Metabolites and tissue distribution of resveratrol in the pig

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Azorín-Ortuño, M., Yáñez-Gascón, M.J., Vallejo, F., Pallarés, F.J., Larrosa, M., Lucas, R., Morales, J.C., Tomás-Barberán, F.A., García-Conesa, M.T. and Espín, J.C. (2011), Metabolites and tissue distribution of resveratrol in the pig. Mol. Nutr. Food Res., 55: 1154-1168. , which has been published in final form at https://doi.org/10.1002/mnfr.201100140 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 Metabolites and tissue distribution of resveratrol in the pig María Azorín-Ortuño1, María J. Yáñez-Gascón1, Fernando Vallejo1, Francisco J. Pallarés2, Mar Larrosa1, Ricardo Lucas3, Juan C. Morales3, Francisco A. Tomás-Barberán1, María T. García-Conesa1, and Juan Carlos Espín1* 1Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain. 2Department of Anatomy and Compared Pathology, School of Veterinary, University of Murcia, 30100 Campus de Espinardo, Murcia, Spain. 3Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas (IIQ), CSIC-Universidad de Sevilla, 41092 Sevilla, Spain. *Correspondence: Juan Carlos Espín, Ph.D., Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain. E-mail: [email protected]; Tel: +34-968-396344; Fax: +34-968- 396213. List of Abbreviations: A, Aorta tissue; B, Bile; Br, Brain; CeC and CeT, Cecum content and tissue, respectively; Cr, Cerebellum; CSF, Cerebrospinal fluid; DC and DT, Duodenum content and tissue; DCC and DCT, Distal colon content and tissue; DH-RES, α,β-dihydroresveratrol (dihydroresveratrol); H, Heart; IC and IT, Ileum content and tissue; J1C and J1T, Jejunum-1 content and tissue; J2C and J2T, Jejunum-2 content and tissue; J3C and J3T, Jejunum-3 content and tissue; JP, Jugular vein plasma; K, Kidneys; LgD, Longissimus dorsi muscle; Ln, Lungs; Lv, Liver; L, Lymph; LDL, Low density lipoprotein; LYN, Superficial inguinal lymphatic node; MF, Mesenteric fat; Ov, Ovaries; PCC and PCT, Proximal colon content and tissue; PF, Perirenal fat; Pn, Pancreas; PP, Portal vein plasma; QqQ, UHPLC-triple quadrupole; RBC, Red blood cells; RC and RT, Rectum content and tissue; RES, transresveratrol (3,5,4’-trihydroxy-trans-stilbene; resveratrol); SF, Subcutaneous fat; SM, Semimembranosus muscle; Sp, Spleen; StC and StT, Stomach content and tissue; U, Urine; UBl, Urinary bladder tissue; Ut, Uterus; WBC, White Blood Cells. Keywords: Bioavailability / disposition / metabolites / polyphenol / dihydroresveratrol 2 Abstract Scope: trans-Resveratrol (RES) and(or) its metabolites exert many in vivo effects. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. Methods and results: Forty-seven tissues, organs and fluids were analyzed at 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. Conclusion: This study contributes to clarify the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects. 3 1 Introduction The natural occurring polyphenol resveratrol (3,5,4´-trihydroxy-trans-stilbene; RES) has been reported to exert many different health-promoting effects including antioxidant, anti-inflammatory, antitumor, anti-platelet aggregation, cardioprotective and aging-delay effects [1]. However, despite the reported beneficial effects, RES is poorly bioavailable as it is rapidly absorbed, metabolized and excreted in humans and different animal models [2-7]. Therefore, this apparent paradox (high activity but low bioavailability) involves that the real bioactive RES metabolites as well as the mechanisms underlying RES properties are not yet fully understood [1, 8]. Most studies regarding RES metabolism have focused on the analysis of RES and derived metabolites in plasma, urine or feces. However, there is a general lack of studies looking at the distribution of RES in organs, fluids or tissues upon oral or intravenous administration [9, 10]. RES tissue distribution has only been reported so far in rodents [11-16, amongst others] and one study in rabbits [17]. It is known that the pig gastrointestinal system and the associated gut microbiota are closer to the human digestive system than that of rodents [18, 19]. Despite research on pigs require large and rather expensive facilities, its use has been reported to be a valuable tool when investigating the metabolism of nutrients as well as phenolic compounds such as phenolic acids [20], flavan-3-ols [21], anthocyanins [22, 23], ellagitannins [24] and quercetin [25, 26]. Recently, we have reported the pharmacokinetics of RES in pigs [7]. In the pig, the metabolic profile of RES in plasma revealed the presence of RES diglucuronide, two isomers of RES sulfoglucuronide, two isomers of RES glucuronide, RES sulfate, and RES, being RES-3-O-glucuronide the most abundant plasma metabolite. Taking into account the lack of studies regarding the tissue distribution of RES in big mammals, physiologically closer to humans than rodents, the aim of this study was to explore extensively the metabolism and tissue distribution of RES in the pig. The results presented here could shed some light on the apparent ‘RES paradox’ (high activity but low bioavailability) since we show that RES and its derived metabolites are widely distributed in different pig tissues and biological fluids, many of them, not previously reported. In addition, the metabolic profile of RES in different organs, tissues and fluids, including some new RES and DH-RES metabolites, are reported here for the first time. 4 2 Materials and Methods 2.1 Chemicals trans-Resveratrol (resveratrol, RES, 3,5,4’-trihydroxy-trans-stilbene, >99% purity) and the internal standard quercetin (>98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Organic solvents such as methanol (MeOH), acetone, acetonitrile, etc. were obtained from Merck (Darmstadt, Germany). Milli-Q system (Millipore Corp., USA) ultra pure water was used throughout this experiment. Tiletamine-zolazepam (Zoletil 50) was purchased from Virbac España S.A. (Esplugues de Llobregat, Spain) and sodium pentobarbital (Dolethal) from Vétoquinol (Alcobendas, Madrid, Spain). 