Identification and Quantification by Targeted Metabolomics of Antibiotic-Responsive Urinary Small Phenolic Molecules Derived from the Intestinal Microbiota in Mice

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Mark E. Obrenovich
George Eugene Jaskiw
Renliang Zhang
Belinda Willard
Curtis J. Donskey


Background: Urinary levels of small molecules generated by the gut microbiota (GMB) constitute potential biomarkers for the state of the GMB. Such metabolites include numerous small phenolic molecules linked to anaerobic bacteria, particularly Clostridium species. Due to multiple technical challenges, however, the relationship between these chemicals and the GMB remains poorly characterized. Improved, high-performance liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis can now reliably separate and quantify low levels of multiple small phenolic molecules and their structural isomers.

Methods: CF-1 (female mice) were treated over 2 consecutive days with either i) vehicle, ii) one of 2 different antibiotic regimens (clindamycin or piperacillin/tazobactam) known to inhibit intestinal anaerobes and promote colonization by Clostridium difficile and other pathogens or iii) an antibiotic (aztreonam) that suppresses facultative Gram-negative bacteria but not enterococci or anaerobes and does not promote pathogen colonization Urine collected 24 hours after the last treatment was analyzed by LC-MS.

Results: We identified over 25 compounds, many of which had not been previously reported in mouse urine. Eleven small phenolic molecules showed significant antibiotic-related changes. Urinary levels of the hydroxyphenylpropionic acids were suppressed by clindamycin and piperacillin/tazobactam treatment, but were elevated in aztreonam-treated mice. In addition, aztreonam treatment was associated with elevated levels of the dihydroxyhydrocinnamic acids.

Conclusions: Profiles of differential changes in urinary small phenolic molecules may provide an index of anaerobic bacterial species in the GMB and could prove useful in monitoring susceptibility to overgrowth of pathogens such as C. difficile.


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Author Biography

George Eugene Jaskiw, Louis Stokes Cleveland DVAMC, Case Western Reserve University School of Medicine

Professor, Department of Psychiatry

Case Western Reserve University School of Medicine


1. Wikoff WR, Anfora AT, Liu J, Schultz PG, Lesley SA, Peters EC, Siuzdak G. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(10):3698-703. PubMed PMID: 19234110. Pubmed Central PMCID: PMC2656143. doi: 10.1073/pnas.0812874106

2. Collins SM, Surette M, Bercik P. The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol. 2012;10(11):735-42.

3. Wishart DS, Feunang YD, Marcu A, Guo AC, Liang K, Vazquez-Fresno R, Sajed T, Johnson D, Li C, Karu N, Sayeeda Z, Lo E, Assempour N, Berjanskii M, Singhal S, Arndt D, Liang Y, Badran H, Grant J, Serra-Cayuela A, Liu Y, Mandal R, Neveu V, Pon A, Knox C, Wilson M, Manach C, Scalbert A. HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Research. 2018;46(D1):D608-d17. PubMed PMID: 29140435. Pubmed Central PMCID: PMC5753273. doi: 10.1093/nar/gkx1089

4. Pultz NJ, Donskey CJ. Effect of antibiotic treatment on growth of and toxin production by Clostridium difficile in the cecal contents of mice. Antimicrobial Agents and Chemotherapy. 2005;49(8):3529-32. PubMed PMID: 16048976. Pubmed Central PMCID: PMC1196291. doi: 10.1128/aac.49.8.3529-3532.2005

5. Pultz NJ, Stiefel U, Subramanyan S, Helfand MS, Donskey CJ. Mechanisms by which anaerobic microbiota inhibit the establishment in mice of intestinal colonization by vancomycin-resistant Enterococcus. J Infect Dis. 2005;191(6):949-56.

6. Jump RL, Polinkovsky A, Hurless K, Sitzlar B, Eckart K, Tomas M, Deshpande A, Nerandzic MM, Donskey CJ. Metabolomics analysis identifies intestinal microbiota-derived biomarkers of colonization resistance in clindamycin-treated mice. PloS One. 2014;9(7):e101267. PubMed PMID: 24988418. Pubmed Central PMCID: PMC4079339. doi: 10.1371/journal.pone.0101267

7. Deshpande A, Pant C, Olyaee M, Donskey CJ. Hospital readmissions related to Clostridium difficile infection in the United States. American Journal of Infection Control. 2018;46(3):346-7. PubMed PMID: 29050906. doi: 10.1016/j.ajic.2017.08.043

