stannett Williams Memorial Foundation (The Mason Foundation) CT9957 and MAS2015F020 and equipment Grants from the Rowden White foundation and State of Victoria.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Paul R. Gooley.

Ethics declarations

This study was approved by the University of Melbourne human research ethics committee (HREC #0723086).

Conflict of interest

There were no conflicts of interest.

Additional information

Data deposition The data for this study has been deposited at MetaboLights (http://www.ebi.ac.uk/metabolights) with Accession No. MTBLS369.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Armstrong, C.W., McGregor, N.R., Lewis, D.P. et al. The association of fecal microbiota and fecal, blood serum and urine metabolites in myalgic encephalomyelitis/chronic fatigue syndrome. Metabolomics 13, 8 (2017). https://doi.org/10.1007/s11306-016-1145-z

Download citation

Keywords

  • Myalgic encephalomyelitis/chronic fatigue syndrome
  • Feces
  • Microbiota
  • Short chain fatty acids
  • Energy metabolism
  • Amino acids