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Institute of Animal Nutrition, Physiology and Metabolism, Christian-Albrechts-University Kiel, D-24098 Kiel, Germany
3To whom correspondence should be addressed. E-mail: cermak{at}aninut.uni-kiel.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSAnimals, diets, and experimental...RESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • flavonoids • bioavailability • quercetin • isoquercitrin • rutin
Flavonoids are a large group of natural polyphenols that are widely distributed in plants and are, therefore, ingested by humans and animals with their regular diet (1). They exert multiple effects on mammalian cells and tissues. Many flavonoids are well known for their antioxidative capabilities and are able to influence several key enzymes in vitro (2). On the basis of epidemiologic studies, a protective effect of plant flavonoids against coronary heart disease has been claimed (3–5). Among the many different flavonoids present in plants, the flavonol quercetin is relatively more abundant (Fig. 1). High concentrations of quercetin are present in tea, apples and onions (6–8) and estimated human daily intake ranges between 10 and 20 mg in Western populations (4,8,9). In plants and plant-derived foods, flavonoids are present largely as conjugates, with the flavonoid aglycone linked to variable sugar moieties by a ß-glycosidic bond (10).
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The aim of the present study was to compare the relative bioavailability of the monoglucoside isoquercitrin (Q3G) and of the glucorhamnoside rutin with that of the aglycone quercetin (Fig. 1). We chose pigs because their gastrointestinal system is similar to that of humans (16). In contrast to the above-mentioned studies, all test flavonoids were administered as part of the same diet to mimic physiologic conditions, but also to avoid possible influences of different food matrices at the same time. In addition, we wanted to study possible effects of the administered amount of flavonoids on their relative bioavailability by comparing the bioavailability of these flavonoids at two different concentrations. In the course of our study, we also wanted to determine the possible influences of food composition on the bioavailability of Q3G.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSAnimals, diets, and experimental...RESULTSDISCUSSIONLITERATURE CITED |
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Quercetin, isoquercitrin (Q3G), rutin, tamarixetin, isorhamnetin and rhamnetin were obtained from Roth, Karlsruhe, Germany; all compounds were of HPLC grade. ß-Glucuronidase/sulfatase (crude enzyme extract from Helix pomatia) was from Sigma-Aldrich AG, Deisenhofen, Germany.
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Animals, diets, and experimental procedures |
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TOPABSTRACTMATERIALS AND METHODSAnimals, diets, and experimental...RESULTSDISCUSSIONLITERATURE CITED |
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Growing pigs (cross-breed, Fa. Schaumann; Wahlstedt, Germany) with a body weight of 30–35 kg were used. Experiments 1 and 2 were performed with male castrated pigs; for Experiment 3, however, only female pigs were available. The pigs were restrictively fed (80% of feed intake consumed ad libitum) with a commercial pig diet based on ground barley, wheat and defatted soy bean meal twice daily. Pigs had free access to tap water.
Experiment 1.
Permanent double-lumen catheters (Cook Deutschland GmbH, Mönchengladbach, Germany) were surgically implanted into the portal and jugular veins of male pigs (n = 4). After 1 wk of postsurgical recovery, the pigs were administered a single dose of 148 µmol quercetin/kg body (equivalent to 50 mg/kg) mixed by hand into 200 g of diet. To achieve fast and complete intake of the test meal, the ground ration was moistened with water. Jugular and portal blood samples were collected at various intervals over a time period of 24 h. After another day ("washout phase"), the pigs were administered a second and third test meal with isomolar amounts of Q3G or rutin, respectively. A 1-d washout phase followed each single test.
Experiment 2.
Male pigs (n = 6) were equipped with a permanent single lumen catheter (Cook Deutschland GmbH, Mönchengladbach, Germany) into the jugular vein. After 1 wk of postsurgical recovery, these pigs received a single dose of 29.6 µmol quercetin/kg (equivalent to 10 mg/kg) mixed into 200 g of their diet (as in Experiment 1). Jugular blood samples were collected over a time period of 24 h. After another "wash out" day, the pigs were administered an isomolar amount of Q3G with their diet and jugular blood was collected again.
Experiment 3.
Female pigs (n = 3) were equipped with a permanent single lumen catheter into the jugular vein. After 1 wk of postsurgical recovery, these pigs were administered Q3G at a dose of 29.6 µmol/kg mixed into 200 g of the diet (as in Experiments 1 and 2). In a further test, the pigs were administered the Q3G mixed into 15 g of ground beef. No further food was offered for the next 5 h. Each of those treatments was repeated once to yield two observations per treatment and pig. Between each experimental procedure, there was a 1-d washout phase.
