European Journal of Nutrition

, Volume 58, Issue 4, pp 1529–1543 | Cite as

Inter-individual variability in the production of flavan-3-ol colonic metabolites: preliminary elucidation of urinary metabotypes

  • Pedro MenaEmail author
  • Iziar A. Ludwig
  • Virginia B. Tomatis
  • Animesh Acharjee
  • Luca Calani
  • Alice Rosi
  • Furio Brighenti
  • Sumantra Ray
  • Julian L. Griffin
  • Les J. Bluck
  • Daniele Del RioEmail author
Original Contribution



There is much information on the bioavailability of (poly)phenolic compounds following acute intake of various foods. However, there are only limited data on the effects of repeated and combined exposure to specific (poly)phenol food sources and the inter-individual variability in their bioavailability. This study evaluated the combined urinary excretion of (poly)phenols from green tea and coffee following daily consumption by healthy subjects in free-living conditions. The inter-individual variability in the production of phenolic metabolites was also investigated.


Eleven participants consumed both tablets of green tea and green coffee bean extracts daily for 8 weeks and 24-h urine was collected on five different occasions. The urinary profile of phenolic metabolites and a set of multivariate statistical tests were used to investigate the putative existence of characteristic metabotypes in the production of flavan-3-ol microbial metabolites.


(Poly)phenolic compounds in the green tea and green coffee bean extracts were absorbed and excreted after simultaneous consumption, with green tea resulting in more inter-individual variability in urinary excretion of phenolic metabolites. Three metabotypes in the production of flavan-3-ol microbial metabolites were tentatively defined, characterized by the excretion of different amounts of trihydroxyphenyl-γ-valerolactones, dihydroxyphenyl-γ-valerolactones, and hydroxyphenylpropionic acids.


The selective production of microbiota-derived metabolites from flavan-3-ols and the putative existence of characteristic metabotypes in their production represent an important development in the study of the bioavailability of plant bioactives. These observations will contribute to better understand the health effects and individual differences associated with consumption of flavan-3-ols, arguably the main class of flavonoids in the human diet.


Polyphenols Green tea catechins Coffee caffeoylquinic acids Colonic microbiota Urinary phenotype Metabotypes 



We thank the volunteers who participated in the study, Polly Page for her key role in study steering and management oversight, and the Volunteer Studies and Clinical Services and Sample Management Teams at MRC EWL for their assistance in the conduction of the study. We acknowledge Prof. Alan Crozier (University of California, Davis, USA) for his help with manuscript revision and data discussion. We are also grateful to Gary Williamson (University of Leeds, UK), Denis Barron (Nestle Research Center, Lausanne, Switzerland), and Takao Yokota (Teikyo University, Japan) for the generous gift of a number of phase II metabolites. Dr. Les Bluck, joint senior author for this work, played a fundamental role in the design of the original study; it is with much sadness that his death prevented him from seeing the research come to fruition.

Author contributions

PM and IL designed and conducted research, analyzed data, performed statistical analysis, and wrote the paper; VT designed and conducted research, analyzed data, and performed statistical analysis; AA performed statistical analysis; LC conducted research; AR, FB, and JLG provided critical review of the manuscript; SR designed and conducted research, and provided critical review; LJB designed research; DDR designed research and had primary responsibility for final content. All authors read and approved the final manuscript.


This work was partially funded by MRC core funding (Physiological Modelling of Metabolic Risk, MC_UP_A090_1005, and Nutrition, Surveys and Studies, MC_U105960384) and University of Parma core funding (FIL 2014-2017). P.M. was partially funded by a Grant of the Postdoctoral Fellowship Program from Fundación Séneca (Murcia Region, Spain). I.A.L. was supported by a postdoctoral fellowship funded by the Spanish Ministry of Economy and Competitiveness (IJCI-2014-20689).

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Supplementary material

394_2018_1683_MOESM1_ESM.docx (2.8 mb)
Supplementary material 1 (DOCX 2909 KB)