8. Obrenovich ME, Tima M, Polinkovsky A, Zhang R, Emancipator SN, Donskey CJ. Targeted Metabolomics Analysis Identifies Intestinal Microbiota-Derived Urinary Biomarkers of Colonization Resistance in Antibiotic-Treated Mice. Antimicrobial Agents and Chemotherapy. 2017;61(8):pii: e00477-17. PubMed PMID: 28584146. Pubmed Central PMCID: PMC5527637. doi: 10.1128/aac.00477-17

9. Curtius HC, Redweik U, Steinmann B, Leimbacher W, Wegmann H. Use of deuterated tyrosine and phenylalanine in the study of catecholamine and aromatic amino acid metabolism. In: Klein ER, Klein PD, editors. Proceedings of the Second International Conference on Stable Isotopes, October 20-23, 1975, Oak Brook, Illinois. Washington, DC: U.S. Energy Research and Development Administration; 1976. p. 385-91.

10. Rampini S, Vollmin JA, Bosshard HR, Muller M, Curtius HC. Aromatic acids in urine of healthy infants, persistent hyperphenylalaninemia, and phenylketonuria, before and after phenylalanine load. Pediatr Res. 1974;8(7):704-9.

11. Williamson G, Clifford MN. Role of the small intestine, colon, and microbiota in determining the metabolic fate of polyphenols. Biochemical Pharmacology. 2017;139:24-39. PubMed PMID: 28322745. doi: 10.1016/j.bcp.2017.03.012

12. Kesli R, Gokcen C, Bulug U, Terzi Y. Investigation of the relation between anaerobic bacteria genus clostridium and late-onset autism etiology in children. J Immunoassay Immunochem. 2014;35(1):101-9.

13. Shaw W. Increased urinary excretion of a 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia spp. in the gastrointestinal tract, in urine samples from patients with autism and schizophrenia. Nutritional Neuroscience. 2010;13(3):135-43. PubMed PMID: 20423563. doi: 10.1179/147683010X12611460763968

14. Shaw W. Clostridia bacteria in the gastrointestinal tract as a major cause of depression and other neuropsychiatric disorders. In: Greenblatt J, Brogan K, editors. Integrative Psychiatry for Depression: Redefining Models for Assessment, Treatment, and Prevention of Mood Disorders. New York, NY: Taylor and Francis; 2016. p. 31-48.

15. Xiong X, Liu D, Wang Y, Zeng T, Peng Y. Urinary 3-(3-Hydroxyphenyl)-3-hydroxypropionic Acid, 3-Hydroxyphenylacetic Acid, and 3-Hydroxyhippuric Acid Are Elevated in Children with Autism Spectrum Disorders. Biomed Res Int. 2016;2016:9485412. PubMed PMID: 27123458. Pubmed Central PMCID: PMC4829699. doi: 10.1155/2016/9485412

16. Fang ZZ, Gonzalez FJ. LC-MS-based metabolomics: an update. Archives of Toxicology. 2014;88(8):1491-502. PubMed PMID: 24710571. doi: 10.1007/s00204-014-1234-6

17. Obrenovich ME, Donskey CJ, Schiefer IT, Bongiovanni R, Li L, Jaskiw GE. Quantification of phenolic acid metabolites in humans by LC-MS: a structural and targeted metabolomics approach. Bioanalysis. 2018;10(19):1591-608. PubMed PMID: 30295550. doi: 10.4155/bio-2018-0140

18. 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(9):e73076. PubMed PMID: 24023812. Pubmed Central PMCID: PMC3762851. doi: 10.1371/journal.pone.0073076

19. Shangari N, Chan TS, O'Brien PJ. Sulfation and glucuronidation of phenols: implications in coenyzme Q metabolism. Methods in Enzymology. 2005;400:342-59. PubMed PMID: 16399359. doi: 10.1016/s0076-6879(05)00020-0

20. Obrenovich ME, Donskey CJ, Schiefer IT, Bongiovanni R, Li L, Jaskiw GE. Quantification of Phenolic Acid Metabolites in Humans by LC-MS – A Structural and Targeted Metabolomics Approach. Bioanalysis.(in press).

21. van de Merbel NC. Quantitative determination of endogenous compounds in biological samples using chromatographic techniques. Trends Anal Chem. 2008;27(10):924-33.

