Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites.

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Metabolic transformations of dietary polyphenols: comparison between in vitro

colonic and hepatic models and in vivo urinary metabolites☆

,

☆☆

,

★

Claudia Vetrani

a,

⁎

, Angela A. Rivellese

a

, Giovanni Annuzzi

a

, Martin Adiels

b, c

, Jan Borén

b

, Ismo Mattila

d

,

Matej Ore

_šič

d

, Anna-Marja Aura

e

a

Department of Clinical Medicine and Surgery,“Federico II” University, Naples, Italy

b

Sahlgrenska Center for Cardiovascular and Metabolic Research/Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden

c_{Department of Mathematical Sciences, University of Gothenburg, Gothenburg, Sweden} d

Steno Diabetes Center, Gentofte, Denmark

e

VTT Technical Research Centre of Finland, Espoo, Finland

Received 28 October 2015; received in revised form 29 February 2016; accepted 7 March 2016

Abstract

Studies on metabolism of polyphenols have revealed extensive transformations in the carbon backbone by colonic microbiota; however, the influence of microbial and hepatic transformations on human urinary metabolites has not been explored. Therefore, the aims of this study were (1) to compare the in vitro microbial phenolic metabolite profile of foods and beverages with that excreted in urine of subjects consuming the same foodstuff and (2) to explore the role of liver on postcolonic metabolism of polyphenols by using in vitro hepatic models. A 24-h urinary phenolic metabolite profile was evaluated in 72 subjects participating in an 8-week clinical trial during which they were randomly assigned to diets differing for polyphenol content. Polyphenol-rich foods and beverages used in the clinical trial were subjected to human fecal microbiota in the in vitro colon model. Metabolites from green tea, one of the main components of the polyphenol-rich diet, were incubated with primary hepatocytes to highlight hepatic conversion of polyphenols. The analyses were performed using targeted gas chromatography with mass spectrometer (GCxGC-TOFMS:colon model; GC-MS: urine and hepatocytes). A significant correlation was found between urinary and colonic metabolites with C1-C3 side chain (P=.040). However, considerably higher amounts of hippuric acid, 3-hydroxybenzoic acid and ferulic acid were detected in urine than in the colon model. The hepatic conversion showed additional amounts of these metabolites complementing the gap between in vitro colon model and the in vivo urinary excretion. Therefore, combining in vitro colon and hepatic models may better elucidate the metabolism of polyphenols from dietary exposure to urinary metabolites.

Keywords: Polyphenols; Metabolism; Phenolic carbon backbone; In vitro colon model; Primary hepatocytes

1. Introduction

Polyphenols is a group of phytochemicals that have been studied

extensively. Polyphenol intake is associated with reduced risk of

cardiovascular disease and diabetes as shown by epidemiological

ﬁndings

[1

–4]

. In addition, some clinical trials have shown the effect of

polyphenol exposure on cardiometabolic risk factors, mainly lipid and

glucose metabolism and oxidative stress

[5

–9]

. Hence, the bene

ﬁts of

polyphenol intake are becoming more and more apparent.

Moreover, the metabolism of polyphenols has been explored. After the

intake, a small amount of the ingested polyphenols are absorbed from

intestine and can be converted to glucuronidated and sulfated conjugates

ScienceDirect

Journal of Nutritional Biochemistry 33 (2016) 111

_–118

Abbreviations: diMeBA, dimethoxybenzoic acid; diOHBA, dihydroxybenzoic acid; diOHPAc, 2-(3´,4´-dihydroxyphenyl)acetic acid; 3,4-diOHPPr, 3-(3´,4´-dihydroxyphenyl)propionic acid; 3,5-diOHBA, 3,5-dihydroxybenzoic acid; 3-OHBA, 3-hydroxybenzoic acid; 3-OHPAc, 2-(3´-hydroxyphenyl) acetic acid; 3-OHPPr, 3-(3´-hydroxyphenyl)propionic acid; 3-PPr, 3-phenylpropionic acid; OHBA, hydroxybenzoic acid; mecatechol, methylcatechol; 4-OHPPr, 3-(4´-hydroxyphenyl)propionic acid.

☆ _{Financial Support: This work was supported by ETHERPATHS project (European Commission Grant FP7-KBBE-222,639) and Ministero dell'Istruzione,}

dell'Università della Ricerca, Rome, Italy (PRIN no. 2010JC WWKM).

☆☆ _{Chemical compounds studied in this article: 3,4-dimethoxybenzoic acid (PubChem CID: 7121); 3,4-dihydroxybenzoic acid (PubChem CID: 72);}

2-(3´,4´-dihydroxyphenyl)acetic acid (PubChem CID: 547); 3-(3´,4´-dihydroxyphenyl)propionic acid (PubChem CID: 348,154); 3,5-dihydroxybenzoic acid (PubChem CID: 7424); 3-hydroxybenzoic acid (PubChem CID: 7420); 4-hydroxybenzoic acid (PubChem CID: 135); 4-methylcatechol (PubChem CID: 9958); benzoic acid (PubChem CID: 243); gallic acid (PubChem CID: 370), ferulic acid (PubChem CID: 445,858); sinapic acid (PubChem CID: 637,775); caffeic acid (PubChem CID: 689,043); 4-coumaric acid (PubChem CID: 637,542); enterodiol (PubChem CID: 115,089); vanillic acid (PubChem CID: 8468); hippuric acid (PubChem CID: 464).

★ _{Conflict of Interest: The authors report no personal or financial conflict of interest arising from the present research and its publication.}

⁎ Corresponding author. Department of Clinical Medicine and Surgery, “Federico II” University, 5, S. Pansini 80131, Naples, Italy. Tel.: 81-7462306; fax +39-81-7462321.

E-mail address:c.vetrani@libero.it(C. Vetrani).

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by the enzymes of intestinal tissue and can enter the bloodstream. In

addition, in the liver, they can be further metabolized to

glucur-onidated, sulfated and glycinated derivatives

[10,11]. Furthermore,

studies on metabolism of polyphenols have revealed extensive

microbial transformations in the carbon backbone by colonic

microbiota

[12,13]. After colonic metabolism, microbial metabolites

are absorbed and have a long residence time (up to 24

–48 h) in the

bloodstream before being excreted in urine

[14,15]. Moreover,

human intervention studies have investigated the metabolism of

single foodstuff or compound group (i.e.

ﬂavan-3-ols, anthocyanins,

phenolic acids)

[15

–20]

. The study presented here is a continuation

of our previous publication

[21]

in which a polyphenol-rich diet was

consumed by subjects having features of metabolic syndrome.

