The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Tissue Distribution of in Pigs after Long-Term Dietary Supplementation1,2

Juliane Bieger,3 Rainer Cermak,4*RalfBlank,3 VincentC.J.deBoer,5 PeterC.H.Hollman,5 Joseph Kamphues,6 and Siegfried Wolffram3

3Institute of Animal Nutrition and Physiology, University of Kiel, 24098 Kiel, Germany; 4Institute of Veterinary Physiology, University of Leipzig, 04103 Leipzig, Germany; 5RIKILT-Institute of Food Safety, Wageningen University and Research Centre, 6708 Wageningen, The Netherlands; and 6Institute of Animal Nutrition, University of Veterinary Medicine Hannover, Foundation,

30173 Hannover, Germany Downloaded from https://academic.oup.com/jn/article/138/8/1417/4750791 by guest on 29 September 2021

Abstract Although the flavonol quercetin is intensively investigated, our knowledge about its bioavailability and possible target organs is far from being complete. The aim of this study was to check the potential of quercetin to accumulate in various tissues after long-term dietary treatment compared with a single treatment with flavonol. Pigs ingested either a single dose of quercetin aglycone (25 mg/kg body weight; Expt. 1) or received the flavonol twice a day at the same dose mixed into their regular meals (i.e 50 mgkg21d21) for 4 wk (Expt. 2). In both experiments, we took plasma and tissue samples 90 min after the final meal and analyzed them using HPLC. Additionally, the specific activity of the enzyme b-glucuronidase was measured in selected tissues. Higher flavonol concentrations than in plasma were found in only the liver (Expt. 1) or the intestinal wall and kidneys (Expt. 2). All tissues except blood plasma contained a variable amount of deconjugated quercetin in the range of 30–100% of total flavonols. However, the specific b-glucuronidase activity was not correlated with the proportions of deconjugated flavonols in the various tissues. Long-term dietary intake of the flavonol did not lead to a greater accumulation in any tissue compared with the single treatment. Flavonol concentrations only exceeded the plasma concentration within organs involved in its metabolism and excretion, including liver, small intestine, and kidneys. J. Nutr. 138: 1417–1420, 2008.

Introduction flavonoids) (5). Conjugated flavonols may have different bio- such as the flavonol quercetin are polyphenols that logical activities compared with the aglycones (6–8). are present as secondary metabolites in plants and occur in most In the same study, data from 3 pigs that had received a plant-derived foodstuffs at various concentrations (1). Several quercetin-containing diet (500 mgkg21d21) for 3 d were also studies have shown a correlation between the consumption of presented (5). Tissue and plasma flavonol concentrations were polyphenol-rich foods and the prevention of chronic diseases, much lower compared with the rats with a similar daily quer- such as coronary heart disease (2–4). Knowledge of the dis- cetin intake (1% diet). However, the length of the feeding period tribution of flavonoids within the body is crucial for a proper was much shorter in the pig experiment. Moreover, pigs were understanding of their biological effects. deprived of food for 8 h before slaughtering, whereas the rats In a previous study, we described the tissue distribution in were killed in a postprandial state (5). Thus, it remains unclear rats of quercetin and its 2 methylated derivatives, whether a longer feeding period leads to a considerably higher and , after long-term feeding of 2 diets containing tissue accumulation of flavonols in pigs, which are considered a either 0.1 or 1% quercetin (;50 or 500 mg/kg body weight, much better model for humans than rats (9). respectively) (5). The highest tissue concentrations of quercetin, Therefore, one aim of the present study was to investigate isorhamnetin, and tamarixetin were detected in the lungs, testes, whether quercetin accumulates in pigs in a tissue-specific and kidneys. The lowest concentrations were found in the brain manner after chronic administration of the flavonol compared and white adipose tissue. Whereas blood plasma contained with a single quercetin dose. Furthermore, b-glucuronidase nearly exclusively conjugated flavonols, several tissues, includ- activity was determined in several tissues. Similar to humans and ing lung, liver, kidney, and testes, contained a rather high rats, blood plasma of pigs contains almost exclusively glucu- proportion of free quercetin and isorhamnetin (4–40% of total ronidated and sulfated metabolites after oral intake of quercetin (10,11). Therefore, any flavonol aglycones present in tissues most likely are liberated by deconjugating enzymes. The hydro- 1 Supported by grant WO 763/2-3 from the German Research Foundation. lysis of glucuronides, the main quercetin metabolites (12,13), is 2 Author disclosures: J. Bieger, R. Cermak, R. Blank, V. C. J. de Boer, P. C. H. Hollman, J. Kamphues, and S. Wolffram, no conflicts of interest. catalyzed by b-glucuronidase, an acid hydrolase expressed in * To whom correspondence should be addressed. E-mail: [email protected] many tissues and body fluids (14). We wanted to compare the leipzig.de. concentrations of quercetin aglycone and the specific b-glucu-