  1. 1.
    Del Rio D, Rodriguez-Mateos A, Spencer JPE, Tognolini M, Borges G, Crozier A (2013) Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid Redox Signal 18(14):1818–1892. Google Scholar
  2. 2.
    Rodriguez-Mateos A, Vauzour D, Krueger CG, Shanmuganayagam D, Reed J, Calani L, Mena P, Del Rio D, Crozier A (2014) Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update. Arch Toxicol 88(10):1803–1853. Google Scholar
  3. 3.
    Bresciani L, Martini D, Mena P, Tassotti M, Calani L, Brigati G, Brighenti F, Holasek S, Malliga D-E, Lamprecht M, Del Rio D (2017) Absorption profile of (poly)phenolic compounds after consumption of three food supplements containing 36 different fruits, vegetables, and berries. Nutrients 9(3):194. Google Scholar
  4. 4.
    Shi Y, Williamson G (2015) Comparison of the urinary excretion of quercetin glycosides from red onion and aglycone from dietary supplements in healthy subjects: a randomized, single-blinded, cross-over study. Food Funct 6:1443–1448. Google Scholar
  5. 5.
    Borel P, Desmarchelier C, Nowicki M, Bott R, Morange S, Lesavre N (2014) Interindividual variability of lutein bioavailability in healthy men: characterization, genetic variants involved, and relation with fasting plasma lutein concentration. Am J Clin Nutr 100(1):168–175. Google Scholar
  6. 6.
    Stalmach A, Mullen W, Barron D, Uchida K, Yokota T, Cavin C, Steiling H, Williamson G, Crozier A (2009) Metabolite profiling of hydroxycinnamate derivatives in plasma and urine after the ingestion of coffee by humans: identification of biomarkers of coffee consumption. Drug Metab Dispos 37(8):1749–1758. Google Scholar
  7. 7.
    Stalmach A, Mullen W, Steiling H, Williamson G, Lean MEJ, Crozier A (2010) Absorption, metabolism, and excretion of green tea flavan-3-ols in humans with an ileostomy. Mol Nutr Food Res 54(3):323–334. Google Scholar
  8. 8.
    Stalmach A, Williamson G, Crozier A (2014) Impact of dose on the bioavailability of coffee chlorogenic acids in humans. Food Funct 5(8):1727–1737. Google Scholar
  9. 9.
    Williamson G, Clifford MN (2017) Role of the small intestine, colon and microbiota in determining the metabolic fate of polyphenols. Biochem Pharmacol 39:24–39. Google Scholar
  10. 10.
    Manach C, Milenkovic D, Van de Wiele T, Rodriguez-Mateos A, de Roos B, Garcia-Conesa MT, Landberg R, Gibney ER, Heinonen M, Tomas-Barberan F, Morand C (2016) Addressing the inter-individual variation in response to consumption of plant food bioactives—towards a better understanding of their role in healthy ageing and cardiometabolic risk reduction. Mol Nutr Food Res. Google Scholar
  11. 11.
    Setchell KD, Clerici C (2010) Equol: history, chemistry, and formation. J Nutr 140(7):1355S–1362S. Google Scholar
  12. 12.
    Bolca S, Possemiers S, Maervoet V, Huybrechts I, Heyerick A, Vervarcke S, Depypere H, De Keukeleire D, Bracke M, De Henauw S, Verstraete W, Van de Wiele T (2007) Microbial and dietary factors associated with the 8-prenylnaringenin producer phenotype: a dietary intervention trial with fifty healthy post-menopausal Caucasian women. Br J Nutr 98(5):950–959. Google Scholar
  13. 13.
    Hazim S, Curtis PJ, Schar MY, Ostertag LM, Kay CD, Minihane AM, Cassidy A (2016) Acute benefits of the microbial-derived isoflavone metabolite equol on arterial stiffness in men prospectively recruited according to equol producer phenotype: a double-blind randomized controlled trial. Am J Clin Nutr 103(3):694–702. Google Scholar
  14. 14.
    Tomás-Barberán FA, González-Sarrías A, García-Villalba R, Núñez-Sánchez MA, Selma MV, García-Conesa MT, Espín JC (2017) Urolithins, the rescue of “old” metabolites to understand a “new” concept: metabotypes as a nexus among phenolic metabolism, microbiota dysbiosis, and host health status. Mol Nutr Food Res. Google Scholar
  15. 15.