Urinary excretion was analyzed for hydroxylated phenylpropionic

and phenylacetic acids, some benzoic acid derivatives and

mamma-lian lignans; the phenolic pro

ﬁle showed a signiﬁcant difference

between the polyphenol-diet-consuming group and the control

group that was achieved by strict control of polyphenol-rich

foodstuff

[21]. In addition, some transformations of carbon backbone

of dietary polyphenols were observed. However, it was not clear

what were the roles of colonic and postcolonic hepatic metabolism in

the carbon backbone transformations of excreted phenolic

metab-olites in urine. Hence, in this study, the same polyphenol-rich

foodstuff used in the clinical trial were subjected to the conversions

in the in vitro colon model to

ﬁnd the correlation between microbial

and urinary metabolites. Furthermore, green tea microbial

metab-olite extract was exposed to the postcolonic hepatic conversions

because microbial phenolic acid metabolites of green tea at 6 h time

point were diverse and the extract could represent a microbial

metabolite pro

_{ﬁle of the polyphenol-rich diet better than any other}

single food or beverage used in the study. The hypothesis of this

study is that transformations of carbon skeleton of dietary

polyphe-nols re

ﬂect both colonic and subsequent hepatic metabolism and this

can be shown in the urinary excretion pro

ﬁle.

2. Materials and methods

2.1. Clinical trial

A description of the clinical trial has been published earlier[21]. Brieﬂy, 86 overweight/ obese subjects with a high cardiovascular and metabolic risk proﬁle were enrolled in the study. They had a waist circumference above the standard cutoff and at least one of the features of the metabolic syndrome according to the NCEP/ATP III criteria[22]. They followed a high-polyphenol diet (HP-diet; 2868 mg/day) or a low-polyphenol control diet (LP-diet, 363 mg/day) for 8 weeks. The two diets were isoenergetic and had the same composition for nutrients. The main dietary sources of polyphenols in the HP-diet were artichokes, fennel, onion, spinach, arugula salad (rucola), orange, dark chocolate bar, decaffeinated coffee and green tea. The intake of alcoholic beverages was not allowed. All foods and beverages were provided to the subjects during the intervention. At baseline and after the intervention, all subjects collected 24-h urine for the evaluation of the excretion of phenolic metabolites. The volunteers received careful instructions and motivation to complete 24-h urine collection. After a 12-h overnight fast, they collected all excreted urine to a 3000-ml plastic urine containers (SARSTEDT s.r.l. Verona, Italy) until the following morning.

The study was conducted according to the guidelines of the Declaration of Helsinki, and all procedures involving human subjects were approved by“Federico II” University Ethics Committee. Written informed consent was obtained from all subjects. The study was registered atwww.clinicaltrials.gov(identiﬁer NCT01154478).

2.2. In vitro colon model

2.2.1. Materials

Onion (cultivated in Finland), fennel (cultivated in The Netherlands), artichoke (cultivated in France), spinach (cultivated in Italy), oranges (cultivated in South Africa), arugula salad (cultivated in Italy), green tea (China Gun Powder, Nordqvist, Helsinki, Finland) and dark cocoa powder (Van Houten, Lebecke Wieze, Belgium) were purchased from a local store in Finland, whereas decaffeinated coffee (Lavazza DeK, Torino, Italy) was purchased in Italy.

The internal standard during the comprehensive proﬁling of small polar metabolites from the colon model was 2-hydroxycinnamic acid (Aldrich Inc., St. Louis, USA). The following compounds were used as standards: benzoic acid, 3-hydroxybenzoic acid (3-OHBA), 3-(4´-hydroxyphenyl)propionic acid (4-OHPPr), 4-methylcatechol (4-mecatechol) and 3-(3´,4´-dihydroxyphenyl)propionic acid (3,4-diOHPPr) were purchased from Aldrich

(Steinheim, Germany); 4-hydroxybenzoic acid (4-OHBA), 2-(3´-hydroxyphenyl)acetic acid (3-OHPAc), 2-(3´,4´-dihydroxyphenyl) acetic acid (3,4-diOHPAc), 4-hydroxycinnamic acid and ferulic acid (methoxy-4-hydroxy-cinnamic acid) were from Sigma (St. Louis, USA); 3-phenylpropionic acid (3-PPr), vanillic acid (3-methoxy-4-hydroxybenzoic acid) and 3,4-dihydroxybenzoic acid (3,4-diOHBA) were from Fluka (Buchs, Switzerland); 3-(3´-hydro-xyphenyl)propionic acid (3-OHPPr) was from Alfa Aesar (Karlsruhe, Germany) and gallic acid was from Extrasynthése (Genay, France). N-Methyl-N-trimethylsilyl triﬂuoroacetamide (MSTFA) from Pierce (Rockford, USA) and methoxyamine hydrochloride (2%) in pyridine (MOX; Pierce, Rockford, USA) were used in the derivatization of the metabolites.

2.2.2. Preparation of foods for the colon model

In order to remove free sugars, orange, onion and fennel were predigested in enzymatic digestion in vitro model and freeze-dried, as described below (2.2.4 Enzymatic in vitro digestion). Other foods or beverages were not predigested in the upper intestinal model to preserve soluble indigestible material. Spinach and arugula salad were directly freeze-dried because they do not contain starch, sugar or protein in excess to disturb microbial metabolism and to avoid loss of polyphenols in the upper intestinal model. Artichoke was cooked for 45 min in acidified (15 ml of 10% acetic acid in 3 L of water) and freeze-dried. Green tea infusion was prepared by incubating 50 g of green tea in 500 ml of boiling water for 5 min andfiltering the tea leaves, which were discarded. Green tea infusion was cooled and freeze-dried. Decaffeinated espresso was prepared using an SIC-certified espresso maker (Morenita Express, Ornavasso, Italy) according to the instructions of use. The coffee from several 3-cup portions were combined and cooled rapidly on ice in the anaerobic chamber to avoid oxidation. Large aliquots of coffee were also frozen and freeze-dried prior to the hydration and dosage for use in the colon model. Commercial dark cocoa powder (Van Houten, Lebbeke-Wiese, Belgium) was used as it is.

2.2.3. Characterization of the foods and beverages

The dry weight (d.w.) of the foods was determined by weighing at least three samples after freeze-drying (when applicable) and the remnant moisture was determined by Karl Fischer titration. The samples were weighed for the colon model experiment on the basis of the absolute d.w.

