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1 PII S (02) Free Radical Biology & Medicine, Vol. 33, No. 2, pp , 2002 Copyright 2002 Elsevier Science Inc. Printed in the USA. All rights reserved /02/$ see front matter Original Contribution THE METABOLIC FATE OF DIETARY POLYPHENOLS IN HUMANS ANDREAS R. RECHNER,* GUNTER KUHNLE,* PAUL BREMNER,* GARY P. HUBBARD, KEVIN P. MOORE, and CATHERINE A. RICE-EVANS* *Antioxidant Research Group, Wolfson Centre for Age-Related Diseases, School of Biomedical Sciences, King s College London, London, UK; Hugh Sinclair Unit of Human Nutrition, Department of Food Science and Technology, University of Reading, Reading, UK; and Centre for Hepatology, Dept. of Medicine, Royal Free Campus, Royal Free and University College Medical School, UCL, London, UK (Received 31 October 2001; Revised 5 March 2002; Accepted 12 April 2002) Abstract Dietary polyphenols are widely considered to contribute to health benefits in humans. However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may contribute toward disease prevention. Although many studies are focusing on the bioavailability of polyphenols through studying their uptake and the excretion of their conjugated forms, few are emphasizing the occurrence of metabolites in vivo formed via degradation by the enzymes of colonic bacteria and subsequent absorption. The purpose of this research was to investigate the relationship between biomarkers of the colonic biotransformation of ingested dietary polyphenols and the absorbed conjugated polyphenols. The results show that the majority of the in vivo forms derive from cleavage products of the action of colonic bacterial enzymes and subsequent metabolism in the liver. Those include the glucuronides of 3-hydroxyphenylacetic, homovanillic, vanillic and isoferulic acid as well as 3-(3-methoxy-4-hydroxyphenyl)-propionic, 3-(3-hydroxyphenyl)-propionic acid, and 3-hydroxyhippuric acid. In contrast, intact conjugated polyphenols themselves, such as the glucuronides of quercetin, naringenin and ferulic, p-coumaric, and sinapic acid were detected at much lower levels. The results suggest that consideration should be given to the cleavage products as having a putative role as physiologically relevant bioactive components in vivo Elsevier Science Inc. Keywords Absorption, Metabolism, Excretion, Naringenin, Quercetin, Dihydroferulic acid, 3-(3-Hydroxyphenyl)- propionic acid, Hydroxycinnamates, 3-Hydroxyhippuric acid, Hippuric acid, Bioavailability, GC-MS, Free radicals INTRODUCTION A wide range of biological effects has been attributed to dietary polyphenols (reviewed in [1 3]), but many studies have ignored the question of their achievable concentrations in the circulation after ingestion as well as the possibility of conjugation and metabolism of polyphenols. Therefore, detailed knowledge concerning the conjugative and metabolic events and resulting plasma levels following the ingestion of a polyphenol-rich diet is crucial for the understanding of their bioactivity and cytoprotective effects. The major dietary families of polyphenols are the flavan-3-ols, flavonols, flavanones, anthocyanins, and hydroxycinnamates. Author correspondence to: Prof. Catherine Rice-Evans, Antioxidant Research Group, Wolfson Centre for Age-Related Diseases, Guy s, King s and St. Thomas s School of Biomedical Sciences, King s College London, Guy s Campus, Hodgkin Building, 3rd Floor, London SE1 9RT, UK; Tel: 44 (0) ; Fax: 44 (0) ; catherine.rice-evans@kcl.ac.uk. Dietary polyphenols are substrates for -glucosidases, UDP-glucuronosyltransferase, or catechol-o-methyltransferase in the small intestine as well as for a number of phase I and II enzymes in the liver [4 7]. In addition, ingested polyphenols are subjected to hydrolysis and degradation in the colon due to the activity of enzymes of the colonic microflora [4 8]. Hydroxycinnamates, which are esterified naturally [9], are not cleaved in the gastric lumen [10,11] nor the small intestine [12], but in the colon by esterase activity of the gut microflora [4,12]. Subsequent absorption of the free hydroxycinnamic acids, p-coumaric, caffeic and ferulic acid, has been reported [13] as well as O-methylation of caffeic acid to ferulic or isoferulic acid, and conjugation to glucuronides or sulphates [10 14]. Degradation by the gut microflora has also been proposed to result in the formation of dihydroferulic and 3-hydroxyhippuric and possibly also hippuric acid [10]. The latter two might derive from the absorbed corresponding phe- 220

2 Metabolism of dietary polyphenols 221 nylpropionic acids following -oxidation to benzoic acids and subsequent glycination in the liver [8,10,15]. The bioavailability and metabolism of individual flavan-3-ols has been studied in animals and humans demonstrating the absorption and elimination of low micromolar amounts of their direct conjugates [16 19]. Factors influencing metabolism and conjugation of flavan-3-ols have been reported from human and animal studies as well as from in vitro models, including the formation of 5- or 7-O-glucuronosyl conjugates and 3 or 4 -O-methyl glucuronides [20 24]. However, degradation in the colon results in the destruction of the flavan structure and the formation of simpler phenolic compounds such as hydroxyphenylvalerolactones and hydroxyphenylpropionic acids [5,20]. Due to their instability at higher ph values the analysis of anthocyanins in body fluids is difficult but absorption and excretion of very low proportions of the intact glycosides has been reported