    Gonzalez-Sarrias A, Garcia-Villalba R, Romo-Vaquero M, Alasalvar C, Orem A, Zafrilla P, Tomas-Barberan FA, Selma MV, Espin JC (2017) Clustering according to urolithin metabotype explains the interindividual variability in the improvement of cardiovascular risk biomarkers in overweight-obese individuals consuming pomegranate: a randomized clinical trial. Mol Nutr Food Res. Google Scholar
  16. 16.
    Selma MV, Gonzalez-Sarrias A, Salas-Salvado J, Andres-Lacueva C, Alasalvar C, Orem A, Tomas-Barberan FA, Espin JC (2017) The gut microbiota metabolism of pomegranate or walnut ellagitannins yields two urolithin-metabotypes that correlate with cardiometabolic risk biomarkers: comparison between normoweight, overweight-obesity and metabolic syndrome. Clin Nutr S0261-5614(17):30103–30106. Google Scholar
  17. 17.
    Tomas-Barberan F, García-Villalba R, Quartieri A, Raimondi S, Amaretti A, Leonardi A, Rossi M (2013) In vitro transformation of chlorogenic acid by human gut microbiota. Mol Nutr Food Res 58(5):1122–1131. Google Scholar
  18. 18.
    Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N, Brighenti F (2010) Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 26(11–12):1110–1116. Google Scholar
  19. 19.
    Brindani N, Mena P, Calani L, Benzie I, Choi SW, Brighenti F, Zanardi F, Curti C, Del Rio D (2017) Synthetic and analytical strategies for the quantification of phenyl-gamma-valerolactone conjugated metabolites in human urine. Mol Nutr Food Res. Google Scholar
  20. 20.
    Curti C, Brindani N, Battistini L, Sartori A, Pelosi G, Mena P, Brighenti F, Zanardi F, Del Rio D (2015) Catalytic, enantioselective vinylogous mukaiyama aldol reaction of furan-based dienoxy silanes: a chemodivergent approach to γ-valerolactone flavan-3-ol metabolites and δ-lactone analogues. Adv Synth Catal 357(18):4082–4092. Google Scholar
  21. 21.
    Westerhuis JA, van Velzen EJJ, Hoefsloot HCJ, Smilde AK (2008) Discriminant Q2 (DQ2) for improved discrimination in PLSDA models. Metabolomics 4(4):293–296. Google Scholar
  22. 22.
    Acharjee A, Finkers R, Visser RG, Maliepaard C (2013) Comparison of regularized regression methods for omics data. Metabolomics 3:126–134. Google Scholar
  23. 23.
    Edmands WMB, Ferrari P, Rothwell JA, Rinaldi S, Slimani N, Barupal DK, Biessy C, Jenab M, Clavel-Chapelon F, Fagherazzi G, Boutron-Ruault MC, Katzke VA, Kühn T, Boeing H, Trichopoulou A, Lagiou P, Trichopoulos D, Palli D, Grioni S, Tumino R, Vineis P, Mattiello A, Romieu I, Scalbert A (2015) Polyphenol metabolome in human urine and its association with intake of polyphenol-rich foods across European countries. Am J Clin Nutr 102(4):905–913. Google Scholar
  24. 24.
    Ludwig IA, Mena P, Calani L, Cid C, Del Rio D, Lean MEJ, Crozier A (2014) Variations in caffeine and chlorogenic acid contents of coffees: what are we drinking? Food Funct 5(8):1718–1726. Google Scholar
  25. 25.
    Zamora-Ros R, Rothwell JA, Scalbert A, Knaze V, Romieu I, Slimani N, Fagherazzi G, Perquier F, Touillaud M, Molina-Montes E, Huerta JM, Barricarte A, Amiano P, Menéndez V, Tumino R, de Magistris MS, Palli D, Ricceri F, Sieri S, Crowe FL, Khaw KT, Wareham NJ, Grote V, Li K, Boeing H, Förster J, Trichopoulou A, Benetou V, Tsiotas K, Bueno-de-Mesquita HB, Ros M, Peeters PH, Tjønneland A, Halkjær J, Overvad K, Ericson U, Wallström P, Johansson I, Landberg R, Weiderpass E, Engeset D, Skeie G, Wark P, Riboli E, González CA (2013) Dietary intakes and food sources of phenolic acids in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Br J Nutr 110(8):1500–1511. Google Scholar
  26. 26.
    Roowi S, Stalmach A, Mullen W, Lean ME, Edwards CA, Crozier A (2010) Green tea flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans. J Agric Food Chem 58(2):1296–1304. Google Scholar
  27. 27.
    van der Hooft JJ, de Vos RC, Mihaleva V, Bino RJ, Ridder L, de Roo N, Jacobs DM, van Duynhoven JP, Vervoort J (2012) Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake. Anal Chem 84(16):7263–7271. Google Scholar
  28. 28.
    Urpi-Sarda M, Monagas M, Khan N, Llorach R, Lamuela-Raventós RM, Jáuregui O, Estruch R, Izquierdo-Pulido M, Andrés-Lacueva C (2009) Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1216(43):7258–7267. Google Scholar