2.2.4. Enzymatic in vitro digestion

Enzymatic in vitro digestion of samples (10.5 g d.w.) was performed as described by Aura et al.[23]with following exceptions: the method was scaled up (7-fold) and the digestion andfiltration were performed at 37°C under anaerobic conditions to prevent oxidation of the phenolic components. Strictly anaerobic conditions were accomplished by operating in an anaerobic chamber (Don Whitley, Shipley, West Yorkshire, UK) using a gas mixture (nitrogen, 80%; hydrogen, 10%; carbon dioxide, 10%) and by using reduced 20 mM sodium phosphate buffer (pH 6.9) by heating the buffer to 80°C and cooling under nitrogen stream and placing the buffer into the anaerobic chamber for 2 days before using for the upper intestinal model. The atmosphere of the chamber was verified by detective strips sensitive for oxygen. Digested and filtrated samples were frozen rapidly and freeze-dried in vacuum.

2.2.5. Incubation in the in vitro colon model

Colon model experiments were performed under strictly anaerobic conditions according to Aura et al.[24,25]. In the colon model, anaerobic conditions were maintained in a same manner as in the upper intestinal model described inSection 2.2.4, but the buffer was 0.11 M carbonate/0.02 M phosphate buffer, pH 5.5, containing cysteine HCl (0.5 g/L) as a reducing agent.

Fecal suspensions (10%, w/v) for each experiment were prepared from freshly passed feces from at least four healthy volunteers who had made a written informed consent. Fecal samples were numbered to hide the identity of the donors. The suspensions werefiltered through a 1-mm sieve and diluted to 10% (w/v). Each solid food was dosed 10 mg d.w./1 ml of fecal suspension, whereas freeze-dried green tea and coffee and dark cocoa powder were dosed 2.5 mg d.w./1 ml of fecal suspension. Triplicate samples were incubated in a water bath at 37°C for 0, 2, 4, 6, 8 and 24 h and stirred magnetically (250 rpm). Aliquots were drawn from the bottles and microbiota was removed by centrifugation (Heraeus, Biofuge Primo R, Thermo Scientific Inc., Waltham, MA, USA; 7000g, 4°C, 10 min) and 0.2-μm PTFE filters (Millipore Corp., Bedford, MA, USA) before extraction of phenolic metabolites from 1 ml of microbe-free fecal water. The samples were extracted twice by ethyl acetate and silylated as described in Aura et al.[23].

2.3. Human primary hepatocyte experiments

2.3.1. Hepatic incubation

Human primary hepatocytes (Biopredic International, Rennes, France) were incubated according to the protocol and media supplied by Biopredic (Rennes, France). Cells were incubated for at least 24 h in fresh maintaining media, after which they were subjected to cell medium or exposure to diluted (1:10) extracts: green tea extract after colonic conversion (converted green tea), fecal microbial extract (no green tea) or green tea extract before colonic conversion (no microbiota) for 8 or 24 h. Fecal microbial extract (no green tea) and green tea extract before colonic conversion (no microbiota) were used as controls.

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To exclude spontaneous chemical conversions in the medium, the medium without hepatocytes with the same extracts and controls were incubated for 8 and 24 h. Cells were washed with PBS (2 ml) twice and 400μl PBS and protease inhibitors (leupeptin, trasylol, PMSF, pepstatin, calpain, EDTA) were added. A total of 50μl were used for cell counting and a total of 350μl were frozen for extraction.

2.3.2. Cell extraction

A mixture of 25 ml Tris–HCl (1 M), 15 ml NaCl (5 M), 5 ml EDTA (0.5 M), 2.5 ml PMSF and 5 ml Triton X-100 was diluted to 100 ml (pH 7.4) and 87.5μl was added to the frozen cells. After three intervals of ultrasound treatment (15 s), 1 ml of methanol and 15μl of 2-hydroxycinnamic acid (1:8) were added and the mixture was vortexed for 1 min and let to rest at room temperature. The lysed hepatocytes and media samples (500μl) were extracted twice with 1 ml methanol. The combined extracts were evaporated to dryness, and 500μl of 0.15 M acetic acid buffer (pH 4.1) with the addition of ascorbic acid (25 mg) was added. Samples were hydrolyzed using β-glucuronidase enzyme from Helix pomatia (10 mg; Sigma G0751-500KU, St. Louis, USA) at 37°C for 16 h and an internal standard (123 ppm 2-hydroxycinnamic acid, Aldrich, St Louis, USA). All samples were extracted twice using 3 ml of ethyl acetate and evaporated to dryness and closed under nitrogenﬂow.

2.4. Analysis of the samples and calculation

Microbial metabolites of foods and beverages from the colon model were analyzed using the two-dimensional gas chromatography coupled with time-of-ﬂight mass detection (GCxGC-TOFMS; Leco Pegasus 4D) instrument as described by Aura et al.[26]. Time course of the food speciﬁc metabolite concentrations (μM) for each food or beverage is shown in Supplement 1. The metabolites from human urine and primary hepatocytes were analyzed using the targeted analysis of microbial metabolites by gas chromatography with mass detection (GC-MS) using SCAN mode as described by Aura and coworkers[26]with authentic standards and 2-hydroxycinnamic acid as the internal standard. Metabolite concentrations in hepatocyte media were expressed in nanomoles per liter (nmol/L) whereas those in urine were expressed as nanomoles per milligram (nmol/mg) creatinine. For further details on the analyses of urinary metabolites, see Vetrani et al.[21].

The estimated daily exposure to polyphenol metabolites from polyphenol-rich food and beverages was calculated as follows: metabolite proﬁle of 6-h time point in the in vitro colon model was chosen and the concentration of each metabolite in fecal control was subtracted. A 6-h time point was selected because polyphenols are fully converted to the diverse microbial metabolites and would represent the likely daily absorption proﬁle of the metabolites from the colon. The concentration of each metabolite was expressed as nanomoles per day (nmol/day).

In addition, the differences between green tea infusions and cocoa samples used in the clinical trial and in the in vitro colon model were taken into account in the calculations. Green tea infusion used in the colon model (50 g green tea/500 ml hot water) was stronger than the infusion used in the clinical trial (12 g green tea/400 ml hot water). Hence, metabolite concentrations from green tea in the colon model were divided by coefficient 3.333 (Fig. 1a). Dark cocoa powder used in the colon model differed from the dark chocolate bar used in the clinical trial and fat and sugar contents needed to be subtracted, respectively, according to the list of the ingredients: 100 g of dark chocolate bar corresponded to 16.7 g of fatless and sugarless cocoa and thus the coefficient for dark chocolate bar is 0.167. Furthermore, 100 g of dark cocoa powder corresponds to 78.6 g fatless and sugarless cocoa and thus the coefficient is 0.786. The concentrations of dark cocoa microbial metabolites were divided by 0.786 (dark cocoa powder) and multiplied by 0.167 (dark chocolate bar) (Fig. 1b).