after ingestion of anthocyanin rich berry or wine extracts [25 29]. The only metabolite associated with dietary intake of anthocyanins described is protocatechuic acid in rats, which is a proposed precursor of vanillic acid [27]. The flavonols, naturally found as glycosides [9], have been intensively studied. The uptake involves a cleavage of the glycosides in the small intestine followed by absorption and metabolism of the aglycone [5,30 33], while some reports indicate an absorption of the intact glycosides [32]. Conjugation of the flavonols to sulfates and glucuronides as well as O-methylation of the catechol group to isorhamnetin and tamaraxetin, in the case of quercetin, have been reported in animal models and human studies [5,7,21,30,33]. Only a small proportion of ingested flavonols is absorbed with an intact flavonol structure [7,30 32]. A number of colonic metabolites have been identified in human urine as simple phenolic acids, such as hydroxyhippuric acids and hydroxyphenylacetic acids [5,34,35]. The flavanones, exclusively found in citrus fruit and tomato [9], predominantly as glycosides, undergo similar metabolic routes to flavonols [5,36,37]. Cleavage of the glycosides in the small intestine followed by absorption and conjugation of the aglycone, mainly as the glucuronide, has been reported in humans as well as in animal models [36 40]. The reported absorption rates of flavanones in humans are much higher ( 10%) than those of other flavonoids [36,38]. The aim of this study was to investigate biomarkers of the biotransformation of ingested dietary polyphenols in humans. The balance between the absorption of dietary polyphenols (from a polyphenol-rich diet) as specific conjugated compounds and their metabolism through colonic degradation, resulting in specific simple phenolic metabolites, was examined and the structural assignments confirmed by gas chromatography with mass spectrometric detection (GC-MS) techniques. Chemicals MATERIAL AND METHODS Ferulic acid, isoferulic acid, o-hydroxyhippuric acid, chlorogenic acid, hippuric acid, and trans-cinnamic acid, cyanidin-3-glucoside, quercetin-3-glucoside, naringenin were obtained from Extrasynthese (Genay, France). Methanol (high-performance liquid chromatography [HPLC] grade), acetonitrile (HPLC grade), acetic acid, acetone were obtained by Rathburn Chemicals LTD (Walkerburn, Scotland). Hydrochloric acid was from BDH Laboratory Supplies (Poole, England). Quercetin, p-coumaric acid, 3-hydroxyphenylacetic acid, vanillic acid, homovanillic acid, caffeine, thymol, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, trichloroacetic acid, N-(t-butyldimethylsilyl)-Nmethyltrifluoroacetamide chlorosilane (TBDMSCl), undecane, and -glucuronidase (EC ) Type L-II from Limpets were purchased from Sigma Chemical Co. (Steinheim, Germany). 3-(4-Hydroxy-3-methoxyphenyl)-propionic acid (dihydroferulic acid) and 3-(3-hydroxyphenyl)- propionic acid were obtained from Lancaster Synthesis Ltd. (Morecambe, UK). Study design Ethical permission was obtained from the University of Reading Ethics Committee. Twenty healthy male and female subjects, aged between 20 and 50 years (mean age years) with a Body Mass Index (BMI) between 18.3 kg/m 2 to 34.0 kg/m 2 (mean BMI kg/m 2 ), were recruited from the Reading area in the UK. Exclusion criteria were pregnancy, any form of known liver disease, diabetes mellitus or a previous myocardial infarction, gall bladder problems or abnormalities of fat metabolism, involvement in a weightreducing dietary regimen, ingestion of any dietary supplements (including dietary fatty acids), regular or vigorous exercisers (more than 3 times/week, 20 min each session), consumption of more than 15 units of alcohol per week. The exclusion criteria were developed to mimic the average population. The 20 subjects were split into four groups containing 5 subjects each. The feeding study and the collection of samples is depicted schematically in Fig. 1. Each group underwent a 3 d flavonoid-free diet, and then a 5 d feeding period. After the 3 d flavonoid-free period subjects were required to attend the Human Investigation Unit at the Hugh Sinclair Human Nutrition Unit to give a baseline blood sample, and consume their first flavonoid-rich meal. For the next 4 d subjects were required to attend the unit at lunchtime to consume their fla-

3 222 A. R. RECHNER et al. Fig. 1. Scheme of the study design. vonoid-rich meal. Every urine sample was collected and pooled every 12 h, daytime collection at 7:00 p.m., nighttime collection at 7:00 a.m. On the last day of feeding, subjects were asked to attend the unit from 9:00 a.m. until 6:00 p.m. During this time the subjects were cannulated, and blood samples (10 ml) were taken throughout the day. The subjects were given a flavonoidrich meal at lunchtime. Baseline blood samples were taken before the meal (0 h), and at 1, 3, and 5 h after the meal. Urine collection also continued. The polyphenol specific meal components were tomato and onion pasta sauce (150 g), pasta (220 g), cooked broccoli (60 g), cherry tomatoes (90 g), cucumber (60 g), continental leaf salad (50 g), pepper salad dressing (4 g), defrosted raspberries (100 g), red grape juice (300 ml) and apple juice (300 ml). The analyzed dietary parameters were 5826 kj (1379 Kcal), 17.6 g protein (5.1%), 30.9 g fat (20.2%), g carbohydrates (74.8%). For other meals the volunteers were requested to adhere to the low-polyphenol diet. HPLC analysis HPLC analysis was undertaken using the Waters system consisting of controller 