0022-3166/08 $8.00 ª 2008 American Society for Nutrition. 1417 Manuscript received 11 February 2008. Initial review completed 26 March 2008. Revision accepted 9 May 2008. ronidase activity in various body tissues and to determine was calculated by dividing the peak maximum of hemoglobin in tissues whether these variables are correlated. with the peak maximum of the respective full blood sample. Flavonol concentrations determined in the respective plasma sample were then multiplied with the fraction of residual blood in tissues and subtracted from the value obtained for the tissue (5). Materials and Methods Chemicals Assay for b-glucuronidase activity. According to a previously and bovine serum albumin were obtained from Carl Roth. published method (18), we determined activity of b-glucuronidase in b-Glucuronidase/sulfatase (crude enzyme extract from Helix pomatia) cell-free extracts prepared from liver, kidney, skeletal muscle (diaphragm was purchased from Sigma-Aldrich. Dye reagent concentrate for protein and longissimus dorsi muscle), and lung tissue obtained from 3 pigs of assay was obtained from Bio-Rad-Laboratories. Expt. 2 by measuring the release of p-nitrophenol from p-nitrophenol glucuronide (10 mmol/L) after a 5-min lag-phase during a period of Animals and diets 25 min at 37C at 400 nm (UV-1602, Shimadzu Europe). One activ- ity unit (U) is defined as the amount of enzyme releasing 1 nmol All experiments were performed with cross-bred male castrated pigs 21 obtained from the Institute of Animal Breeding of the University of Kiel. p-nitrophenolmin at 37C and pH 7.2. Experiments were performed in accordance with the German animal welfare law. Statistics Downloaded from https://academic.oup.com/jn/article/138/8/1417/4750791 by guest on 29 September 2021 Flavonol concentrations and tissue-specific activities of b-glucuronidase Expt. 1 (short-term feeding). Three pigs (body weight ;70 kg) were are presented as means 6 SEM. For data evaluation and analyses, we individually housed and adapted to a commercial diet for fattening pigs used GraphPad Prism 4 (GraphPad Software). Differences between mainly composed of wheat, barley, rye, and defatted soybean meal (diet concentrations of total flavonols (sum of quercetin, isorhamnetin, and no. 4300, Plambeck) containing (per kg diet) 165 g crude protein, 21 g tamarixetin from enzymatically treated samples) were evaluated using crude fat, 53 g crude fiber, 12.6 MJ metabolizable energy, and 897 g dry ANOVA with subsequent pairwise comparisons of group means with matter. Vitamins and minerals were supplemented according to the Dunnett’s multiple comparison test. Because plasma is considered as the recommendations of the German Society of Nutritional Physiology (15). central compartment from which distribution into tissues occurs, tissue Tap water was consumed ad libitum by nipple drinkers. On the concentrations were compared with plasma concentrations. The rela- experimental day, pigs consumed 1 meal consisting of the basal diet tionship between concentration of aglycone in organs and tissue specific supplemented with a dose of 25 mg quercetin/kg body weight. Then b-glucuronidase activity was investigated with Pearson correlation anal- 90 min later, pigs were killed and samples (various body tissues, blood) ysis. We used the Tukey-Kramer test for unbalanced data for analysis of were taken. differences between Expt. 1 and Expt. 2. The level of significance was set at P , 0.05. Expt. 2 (long-term feeding). Seven pigs (initial body weight 33–37 kg) were kept under similar conditions and fed restrictively (80% of voluntary feed intake) twice daily over a period of 4 wk with the same Results diet as in Expt. 1 (final body weight 48–56 kg). Quercetin aglycone was At 90 min after ingestion of a single quercetin-containing meal added to each meal at a dosage of 25 mg/kg body weight, resulting in a (Expt. 1), the total flavonol concentration in liver was higher total daily intake of 50 mg/kg. At the end of the feeding period, pigs were killed by exsanguination after anesthesia (ketamine and azaperone) 60– than in plasma (Table 1). In all samples analyzed, quercetin was 90 min after the last morning feeding. the main metabolite (Table 1). The proportions of aglycones were not determined in this experiment. 2 2 Experimental procedures After feeding a quercetin-containing diet (50 mgkg 1d 1) Preparation of samples. During exsanguination, blood samples (2 over a period of 4 wk (Expt. 2), colon, kidney, and jejunum times 10 mL) from each pig were immediately collected into heparinized contained higher total flavonol concentration than plasma (Table tubes. Tissue samples (;10 g each) were obtained from kidneys, liver, 2). Flavonol concentrations in all other investigated tissues, mid-jejunum, skeletal muscle (diaphragm and longissimus dorsi muscle), including liver, did not differ from plasma. Lungs, white adipose and lung tissue (Expt. 1 and 2) as well as from proximal colon, white tissue, muscles, and brain did not accumulate flavonols after the adipose tissue, mesentery, intestinal lymph nodes, and brain (only in 4-wk treatment. In all tissues, quercetin accounted for 67–98% of Expt. 2). Samples were immediately frozen on dry ice and stored at –70C until further preparation. Plasma was separated from 1 of the 2 blood samples and stored as described above. The 2nd blood sample and tissue samples were lyophilized, frozen in liquid nitrogen, and ground TABLE 1 Quercetin, isorhamnetin, and tamarixetin under constant cooling using a mill (Jahnke and Kunkel Analysenmu¨ hle, concentrations in tissues of pigs fed a single IKA Labortechnik) or a mortar. All tissue samples were weighed before quercetin dose (25 mg kg21) (Expt. 1)1,2 and after lyophilization and stored in airtight containers at 270C until analysis. Sum of Quercetin Isorhamnetin Tamarixetin flavonols Tissue extractions and HPLC analyses. Two aliquots of plasma (980 mL) or tissue [100 mg suspended in 980 mL of saline (150 mmol/L NaCl)] nmol/g wet tissue were spiked with 20 mL of internal standard (50 mg /L Liver 6.20 6 1.74 1.51 6 0.09 0.89 6 0.18 8.60 6 1.84* methanol) and acidified with acetic acid (0.583 mol/L) to a final pH of 5. Kidney 2.84 6 0.90 0.48 6 0.24 0.64 6 0.08 3.96 6 1.14 To determine the fraction of conjugated and free compounds, 1 of the 2 Jejunum 2.36 6 1.99 0.32 6 0.25 0.13 6 0.10 2.69 6 2.23 plasma and tissue samples were treated with b-glucuronidase/sulfatase. Lung 0.39 6 0.35 0.04 6 0.01 0.02 6 0.01 0.57 6 0.31 All samples were incubated for 60 min at 37C. Further treatment of Muscle (longissimus 0.11 6 0.005 0.01 6 0.002 n.d.3 0.14 6 0.003 samples and analysis by HPLC were performed according to previously dorsi) published methods (11,16,17). Plasma, mmol/L 1.48 6 0.16 0.06 6 0.03 0.08 6 0.006 1.63 6 0.15