Finally, the daily dose (g/portion) and the frequency (portion/week) of the foods and beverages used in the trial[21]were taken into account and the total concentration (nmol/day) of each metabolite was calculated as the sum of concentrations from all the foodstuff.

2.5. Statistical analysis

The statistics of the metabolites concentrations (μM) in the colon model in triplicates were analyzed using MatLab Version R2008b and two-Way ANOVA with repeated measures using a Bonferroni adjustment. The metabolite was considered a foodstuff related one, when the response differed signiﬁcantly (Pb.05) from the fecal control (no added substrate).

For primary hepatocytes experiments, post hoc tests were applied to distinguish signiﬁcant (Pb.05) differences between extracts of green tea after colonic conversion (converted green tea) and controls: green tea before colonic conversion (no microbiota) or fecal control (no green tea) and between the corresponding responses of hepatocytes and of the media without hepatocytes.

Bivariate associations between estimated and urinary metabolites were analyzed by Pearson's correlation coefﬁcients and a P value b.05 was considered signiﬁcant. These analyses were performed according to standard methods using the Statistical Package for Social Sciences software version 21.0 (SPSS/PC; SPSS, Chicago, IL, USA).

3. Results

3.1. Clinical trial

Seventy-eight subjects completed the trial but only 72 participants

provided 24-h urine samples and were considered for the analyses: 37

subjects for the LP-diet and 35 for the HP-diet

[21]. Complementary to the

previous publication

[21], the urinary metabolite pro

ﬁles detected after

the HP-diet and the absolute changes from baseline values are reported in

Table 1. A signi

ﬁcantly higher concentrations were found for colonic

metabolites with C2-C3 side chain (hydroxylated phenylacetic and

phenylpropionic acids), but not for C1 side chain (benzoic acid).

Hippuric acid and 3-hydroxybenzoic acid represented C1 side chain

and ferulic acid hydroxycinnamic acids showing signi

ﬁcantly higher

concentrations in urinary excretion in the HP group than in the LP group.

3.2. In vitro colon model

The time course of microbial metabolite formation from each food

or beverage is shown in Supplement 1.

The calculated concentration of phenolic microbial metabolites is

shown in

Table 2. The abundant metabolites (

N5000 nmol/24 h) in

the calculated concentrations and their source foods were 3-(3

′-hydroxyphenyl)propionic acid (3-OHPPr: artichoke, orange and

coffee), 3-phenylpropionic acid (3-PPr: artichoke, orange and green

tea), 3-(3

′,4′-dihydroxyphenyl)propionic acid (3,4-diOHPPr: coffee,

arugula salad and fennel), 2-(3

′,4′-dihydroxyphenyl)acetic acid

(3,4-diOHPAc: arugula salad, spinach and onion), 3-(4

′-hydroxyphenyl)-propionic acid (4-OHPPr: artichoke and orange), benzoic acid (BA:

orange, onion and fennel) and

ﬁnally 2-(3-hydroxyphenyl)acetic acid

(3-OHPAc: cocoa powder). Metabolites showing low calculated

average concentrations (

b5000 nmol/day) were 3-hydroxybenzoic

acid (3-OHBA: artichoke, fennel, arugula salad and spinach), sinapic

acid (arugula salad), 4-coumaric acid (spinach), 3,4-dihydroxybenzoic

acid (3,4-diOHBA: spinach, cocoa powder and fennel),

4-hydroxy-benzoic acid (4-OHBA: artichoke, fennel and cocoa powder), 4-methyl

catechol (coffee, artichoke and cocoa powder), ferulic acid (cocoa and

spinach), gallic acid (fennel and onion), vanillic acid (arugula salad)

and

ﬁnally enterodiol (orange, onion and green tea). Ferulic acid and

3-OHBA showed low concentrations in the 6-h metabolite pro

ﬁle

taking into account the dose and frequency of their precursor

foodstuff: cocoa and spinach, as well as artichoke, fennel, arugula

salad and spinach, respectively.

A moderate but signi

ﬁcant correlation between average calculation

of 6-h colonic metabolites from in vitro colon model and 24-h urinary

excretion in vivo was observed (r = 0.280, P = .040;

Fig. 2). Since most

of the 3-PPR and 4-OHBA originated from the fecal background

(Supplement 1), these metabolites were excluded from the

correla-tion analysis as a nonrelevant artifact.

3.3. Human primary hepatocytes

When hepatocytes were incubated with green tea extract after colonic

conversion (converted green tea), signi

ﬁcant concentrations of 3-OHBA,

3,4-diOHPPr, ferulic acid and hippuric acid were formed as postcolonic

hepatic metabolites (Fig. 3). The same metabolites exhibited low

concentrations or were completely absent from the control incubations:

green tea extract before colonic conversion (no microbiota) or fecal control

(no green tea) or in the absence of hepatocytes (media with each extract).

4. Discussion

This study showed that the microbial polyphenol metabolism of

the occurring in the carbon backbone of the phenolic compounds is

partly due to microbial conversion and partly by postcolonic hepatic

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conversions. A signi

ﬁcant correlation was found between the

metabolite pro

_{ﬁles in 24-h urine from the clinical trial}

[21]

and the

estimated average of each metabolite formed in the in vitro colon

model (Table 2). Microbial metabolites formed from green tea in 6 h in

the in vitro colon model were chosen as representatives of microbial

metabolites from all food and beverages to the hepatic incubation

study. This was considered the best choice because green tea is soluble

in medium and its microbial metabolites were diverse and typical for a

polyphenol-rich diet.

The colonic model pro

ﬁle contained benzoic acid but no hippuric

acid. In contrast, the urinary excretion showed signi

ﬁcantly higher

hippuric acid concentrations, but no difference in benzoic acid

concentrations between high- and low-polyphenol groups

[21].

Hippuric acid is a hepatic glycine conjugate of benzoic acid

[27]

and

its high levels in urine are consistent with a previous study showing

increased excretion of hippuric acid in urine after green tea intake

[28].

Hippuric acid can be a metabolite in hepatocytes from colonic

microbial metabolites: benzoic acid as such or from phenylpropionic

acid (3-PPr, in abundance in the colon model) or phenylacetic acid

(not analyzed), the side chain of which can be shortened by

β-oxidation

[29,30]. The resulting benzoic acid is

ﬁnally glycinated to

hippuric acid, as has been reported earlier

[27,28,31]. Thus, the gap

between urinary excretion in vivo and colonic conversions in vitro

concerning benzoic acid and hippuric acid could be explained by the

hepatic metabolites.