600, autosampler 717 plus, photodiode array detector 996, on-line degasser. Samples were analyzed on a Novo-Pak C18 column, mm, with 4 m particle size and a guard column of the same material mm. Mobile phase A consisted of methanol/water/5n HCl (5/94.9/0.1 v/v/v) and mobile phase B of acetonitrile/water/5n HCl (50/49.9/0.1 v/v/v). The gradient applied was as follows: from 0 to 5 min 100% A, from 5 to 40 min to 50% A and 50% B, from 40 to 60 min to 100% B, from 60 to 65 min 100% B, and from 65.1 min 100% A. Run time was 70 min followed by a 10 min delay prior to the next injection. Injection volume was 50 l for urine samples and 100 l for plasma samples. Components in urine and plasma were identified according to retention time, ultraviolet (UV)/ visible spectra and spiking with commercially available relevant standards (naringenin, quercetin, ferulic acid, isoferulic acid, p-coumaric acid, dihydroferulic acid, 3-(3-hydroxyphenyl)-propionic acid, 3-hydroxyphenylacetic acid, homovanillic acid, vanillic acid, and hippuric acid). The complex polyphenolic composition of the meal, in combination with a lack of authentic standards, led to the classification and quantification of the peaks according to their spectral characteristics as hydroxycinnamates, anthocyanins, flavonols, flavan-3-ols, and flavanones. Hydroxycinnamates (14 peaks) were quantified relative to chlorogenic acid, anthocyanins (11 peaks) relative to cyanidin-3-glucoside, flavonols (13 peaks) relative to quercetin-3-glucoside, flavan-3-ols (2 peaks) relative to ( )-catechin and flavanones (2 peaks) relative

4 Metabolism of dietary polyphenols 223 Table 1. Standards for GC/MS Identification Compound MW TMW BP Fragment Ions 3-Hydroxyphenylacetic acid (3-hydroxyphenyl)-propionic acid (4-hydroxyphenyl)-propionic acid Vanillic acid Homovanillic acid Dihydroferulic acid p-coumaric acid o-hydroxyhippuric acid Isoferulic acid Ferulic acid Sinapic acid Hippuric acid Derivatization with TBDMS adds 114 mass units to every hydroxyl- and carboxyl group, and typically fragments with loss of 15 mass units (loss of methyl group) and loss of 57 mass units (loss of butyl group). Table shows major peaks of the standards used. MW-molecular weight of each compound, TMW-total molecular weight post-derivatization. BP base peak (i.e., the most abundant fragment ion) and fragment ions other abundant signals of each compound. to naringenin. All standard curves were obtained from the authentic standard compounds. 3- and 4-hydroxyhippuric acids were quantified relative to 3- and 4-hydroxybenzoic acids, respectively. Wavelengths used for quantification were: quercetin (370 nm), ferulic, isoferulic, p-coumaric acid (310 nm), naringenin, dihydroferulic, homovanillic acid (280 nm), vanillic acid (260 nm urine, 295 nm plasma), 3-hydroxyphenylacetic and 3-(3-hydroxyphenyl)-propionic acid (272 nm), 3-hydroxyhippuric acid (288 nm), 4-hydroxyhippuric acid (254 nm), hippuric acid (225 nm). Limits of quantification in plasma were 2.5 nmol/l for p-coumaric acid, 5 nmol/l for ferulic and isoferulic acid and naringenin, 10 nmol/l for vanillic acid and quercetin, 20 nmol/l for phenylpropionic acids. Coefficient of variance for all standards was 5%. GC-MS analysis Standard solutions were prepared as follows: 1 mg of each standard compound was dissolved in 50 l dry acetonitrile and 20 l N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) containing 1% TBDM- SCl. After 30 min, 10 l of the derivatized samples were dried under nitrogen and dissolved in 20 l undecane. This stock solution was diluted prior to GC-MS analysis. The samples were purified as described before and dried under nitrogen. The samples were subsequently derivatized with TBDMS as described above. The samples and standards were analyzed on a Fisons GC8000 gas chromatograph, applying a DB-1701 column (column length for plasma 12 m and for urine 15 m, resulting in different retention times) and a Fisons Trio 1000 using EI positive and full scan mode. 1 l of sample was injected and the following temperature gradient applied: 0 1 min: 150 C, 1 16 min: 20 C/min. The major fragment ions for the derivatized standards are shown in Table 1. Sample preparation Meals. Whole meal samples were homogenized with a food processor, freeze-dried, and aliquots extracted with 70% methanol, followed by HPLC analysis of the supernatant after centrifugation. Plasma. L-Ascorbic acid (5 mg) and 200 l of20% trichloracetic acid in methanol were added to 1 ml of plasma for deproteinization, extracted with 1 ml of acetone and centrifuged. The supernatant was freeze-dried and the resulting residue dissolved in 250 l of20% methanol for HPLC analysis. For -glucuronidase treatment 1 ml of plasma was acidified with 5 l acetic acid prior to the addition of -glucuronidase and incubation for 2hat37 C. Following the -glucuronidase treatment deproteinization and extraction was undertaken as described above. trans- Cinnammic acid was used as internal standard. The precision of the method was 95%, intra run variance was 15%, the recovery was 80% and after -glucuronidase treatment 75%. Urine. Untreated urine samples were filtered through a 0.45 m membrane prior to HPLC analysis. For -glucuronidase treatment 2 ml of urine was acidified with 5 l acetic acid prior to the addition of -glucuronidase and incubation for 2 h at 37 C. trans-cinnamic acid was used as internal standard. The precision of the method was 95%, intrarun variance was 15% and interrun variance 30% while the recovery after -glucuronidase was 90%.