Correction for residual blood. As previously described (5), hemoglo- 1 Values are means 6 SEM, n ¼ 3. *Different from plasma, P , 0.05. bin was determined in the supernatant of homogenized tissue and in full 2 All concentrations represent total concentrations of aglycones plus hydrolyzed blood samples with a spectrophotometer at a wave length of 540 nm conjugates and are corrected for residual blood. (UV-1602, Shimadzu Europe). The fraction of residual blood in tissues 3 n.d., Not detected.

1418 Bieger et al. TABLE 2 Quercetin, isorhamnetin, and tamarixetin concentrations in tissues of pigs fed 50 mg quercetin kg21d21 for 4 wk (Expt. 2)1,2

Quercetin Aglycone Isorhamnetin Aglycone Tamarixetin Aglycone Sum of flavonols

nmol/g wet tissue % nmol/g wet tissue % nmol/g wet tissue % nmol/g wet tissue Colon 13.92 6 4.16 100 0.14 6 0.08 100 0.14 6 0.05 100 14.20 6 4.18* Kidney 6.31 6 1.14 29 1.04 6 0.11 56 1.11 6 0.16 60 8.46 6 1.39* Jejunum 5.51 6 1.87 89 1.19 6 0.46 92 0.51 6 0.20 78 7.21 6 2.47* Liver 2.83 6 0.54 93 0.92 6 0.19 86 0.49 6 0.12 94 4.24 6 0.82 Lymph node 2.23 6 0.16 46 0.11 6 0.02 91 0.06 6 0.01 100 2.42 6 1.37 Mesentery 0.57 6 0.20 100 0.05 6 0.02 100 0.06 6 0.04 33 0.68 6 0.26 Lung 0.28 6 0.07 100 0.03 6 0.01 100 0.02 6 0.03 100 0.34 6 0.07 White adipose tissue 0.17 6 0.03 59 0.01 6 0.003 50 0.02 6 0.01 20 0.23 6 0.03 Muscle (diaphragm) 0.12 6 0.02 100 0.02 6 0.003 50 0.006 6 0.001 100 0.14 6 0.03 Muscle (longissimus dorsi) 0.09 6 0.007 44 0.01 6 0.002 30 0.004 6 0.003 75 0.10 6 0.01 Downloaded from https://academic.oup.com/jn/article/138/8/1417/4750791 by guest on 29 September 2021 Brain 0.02 6 0.01 100 0.001 6 0.001 n.d.3 n.d. n.d. 0.03 6 0.06 Plasma, mmol/L 0.67 6 0.11 ,1 0.05 6 0.01 8 0.06 6 0.01 7 0.81 6 0.12

1 Values are means 6 SEM, n ¼ 7. *Different from plasma, P , 0.05. 2 All concentrations represent total concentrations of aglycones plus hydrolyzed conjugates and are corrected for residual blood. 3 n.d., Not detected. total flavonols (Table 2). The difference in total flavonol In Expt. 1 and 2, plasma concentrations of total flavonols concentration between Expt. 1 and Expt. 2 was significant for never exceeded 2 mmol/L (Tables 1 and 2). At the time point of only liver with a higher value after a single quercetin-containing sampling (90 min after ingestion of the quercetin-enriched meal. meal), plasma concentrations in the range of peak levels can be All tissues investigated in Expt. 2 contained a variable expected (11,17). In comparison, the plasma levels in rats with fraction of free quercetin, isorhamnetin, and tamarixetin (Table a similar dietary intake of quercetin (0.1% quercetin diet, 2). Colon, mesentery, diaphragm, lungs, and brain contained corresponding to ;50 mgkg21 body weightd21) exceeded 20 only deconjugated quercetin (100%); in liver and jejunum, the mmol/L (5). Similarly, we observed the same discrepancy in fraction of free quercetin accounted for ;90%. The fraction of plasma concentrations between rats and pigs after a 10-fold free quercetin in the other tissues was between 30 and 60%. In higher intake of quercetin (;500 mgkg21 body weightd21)ina contrast, plasma contained hardly any nonconjugated quercetin. previous study (5). Thus, the differences in plasma concentra- Fractions of free and conjugated forms of isorhamnetin and tions between pigs and rats seem to be due to