3-OHBA and ferulic acid were also formed in the hepatocytes after

incubation with converted green tea and were absent from the media

without hepatocytes or controls without microbiota. The same

metabolites showed signi

ﬁcantly higher concentrations in the urine

after the polyphenol-rich diet in the clinical trial

[21]. In addition to

benzoic acid, 3-OHBA may be a

β-oxidation product, but in this case

from of 3-OHPPr. Moreover, ferulic acid (4

′-hydroxy-3′-methoxy

cinnamic acid) can be formed from 4-OHPPr via methoxylation at

position 3

′ or via methylation and hydroxylation at the positions 3′

and 4

′, respectively, from 3-OHPPr. In addition, oxidation reaction is

required to complete the structure of hydroxycinnamic acids (double

bond in the side chain). Thus, the hepatic incubation could also

ﬁll in

the gap between human urinary excretion and the in vitro colon model

in respect to 3-OHBA and ferulic acid. Thus, the results obtained in the

present study suggest that liver has a role in the postcolon metabolism

prior to excretion to urine.

Fig. 1. Equations used in the correction of green tea (a) and dark chocolate (b) before the calculation for the estimation.

Table 1

Phenolic metabolites excreted in 24-h urine after an 8-week consumption of high-polyphenol diet and their absolute changes from baseline levels[21]

Metabolitesa

8-Week concentration Absolute change after the intervention Mean SD Mean SD 3,4-diOHPPr 0.141 0.16 ** 0.070 0.13 *** 3-OHPPr 0.115 0.12 ** 0.062 0.12 *** 3-PPr 0 0 0 0 4-OHPPr 0.002 0.00 0 0 3,4-diOHPAc 0.100 0.07 ** 0.002 0.09 *** 3-OHPAc 0.330 0.25 ** 0.222 0.22 *** 3,4-diOHBA 0.025 0.01 ** 0.010 0.01 *** 3,5-diOHBA 0.021 0.01 0.002 0.02 3,4-diMeBA 0.013 0.01 0 0.01 3-OHBA 0.014 0.01 ** 0.008 0.01 *** 4-OHBA 0.059 0.04 −0.019 0.06 Benzoic acid 0.063 0.13 ** 0.012 0.08 4-methylCatechol 0.067 0.07 ** 0.004 0.12 *** Gallic acid 0.004 0.00 ** 0.004 0.00 *** Ferulic acid 0.134 0.08 ** 0.034 0.08 *** Sinapic acid 0.100 0.09 ** 0.044 0.10 Caffeic acid 0.063 0.04 ** 0.032 0.04 *** 4-coumaric acid 0.003 0.00 0.001 0.00 Enterodiol 0.001 0.00 * 0.001 0.00 Enterolactone 0.012 0.01 ** 0.005 0.01 *** Vanillic acid 0.112 0.07 0.007 0.10 *** Hippuric acid 0.023 0.02 ** 0.010 0.02 *** 3,4-diOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHPPr: 3-hydroxyphenylpropio-nic acid; 3-PPr: 3-phenylpropio3-hydroxyphenylpropio-nic acid; 4-OHPPr: 4-hydroxyphenylpropio3-hydroxyphenylpropio-nic acid; diOHPAc: dihydroxyphenylacetic acid; 3-OHPAc: 3-hydroxyphenylacetic acid; diOHBA: dihydroxybenzoic acid; 3,5-diOHBA: 3,5-dihydroxybenzoic acid; 3,diMeBA: 3,dimethoxybenzoic acid; 3-OHBA: 3-hydroxybenzoic acid; OHBA: 4-hydroxybenzoic acid.

*Pb.05 vs. baseline (Wilcoxon test); **Pb.05 vs. baseline (Wilcoxon test) and low-polyphenol diet (Mann–Whitney test); ***Pb.05 vs. low-low-polyphenol diet (Mann– Whitney test).

a

All metabolites are expressed as nanomoles per milligram (nmol/mg) creatinine, except for hippuric acid (μmol/mg creatinine).

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Several major metabolites of dietary phenolic compounds were

common to both colon model and urinary excretion such as 3-OHPAc,

3,4-diOHPPr, 3-OHPPr and 3,4-diOHPAc, which represent the major

metabolites of

ﬂavonoids and phenolic acids

[9]

and were the main

metabolites contributing to the in vitro/in vivo correlation. However,

3-PPR and 4-OHBA were excluded from the correlation analysis

because their concentration in the fecal background was high and they

most likely originated from the diets of the donors and may originate

from several precursors. 3-PPR is considered as nonspeci

ﬁc artifact

due to its higher presence in the fecal background than in the presence

of the precursor foodstuff, as shown in experiments 1

–4 (Supplement

1). The colon model does not take into account further metabolism of

the compounds by tissues, but especially 3-PPr, regardless of its origin,

could serve as a precursor of hepatic metabolites benzoic acid and

hippuric acid, as mentioned above.

The results presented in this study showed the structural

transformations occurring in the carbon backbone of the phenolic

compounds, excluding glucuronide conjugates, which would be

hydrolyzed by the colonic microbiota. Therefore, H. pomatia snail

glucuronidase was used in this study in the hydrolysis of urine and

hepatic samples. It is possible that sulfate conjugates are not

suf

ﬁciently cleaved in the preparation of urine or hepatocyte samples

and esterase activities may additionally cleave oxygen bridges, or

other oxidative reactions may also occur by H. pomatia enzyme.

Esterase side activities have been shown for H. pomatia enzyme

mixture when chlorogenic acid was not found in urinary or plasma

samples hydrolyzed with H. pomatia

[32

–35]

. However, it has been

shown that intestine has also esterases, which may cause similar

hydrolysis of ester or ether bonds

[36

–37]

. In addition, microbiota has

a strong capacity to deconjugate phenolic compounds

[24]. Ferulic

acid was found (with methoxyl functional group) in the incubation

with hepatocytes and in urinary samples despite the use of the same

glucuronidase enzyme at the same pH 4.1. Therefore, a limit of this

study is the inadequate hydrolysis of sulfate conjugates, if H. pomatia

enzyme mixture did not contain suf

ﬁcient amount of sulfatase or the

operating pH was not optimal, which may have caused

underestima-tion of sulfated metabolites, which also can explain why the

correlation is only moderate, even though it is signi

ﬁcant.