5 224 A. R. RECHNER et al. Plasma and urine samples for GC-MS analysis A C-18 cartridge (Waters Sep-Pak, Milford, MA, USA) was washed with 3 ml methanol and conditioned with 6 ml water/methanol/acetic acid (94/5/1 v/v/v) prior to the application of plasma, untreated and -glucuronidase treated. Following the plasma application the cartridge was washed with 6 ml water/methanol/acetic acid (94/5/1 v/v/v) and the polyphenols were eluted from the cartridge material with 3 ml methanol/water/acetic acid (60/39/1 v/v/v). The Solid Phase Extraction (SPE) extract was freeze-dried and the residue was dissolved in 1 ml water/methanol/acetic acid (94/5/1 v/v/v), which was then extracted 3 times with 1 ml of ethylacetate. The ethylacetate extract was blown to dryness under a nitrogen gas stream. The dry residue was used for GC-MS analysis. A volume of 10 ml of urine, untreated and -glucuronidase treated, was acidified with 200 l acetic acid and extract 3 times with 10 ml ethylacetate. The ethylacetate extract was blown to dryness under a nitrogen gas stream. The residue was dissolved in 1 ml water/methanol/acetic acid (94/5/1 v/v/v) and the solution was applied to a C-18 cartridge (Waters Sep-Pak), which was prepared as described above. The cartridge was then washed with 6 ml water/methanol/acetic acid (94/5/1 v/v/v) and the polyphenols were eluted from the cartridge material with 3 ml methanol/water/acetic acid (60/39/1 v/v/v). The SPE extract was freeze-dried and the residue was used for GC-MS analysis. RESULTS Urine samples were collected on 12 h cycles during 6 d of the study, starting with a flavonoid-free sample prior to the 5 d feeding period, and analyzed by HPLC pre- and post- -glucuronidase treatment. A representative HPLC chromatogram of a urine sample post- glucuronidase treatment (day sample during the feeding period) is shown in Fig. 2, with elution profiles of components detected at 225, 260, 280, and 320 nm. The identity of the phenolic acids in urine post- glucuronidase treatment was confirmed by GC-MS analysis. The following compounds (Fig. 3) were identified directly by comparing the mass spectra and retention time with standard data (shown in Table 1): ferulic acid, isoferulic acid, sinapic acid, homovanillic acid, 3-hydroxyphenylacetic acid, hippuric acid, and vanillic acid as well as two different hydroxyhippuric acids with identical spectra but different retention times (Fig. 3). The signals for sinapic acid and isoferulic acid were superposed by noise signals (Fig. 3). However, using the AMDIS-software (Automated Mass Spectral Deconvolution and Identification System, National Institute of Standard and Technology, Gaithersburg, MD, USA), it has been possible to identify those compounds. The proposed hydroxyhippuric acids might be assumed to be 3-hydroxyhippuric acid and 4-hydroxyhippuric acid. All identified metabolites were detected at low levels in basal urine samples at the end of the wash-out period prior to consumption (Table 2). The flavonoids naringenin and quercetin; the hydroxycinnamates ferulic, sinapic, and isoferulic acid; and the phenolic acid 3-hydroxyphenylacetic acid were identified as urinary metabolites post -glucuronidase treatment [mean total amounts excreted ranging from 1.02 mg (for quercetin) to mg (for 3-hydroxyphenylacetic acid)] and appeared as new peaks suggesting their presence as glucuronides after ingestion of a polyphenol-rich meal. Further urinary metabolites (in untreated and treated samples) such as vanillic [mean total amounts excreted mg] and homovanillic acid [mean total amounts excreted mg], both predominantly from enzyme treatment, as well as 3-hydroxyhippuric [mean total amounts excreted mg], 4-hydroxyhippuric [mean total amounts excreted mg] and hippuric acid [mean total amounts excreted mg] were identified as urinary metabolites associated with the high intake of polyphenols, showing an increased excretion after ingestion of a polyphenol-rich meal. The excreted amounts of ferulic, isoferulic, sinapic, vanillic, homovanillic, 3-hydroxyhippuric, 4-hydroxyhippuric and hippuric acid were roughly doubled during a 12 h cycle during the period of polyphenol-rich meal ingestion (Table 2). The observed increase in the excretion of hippuric acids, which totals approximately mg/d, exceeded in quantitative terms all conjugates and other metabolites together ( 20 mg/d) by far and pointed towards the final metabolic fate of most polyphenols. The excretion of naringenin, quercetin, and the later appearing 3-hydroxyphenylacetic acid reached sustained levels during the food supplementation period. Figure 4 shows the 12 h cycling of excreted polyphenols metabolites as a function of time over the 5 d of consumption. The time point of meal ingestion is indicated for clarity. As indicated, all metabolites were excreted in higher amounts in the urine samples of the 12 h cycle including the meal ingestion. The greatest differences between day and night sample were observed for naringenin, ferulic, and isoferulic acid indicating the faster absorption, metabolism, and elimination of these compounds. In contrast, the excretion profile of the colonic metabolite 3-hydroxyphenylacetic acid was delayed implying a prolonged pathway of formation, absorption, metabolism and elimination. Plasma samples were drawn prior to the feeding period, prior to the supplementation and 1, 3, and 5 h post-supplementation on the fifth day of the feeding