species differences tamarixetin roughly followed the distribution pattern of quer- and not to the different sampling points in the previous study. cetin (Table 2). Most interestingly, plasma concentrations after long-term Tissue-specific b-glucuronidase activity was determined in feeding (Expt. 2) were not higher than after intake of a single tissue samples obtained from lung, liver (high proportion of quercetin-containing meal (Expt. 1). Although comparisons aglycones), kidneys (low proportion of aglycones), and muscles between treatments should be interpreted with care, this finding from 3 pigs in Expt. 2. The variation between pigs was indicates that quercetin does not accumulate in plasma after considerable. b-Glucuronidase activity in kidneys, lungs, and dietary administration over several weeks. This is in accordance liver were (mean value, n ¼ 3): 144 6 31.2, 125 6 21.2, and with the short plasma half-life of quercetin of ;4 h in pigs (11), 96.9 6 11.7 U/mg protein, respectively. In the muscle samples, because the quercetin administrations in the long-term feeding specific activity was 38.6 6 7.3 U/mg protein (diaphragm) and experiments were separated by a period of ;3 times the elim- 33.9 6 4.6 U/mg protein (longissimus dorsi muscle), respec- ination half-life. tively. The proportion of aglycones was not correlated with the Liver (Expt. 1), kidneys, and intestinal tissues (Expt. 2) specific b-glucuronidase activity in the various tissues. contained higher flavonol levels than plasma. Although data from intestinal tissues have to be interpreted with caution due to a possible contamination with ingesta (despite intensive rinsing), Discussion the mucosa of the gut is centrally involved in the metabolism and excretion of quercetin (12,19). Similarly, the liver and kidneys After oral intake of quercetin or quercetin glycosides, quercetin are involved in flavonol metabolism and excretion. and its methylated metabolites isorhamnetin and tamarixetin Concentrations of flavonols in skeletal muscle were not appear in the circulation of rats and humans almost exclusively higher after long-term feeding in comparison to short-term in their conjugated form, i.e. as glucuronides and/or sulfates feeding. Thus, muscles did not accumulate quercetin even after (10). This was also shown for pigs (11). Even portal plasma did several weeks but rather appeared to reflect intake of quercetin not contain any detectable amounts of free quercetin after oral with the last meal. With regard to the discussion of any direct administration of the flavonol (17). Thus, quercetin is more or antioxidant effect of flavonols in meat, relevant for post mortem less completely conjugated during its absorption. In contrast storage and stability, the very low concentrations in muscle and to rats, in which nearly one-half of the absorbed amount of fat must be considered. Furthermore, the previously high fla- quercetin is methylated to isorhamnetin, the proportion of vonol concentrations in lungs of rats (5) was not confirmed in isorhamnetin in pig and human plasma is rather low (11,17). the present study with pigs. This was confirmed in the present study, where isorhamnetin Among the various organs investigated, the brain had the and tamarixetin represented ,20% of total plasma flavonols. lowest flavonol concentration. This is in agreement with other