Diets.
The commercial standard diet was provided by Fa. Plambeck, Brügge, Germany. It contained (per kg diet) 162 g crude protein, 12 g crude fat, 122 g neutral detergent fiber, 16.3 MJ gross energy and 897 g dry matter. Vitamins and minerals were supplemented according to the recommendations of the German Society of Nutritional Physiology (17). The meat for the test meal in Experiment 3 was obtained from a local supermarket and contained (per kg diet) 205 g crude protein, 95 g crude fat, 0 g fiber, 8.4 MJ gross energy and 312 g dry matter.
Processing of blood samples and HPLC analysis.
Blood samples (8 mL) were drawn into heparinized containers and immediately centrifuged (1500 x g, 10 min, 4°C). Plasma was stored at -70°C until analysis. Further processing was performed as described previously (18). Aliquots (2 x 800 µL) of each plasma sample were spiked with 15 µL rhamnetin (internal standard, 10 mg/L methanol). One of the aliquots was treated with 5.8 x 106 U/L ß-glucuronidase/2.6 x 105 U/L sulfatase (crude enzyme extract from Helix pomatia) for cleavage of all ester-bonds of glucuronides/sulfates. HPLC analysis was performed according to the method described by Hollman et al. (19) with minor modifications. The column oven was set at 30°C. Calibration curves for quercetin, kaempferol, isorhamnetin and tamarixetin were obtained by the addition of these flavonols from methanolic stock solutions to flavonol-free pig plasma, with rhamnetin serving as an internal standard. All samples were corrected for contamination with quercetin and kaempferol by the crude enzyme extract.
Statistics.
Data are presented as mean values ± SEM. The area under the curve (AUC) was determined according to the trapezoidal rule. In Experiment 1, ANOVA with the Bonferroni post-test was used to compare AUC. In Experiments 2 and 3, the paired t test was used. A P-value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSAnimals, diets, and experimental...RESULTSDISCUSSIONLITERATURE CITED |
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0 after 24 h. Total bioavailability after administration of the glucoside was 50% higher than after administration of the aglycone (Table 1).
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In all of the above-mentioned experiments, portal blood samples were drawn in parallel to the jugular blood samples. However, due to clogging of portal catheters in some pigs, we obtained corresponding jugular and portal blood samples in only two pigs after administration of quercetin or Q3G. Thus, no statistical comparisons were performed due to the small number of observations. In these two pigs, plasma concentration of the main metabolite quercetin was always somewhat higher in the portal than in the jugular plasma. The respective relative proportions of quercetin, isorhamnetin and tamarixetin in portal plasma were comparable to those in the jugular plasma. No free quercetin, isorhamnetin or tamarixetin was detected in portal plasma samples before treatment with ß-glucuronidase/sulfatase (data not shown).
In Experiment 2, bioavailability from Q3G at the lower dose of 29.6 µmol/kg was significantly greater than from quercetin (Table 2). Relative proportions of the main metabolites, quercetin, isorhamnetin and tamarixetin, were similar, as in the experiment with the higher concentration.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSAnimals, diets, and experimental...RESULTSDISCUSSIONLITERATURE CITED |
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The major metabolite in plasma samples, irrespective of the type of flavonol or concentration administered, was always quercetin (71–87%). Additional metabolites detected were two methylated derivatives of this flavonol, tamarixetin and isorhamnetin. Because all of these flavonols were detectable in plasma samples only after enzymatic treatment, the circulating forms are conjugates, i.e., glucuronides and sulfates.
Although our data from portal blood are limited due to technical problems (i.e., obstruction of catheters), we can state that all administered flavonols must have undergone nearly complete metabolism in the intestinal mucosa because no free flavonols were detectable in portal plasma. At least for humans and rats, catechol-o-methyltransferase and UDP-glucuronosyltransferase activities were demonstrated in the mucosa of the small and large intestine. These intestinal enzymes were able to metabolize different flavonoids (20,21). Thus, the intestine possesses the capability to methylate and glucuronidate flavonols.
In examining the relative bioavailability of the different quercetin glycosides and quercetin aglycone, we obtained the following results at the higher dosage (148 µmol/kg). First, the glucorhamnoside rutin had the lowest relative bioavailability and was not absorbed before 3 h after intake, thus indicating that microbial degradation in the terminal ileum or in the large intestine is a prerequisite for absorption. Second, metabolites of both the aglycone and the monoglucoside Q3G appeared within 1 h in the systemic circulation, indicating absorption from the upper small intestine. Third, relative bioavailability from the monoglucoside seemed to be considerably higher than from the aglycone.