6 Metabolism of dietary polyphenols 225 Fig. 2. Representative chromatogram of urine after -glucuronidase treatment (day sample during the period of polyphenol-rich meal ingestion) at 225, 260, 280, and 320 nm. (1) 4-hydroxyhippuric acid [Retention time (RT): 13.7 min], (2) 3-hydroxyhippuric acid [RT: 15.3 min], (3) hippuric acid [RT: 20.9 min], (4) 3-hydroxyphenylacetic acid [RT: 25.6 min], (5) vanillic acid [RT: 26.7 min], (6) homovanillic acid [RT: 28.2 min], (7) naringenin [RT: 54.0 min], (8) ferulic acid [RT: 36.9 min], (9) isoferulic acid [RT: 38.9 min], (10) quercetin [51.8 min]. period. HPLC analysis of a representative plasma extract (post- -glucuronidase treatment) at 280, 295, 320, and 370 nm and the associated UV-spectra of identified compounds are shown in Fig. 5. Two flavonoid conjugates deriving from ingested flavonoids were identified in plasma. In addition, three hydroxycinnamic acid conjugates and three phenolic acid metabolites originating or deriving from the polyphenols ingested were tentatively assigned. These assignments, pre- and post- -glucuronidase treatment, were confirmed by GC-MS analysis. Separation and mass spectra confirmed the presence of 3-hydroxyphenylpropionic acid and dihydroferulic acid (Fig. 6a), whereas post-glucuronidase treatment, the following compounds were identified using GC-MS: 3-hydroxyphenylpropionic acid, vanillic acid, ferulic acid, and isoferulic acid and dihydroferulic acid (Figs. 6a and 6b). p-coumaric acid provided a very weak signal and could not be identified unequivocally. In plasma untreated with -glucuronidase only the phenolic acids 3-(3-methoxy-4-hydroxyphenyl)-propionic acid (dihydroferulic acid) and 3-(3-hydroxyphenyl)- propionic acid were detected after ingestion of the polyphenol-rich meal (Figs. 5 and 6). Following -glucuronidase treatment the flavonoids naringenin and quercetin and the hydroxycinnamates ferulic, isoferulic and p-coumaric acids and the hydroxybenzoic acid

7 226 A. R. RECHNER et al. Fig. 3. Mass spectra of the TBDMS derivates of ferulic acid, isoferulic acid, sinapic acid, 3-hydroxyphenylacetic acid, homovanillic acid, vanillic acid and hydroxyhippuric acid, hippuric acid as detected in urine. The signals of sinapic acid and isoferulic acid were overlaid by other compounds but could be identified using AMDIS. The magnified face m/z indicate the major signals for the respective compound detected in urine. The compounds were identified by comparing the mass spectra with the spectra of the respective standards (not shown). The respective molecular weights and major fragments are listed in Table 1. vanillic acid were detected in plasma implying an in vivo presence as glucuronides (Figs. 5 and 6). The quantification of plasma metabolites and conjugates as a concentration-time profile is represented in Fig. 6. The conjugated compounds, quantified as free flavonoids and phenolic acids following -glucuronidase treatment, showed a maximum plasma concentration between 1 and 3 h after ingestion of the polyphenol-rich meal, whereas the two other identified metabolites, dihydroferulic and 3-(3-hydroxyphenyl)-propionic acid, were peaking between 3 and 5 h (Fig. 7). The mean concentrations of each of the eight identified metabolites (post- -glucuronidase treatment) and the range of concentrations found are summarized in Table 3. Great individual differences in plasma concentrations and detected metabolites were apparent among the 20 volun-