Tissue distribution of quercetin in pigs 1419 studies and shows that the blood brain barrier efficiently limits 2. Middleton E, Kandaswami C, Theoharides TC. The effects of plant flavonol uptake into the central nervous system (5). flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000;52:673–751. The occurrence of flavonols in mesenterial lymph nodes as 3. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary found in Expt. 2 might suggest a possible transport of these antioxidant flavonoids and risk of coronary heart disease: the Zutphen substances via the lymph as previously discussed (20). Elderly Study. Lancet. 1993;342:1007–11. Except for blood plasma, all investigated organs contained a 4. Kris-Etherton PM, Lefevre M, Beecher GR, Gross MD, Keen CL, considerable proportion of deconjugated quercetin, ranging Etherton TD. 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Ausschuss fu¨r Bedarfsnormen der Gesellschaft fu¨rErna¨hrungsphysiologie. In conclusion, this study demonstrated that pigs did not (1987) Energie- und Na¨hrstoffbedarf Landwirtschaftlicher Nutztiere Nr. 4 accumulate quercetin in most tissues after a long-term and high- Schweine. DLG-Verlag, Frankfurt a.M., Germany. dose dietary intake of the flavonol. Only organs involved in 16. Hollman PCH, van Trijp JMP, Buysman MNCP. Fluorescence detection of flavonols in HPLC by postcolumn chelation with aluminium. Anal flavonol metabolism and excretion, including small intestine, Chem. 1996;68:3511–5. liver, and kidneys, contained significantly higher flavonol con- 17. Cermak R, Landgraf S, Wolffram S. The bioavailability of quercetin in centrations than plasma. Thus, the latter organs should be pigs depends on the glycoside moiety and on dietary factors. J Nutr. considered as primary targets of potential beneficial effects. The 2003;133:2802–7. different tissues contained variable amounts of deconjugated 18. O’Leary KA, Day AJ, Needs PW, Sly WS, OÕBrien NM, Williamson G. quercetin. However, the concentration of the aglycone in sev- glucuronides are substrates for human liver b-glucuronidase. FEBS Lett. 2001;503:103–6. eral organs did not correlate to the tissue-specific activity of 14 b 19. Ueno I, Nakano N, Hirono I. Metabolic fate of [ C]quercetin in the -glucuronidase, at least under the conditions of this study. ACI rat. Jpn J Exp Med. 1983;53:41–50. 20. Murota K, Terao J. Quercetin appears in the lymph of unanesthetized Acknowledgments rats as its phase II metabolites after administered into the stomach. FEBS Lett. 2005;579:5343–6. We thank Petra Schulz and Maike Ju¨ rgensen for their valuable 21. Gu L, House SE, Prior RL, Fang N, Ronis MJJ, Clarkson TB, Wilson ME, technical assistance. Badger TM. Metabolic phenotype of isoflavones differ among female rats, pigs, monkeys, and women. 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1420 Bieger et al.