2.2 Synthesis of resveratrol metabolites trans-Resveratrol-3-O-β-D-glucuronide (RES glucuronide, >95% purity) was prepared from RES following a reported synthesis [27]. α,β-Dihydroresveratrol (DH-RES, >95% purity) was synthesized as described earlier by catalytic hydrogenation of RES [28]). Finally, the metabolite α,β-dihydroresveratrol- 3-O-β-D-glucuronide (DH-RES glucuronide) was prepared by hydrogenation of the same protected resveratrol 3-O-β-D-glucurunopyranosyl intermediate used in the preparation of resveratrol-3-O-β-D- glucuronide (3, 4’-O-tert-butyl-5-O-(2,3,4-tri-O-acetyl-β-D-glucurunopyranosyl) resveratrol). Final deprotection to obtain DH-RES glucuronide was carried out in two steps, silyl deprotection with tetrabutylammonium fluoride in tetrahydrofuran and acetyl cleavage with sodium carbonate in a methanol-water mixture. Purity was >95%. dH (300 MHz, D2O) 6.96 (d, J = 8.0Hz, 2 H, Harom), 6.68 (d, J = 8.1 Hz, 2 H, Harom), 6.44, 6.40, 6.31 (3s, 3 H, Harom), 4.80 (d, J = 7.0 Hz, 1 H, H-1), 3.74 (m, 1 H, H-5), 3.52 (m, 3 H, H-3, H-4, H-2), 2.75 (s, 4 H, 2x CH2Ar); d13C (75 MHz, D2O) 176.6 (C=O), 160.2, 159.2, 156.4, 145.5, 134.0, 130.5, 116.1, 110.9, 109.5, 102.9, 102.5 (C-1), 77.7, 76.5, 74.6, 73.5 (C-2, C- 3, C-4, C-5), 39.5, 37.9 (CH2Ar). ESI-MS (ES-): Calcd. for C20H22O9 (M-H) 405.1, found: 405.0. All reactions were monitored by TLC on precoated Silica-Gel 60 plates F254 (Merck), and detected by heating with Mostain (500 mL of 10% H2SO4, 25g of (NH4)6Mo7O24•4H2O, 1g Ce(SO4)2•4H2O). Products were purified by flash chromatography with Merck Silica gel 60 (200-400 mesh). High resolution FAB (+) mass spectral analyses was obtained on a Micromass AutoSpec-Q spectrometer 5 (Micromass, Manchester, UK). NMR spectra were recorded on either a Bruker Avance 300 or ARX 400 or Bruker Avance DRX 500 MHz [300 or 400 MHz (1H), 75 or 100 (13C) (Bruker Biospin GmbH, Rheinstetten, Germany), at room temperature for solutions in CDCl3, D2O or CD3OD. Chemical shifts are referred to the solvent signal. Further NMR experiments (COSY, TOCSY, ROESY, and HMQC) were done when necessary to assign the compound. Data were processed using manufacturer software, raw data were multiplied by shifted exponential window function prior to Fourier transform, and the baseline was corrected using polynomial fitting. 2.3 Animals and study design All experiments were in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Council 86/609/EC). Experimental design, included in the Spanish National Research Project BFU2007-60576, was approved by the Ethics Committee of the University of Murcia (Murcia, Spain) and by the Bioethics Committee-CSIC (Madrid, Spain). Five female pigs (cross-breed 25% Landrace x 25% Large White x 50% Duroc) were provided by the Veterinary Teaching Farm of the University of Murcia (sanitary registry number 302315). Housing and animal interventions were carried out in the same Teaching Farm. The pigs (80 ± 8 kg and 7 months old) were fasted overnight with free access to water. Animals were tranquilized with tiletamine-zolazepam (3.125 mg/kg body weight) to allow animal handling during intragastric RES administration. The dose of RES was prepared as previously described and adjusted to 6.25 mg/kg body weight (500 mg for an 80 kg animal). To determine the actual final dose, the amount of RES remaining in the probe after intragastric administration was evaluated [7]. One animal was used as control and received only water. All animal interventions were carried out at the same hour in the morning. Six hours after RES administration, the pigs were sacrificed using an overdose of sodium pentobarbital and complete necropsies were carried out. All organs, the content of hollow organs as well as different body fluids were weighted and measured. The following samples were obtained: tissue and content from stomach, duodenum, jejunum (3 segments), ileum, cecum, proximal colon, distal colon and rectum, plasma from jugular and portal veins, urine, lymph, bile, cerebrospinal fluid, superficial inguinal lymphatic node, brain, cerebellum, heart, liver, kidney, lung, spleen, pancreas, uterus, urinary bladder, 6 ovary, three adipose (perirenal, mesenteric and subcutaneous) and two skeletal muscle tissue types (Longissimus dorsi and Semimembranosus), erythrocytes, white blood cells and serum LDL particles. The organs were thoroughly rinsed with PBS to avoid external blood contamination. Blood samples were collected just before euthanasia from the jugular vein and immediately after the sacrifice from the portal vein in BD Vacutainer lithium heparin tubes (BD, Franklin Lakes, NJ). To separate the plasma, the collected blood samples were immediately centrifuged at 3000g for 10 min at 4 ºC in a Sigma 1-13 microcentrifuge (Braun Biotech. Int., Melsungen, Germany). The plasma samples were immediately frozen at -80 ºC for further analyses. Erythrocytes were isolated from pig blood (4 