Results from the in vitro colon model showed microbial metabolite

pro

ﬁles for phenolic acids as has been shown for the majority of

polyphenols or polyphenol-rich foods

[12,38

–41]

. Moreover, as

suggested by previous studies

[39,41], hydroxylated phenylpropionic

and benzoic acids were detected as metabolites of coffee. Finally, small

amounts of enterodiol but no enterolactone were detected in the

present study as metabolites from plant lignans

[11]. Enterolactone

formation is suppressed in the colon model in vitro, partly because of

low pH, as reported previously

[42], and partly because the foodstuff in

the presented study were chosen as typical Mediterranean foods rich

in polyphenols, not as good sources of plant lignans. Enterolactone

formation was not in the focus of this study but was monitored from all

the samples. In contrast, the formation of phenolic acid metabolites is

Table 2

Calculated concentrations of known phenolic microbial metabolites after a 6-h incubation in the in vitro colon model using human fecal suspension (10%, w/v) from polyphenol-rich foodstuff and dose and frequency data from the human trial[21]

Metabolites Onion Fennel Arugula salad

Artichoke Spinach Orange Green tea Coffee Dark cocoa Sum

Human study setup g/portion 200 200 90 325 150 190 12 28 25

portion/week 3 2 7 2 2 7 7 7 7 3,4-diOHPPr 571 2565 8505 3142 524 647 15.1 21,319 149 37,436 3-OHPPr 1884 1511 0 392,191 6314 32,105 701 138,649 2167 575,522 4-OHPPr 403 300 0 16,271 1061 1689 15.5 0 483 20,223 3-PPr 0 0 0 165,176 0 6811 5989 0 0 177,976 3,4-diOHPAc 3294 444 14,060 0 6520 285 438 0 396 25,437 3-OHPAc 0 0 0 386 0 258 448 0 5269 6361 3,4-diOHBA 9 114 0 0 826 46.2 0 3.89 344 1343 3-OHBA 81.6 216 190 1331 130 87.2 9.46 9.5 82.8 2137 4-OHBA 26.1 194 7 887 0 0 4.45 31.1 253 1403 Benzoic acid 4417 2346 0 1842 0 4519 49.6 280 530 13,984 4-methylcatechol 18.2 0 0 401 0 20.3 75.9 403 199 1118 Gallic acid 235 303 0 0 0 7.37 13.5 0 17.6 576 Ferulic acid 0 0 10.1 0 181 8.71 0.63 0 398 599 Sinapic acid 0 0 1714 0 0 149 0 0 0 1863 4-coumaric acid 0 0 44.6 0 1481 3.30 6.62 0 247 1783 Enterodiol 17.5 0 0 0 0 56.4 5.03 0 0 79 Vanillic acid 21.6 51.9 307 0 0 86.8 0 0 38.7 507

3,4-diOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHPPr: 3-hydroxyphenylpropionic acid; 4-OHPPr: 4-hydroxyphenylpropionic acid; 3-PPr: 3-phenylpropionic acid; 3,4-diOHPAc: 3,dihydroxyphenylacetic acid; 3-OHPAc: 3-hydroxyphenylacetic acid; 3,diOHBA: 3,dihydroxybenzoic acid; 3-OHBA: 3-hydroxybenzoic acid; OHBA: hydroxybenzoic acid; 4-mecatechol: 4-methylcatechol.

Fig. 2. Correlation between urinary metabolites concentrations and phenolic metabolites estimated from the incubation in the in vitro colon model.3,4-diOHBA: 3,4-dihydroxybenzoic acid; 3,4-diOHPAc: 3,4-dihydroxyphenylacetic acid; 3,4-diOHPPr: 3,4-dihydroxyphenylpro-pionic acid; OHBA: hydroxybenzoic acid; OHPAc: hydroxyphenylacetic acid; 3-OHPPr: 3-hydroxyphenylpropionic acid; 4-Cou: 4-coumaric acid; 4-MeCat: 4-metylcatechol; 4-OHBA: 4-hydroxybenzoic acid; BA: benzoic acid; GA: gallic acid; FA: ferulic acid; SA: sinapic acid; ED: enterodiol; VA: vanillic acid.

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not affected by the accumulation of the metabolites

[42]. Some of the

main metabolites of green tea, namely valeric acid and valerolactone

derivatives

[43,44], were unfortunately not included in the analyses.

5-(3´-Hydroxyphenyl) valeric acid and 5-(3´,4´-dihydroxyphenyl)

valeric acid were detected as microbial metabolites from (+)-catechin

and (

−)-epicatechin stereoisomers using GCxGC-TOFMS

metabolo-mics

[43]; however, the authentic standards of valeric acid and

valerolactone derivatives were not available by the time of this work.

In addition, gallic acid, surely abundant in green tea, is metabolized

quickly and it had a minor weight in the average calculations.

The comparison of in vivo and in vitro metabolite pro

ﬁles is

challenging due to high individual variability of all colonic metabolites

[45], which can partly be caused because of individual variation of

microbiota, consisting of over 1000 different species

[46 ,47].

However, the conversion differences are not extensive because the

functional genes within colonic microbiota do not differ between

subjects as strongly as the microbial composition due to a

“functional

core

” of the microbiome

[46,47]. This was described in a study of a

cohort of individuals where at least 40% of the microbial genes were

shared by half of the individuals within the cohort

[46]. Most likely

reason of the difference between the urinary excretion and calculated

colonic metabolite pro

ﬁle is that people had the possibility to vary the

intake within a week and still be compliant within the frames of the

diet, but foods introduced to the colon model were de

ﬁned and

calculated according to averages of the instructed frequency and dose.

However, the 6-h incubation time may not give the correct metabolite

pro

ﬁle for the average. The effect of food matrix in onion and fennel

was apparent, showing slower release and consequent conversion

than isolated compounds or beverages would give. Therefore, the

calculations may have showed more relevant metabolite pro

ﬁle for

foods than for beverages.

Different colonic communities share general metabolic activities,

which convert food components to speci

ﬁc metabolite proﬁles

[48]. It

has also been shown that several phenolic precursors in foods are

converted to a relatively small number of colonic phenolic acids and

lactones that represent the predominant microbial metabolites

[44].

The in vitro colon model was performed with a pooled human fecal

suspension from several donors to make results repeatable and to

diminish this interindividual variation in vitro

[48]. Such an approach

was chosen because metabolites speci

ﬁc for each food or beverage

could be distinguished from the fecal control (with no foods or

beverages), designating the metabolite pro

ﬁle from the

polyphenol-rich diet, when the standard deviations are low for signi

ﬁcant

metabolites of the diet. If all the foods were incubated with feces

from 5 to 10 even 20 individuals, which would have been adequate for

statistics, the standard deviations would have been as high as in the

urinary excretion, and speci

ﬁc food-related metabolites could not

have been identi

ﬁed. The perspective would have been in individual

responses without the possibility to identify the metabolites for each

food. Using several donors for each food, the number of samples and

metabolites would also have been too many considering the

dimensions of the study. Therefore, the pooled fecal suspensions

showed the signi

ﬁcant metabolite proﬁle for each foodstuff, and the

24-h urinary excretion showed the natural individual variation in vivo,

which served the approach of the study in an optimal manner.