22. Wu Y, Li L. Development of isotope labeling liquid chromatography-mass spectrometry for metabolic profiling of bacterial cells and its application for bacterial differentiation. Analytical Chemistry. 2013;85(12):5755-63. PubMed PMID: 23495969. doi: 10.1021/ac400330z

23. Vostalova J, Galandakova A, Palikova I, Ulrichova J, Dolezal D, Lichnovska R, Vrbkova J, Rajnochova Svobodova A. Lonicera caerulea fruits reduce UVA-induced damage in hairless mice. Journal of Photochemistry and Photobiology B: Biology. 2013;128:1-11. PubMed PMID: 23974431. doi: 10.1016/j.jphotobiol.2013.07.024

24. Goodwin BL, Ruthven CR, Sandler M. Gut flora and the origin of some urinary aromatic phenolic compounds. Biochemical Pharmacology. 1994;47(12):2294-7. PubMed PMID: 8031324.

25. Gasperotti M, Masuero D, Guella G, Mattivi F, Vrhovsek U. Development of a targeted method for twenty-three metabolites related to polyphenol gut microbial metabolism in biological samples, using SPE and UHPLC-ESI-MS/MS. Talanta. 2014;128:221-30. PubMed PMID: 25059152. doi: 10.1016/j.talanta.2014.04.058

26. Khanal R, Howard LR, Prior RL. Urinary excretion of phenolic acids in rats fed cranberry, blueberry, or black raspberry powder. Journal of Agricultural and Food Chemistry. 2014;62(18):3987-96. PubMed PMID: 24180593. doi: 10.1021/jf403883r

27. Armstrong MD, Chao FC, Parker VJ, Wall PE. Endogenous formation of hippuric acid. Proceedings of the Society for Experimental Biology and Medicine. 1955;90(3):675-9. PubMed PMID: 13289873.

28. Bernhard K, Vuilleumier JP, Brubacher G. Zur Frage der Entstehung der Benzoesäure im Tierkörper. Helvetica Chimica Acta. 1955;38(6):1438-44. doi: 10.1002/hlca.19550380616

29. Breen KJ, Bryant RE, Levinson JD, Schenker S. Neomycin absorption in man. Studies of oral and enema administration and effect of intestinal ulceration. Annals of Internal Medicine. 1972;76(2):211-8. PubMed PMID: 5009591.

30. Baba S, Furuta T, Fujioka M, Goromaru T. Studies on drug metabolism by use of isotopes XXVII: urinary metabolites of rutin in rats and the role of intestinal microflora in the metabolism of rutin. J Pharm Sci. 1983;72(10):1155-8.

31. Ottaviani JI, Borges G, Momma TY, Spencer JP, Keen CL, Crozier A, Schroeter H. The metabolome of [2-(14)C](-)-epicatechin in humans: implications for the assessment of efficacy, safety, and mechanisms of action of polyphenolic bioactives. Scientific Reports. 2016;6:29034. PubMed PMID: 27363516. Pubmed Central PMCID: PMC4929566. doi: 10.1038/srep29034

32. Borges G, van der Hooft JJJ, Crozier A. A comprehensive evaluation of the [2-14C](-)-epicatechin metabolome in rats. Free Radical Biology and Medicine. 2016;99:128-38. PubMed PMID: 27495388. doi: 10.1016/j.freeradbiomed.2016.08.001

33. Nakagawa Y, Shetlar MR, Wender SH. Urinary products from quercetin in neomycin-treated rats. Biochimica et Biophysica Acta. 1965;97:233-41. PubMed PMID: 14292832.

34. Griffiths LA. Studies on flavonoid metabolism. Identification of the metabolities of (+)-catechin in rat urine. Biochemical Journal. 1964;92(1):173-9. PubMed PMID: 5840958. Pubmed Central PMCID: PMC1215455.

35. Hanske L, Loh G, Sczesny S, Blaut M, Braune A. The bioavailability of apigenin-7-glucoside is influenced by human intestinal microbiota in rats. Journal of Nutrition. 2009;139(6):1095-102. PubMed PMID: 19403720. doi: 10.3945/jn.108.102814

36. Claus SP, Tsang TM, Wang Y, Cloarec O, Skordi E, Martin FP, Rezzi S, Ross A, Kochhar S, Holmes E, Nicholson JK. Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes. Molecular Systems Biology. 2008;4:219. PubMed PMID: 18854818. Pubmed Central PMCID: PMC2583082. doi: 10.1038/msb.2008.56

37. Goldin BR, Peppercorn MA, Goldman P. Contributions of host and intestinal microflora in the metabolism of L-dopa by the rat. Journal of Pharmacology and Experimental Therapeutics. 1973;186(1):160-6. PubMed PMID: 4723308.