mL) according to Nakamura et al. [29]. White blood cells (WBC) were isolated from pig heparinized blood (3 mL). Blood was diluted 1:1 with PBS and processed by density gradient centrifugation with Histopaque-1077 and Histopaque-1119 (Sigma- Aldrich, Madrid, Spain) according to the manufacturer instructions. Urine, bile, lymph and cerebrospinal fluid were collected by puncture, with sterile syringes and needles, from the urinary bladder, gallbladder, the cisterna chyli and the subarachnoid space through the atlantooccipital articulation, respectively. After collection, all fluid samples were placed into sterile containers and frozen at -80 ºC for further analyses. LDL isolation from 4 mL of pig serum was carried out according to the protocol of Vieira et al. [30] but using 68,000 rpm for 90 min at 4 ºC in a Beckman 70.1 Ti rotor. 2.4 Sample preparation Plasma (300 µL), lymph (150 µL), cerebrospinal fluid (200 µL) and bile (100 µL) samples were extracted using acetonitrile and processed according to Azorín-Ortuño et al. [7]. The supernatant was evaporated under vacuum in a SpeedVac Concentrator Savant SPD121P (Thermo Scientific, Alcobendas, Spain). The pellet was re-dissolved in 150 µL methanol. Methanolic samples were diluted 1:1 (v:v) with acidified (0.2% formic acid) ultrapure Milli-Q water (Millipore Corp., Bedford, MA), filtered through a 0.45 µm membrane filter Millex-HV13 (Millipore Corp.) and an aliquot was analyzed by HPLC. Whole blood (300 µL) was processed according to Biasutto et al. [31]. 7 Urine samples were diluted 5-fold with ultrapure water, filtered through the Millex filter and injected into the HPLC equipments. The content (1 g) of the stomach, duodenum, jejunum (3 segments), ileum, cecum, proximal colon, distal colon and rectum were processed according to Espín et al. [24]. The skeletal muscle tissues (Longissimus dorsi and Semimembranosus), erythrocytes, WBC as well as the organs (tissues from the above parts of the gastrointestinal tract, superficial inguinal lymphatic node, brain, cerebellum, heart, liver, kidneys, lungs, spleen, pancreas, uterus, urinary bladder, aorta and ovaries) were processed (0.5 g) according to Azorín-Ortuño et al. [32]. Adipose (perirenal, mesenteric and subcutaneous) tissues (1 g) were processed following the protocol of González et al. [33]. LDL particles (700 µL) with a mean protein content of 0.9 mg/mL protein were extracted according to de la Torre-Carbot et al. [34]. In all the cases, sample manipulation was performed avoiding the direct light exposure to prevent the possible photochemical isomerization of trans-resveratrol to the cisform. The internal standard quercetin (20 µM) was routinely used in all the samples. 2.5 LC-MS/MS analyses The identification of RES and derived metabolites was achieved by HPLC-DAD-ESI-MS/MS, UHPLC- triple quadrupole (QqQ) MS detection and HPLC-Q-TOF. The LC-DAD-ESI ion trap system was a 1100 series HPLC-DAD device (Agilent Technologies, Waldbronn, Germany) equipped with an ion-trap mass spectrometer (Agilent). The heated capillary and voltage were maintained at 350 ºC and 4 kV, respectively. Mass scan (MS) and MS/MS daughter spectra were measured from m/z 100 up to 800 m/z. Collision-induced fragmentation experiments were performed in the ion trap using helium as collision gas, and the collision energy was set at 50%. Mass spectrometry data were acquired in the negative ionization mode. Chromatographic separations were achieved on a 250 x 4 mm i.d., 5 µm, C18 Mediterranea Sea column (Teknokroma, Barcelona, Spain) using water:formic acid (99:1, v:v) (A) and acetonitrile (B) as the mobile phases at a flow rate of 1 mL/min. The gradient started with 5% B in A to reach 55% B at 30 min, 90% B at 31 min for 5 min and 8 returning to the initial conditions (5% B). For quantifying DH-RES and derived metabolites in UV, the conditions were the same but using a 250 x 4.6 mm i.d., 3 µm, C18 100 Å Phenomenex column (Phenomenex®, Torrance, CA, USA). The samples were diluted in water (1:1) and different sample volumes (from 50 to 80 µL, depending on the sample) were injected. The UHPLC-MS triple quadrupole system (QqQ) consisted of a 1290 Infinity HPLC series (Agilent) equipped with a 6460 series triple quadrupole mass spectrometer (Agilent). Chromatographic separations were carried out at room temperature on a 100 x 3 mm i.d., 2.7 µm, C18 Poroshell 120 column (Agilent) using water:formic acid (99.9:0.1, v:v) (A) and acetonitrile:formic acid (99.9:0.1, v:v) (B) as the mobile phases at a flow rate of 0.6 mL/min. The gradient started with 15% B in A to reach 50% at 15 min, 90% at 18 min for 5 min and returning to the initial conditions (15% B). The injected sample volume was 10 µL. The optimum mass spectrometer parameters for detection of the available metabolites (DH-RES, RES and their corresponding glucuronides) were optimized connecting directly the column inlet to the Jet Stream source. The source parameters were the following: capillary voltage -3500 V, charging potential -500 V, nebulizer pressure 40 (psi), auxiliary gas heated to 275 °C at a flow rate of 9000 cm3/min. MS data were collected in multiple reaction monitoring (MRM) mode by monitoring specific transitions of parent and product ions for each metabolite: RES triglucuronide 755/227, RES diglucuronide 579/227, RES glucuronide 403/227, RES sulfoglucuronide 483/227, RES trisulfate 467/227, RES disulfate 387/227, RES sulfate 307/227, RES 227/185, DH-RES triglucuronide 