5. Conclusion

The hypothesis of this study was that urinary excretion pro

ﬁle

re

_{ﬂects colonic and subsequent hepatic transformations of carbon}

skeleton of dietary polyphenols. This hypothesis was veri

ﬁed in our

study by complementary concentrations of postcolonic hepatic

Fig. 3. Hepatic conversion of microbial metabolites from green tea (light-gray bars: green tea extract converted by microbes— converted green tea) in the presence of human primary hepatocytes (Hepatocytes) as compared with control extracts (medium-dark-gray bars: cell medium, black bars: microbial control— no green tea, dark-gray bars: nonconverted green tea— no microbiota) and in absence of hepatocytes (No Hepatocytes). Of the analyzed metabolites, 3,4-DiOHPPr, 3-OHBA, ferulic acid and hippuric acid were signiﬁcantly higher in the presence of converted green tea extract and hepatocytes than in all the controls (⁎⁎⁎Pb.001).3,4-DiOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHBA: 3-hydroxybenzoic acid.

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metabolites in urinary excretion, showing also that colonic

conver-sions reach only moderate but still signi

ﬁcant correlation with urinary

metabolite pro

ﬁle. Combination of in vitro colon and hepatic models

could be used as a predictive model in

ﬁnding foodstuff or diet-speciﬁc

metabolites in human body

ﬂuids.

Authorship

The authors' responsibilities were as follows: CV and AMA wrote

the manuscript with input from all authors and were responsible for

statistical aspects of the study; CV, AMA, AAR and GA conceived and

designed the study, IM performed metabolites analyses with the input

of MO; AMA, MA and JB carried out in vitro experiments. CV, AAR and

GA were involved in the clinical trial. CV, AAR and AMA had primary

responsibility for

ﬁnal content. All authors revised the manuscript and

approved the

ﬁnal version.

Acknowledgments

Food for the clinical trial was kindly supplied by Lavazza, Torino,

Italy (coffee); Nestlé, Vevey, Switzerland (chocolate); Parmalat S.p.A.,

Parma, Italy (juice); Zuegg S.p.A., Verona, Italy (jam); Pompadour Tè

S.r.l., Bolzano, Italy (green tea); Coop. Nuovo Cilento s.c.r.l., San Mauro

Cilento, Salerno, Italy (extra virgin olive oil).

We gratefully acknowledge the laboratory work done by Maria Heyden

and Annika Lundqvist in the hepatocyte study; Annika Majanen, Siv

Matomaa, Kari Lepistö, Niina Torttila and Airi Hyrkäs in the execution of the

metabolical in vitro colon model and the analyses of the metabolites; and

Angela Giacco and Marilena Vitale for dietary counseling.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

http://dx.

doi.org/10.1016/j.jnutbio.2016.03.007.

References

[1]Tresserra-Rimbau A, Rimm EB, Medina-Remón A, Martínez-González MA, de la Torre R, Corella D, et al. Inverse association between habitual polyphenol intake and incidence of cardiovascular events in the PREDIMED study. Nutr Metab Cardiovasc Dis 2014;24:639–47.

[2]Hollman PC, Geelen A, Kromhout D. Dietary flavonol intake may lower stroke risk in men and women. J Nutr 2010;140:600–4.

[3]Wedick NM, Pan A, Cassidy A, Rimm EB, Sampson L, Rosner B, et al. Dietary flavonoid intakes and risk of type 2 diabetes in US men and women. Am J Clin Nutr 2012;95:925–33.

[4]Liu YJ, Zhan J, Liu XL, Wang Y, Ji J, He QQ. Dietary flavonoids intake and risk of type 2 diabetes: a meta-analysis of prospective cohort studies. Clin Nutr 2014;33: 59–63.

[5]Chiva-Blanch G, Urpi-Sarda M, Ros E, Valderas-Martinez P, Casas R, Arranz S, et al. Effects of red wine polyphenols and alcohol on glucose metabolism and the lipid profile: a randomized clinical trial. Clin Nutr 2013;32:200–6.

[6]Annuzzi G, Bozzetto L, Costabile G, Giacco R, Mangione A, Anniballi G, et al. Diets naturally rich in polyphenols improve fasting and postprandial dyslipidemia and reduce oxidative stress: a randomized controlled trial. Am J Clin Nutr 2014;99: 463–71.

[7]Hernáez Á, Fernández-Castillejo S, Farràs M, Catalán Ú, Subirana I, Montes R, et al. Olive oil polyphenols enhance high-density lipoprotein function in humans: a randomized controlled trial. Arterioscler Thromb Vasc Biol 2014;34:2115–9. [8]Ibero-Baraibar I, Abete I, Navas-Carretero S, Massis-Zaid A, Martinez JA, Zulet MA.

Oxidised LDL levels decreases after the consumption of ready-to-eat meals supplemented with cocoa extract within a hypocaloric diet. Nutr Metab Cardiovasc Dis 2014;24:416–22.

[9]Bozzetto L, Annuzzi G, Pacini G, Costabile G, Vetrani C, Vitale M, et al. Polyphenol-rich diets improve glucose metabolism in people at high cardiometabolic risk: a controlled randomised intervention trial. Diabetolo-gia 2015;58:1551–60.

[10]Aura AM. Microbial metabolism of dietary phenolic compounds in the colon. Phytochemistry Rev 2008;7:407–29.

[11]Del Rio D, Rodriguez-Mateos A, Spencer JP, Tognolini M, Borges G, Crozier A. Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid Redox Signal 2013;18: 1818–92.

[12]Serra A, Macià A, Romero MP, Reguant J, Ortega N, Motilva MJ. Metabolic pathways of the colonic metabolism of flavonoids (flavonols, flavones and flavanones) and phenolic acids. Food Chem 2012;130:383–93.

[13]Aura AM. In vitro colon model to study metabolic syndrome. In: Oresic M, Vidal-Puig A, editors. A Systems Biology approach to study metabolic syndrome. Switzerland: Springer International Publishing; 2014. p. 251–78.

[14]Kuijsten A, Arts IC, Vree TB, Hollman PC. Pharmacokinetics of enterolignans in healthy men and women consuming a single dose of secoisolariciresinol diglucoside. J Nutr 2005;135:795–801.

[15]Seeram NP, Henning SM, Zhang Y, Suchard M, Li Z, Heber D. Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. J Nutr 2006;136:2481–5.

[16]Clifford MN, van der Hooft JJ, Crozier A. Human studies on the absorption, distribution, metabolism, and excretion of tea polyphenols. Am J Clin Nutr 2013; 98:1619S–30S.