8 Table 2. Mean Amounts and Ranges (n 20) of the Identified Metabolites of Ingested Polyphenols in 12 h Urine (after -glucuronidase Treatment) During the Study Naringenin Quercetin Ferulic acid Isoferulic acid Sinapic acid Vanillic acid Homovanillic acid 3-hydroxyphenyl-acetic acid 3-hydroxyhippuric acid a 4-hydroxyhippuric acid b Hippuric acid h (night) 0.02 (nd 0.30) 24 h (day) c 1.13 (nd 2.04) 36 h (night) 0.41 (nd 1.01) 48 h (day) 1.14 ( ) 60 h (night) 0.38 ( ) 72 h (day) c 1.11 ( ) 84 h (night) 0.45 ( ) 96 h (day) c 1.12 ( ) 108 h (night) 0.39 ( ) 120 h (day) c 1.41 ( ) 132 h (night) 0.32 ( ) 144 h (day) 0.05 (nd 0.35) Total 7.9 ( ) n.d. (n.d. 0.02) 0.07 (nd 0.26) 0.06 (nd 0.29) 0.09 (nd 0.34) 0.10 (nd 0.93) 0.10 (nd 0.34) 0.14 (nd 0.90) 0.13 (nd 0.40) 0.13 (nd 1.14) 0.13 (nd 0.28) 0.07 (nd 0.24) 0.01 (nd 0.07) 1.0 (nd 3.6) 0.21 ( ) 0.53 (nd 1.16) 0.27 (nd 0.99) 0.52 ( ) 0.26 ( ) 0.58 ( ) 0.38 ( ) 0.61 ( ) 0.30 ( ) 0.65 ( ) 0.33 ( ) 0.13 (nd 0.41) 4.8 ( ) 0.11 (n.d. 1.02) 0.25 (nd 0.88) 0.12 (nd 0.77) 0.25 ( ) 0.11 (nd 0.33) 0.25 ( ) 0.16 ( ) 0.32 ( ) 0.13 ( ) 0.28 ( ) 0.11 ( ) 0.05 (nd 0.26) 2.2 ( ) 0.10 (nd 0.73) 0.28 ( ) 0.07 (nd 0.37) 0.21 (nd 0.52) 0.17 (nd 0.89) 0.31 ( ) 0.10 ( ) 0.27 ( ) 0.10 ( ) 0.32 ( ) 0.16 ( ) 0.06 (nd 0.6) 2.1 ( ) 1.8 (nd 11.4) 1.4 (nd 4.4) 1.4 (nd 12.3) 1.8 ( ) 1.1 ( ) 1.4 ( ) 2.2 ( ) 2.8 ( ) 1.8 ( ) 2.2 ( ) 1.2 (nd 4.7) 0.6 (nd 7.2) 19.8 ( ) 1.2 (n.d. 2.8) 2.4 (nd 8.0) 2.1 (nd 6.9) 2.7 (nd 6.3) 2.3 (nd 9.4) 3.4 (nd 8.6) 2.2 (nd 6.1) 3.2 (nd 9.9) 2.3 (nd 7.3) 3.2 (nd 15.1) 2.7 (nd 9.9) 1.7 (nd 5.0) 29.2 (nd 68.1) 0.2 (n.d. 1.6) 0.7 (nd 1.9) 2.0 (nd 18.4) 4.8 (nd 13.3) 4.4 (nd 35.7) 7.1 (nd 15.8) 4.2 (nd 24.4) 7.5 (nd 14.0) 5.7 (nd 22.6) 7.0 (nd 16.6) 5.8 (nd 52.7) 5.9 (nd 32.9) 55.1 (nd 174.8) 6.5 ( ) 11.3 (nd 44.1) 11.4 (nd 52.8) 17.7 ( ) 12.8 ( ) 15.6 ( ) 13.1 ( ) 16.5 ( ) 12.4 ( ) 16.2 ( ) 16.7 ( ) 9.5 (nd 36.1) ( ) 4.1 ( ) 8.6 (nd 19.4) 7.8 (0 34.0) 8.2 ( ) 6.7 ( ) 9.0 ( ) 6.8 ( ) 10.3 ( ) 7.2 ( ) 10.4 ( ) 5.5 ( ) 4.5 (nd 24.5) 89.0 ( ) 249 (21 561) 307 (39 701) 285 (87 502) 391 (93 891) 277 ( ) 382 (34 664) 325 (82 700) 427 ( ) 315 ( ) 364 ( ) 377 ( ) (nd 361) 3895 ( ) a Quantified relative to 3-hydroxybenzoic acid. b Quantified relative to 4-hydroxybenzoic acid. c Ingestion of a polyphenol-rich meal. nd not detected.

9 228 A. R. RECHNER et al. Fig. 4. Time profile of the urinary excretion (mean amounts in mg, n 20) of 10 identified urinary metabolites after -glucuronidase treatment. The periods of polyphenol consumption are indicated by the gridlines. teers. The glucuronides of naringenin, quercetin, ferulic, isoferulic and vanillic acid as well as dihydroferulic and 3-(3-hydroxyphenyl)-propionic acid were detected in the majority of the 20 volunteers, whereas p-coumaric acid glucuronide was less prominent. The two identified hydroxyphenylpropionic acids reach on average approximately twice the maximum plasma concentration of the glucuronides of quercetin, naringenin, ferulic, isoferulic and vanillic acid, which achieved a maximum plasma concentration of nmol/l. The plasma levels of p-coumaric acid glucuronide appeared to be lower (more than an order of magnitude less). The conjugates of quercetin, ferulic acid and vanillic acid and the metabolites 3-(3-methoxy-4-hydroxyphenyl)-propionic acid (dihydroferulic acid), and 3-(3-hydroxyphenyl)-propionic acid were detected in a number of basal plasma samples. For both body fluids, great interindividual differences in the plasma concentration and amounts excreted in urine of the identified conjugates and metabolites were observed mirroring significant individual variation in absorption and metabolism of polyphenols. The analytical results reveal that the ingestion of a polyphenol-rich meal alters the profile of human plasma and urine significantly. New compounds were detected in both body fluids post-ingestion, each metabolized or conjugated to different extents, with glucuronidation, O-methylation and colonic degradation being the most prominent biotransformation identified. For each week of the study, the meal ingested on the blood sampling day was freeze-dried in order to determine the polyphenol intake. The methanolic extract of an aliquot of the homogenized meal was subjected to HPLC analysis, which revealed a highly complex polyphenol pattern. Identification was undertaken in terms of retention time of known standards, spiking with a standard of the suspected compound and photodiode array detection to confirm the assignments spectroscopically. Some major and minor peaks were identified including chlorogenic acid, cyanidin-3-glucoside, quercetin-3-rutinoside, quercetin-3-glucoside, naringenin, ( )-epicatechin. Other peaks, for which standards are commercially unavailable, were tentatively assigned as cyanidin-3-sophoroside, 3- and 4-caffeoylquinic acid, quercetin-3,4 -diglucoside, etc., or generally classified as a member of a specific phenolic family, hydroxycinnamate, anthocyanin or flavanol according to their UV-spectra. Those classified peaks were quantified in order to give an approximation of composition relative to the main polyphenol of its class, namely chlorogenic acid for the hydroxycinnamates, quercetin-3-glucoside for the flavonols, cyanidin-3-glucoside for the anthocyanins, ( )- epicatechin for the flavan-3-ols and naringenin for the flavanones. The resulting estimate for the polyphenol intake during the