757/229, DH-RES diglucuronide 581/229, DH-RES glucuronide 405/229, DH-RES sulfoglucuronide 485/229, DH-RES trisulfate 469/229, DH-RES disulfate 389/229, DH-RES sulfate 309/229 and DH-RES 229/187. The Q-TOF equipment consisted of a HPLC-DAD-MS system (1200 series, Agilent) equipped with a UHD Accurate-Mass Q-TOF (6540A series, Agilent). The column, flow rate, injected volume and gradient conditions were the same as those described above for the UHPLC-QqQ equipment. The optimum mass spectrometer parameters for detection of both RES and DHR-RES and their corresponding 3-O-glucuronides (at 80 µM) were optimized, connecting directly the column inlet to the Jet Stream source. The source parameters were the following: capillary voltage -3500 V, charging potential -500 V, nebulizer pressure 40 (psi), auxiliary gas heated to 275 °C and a flow rate of 9000 cm3/min. The parameters of the mass spectrometer were similar to those optimized in the UHPLC-QqQ 15 A number of studies dealing with the bioavailabiliy and metabolism of the molecule trans-resveratrol (17, RES) in humans and in rodents suggest that RES has a low bioavailability as it is rapidly metabolized and excreted, mainly via urine [5, 9, 10, 36]. These studies report both inter- and intraspecies differences together with a high variability in the metabolic profiles which may be caused by different variables such as gender, age, polymorphisms (phase I and II enzymes, transporters, etc.), different administration procedures, sample processing, analytical protocols, type of drugs and protocols used in anesthesia, etc as recently discussed [7]. The main RES-derived metabolites identified so far in humans and rodents are the sulfates, disulfates, trisulfates, glucuronides, diglucuronides and sulfoglucuronides derivatives as well as the microbiota-derived DH-RES and its corresponding sulfate and glucuronide conjugates [2, 6, 9, 10, 14, 16, 36-38]. The recovery of RES metabolites in previous studies, after oral RES administration, was very variable [36]. This depended on the dose and route of administration, the analytical approach, the time of analysis post-administration as well as the organs and fluids finally analyzed. The highest recoveries reported (higher than 95%) were attained by measuring total radioactivity in both urine and feces [10, 36]. Besides this amount of information, there is a lack of studies looking at RES and RES metabolites presence and specific distribution in most organs and tissues and thus the aim of the present study was to explore more in depth the metabolism of intragastrically administered RES in the pig. A complete study regarding the tissue distribution of RES metabolites, at different time-points, after RES administration is not common although achievable in rodents. Recently, a detailed study on the gastrointestinal or transit kinetics of RES in the GIT at different time-points after RES administration, has been reported in mice [39]. However, given the size and characteristics of the pig as an animal model and the high-cost facilities required for the experimentation with this animal, it is reasonable to choose only one postingestion time. Our approach included a systematic analysis at a single time-point post-ingestion using different equipments. We finally chose 6 h as post-ingestion time analysis to increase the chances of finding microbiota-derived metabolites (DH-RES), based on previous studies carried out in the pig with other polyphenols [20] as well as on the data obtained for RES in our previous mice study [39]. Under this approach, we focused on the identification of possible new in vivo reservoirs for RES or derived metabolites. This could cover, at least partially, the gap regarding the lack of studies of metabolism and 16 tissue distribution of RES in animals (such as the pig) physiologically much closer to humans than rodents. Bearing in mind all the above, we admit that this is a specific picture of RES and derived metabolites distribution in the pig 6 h after a single intragastric RES administration and therefore, the distribution as well as the type and amount of metabolites could differ at other time-points or upon chronic consumption. Most of the metabolites identified in the present study are in agreement with those previously published in the literature. However, we have identified a number of new RES and DH-RES-derived metabolites, i.e. the metabolites 3 (DH-RES diglucuronide), 4 (DH-RES sulfoglucuronide), 9 (RES ribosyl-sulfate) and 16 (DH-RES disulfate) (Table 1). Regarding the compound 9, the occurrence of ribosyl conjugates, as another mechanism of xenobiotic metabolism in mammals, has been reported in several occasions on N-containing xenobiotics, and the N-ribosyl derivatives were tentatively described. In some cases, combination of this ribosyl substitution with the addition of hydroxyl-sulphate has also been reported [40]. Our data suggest that this minor metabolite is a ribosyl derivative of RES by conjugation in one of the phenolic hydroxyls, and in addition, there is a sulphate conjugation on another phenolic hydroxyl. Therefore, this is the first time that pentosyl (most probably ribosyl) conjugates of phenolics, RES in this case, are reported in nature. The RES-microbiota derived metabolite dihydroresveratrol (18, DH-RES) and its conjugates have been largely underestimated. In 2004, Walle et al. [4] already highlighted the possible importance of these microbial compounds in