[17]Actis-Goretta L, Lévèques A, Rein M, Teml A, Schäfer C, Hofmann U, et al. Intestinal absorption, metabolism, and excretion of (−)-epicatechin in healthy humans assessed by using an intestinal perfusion technique. Am J Clin Nutr 2013;98:924–33. [18]Czank C, Cassidy A, Zhang Q, Morrison DJ, Preston T, Kroon PA, et al. Human

metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a (13)C-tracer study. Am J Clin Nutr 2013;97:995–1003.

[19]Renouf M, Marmet C, Giuffrida F, Lepage M, Barron D, Beaumont M, et al. Dose– response plasma appearance of coffee chlorogenic and phenolic acids in adults. Mol Nutr Food Res 2014;58:301–9.

[20]Vitaglione P, Donnarumma G, Napolitano A, Galvano F, Gallo A, Scalfi L, et al. Protocatechuic acid is the major human metabolite of cyanidin-glucosides. J Nutr 2007;137:2043–8.

[21]Vetrani C, Rivellese AA, Annuzzi G, Mattila I, Meudec E, Hyötyläinen T, et al. Phenolic metabolites as compliance biomarker for polyphenol intake in a randomized controlled human intervention. Food Res Int 2014;68:233–8. [22]National Cholesterol Education Program (NCEP) Expert Panel on Detection,

Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final reportCirculation 2002;106:3143–421.

[23]Aura AM, Härkönen H, Fabritius M, Poutanen K. Development of an in vitro enzymatic digestion method for removal of starch and protein and assessment of its performance using rye and wheat breads. J Cereal Sci 1999;29:139–52. [24]Aura AM, O'Leary KA, Williamson G, Ojala M, Bailey M, Puupponen-Pimiä R, et al. Quercetin derivatives are deconjugated and converted to hydroxyphe-nylacetic acids but not methylated by human fecal flora in vitro. J Agric Food Chem 2002;50:1725–30.

[25]Aura AM, Martin-Lopez P, O'Leary KA, Williamson G, Oksman-Caldentey KM, Poutanen K, et al. In vitro metabolism of anthocyanins by human gut microflora. Eur J Nutr 2005;44:133–42.

[26]Aura AM, Mattila I, Hyötyläinen T, Gopalacharyulu P, Cheynier V, Souquet JM, et al. Characterization of microbial metabolism of Syrah grape products in an in vitro colon model using targeted and non-targeted analytical approaches. Eur J Nutr 2013;52:833–46.

[27]Gonthier MP, Verny MA, Besson C, Rémésy C, Scalbert A. Chlorogenic acid bioavailability largely depends on its metabolism by the gut microflora in rats. J Nutr 2003;133:1853–9.

[28]Mulder TP, Rietveld AG, van Amelsvoort JM. Consumption of both black tea and green tea results in an increase in the excretion of hippuric acid into urine. Am J Clin Nutr 2005;81:256S–60S.

[29]Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SK, et al. Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products. Free Radic Biol Med 2004;36:212–25.

[30] Chesson A, Provan GJ, Russell WR, Scobbie L, Richardson AJ, Stewart C. Hydroxycinnamic acids in the digestive tract of livestock and humans. J Sci Food Agric 1999;79:373–8.

[31]Pagella JH, Chen XB, MacLeod NA, Orskov ER, Dewey PJ. Excretion of benzoic acid derivatives in urine of sheep given intraruminal infusions of 3-phenylpropionic and cyclohexane carboxylic acids. Br J Nutr 1997;77:577–92.

[32]Cremin P, Kasim-Karakas S, Waterhouse AL. LC/ES-MS detection of hydroxycin-namates in human plasma and urine. J Agric Food Chem 2001;49:1747–50. [33]Ito H, Gonthier M-P, Manach C, Morand C, Mennen L, Rémésy C, et al. Polyphenol

levels in human urine after intake of six different polyphenol-rich beverages. Br J Nutr 2005;94:500–9.

[34]Manach C, Williamson G, Morand C, Scalbert A, Rémésy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81:230S–42S.

[35]Monteiro M, Farah A, Perrone D, Trugo LC, Donangelo C. Chlorogenic acid compounds from coffee are differentially absorbed and metabolized in humans. J Nutr 2005;137:2196–201.

[36]Andreasen MF, Kroon PA, Williamson G, Garcia-Conesa MT. Intestinal release and uptake of phenolic antioxidant diferulic acids. Free Radic Biol Med 2001;31: 304–14.

[37]Kern SM, Bennett RN, Mellon FA, Kroon PA, Garcia-Conesa MT. Absorption of hydroxycinnamates in humans after high-bran cereal consumption. J Agric Food Chem 2003;51:6050–5.

[38]Dall'Asta M, Calani L, Tedeschi M, Jechiu L, Brighenti F, Del Rio D. Identification of microbial metabolites derived from in vitro fecal fermentation of different polyphenolic food sources. Nutrition 2012;28:197–203.

(8)

[39]Gonthier MP, Remesy C, Scalbert A, Cheynier V, Souquet JM, Poutanen K, et al. Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro. Biomed Pharmacother 2006;60:536–40. [40]Roowi S, Stalmach A, Mullen W, Lean ME, Edwards CA, Crozier A. Green tea

flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans. J Agric Food Chem 2010;58:1296–304.

[41]Marmet C, Actis-Goretta L, Renouf M, Giuffrida F. Quantification of phenolic acids and their methylates, glucuronides, sulfates and lactones metabolites in human plasma by LC–MS/MS after oral ingestion of soluble coffee. J Pharm Biomed Anal 2014;88:617–25.

[42]Aura AM, Oikarinen S, Mutanen M, Heinonen SM, Adlercreutz HC, Virtanen H, et al. Suitability of a batch in vitro fermentation model using human faecal microbiota for prediction of conversion of flaxseed lignans to enterolactone with reference to an in vivo rat model. Eur J Nutr 2006;45:45–51.

[43]Aura A-M, Mattila I, Seppänen-Laakso T, Miettinen J, Oksman-Caldentey KM, Orešič M. Microbial metabolism of catechin stereoisomers by human faecal

microbiota: comparison of targeted analysis and a non-targeted metabolomics method. Phytochem Lett 2008;1:18–22.

[44]Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N, Brighenti F. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 2010;26:1110–6.

[45]Cerdá B, Periago P, Espín JC, Tomás-Barberán FA. Identification of urolithin a as a metabolite produced by human colon microflora from ellagic acid and related compounds. J Agric Food Chem 2005;53:5571–6.

[46]Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010; 464:59–65.

[47]The human microbiome Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207–14.

[48]Jacobs DM, Gaudier E, van Duynhoven J, Vaughan EE. Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics. Curr Drug Metab 2009;10:41–54.