In addition to the significantly higher bioavailability from Q3G at the lower concentration in Experiment 2, Q3G tended to be (P = 0.07) more bioavailable at the higher concentration in Experiment 1. The lack of significance likely was attributable to the small number of pigs (n = 4) in Experiment 1. Taken together, the relative bioavailabilities of quercetin and Q3G appear not to be substantially influenced by the dose, at least over the concentration range we administered in the present study.
All of these observations are in accordance with previous studies conducted with the same glycosides in humans (11–14) and rats (15,22). In a study with rats that were administered 245 µmol/kg as quercetin, Q3G or rutin as part of their diet, the authors stated that the relative bioavailabilities of Q3G and rutin were 184 and 25%, respectively, compared with that of the aglycone (22). In ileostomy volunteers, Hollman et al. (23) came to the same conclusions concerning the relative absorption of quercetin from the aglycone, rutin, or from an onion supplement rich in quercetin glucosides. However, the absorption of quercetin was not measured directly but defined as the disappearance from the ileostomy effluent (23).
The reason for the favored absorption of the monoglucoside remains to be determined. In considering uptake across the intestinal brush border membrane as the rate-limiting step in intestinal absorption of flavonols, two hypotheses have been proposed (24,25). Q3G (but not rutin) is a potential substrate for the lactase phloridzin hydrolase in the brush border membrane (26–28). It is assumed that the hydrophilic monoglucoside concentrates at the brush border membrane and might be deglycosylated by this enzyme. The lipophilic aglycone is thought to be able to penetrate the apical enterocyte membrane by passive diffusion. Consequently, the higher concentration of quercetin originating adjacent to the apical membrane would result in a higher diffusion rate at this site and, thus, in greater absorption of the flavonol from Q3G. The other possible explanation for the higher absorption from quercetin monoglucosides is carrier-mediated uptake of these compounds by the intestinal sodium-dependent glucose transporter-1 (SGLT1). Several in vitro studies have indicated a role of SGLT1 in mucosal uptake of quercetin glucosides (25,29,30), although recent in vivo studies with rats point to a major role of lactase phloridzin hydrolase (31,32).
After administration of Q3G, its main plasma metabolite, quercetin, showed a second larger plasma peak at 210 min (Fig. 3). We assume that only part of the Q3G was absorbed in the upper gastrointestinal tract (corresponding to the first peak); the rest was deglycosylated either by microbial enzymes or by intestinal ß-glucosidases in the more distal segments, resulting in the second delayed absorption maximum.
Interestingly, Q3G was significantly better absorbed when it was administered with a small amount of ground beef than with the standard diet. It is possible that the minimal amount of glucose in meat and the more favorable Q3G-glucose ratio present under these conditions might explain this finding. If Q3G were transported by SGLT1, a higher Q3G-glucose ratio would facilitate uptake of the flavonol glucoside. Another possible reason for the preferential Q3G absorption, however, might be the fat content of the meat. A recent study demonstrated that lipids can enhance the absorption of quercetin (33). However, we cannot exclude the possibility that the smaller amount of this test meal (ground beef) and, thus, the higher concentration of Q3G within the diet affected the bioavailability. Irrespective of the cause of the preferential absorption of Q3G from meat than from the standard diet, it is evident that the bioavailability of flavonols depends not only on their chemical form, but also, to a great extent, on various dietary factors.
In summary, we have shown that the flavonol, quercetin, and quercetin glycosides are metabolized in the intestinal mucosa. The main metabolites with an intact flavonol structure are conjugated derivatives of quercetin, isorhamnetin and tamarixetin. When administered as part of a normal pig diet, total bioavailability from Q3G is higher than from quercetin aglycone. However, total bioavailability of Q3G is clearly dependent on food composition because administration with meat significantly enhanced absorption. Our results indicate that the bioavailability of quercetin from quercetin glycosides is determined by the chemical form of the flavonols and, to a large extent, by dietary factors.
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FOOTNOTES |
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2 Supported by a grant from the Deutsche Forschungsgemeinschaft (no. WO 763/2–1 and -2).
4 Abbreviations used: AUC, area under the curve; cmax, maximal plasma concentration; Q3G, quercetin-3-O-glucoside; Q4G, quercetin-4'-O-glucoside; rutin, quercetin-3-O-glucorhamnoside; SGLT, sodium-dependent glucose transporter; tmax, time at maximal plasma concentration.
Manuscript received 5 May 2003. Initial review completed 2 June 2003. Revision accepted 25 June 2003.
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LITERATURE CITED |
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