the RES metabolism. This underestimation has been due to the use of UV detection at 305 nm or 320 nm (maxima of absorbance for RES spectrum) whereas the maximum UV for DH-RES is 276 nm, a bell-shaped spectrum with very low absorption at either 305 or 320 nm [10, 41]. In addition, the molar absorptivity of DH-RES-like metabolites is significantly lower than that of RES-like metabolites which hampers their detection. In our study, the levels of DH-RES and its conjugates in the pig GIT from stomach to ileum were very low although a number of DH-RES metabolites (8, 12, 14, 18) were detected (Table 2). In contrast, RES metabolites were very abundant, specially in the ileum (Table 2). It is also noticeable the presence of a relatively high concentration of RES (17) in the stomach content 6 h after RES administation as well as the occurrence of cis-RES (19) and conjugates from both RES (5, 7, 13) and, to a lesser extent, from 17 DH-RES (12, 14). The presence of RES conjugates in the stomach could be related to the previously reported ability of gastric cells to metabolize phenolics, including RES [42]. However, the presence of a minor amount of microbial DH-RES metabolites in the stomach content cannot be easily explained. A tentative explanation could be the presence of a small amount of microbial groups from stomach to ileum involved in the formation of DH-RES from RES. The abundance of RES conjugates in the pig ileum could be due to the biliar secretion of the enterohepatic circulation together with an active efflux of conjugates to the lumen content (Table 2) which has been reported in previous studies [4, 43]. However, the RES profile changed from the ileum (with abudant conjugates such as sulfoglucuronides, glucuronides and sulfates) to cecum (mainly aglycones and higher amount of DH-RES than RES) (Table 3, Figure 2). Although the distribution of the microbiota along the GIT involved in the conversion from RES (17) to DH-RES (18) is not known yet, the above changes could be explained by the significant increase in the cecum of the microbial groups involved in the production of DH-RES. Moreover, the lower presence of conjugates in the cecum and colon could be due to a decrease in the efflux of metabolites to the lumen caused by a lower amount of the RES transporters BCRP and MRP2, as previously reported in the rat [44]. The expression of these transporters has been reported to be maximal in the ileum [45]. Finally, another reason to explain the change in the metabolic profile from ileum to cecum could be the substantial increase in the glucuronidase and sulfatase activities associated to the large intestine microbiota [45]. The predominant presence of DH-RES in the pig large intestine is in agreement with Alfaras et al. [46] who identified DH-RES as the most abundant metabolite in the colon rat upon oral RES administration. This is specially relevant considering the colon cancer chemopreventive activities attributed to RES. Recently, the distribution of RES and some of its conjugates has been reported in colorectal tissue of patients that consumed eight daily doses of resveratrol [47]. Interestingly, RES and some conjugates (glucuronides, sulfates and sulfoglucuronide) were detected in that study. However, the possible occurrence of DH-RES and derived conjugates was not explored. Therefore, the presence of DH-RES and derived metabolites cannot be discarded in the colorrectal tissue of the patients enrolled in the above study, which could add to the controverted issue of the identification of the actual molecules responsible for the biological activity attributed to RES. 18 In the present pig study, the occurrence of metabolites in the bloodstream was low at this postingestion time (Table 4), in accordance with previous pharmacokinetic studies in both humans [6] and pigs [7]. The portal vein mainly drains blood from the GIT to capillary beds in the liver. Accordingly, the amount of RES metabolites in plasma from the portal vein (PP) approximately doubled the amount of metabolites present in jugular vein plasma (JP). In addition, the relative contribution of RES (17) to the metabolic profile was also more relevant in the case of PP than in JP (Table 4). A rich metabolic profile with high concentration of RES metabolites was found in the pig bile (B) which reinforced the active enterohepatic circulation of RES (Table 4). In contrast, the presence of DHRES metabolites in bile was much lower (Table 4) which could be due either to a low enterohepatic circulation or to the post-ingestion time analysis (6 h), probably not enough for producing DH-RES metabolites. Both possibilities could explain the low amount of DH-RES metabolites detected in the content of the GIT from stomach to ileum after 6 h of intragastric RES administration (Table 2). Interestingly, we also report here for the first time the metabolic profile of RES in the lymph which approximately matched that of plasma (Table 4). Although the lymphatic system plays an important role in the absorption of dietary lipophilic compounds, it is not usually investigated in metabolic studies with polyphenols, which many of them are amphiphilic molecules. Our results are in agreement with those reported by Terao and Murota [48] that detected quercetin metabolites mostly in the rat lymph after intragastric administration of quercetin. The authors also supported their results by the ability of the stomach to metabolize quercetin [42, 48], which was in