38. Borud O, Midtvedt T, Gjessing LR. Urinary phenolic compounds in gnotobiotic and conventional rats on a free diet, and before and after L-DOPA loading on a milk diet. Acta Pharmacologica et Toxicologica. 1971;30(3):185-92. PubMed PMID: 5003294.

39. Stalmach A, Edwards CA, Wightman JD, Crozier A. Colonic catabolism of dietary phenolic and polyphenolic compounds from Concord grape juice. Food & Function. 2013;4(1):52-62. PubMed PMID: 22961385. doi: 10.1039/c2fo30151b

40. Clifford MN, Jaganath IB, Ludwig IA, Crozier A. Chlorogenic acids and the acyl-quinic acids: discovery, biosynthesis, bioavailability and bioactivity. Natural Product Reports. 2017;34(12):1391-421. PubMed PMID: 29160894. doi: 10.1039/c7np00030h

41. Olthof MR, Hollman PC, Katan MB. Chlorogenic acid and caffeic acid are absorbed in humans. Journal of Nutrition. 2001;131(1):66-71. PubMed PMID: 11208940.

42. Pekkinen J, Rosa NN, Savolainen OI, Keski-Rahkonen P, Mykkanen H, Poutanen K, Micard V, Hanhineva K. Disintegration of wheat aleurone structure has an impact on the bioavailability of phenolic compounds and other phytochemicals as evidenced by altered urinary metabolite profile of diet-induced obese mice. Nutrition & Metabolism. 2014;11(1):1. PubMed PMID: 24383425. Pubmed Central PMCID: PMC3891979. doi: 10.1186/1743-7075-11-1

43. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Research. 2017;45(D1):D353-d61. PubMed PMID: 27899662. Pubmed Central PMCID: PMC5210567. doi: 10.1093/nar/gkw1092

44. Phipps AN, Stewart J, Wright B, Wilson ID. Effect of diet on the urinary excretion of hippuric acid and other dietary-derived aromatics in rat. A complex interaction between diet, gut microflora and substrate specificity. Xenobiotica. 1998;28(5):527-37. PubMed PMID: 9622854. doi: 10.1080/004982598239443

45. Envigo. Teklad Global 18% Protein Extruded Rodent Diet (Sterilizable) 2018 [cited 2018 08/08/2018]. Diet composition]. Available from: .

46. Xu B, Chang SK. Characterization of phenolic substances and antioxidant properties of food soybeans grown in the North Dakota-Minnesota region. Journal of Agricultural and Food Chemistry. 2008;56(19):9102-13. PubMed PMID: 18781761. doi: 10.1021/jf801451k

47. Zhao Z, Egashira Y, Sanada H. Phenolic antioxidants richly contained in corn bran are slightly bioavailable in rats. Journal of Agricultural and Food Chemistry. 2005;53(12):5030-5. PubMed PMID: 15941352. doi: 10.1021/jf050111n

48. Mattila P, Pihlava JM, Hellstrom J. Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products. Journal of Agricultural and Food Chemistry. 2005;53(21):8290-5. PubMed PMID: 16218677. doi: 10.1021/jf051437z

49. Mateo Anson N, Aura AM, Selinheimo E, Mattila I, Poutanen K, van den Berg R, Havenaar R, Bast A, Haenen GR. Bioprocessing of wheat bran in whole wheat bread increases the bioavailability of phenolic acids in men and exerts antiinflammatory effects ex vivo. Journal of Nutrition. 2011;141(1):137-43. PubMed PMID: 21106920. doi: 10.3945/jn.110.127720

50. Aura AM. Microbial metabolism of dietary phenolic compounds in the colon. Phytochemistry Reviews. 2008;7(3):407-29.

51. Bondia-Pons I, Aura A-M, Vuorela S, Kolehmainen M, Mykkanen H, Poutanen K. Rye phenolics in nutrition and health. Journal of Cereal Science. 2009;49:323–36. doi: 10.1016/j.jcs.2009.01.007

52. Andreasen MF, Kroon PA, Williamson G, Garcia-Conesa MT. Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals. Journal of Agricultural and Food Chemistry. 2001;49(11):5679-84. PubMed PMID: 11714377.