  29. 29.
    Urpi-Sarda M, Boto-Ordóñez M, Queipo-Ortuño MI, Tulipani S, Corella D, Estruch R, Tinahones FJ, Andres-Lacueva C (2015) Phenolic and microbial-targeted metabolomics to discovering and evaluating wine intake biomarkers in human urine and plasma. Electrophoresis 36(18):2259–2268. Google Scholar
  30. 30.
    Ulaszewska MM, Trost K, Stanstrup J, Tuohy KM, Franceschi P, Chong MFF, George T, Minihane AM, Lovegrove JA, Mattivi F (2016) Urinary metabolomic profiling to identify biomarkers of a flavonoid-rich and flavonoid-poor fruits and vegetables diet in adults: the FLAVURS trial. Metabolomics 12(2):1–22. Google Scholar
  31. 31.
    Meselhy MR, Nakamura N, Hattori M (1997) Biotransformation of (−)-epicatechin 3-O-gallate by human intestinal bacteria. Chem Pharm Bull 45(5):888–893. doiGoogle Scholar
  32. 32.
    Ward NC, Croft KD, Puddey IB, Hodgson JM (2004) Supplementation with grape seed polyphenols results in increased urinary excretion of 3-hydroxyphenylpropionic acid, an important metabolite of proanthocyanidins in humans. J Agric Food Chem 52(17):5545–5549. Google Scholar
  33. 33.
    van Velzen EJJ, Westerhuis JA, Grün CH, Jacobs DM, Eilers PHC, Mulder TP, Foltz M, Garczarek U, Kemperman R, Vaughan EE, van Duynhoven JPM, Smilde AK (2014) Population-based nutrikinetic modeling of polyphenol exposure. Metabolomics 10:1059–1073. Google Scholar
  34. 34.
    Cueva C, Sánchez-Patán F, Monagas M, Walton GE, Gibson GR, Martín-Álvarez PJ, Bartolomé B, Moreno-Arribas MV (2013) In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites. FEMS Microbiol Ecol 83(3):792–805. Google Scholar
  35. 35.
    Muñoz-González I, Jiménez-Girón A, Martín-Álvarez PJ, Bartolomé B, Moreno-Arribas MV (2013) Profiling of microbial-derived phenolic metabolites in human feces after moderate red wine intake. J Agric Food Chem 61(39):9470–9479. Google Scholar
  36. 36.
    Takagaki A, Nanjo F (2015) Bioconversion of (−)-epicatechin, (+)-epicatechin, (−)-catechin, and (+)-catechin by (−)-epigallocatechin-metabolizing bacteria. Biol Pharm Bull 38(5):789–794. Google Scholar
  37. 37.
    Gonthier MP, Donovan JL, Texier O, Felgines C, Remesy C, Scalbert A (2003) Metabolism of dietary procyanidins in rats. Free Radic Biol Med 35(8):837–844. Google Scholar
  38. 38.
    Kutschera M, Engst W, Blaut M, Braune A (2011) Isolation of catechin-converting human intestinal bacteria. J Appl Microbiol 111(1):165–175. Google Scholar
  39. 39.
    Zamora-Ros R, Knaze V, Rothwell JA, Hémon B, Moskal A, Overvad K, Tjønneland A, Kyrø C, Fagherazzi G, Boutron-Ruault M-C, Touillaud M, Katzke V, Kühn T, Boeing H, Förster J, Trichopoulou A, Valanou E, Peppa E, Palli D, Agnoli C, Ricceri F, Tumino R, de Magistris MS, Peeters PHM, Bueno-de-Mesquita HB, Engeset D, Skeie G, Hjartåker A, Menéndez V, Agudo A, Molina-Montes E, Huerta JM, Barricarte A, Amiano P, Sonestedt E, Nilsson LM, Landberg R, Key TJ, Khaw K-T, Wareham NJ, Lu Y, Slimani N, Romieu I, Riboli E, Scalbert A (2016) Dietary polyphenol intake in Europe: the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Eur J Nutr 55(4):1359–1375. Google Scholar
  40. 40.
    Bai W, Wang C, Ren C (2014) Intakes of total and individual flavonoids by US adults. Int J Food Sci Nutr 65(1):9–20. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Human Nutrition Unit, Department of Food and DrugsUniversity of ParmaParmaItaly
  2. 2.Food Technology DepartmentUniversitat de Lleida-Agrotecnio CenterLleidaSpain
  3. 3.UK Medical Research Council Elsie Widdowson Laboratory (formerly MRC Human Nutrition Research)CambridgeUK
  4. 4.Department of BiochemistryUniversity of CambridgeCambridgeUK
  5. 5.Institute of Cancer and Genomic Sciences, Centre for Computational BiologyUniversity of BirminghamBirminghamUK
  6. 6.Institute of Translational MedicineUniversity Hospitals Birmingham NHS Foundation TrustBirminghamUK

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