accordance with our study with the presence of RES metabolites in the stomach. On the contrary, Chen et al. [49] mainly detected unmetabolized quercetin and rutin in the lymph upon intraduodenally administration of these flavonoids. The conclusions drawn by these authors have been previously questioned due to the methodological approach followed in that study [50]. As expected, the presence of RES and DH-RES metabolites in systemic organs and tissues was very small and amounted up to 0.5% of the initial dose of RES administered (Table 5; Figure 1). RES and derived metabolites were already extensively distributed in the pig body 6 h after RES administation with the highest amounts detected in the kidneys (K) and the liver (Lv). The main compound detected in pig brain was RES (17) in accordance to previous studies on rats [15]. This is of interest in relation to the 19 modulation of several blood flow variables in humans 45 min after RES (500 mg) intake [51] and indicates that RES itself could be the bioactive molecule in brain. Although the RES amount found in the pig brain was very low (0.18 µg/g; 22 µg in the entire brain), the detection of higher amounts cannot be discarded at shorter post-ingestion times. The occurrence of RES and derived metabolites (including DH-RES and its conjugates) in perirenal, mesenteric and subcutaneous fats (PF, MF and SF, respectively) as well as in skeletal muscle such as Longissimus dorsi (LgD) and Semimembranosus (SM) muscles (Table 5) is important due to the increasing evidence of the link between RES intake and fat mobilization [52] as well as the attenuation of sarcopenia [53]. The most abundant ciculating RES metabolites have been reported to be RES 3-O-sulfate in humans [6] and RES 3-O-glucuronide in pigs [7]. This is noticeable taking into account the physiological similarities between both species. In the present study, RES 3-O-glucuronide (compound 10), was the most abundant metabolite in plasma form jugular and portal veins (Table 4) which confirmed the previous report [7]. In addition, compound 10 was also the main metabolite in systemic organs and urine (Tables 4 and 5). In contrast, RES sulfate (compound 13) was the most abundant metabolite in the lumen of the pig GIT, especially in the ileum, followed by RES sulfoglucuronide isomer-2 (compound 6). A possible explanation could lie in the pig ATP-binding cassette transporters, involved in the disposal of resveratrol [54], which could be especiallly efficient in the efflux of sulfate conjugates from the enterocyte to the GIT lumen. This would decrease the absorption and systemic distribution of RES sulfates as well as to increase their accumulation in the pig intestine, in agreement with the results found in our study. In summary, we report here a comprehensive study regarding the metabolism and tissue distribution of RES in the pig, an animal physiologically close to humans. This is the first report regarding the occurrence of RES and DH-RES metabolites in aorta tissue (A), urinary bladder tissue (UBl), cerebellum (Cr), ovaries (Ov), uterus (Ut), pancreas (Pn), fat (PF, MF and SF) and muscle (LgD and SM). In addition, new RES and DH-RES metabolites are also reported here for the first time. It should be noted that the specific RES and DH-RES metabolites distribution described here could be different at other post-ingestion time-points. For example, shorter post-ingestion time analyses such as 1 h (approximately 20 coincident with plasma Tmax) could yield lower DH-RES metabolites (if any), but possibly a higher RES metabolites detection in systemic organs. The research for reaching this challenging goal is warranted. The information included in this study may be very useful to move forward in the knowledge about trans-resveratrol bioactivity and beneficial health effects as well as in the understanding of the mechanisms underlying its properties. 21 Acknowledgements This work has been supported by the Projects CICYT-BFU2007-60576, Fundación Seneca de la Region de Murcia (grupo de excelencia GERM 06, 04486) and Consolider Ingenio 2010, CSD2007-00063 (Fun- C-Food). Authors are grateful to Dr. Alberto Quiles and Dr. Cándido Gutiérrez from the Veterinary School (Univ. Murcia, Spain) and José Luis Pineda from Proquimur S.L. (Murcia, Spain) for their assistance in some parts of the study. M.A.O. is holder of a predoctoral JAE grant from CSIC and M.L. and R.L. of a JAE-DOC contract from CSIC (Spain). Conflict of interest statement All authors declare no conflict of interests. 22 REFERENCES [1] Baur, J. A., Sinclair, D. A. Therapeutic potential or resveratrol: the in vivo evidence. Nature Rev. 2006, 5, 493-506. [2] Marier, J. F., Vachon, P., Gritsas, A., Zhang, J. et al., Metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linkedrat model. J. Pharmacol. Exp. Ther. 2002, 302, 369-373. [3] Goldberg, D. M., Yan, J., Soleas, G. J. Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clin. Biochem. 2003, 36, 79-87. [4] Walle, T., Hsieh, F., DeLegge, M. H., Oatis, J. E., Jr., Walle, K. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab. Dispos. 