study is shown in Table 4. The majority of identified polyphenols ingested were flavonols ( mg/meal), hydroxycinnamates ( mg/meal) and anthocyanins ( mg/meal), while the levels ingested of flavan-3-ols ( mg/meal) and flavanones ( mg/meal) were relatively small. DISCUSSION The study clearly indicates the differential amounts of polyphenol-specific metabolites derived from colonic degradation versus intestinal absorption. The majority of identified components derived from dietary polyphenols are the colonic metabolites (average increase over the feeding period for the conjugates of 1.23 mg/12 h vs mg/12 h for the colonic metabolites). The glucuronides of naringenin and quercetin, as detected in plasma as well as in urine, were the only conjugates of intact flavonoids detectable at this level of dietary supplementation. With respect to the approximate dose of quercetin and naringenin glycosides ingested, the levels and their variability found in plasma and urine and the pharmacokinetic behavior are consistent with the reported observations of others [30 32,36 38]. Glucuronides of hydroxycinnamic acids were detected in plasma, namely p-coumaric, ferulic and isof-

10 Metabolism of dietary polyphenols 229 Fig. 5. Representative chromatogram [20 60 min] of an extract of plasma after -glucuronidase treatment (3 h postingestion sample) at 280, 295, 320 and 370 nm (1) vanillic acid [RT: 28.6 min], (2) dihydroferulic acid [RT: 35.1 min], (3) 3-(3-hydroxyphenyl)-propionic acid [RT: 36.4 min], (4) naringenin [RT: 55.3 min], (5) p-coumaric acid [RT: 36.0 min], (6) ferulic acid [RT: 37.8 min], (7) isoferulic acid [RT: 39.5 min], (8) quercetin [RT: 52.4 min].

11 230 A. R. RECHNER et al. Fig. 6. Spectra of the TBDMS-derivatized compounds from plasma (a.) and -glucuronidase treated plasma (a. b.). Magnified face numbers m/z indicate the major signals of the respective compounds, as the spectra are overlaid by noise. erulic acids, and in urine, namely ferulic and isoferulic acid. The hydroxycinnamic acids p-coumaric and ferulic acid are conjugated after or during absorption of the free acids following the cleavage of the natural esters, which most likely occurs in the colon [12]. Ferulic acid may derive from dietary sources per se or from biotransformation of caffeic acid derived from caffeic acid esters after cleavage from quinic acid or glucose. This process could also result in the formation of isoferulic acid, a nondietary hydroxycinnamate, which was detected in a previous study as a biomarker of the intake of chlorogenic acids from consumption of coffee [10]. The colonic microflora might be responsible for the O-methylation of hydroxycinnamic acids to yield the identified metabolites [41]. Alternatively caffeic acid itself maybe absorbed, after cleavage of the quinic acid from chlorogenic acids, and subsequently be O-methylated in the liver. A proposal for the conjugation and metabolism of

12 Metabolism of dietary polyphenols 231 Fig. 7. Time course of the plasma levels (mean levels, n 18) of the eight identified metabolites, after -glucuronidase treatment, following the consumption of a polyphenol-rich meal. the detected flavonoids and hydroxycinnamic acids is summarized in Fig. 8. Dihydroferulic acid (3-(3-methoxy-4-hydroxyphenyl)-propionic acid) detected in plasma has been reported as a urinary metabolite of flavonols [5,35], such as quercetin, and of hydroxycinnamates [10,42], such as caffeic and ferulic acid derivatives. It has also been proposed as precursor of vanillic acid [10], which was detected in plasma and urine. Vanillic acid has been shown from animal studies to derive from the colonic degradation of flavan-3-ols and flavonols [5]. The differences in conjugation and concentration-time profiles in plasma suggest a different metabolic pathway for the formation of dihydroferulic and 3-(3-hydroxyphenyl)-propionic acid. 3-(3- Hydroxyphenyl)-propionic acid is a known metabolite of the colonic degradation of flavonols, flavan-3-ols, flavanones (hesperetin) and hydroxycinnamates [4,5]. It is also been proposed as the potential precursor of 3-hydroxyhippuric acid [8], which was also detected in urine and showed an association with the high polyphenol intake. In contrast to the other identified metabolites, dihydroferulic and 3-(3-hydroxyphenyl)-propionic acid appeared unconjugated in plasma accumulating to maximal plasma concentrations at a later time point supporting the notion of formation via degradation and metabolism of flavonoids and hydroxycinnamates in the colon. Both hydroxyphenylacetic acids have been identified as metabolites of quercetin and isorhamnetin derivatives in studies in animals ingesting pure compounds, implying degradation by the colonic microflora [5,8,34,35]. The delayed urinary