53. Teuchy H, Van Sumere CF. The metabolism of (1- 14 C) phenylalanine, (3- 14 C) cinnamic acid and (2- 14 C) ferulic acid in the rat. Archives Internationales de Physiologie et de Biochimie. 1971;79(3):589-618. PubMed PMID: 4107877.

54. Behr C, Kamp H, Fabian E, Krennrich G, Mellert W, Peter E, Strauss V, Walk T, Rietjens I, van Ravenzwaay B. Gut microbiome-related metabolic changes in plasma of antibiotic-treated rats. Archives of Toxicology. 2017;91(10):3439-54. PubMed PMID: 28337503. doi: 10.1007/s00204-017-1949-2

55. Zheng X, Xie G, Zhao A, Zhao L, Yao C, Chiu NH, Zhou Z, Bao Y, Jia W, Nicholson JK, Jia W. The footprints of gut microbial-mammalian co-metabolism. Journal of Proteome Research. 2011;10(12):5512-22. PubMed PMID: 21970572. doi: 10.1021/pr2007945

56. Swann JR, Tuohy KM, Lindfors P, Brown DT, Gibson GR, Wilson ID, Sidaway J, Nicholson JK, Holmes E. Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats. Journal of Proteome Research. 2011;10(8):3590-603. PubMed PMID: 21591676. doi: 10.1021/pr200243t

57. Lee SH, An JH, Park HM, Jung BH. Investigation of endogenous metabolic changes in the urine of pseudo germ-free rats using a metabolomic approach. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2012;887-888:8-18. PubMed PMID: 22300547. doi: 10.1016/j.jchromb.2011.12.030

58. Pekkinen J, Rosa-Sibakov N, Micard V, Keski-Rahkonen P, Lehtonen M, Poutanen K, Mykkanen H, Hanhineva K. Amino acid-derived betaines dominate as urinary markers for rye bran intake in mice fed high-fat diet--A nontargeted metabolomics study. Molecular Nutrition & Food Research. 2015;59(8):1550-62. PubMed PMID: 25944556. doi: 10.1002/mnfr.201500066

59. Gu L, House SE, Rooney L, Prior RL. Sorghum bran in the diet dose dependently increased the excretion of catechins and microbial-derived phenolic acids in female rats. Journal of Agricultural and Food Chemistry. 2007;55(13):5326-34. PubMed PMID: 17536823. 10.1021/jf070100p

60. Wang J, Hodes GE, Zhang H, Zhang S, Zhao W, Golden SA, Bi W, Menard C, Kana V, Leboeuf M, Xie M, Bregman D, Pfau ML, Flanigan ME, Esteban-Fernandez A, Yemul S, Sharma A, Ho L, Dixon R, Merad M, Han MH, Russo SJ, Pasinetti GM. Epigenetic modulation of inflammation and synaptic plasticity promotes resilience against stress in mice. Nat Commun. 2018;9(1):477. PubMed PMID: 29396460. Pubmed Central PMCID: PMC5797143. doi: 10.1038/s41467-017-02794-5

61. Lees HJ, Swann JR, Wilson ID, Nicholson JK, Holmes E. Hippurate: the natural history of a mammalian-microbial cometabolite. Journal of Proteome Research. 2013;12(4):1527-46. PubMed PMID: 23342949. doi: 10.1021/pr300900b

62. Yap IK, Li JV, Saric J, Martin FP, Davies H, Wang Y, Wilson ID, Nicholson JK, Utzinger J, Marchesi JR, Holmes E. Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse. Journal of Proteome Research. 2008;7(9):3718-28. PubMed PMID: 18698804. doi: 10.1021/pr700864x

63. Liu H, Tayyari F, Edison AS, Su Z, Gu L. NMR-based metabolomics reveals urinary metabolome modifications in female Sprague-Dawley rats by cranberry procyanidins. The Journal of Nutritional Biochemistry. 2016;34:136-45. PubMed PMID: 27309592. doi: 10.1016/j.jnutbio.2016.05.007

64. Shaw KNF, Gutenstein M, Jepson JB, editors. Intestinal flora and diet in relation to m-hydroxyphenyl acids of human urine. Fifth International Congress of Biochemistry; 1961; Moscow. New York: Pergamon Press; 1963.

65. Harrison CA, Laubitz D, Midura-Kiela MT, Jamwal DR, Besselsen DG, Ghishan FK, Kiela PR. Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis. Journal of Crohn's & Colitis. 2019;13(1):115-26. PubMed PMID: 30252029. Pubmed Central PMCID: PMC6302957. doi: 10.1093/ecco-jcc/jjy144

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