2004, 32, 1377-1382. [5] Wenzel, E., Somoza, V. Metabolism and bioavailability of trans-resveratrol. Mol. Nutr. Food Res. 2005, 49, 472-481. [6] Boocock, D. J., Faust, G. E., Patel, K. R., Schinas, A. M. et al., Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer Epidemiol. Biomarkers Prev. 2007, 16, 1246-52. [7] Azorín-Ortuño, M., Yáñez-Gascón, M. J., Pallarés, F. J., Larrosa, M. et al., Pharmacokinetic study of trans-resveratrol in adult pigs. J. Agric. Food Chem. 2010, 58, 11165-11171 [8] Gesher, A. J., Steward, W. P. Relationship between mechanisms, bioavailability and preclinical chemopreventive efficacy of resveratrol: a conundrum. Cancer Epidemiol. Biomarkers Prev. 2003, 12, 953-957. [9] Andrés-Lacueva, C., Urpí-Sardá, M., Zamora-Ros, R., Lamuela-Raventós, R. M., in: Fraga, C G. (Ed.), Plant phenolics and human health: biochemistry, nutrition and pharmacology, John Wiley & Sons, Hoboken 2009, pp. 265-299. [10] Walle, T. Bioavailability of resveratrol. Ann. N. Y. Acad. Sci. 2011, 1215, 9-15. [11] Bertelli, A. A., Giovannini, L., Stradi, R., Bertelli, A., Tillement, J. P. Plasma, urine, and tissue levels of trans- and cis-resveratrol (3,4’,5-trihydroxystilbene) after short-term or prolonged administration of red wine to rats. Int. J. Tissue React. 1996, 18, 67-71. 23 [12] Vitrac, X., Desmouliére, A., Brouillaud, B., Krisa, S. et al., Distribution of [14C]-trans-resveratrol, a cancer chemopreventive polyphenol, in mouse tissues after oral administration. Life Sci. 2003, 72, 2219-2233. [13] Sale, S., Verschoyle, R. D., Boocock, D., Jones, D. J. et al., Pharmacokinetics in mice and growthinhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4'-tetramethoxystilbene. Br. J. Cancer 2004, 90, 736-44. [14] Wenzel, E., Soldo, T., Erbersdobler, H., Somoza, V. Bioactivity and metabolism of transresveratrol orally administered to Wistar rats. Mol. Nutr. Food Res. 2005, 49, 482-494. [15] El-Mohsen, M., Bayele, H., Kuhnle, G., Gibson, G. et al., Distribution of [3H]-trans-resveratrol in rat tissues following oral administration. Br. J. Nutr. 2006, 96, 62-70. [16] Juan, M. E., Maijó, M., Planas, J. M. Quantification of trans-resveratrol and its metabolites in rat plasma and tissues by HPLC. J. Pharm. Biomed. Anal. 2010, 51, 391-398. [17] Asensi, M., Medina, I., Ortega, A., Carretero, J. et al., Inhibition of cancer growth by resveratrol is related to its low bioavailability. Free Radic. Biol. Med. 2002, 33, 387-398. [18] Guilloteau, P., Zabielski, R., Hammon, H. M., Metges, C. C. Nutritional programming of gastrointestinal tract development. Is the pig a good model for human? Nutr. Res. Rev. 2010, 23, 4- 22. [19] Hein, E. M., Rose, K., van’t Slot, G., Friedrich, A. W., Humpf, H.-U. Deconjugation and degradation of flavonols glycosides by pig cecal microbiota characterized by fluorescence in situ hybridization (FISH). J. Agric. Food Chem. 2008, 56, 2281-2290. [20] Wu, X., Pittman III, H. E., Hager, T., Hager, A. et al., Phenolic acids in black raspberry and in the gastrointestinal tract of pigs following ingestion of black raspberry. Mol. Nutr. Food Res. 2009, 53, S76-S84. [21] van’t Slot, G., Humpf, H.-U. Degradation and metabolism of catechin, epigallocatechin-3-gallate (EGCG), and related compounds by the intestinal microbiota in the pig cecum model. J. Agric. Food Chem. 2009, 57, 8041-8048. 24 [22] Wu, X., Pittman, H. E., Prior, R. L. Fate of anthocyanins and antioxidant capacity in contents of the gastrointestinal tract of weanling pigs following black raspberry consumption. J. Agric. Food Chem. 2006, 54, 583-589. [23] Milbury, P. E., Kalt, W. Xenobiotic metabolism and berry flavonoid transport across the bloodbrain barrier. J. Agric. Food Chem. 2010, 58, 3950-3956. [24] Espín, J. C., González-Barrio, B., Cerdá, B., López-Bote, C. et al., The Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans. J. Agric. Food Chem. 2007, 55, 10476–10485. [25] De Boer, V. C. J., Dihal, A. A., van der Woude, H., Arts, I. C. W. et al., Tissue distribution of quercetin in rats and pigs. J. Nutr. 2005, 135, 1718-1725. [26] Bieger, J., Cermak, R., Blank, R., de Boer, V. C. J. et al., Tissue distribution of quercetin in pigs after long-term dietary supplementation. J. Nutr. 2008, 138, 1417-1420. [27] Lucas, R., Alcantara, D., Morales, J. C. A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol and hydroxytyrosol. Carbohydrate Res. 2009, 344, 1340-1346. [28] Stivala, L. A., Savio, M., Carafoli, F., Perucca, P. et al., Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J. Biol. Chem. 2001, 276, 22586–22594. [29] Nakamura, T., Bratton, D. L., Murphy, R. C. Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry. J. Mass Spectrom. 1997, 32, 888-896. [30] Vieira, O. V., Laranjinha, J. A., Madeira, V. M., Almeida, L. M. Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants. J. Lipid Res. 1996, 37, 2715-2721. [31] Biasutto, L., Marotta, E., Garbisa, S., Zoratti, M., Paradisi, C. Determination of quercetin and resveratrol in whole blood-implications for bioavailability studies. Molecules 2010, 15, 6570- 6579. 