elimination of 3-hydroxyphenylacetic acid during the 5 d period of polyphenol-rich meal ingestion in this study also suggests a prolonged metabolism in humans involving the colonic microflora. The increase in the amounts of homovanillic acid excreted (also a well-known metabolite of DOPA [43]) can also be associated with the polyphenol-rich diet due to its derivatization from the degradation of flavonols and other flavonoids. The association between a high polyphenol intake during the period of polyphenol-rich meal ingestion and the excretion of the urinary metabolites hippuric, 3- hydroxyhippuric and 4-hydroxyhippuric acids points to- Table 3. Mean Plasma Levels (nmol/l) and Ranges of the Identified Metabolites of Ingested Polyphenols (Post -glucuronidase treatment) During the Study (n 18) Naringenin Quercetin Ferulic acid Isoferulic acid p-coumaric acid Vanillic acid Dihydroferulic acid 3-(3-hydroxyphenyl)- propionic acid Basal (pre-study) nd 0.4 (nd 7) 0 h (pre-meal) nd 12.5 (nd 72) 1 h (23 121) (nd 202) 3 h (15 116) (nd 317) 5 h (nd 75) (nd 366) 0.3 (nd 5) 1.3 (nd 18) 62.4 (22 130) 66.9 (27 106) 35.0 (16 64) nd nd 3.8 (nd 30) 0.1 nd 2.6 (nd 3) (nd 19) (14 77) (nd 7) (20 123) (15 113) (nd 12) (30 111) (9 38) (nd 6) (9 71) 4.5 (nd 58) 25.7 (nd 161) 31.8 (nd 114) (nd 579) (nd 572) 30.7 (nd 150) 58.0 (nd 306) 42.9 (nd 276) (nd 380) (nd 362) nd not detected.

13 232 A. R. RECHNER et al. Table 4. Estimated Intake (in mg) of Polyphenols with One Polyphenol-rich Meal During the Study (4 Weeks of 5 Volunteers Each) Total Flavan-3-ols (as catechin) Total Flavonols (as quercetin-3- glucoside) Total Flavanones (as naringenin) Total Anthocyanins (as cyanidin- 3-glucoside) Total Hydroxycinnamates (as chlorogenic acid) Total Polyphenol Intake Meal week Meal week Meal week Meal week Mean SEM wards the colon as a major catabolic site for dietary polyphenols. Other researchers detected similar associations following a high polyphenol dietary intervention with hippuric acid derived from tea, coffee, or plant polyphenols [10,15,42,44] as well as with 3-hydroxyhippuric acid derived from coffee hydroxycinnamates [10]. The proposed formation of the hippuric acids is shown in Fig. 9. The hypothesis that these urinary metabolites derive from biotransformation of dietary polyphenols in the colon to 3-(3- or 4-hydroxyphenyl)-propionic and 3-phenylpropionic acid followed -oxidation and glycination in the liver Fig. 8. Schematic representation of the proposed metabolism of naringenin, flavonols, and hydroxycinnamic acids [4,5,8,10].

14 Metabolism of dietary polyphenols 233 Fig. 9. Schematic representation of the proposed formation of hippuric acids from polyphenols [4,5,8,10,15,42,44]. prior to their excretion is supported by the detection of 3-(3-hydroxyphenyl)-propionic acid, the proposed precursor of 3-hydroxyhippuric acid, in plasma. Low basal levels of ferulic and isoferulic acid as well as vanillic acid were detectable in urine presupplementation In a small number of volunteers low levels of the metabolites quercetin glucuronide, vanillic acid glucuronide, ferulic acid glucuronide, dihydroferulic acid and 3-(3-hydroxyphenyl)-propionic acid were detectable in the basal plasma level indicating a long half life in the human body or another dietary source (flour product such as bread, pastry, etc., or flavoring with vanillin) or metabolic origin. The formation of phenolic degradation products and conjugated polyphenols will considerably influence the bioactivities and properties in vivo compared to their unconjugated forms [45]. In addition, the low levels detected, combined with the predisposition of conjugates to rapid elimination, question the function and modes of action of polyphenols in the human body. The urgent issue to be addressed is whether the identified conjugates and metabolites are physiologically relevant or can only be regarded as biomarkers of a polyphenol-rich diet. Acknowledgements The authors would like to thank the Food Standards Agency UK (project no. N04017) for financial support and Dr. Louise Bourne for her participation in the organization of the human intervention study. The authors also acknowledge the Biotechnology and Biological Sciences Research Council for a JREI grant for mass spectrometry facilities (18/JE514264) and the European Union for collaborative research funding (QLKT ). REFERENCES [1] Rice-Evans, C. A.; Packer, L., eds. Flavonoids in health and disease. New York: Marcel Dekker Inc.; [2] Kumpulainen, J. T.; Salonen, J. T., eds. Natural antioxidants and food quality in atherosclerosis and cancer prevention. Cambridge: The Royal Society of Chemistry; [3] Ho, C. T.; Lee, C. V.; Huang, M. T., eds. Phenolic compounds in

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