31 Table 3. Total amount (mg) of RES and derived metabolites in the gastrointestinal tract (GIT) from cecum to rectum 6 h after intragastric RES administration. Values (quantified using UV detection) are expressed as total mg (mean ± SE) considering the total weight (g) of each lumen content and tissue. In QqQ, when not quantified, the relative abundance of each metabolite was scored as high (+++); medium (++) and low (+) (these values compare the abundance for the same metabolite in the different tissues but not among different metabolites); ND, not detected in UV. 10 RES-3-O-glucuronide; 12 DH-RES glucuronide; 13 RES sulfate; 14 DH-RES sulfate; 17 RES; 18 DH-RES; 19 cis-RES; n = 4 except in an = 1, bn = 2 and cn = 3. Lumen Content Tissue GIT part (continued) (g) 17 18 QqQ 10 13 17 18 QqQ Cecum (Ce) WC: 342.5±102.8 WT: 175±25 14.06±4.75 26.40±11.81 13 (+) 14 (++) 19 (+) 0.02±0.01 0.05±0.02 0.68±0.31 0.64±0.06 12 (0.007)a 14 (+) 19 (+) Proximal colon (PC) WC: 445±105 WT: 330±10 6.42±5.96c 13.55±8.87c 13 (++) 14 (++) 19 (++) ND 0.063±0.008 0.54±0.27 1.52±0.34 10 (0.02)a 12 (0.008)a 14 (++) 19 (+) Distal colon (DC) WC: 165±65 WT: 310±90 0.79±0.02b 1.92±0.97b 13 (+) 14 (+) 19 (+) ND ND 0.06±0.01 1.12a 10 (0.015)a 12 (++) 13 (++) 14 (+) 19 (+) Rectum (R) WC: 40±20 WT: 85±15 0.26a 2.67a 13 (+) 14 (+) 19 (+) ND ND 0.01±0.004 0.52a 13 (+) 14 (+) Total metabolites (lumen): 66.07±32.4 Total metabolites (tissues): 5.29±1.05 32 Table 4. Concentration of RES and derived metabolites in different fluids, LDL and blood cells of pigs 6 h after intragastric RES administration. Fluids 1 2 5 6 7 9 10 13 17 QqQ Urine (U) (1172±262) mL Det 2.36±0.12 (2.79±0.53) 0.41±0.02 (0.49±0.09) 3.06±0.53 (3.72±1.01) Det Det 21.38±3.62 (24.11±0.97) 2.99±1.25 (3.83±1.59) Det 3 (+); 4 (+); 8 (++); 12 (0.85)a; 14 (+++) 15 (++); 16 (++) 18 (0.52)a; 19 (++) Bile (B) (42.0±9.5) mL Det 9.60±2.60 (0.40±0.11) 9.21±4.00 (0.39±0.17) 33.75±13.81 (1.42±0.58) 13.40±2.92 (0.56±0.12) 7.55±2.44 (0.32±0.10) 28.63±1.08 (1.20±0.05) 26.70±9.53 (1.12±0.40) 2.22±0.22 (0.10±0.01) 4 (+); 8 (++) 12 (0.10)a; 14 (+++); 15 (++); 16 (++) Portal vein plasma (PP) ND ND ND ND ND ND 0.67±0.27 1.67±0.67 µM 0.17±0.04 0.56±0.13 µM 0.11±0.03 0.47±0.12 µM 7 (++); 12 (0.44; 1.1 µM)a; 14 (++) Jugular vein plasma (JP) ND Detb Detb ND ND ND 0.28±0.05 0.69±0.13 µM 0.08±0.02 0.24±0.05 µM ND 7 (+); 12 (0.09; 0.22 µM)a; 14 (+) 17 (0.012; 0.05 µM) 19 (+) Lymph (L) ND ND ND ND ND ND 0.10±0.02 0.25±0.05 µM 0.05±0.002b, Deta 0.17±0.01 µMb 0.024, Deta 0.1 µM 12 (0.21)a; 14 (+) 19 (+) Cerebrospinal fluid (CSF) ND ND ND ND ND ND ND ND ND 13 (+); 17 (+) 19 (+) LDL (ng/mL) ND ND ND ND ND ND Det ND Det 10 (+)a; 12 (+) 17 (++); 19 (+) Red Blood Cells (RBC) (ng/mL) ND ND ND ND ND ND ND ND ND 13 (+); 14 (+) 17 (+); 19 (+) White Blood Cells (WBC) (ng/mL) ND ND ND ND ND ND ND ND Det 13 (+); 17 (++) 19 (+) Total metabolites (mg): 36.33±5.94 (urine) and 5.60±1.54 (bile) Values (quantified using UV detection) are expressed as the mean ± SE µg/mL. In PP, JP and L, the values are also expressed as µM. LDL, RBC and WBC values are expressed as ng/mL. In the case of urine and bile, the total volume of the fluid was known and the values expressed between parenthesis are total mg (mean ± SE). In QqQ, when not quantified, the relative abundance of each metabolite was scored as high (+++); medium (++) and low (+) (these values compare the abundance for the same metabolite in the different fluids but not among different metabolites); Det, detected but not quantified; ND, not detected in UV. 1 RES diglucuronide-1; 2 RES diglucuronide-2; 5 RES sulfoglucuronide-1; 6 RES sulfoglucuronide-2; 7 RES trisulfate; 8 DH-RES trisulfate; 9 RES ribosyl sulfate; 10 RES-3-O-glucuronide; 12 DH-RES glucuronide; 13 RES sulfate; 14 DH-RES sulfate; 15 RES disulfate; 16 DH-RES disulfate; 17 RES; 18 DH-RES; 19 cis-RES; n = 4 except in an = 1, bn = 2 and cn = 3. 33 Table 5. RES and derived metabolites distribution in the different pig organs and tissues examined 6 h after intragastric RES administration. Organ and tissue Weight (g) 10 13 17 QqQ Kidneys (K) 293±26 0.610±0.207 0.167±0.095 0.020±0.014c 5+6 (+); 12 (0.014)a; 14 (+) Liver (Lv) 1603±26 0.516±0.215 0.365±0.145c 0.185±0.057c 4 (+); 5+6 (++); 7 (++) 12 (0.020)a; 14 (++), 19 (+) Lungs (Ln) 483±17 0.038 ± 0.006 0.026±0.004b, Deta 0.042±0.014b, Deta 7 (+); 12 (0.008)a; 14 (+) 19 (+) Cerebellum (Cr) 12±1 0.053 ND 0.001 5+6 (+); 7 (+); 12 (0.006) a 13 (++); 14 (+++); 19 (+) Heart (H) 420±37 0.020±0.004b, Detb 0.011 Detb 7 (+); 12 (0.006)a; 14 (+) Brain (Br) 118±5 ND ND 0.022, Deta 10 (+); 13 (+) Aorta Tissue (A) 38±4 0.019±0.003 0.006, Detb 0.002, Detb 7 (+); 12 (0.005)a; 14 (+) Ovaries (Ov) 40±5 0.008 ± 0.001 0.004±0.0003 0.001±0.0002c, Deta 5+6 (+); 7 (+); 12 (0.002)a 14 (+) Uterus (Ut) 88±4 0.009±0.001c 0.005, Deta 0.001, Deta − Urinary Bladder tissue (UBl) 60±10 0.012 Deta Deta 14 (++) Pancreas (Pn) 103±8 Detb Deta 0.005 4 (+); 5+6 (+); 10 (0.001)a Spleen (Sp) 122±14 Detc Detb Deta 10 (0.005)a; 12 (0.001)a 14 (+) Total metabolites (mg) = 2.21 ± 0.77 Lymph Node (LYN)* − 0.057, Deta Detb Detb 12 (0.07)a; 14 (++) Perirenal Fat (PF)* − Deta ND 0.089±0.022 10 (0.017)a; 12 (0.023)a 13 (++); 14 (++); 19 (+) Mesenteric Fat (MF)* − 0.025, Deta ND 0.015, Deta 12 (0.120)a; 13 (+); 14 (++) 19 (+) Subcutaneous Fat (SF)* − Detb ND 0.019, Deta 10 (0.020)a; 12 (0.030)a 13 (+) Longissimus dorsi (LgD)* − ND ND 0.02±0.003b 10 (0.01)a; 12 (+) 13 (++); 14 (++); 19 (++) Semimembranosus Muscle (SM)* − ND ND ND 13 (+); 14 (+) Results (quantified using UV detection) are expressed as total mg taking into account the weight of the organ or tissue (mean±SE). *Results are expressed as µg/g (the total weight was not determined). Det., detected but not quantified. ND, not detected in UV. In QqQ, when not quantified, the relative abundance of each metabolite was scored as high (+++); medium (++) and low (+) (these values only compare the abundance for the same metabolite in the different organs or tissues but not among different metabolites). 4 DH-RES sulfoglucuronide; 5 RES sulfoglucuronide-1; 6 RES sulfoglucuronide-2; 7 RES trisulfate; 10 RES-3-O-glucuronide; 12 DH-RES glucuronide; 13 RES sulfate; 14 DH-RES sulfate; 17 RES; 19 cis-RES; n=4 except in an=1, bn=2, cn=3.