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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.
| ABSTRACT |
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KEY WORDS: flavonoids bioavailability quercetin fat pigs
The flavonol quercetin is one of the most prevalent flavonoids found in edible plants. In plants and subsequently in plant-derived foods, quercetin is present mainly as glycosides (1,2). It has been shown repeatedly that the sugar moiety is a major determinant governing the intestinal absorption of quercetin (36). In addition to the chemical form of the flavonol, the applied vehicle or the composition of the diet seems to have a substantial effect on quercetin bioavailability (710). In a previous study by our group, the bioavailability of quercetin from quercetin-3-O-glucoside (Q3G)4 was significantly enhanced when Q3G was administered to pigs in a meal rich in fat and low in carbohydrates, compared with a standard carbohydrate-rich pig diet (3). One reason for this finding could have been the difference in fat concentration in the experimental diets because lipids in combination with emulsifiers were shown to enhance the absorption of orally administered quercetin in rats (8). Therefore, in this study we investigated the effect of the dietary fat content on the oral bioavailability of quercetin in pigs. Quercetin was administered either as the rather lipophilic aglycone or as the more water-soluble Q3G in diets with differing fat contents.
| MATERIALS AND METHODS |
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Processing of plasma samples and HPLC analysis. Blood samples were drawn into heparinized containers, immediately centrifuged (1500 x g, 10 min, 4°C) and stored at 70°C until analysis by HPLC as described previously (3,12,13). All samples were treated enzymatically with ß-glucuronidase/sulfatase before the extraction of flavonol compounds.
All flavonols were obtained from Carl Roth GmbH. ß-Glucuronidase/sulfatase type H-2 (crude enzyme extract from Helix pomatia) was purchased from Sigma-Aldrich AG.
Statistical analysis. The area under the plasma concentration-time curve (AUC) was determined according to the linear trapezoidal rule. For each pig and treatment, total bioavailability (AUCtotal) was calculated by adding up the AUC values of quercetin and its metabolites with an intact flavonol structure (isorhamnetin and tamarixetin). AUC data, maximal plasma concentrations (cmax), those at 480 (c480) and 720 min (c720) after ingestion of the test meal, and time at maximal plasma concentration (tmax) values were analyzed with the MIXED model procedure from SAS (Version 8.2, SAS Institute) based on the model: Yi,j,k = µ + Di + Fj + (D x F)i,j + Ak + ei,j,k where µ = mean, Di = diet (i = 3, 17, or 32% fat diet), Fj = administered flavonol (j = quercetin aglycone or Q3G), (D x F) = diet x administered flavonol interaction, Ak = animal, and ei,j, k = residual error. The individual pig was treated as a random factor. AUC data, cmax, c480, c720 and tmax values are presented as least-squares means (LSM) ± SEM. A P-value < 0.05 was considered significant.
| RESULTS |
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After the intake of quercetin aglycone with the 3% fat diet, the mean peak plasma concentration (cmax) was reached
100 min (tmax) after intake (Table 2). When the flavonol was administered together with the 17 or 32% fat diets, plasma concentrations of quercetin rose more sharply (Fig. 1) and reached their peak levels significantly earlier (Table 2). Thereafter, quercetin levels with the 3% fat diet decreased continuously until they were below the detection limit after 24 h (Fig. 1). The elimination of quercetin was clearly delayed after its application with the fat-enriched diets; 480 min after intake (c480) with the 32% fat diet, the quercetin plasma concentration (0.054 ± 0.011 µmol/L, n = 7) was higher than the value obtained with the 3% fat diet (0.015 ± 0.011 µmol/L, n = 7, P < 0.05). However, the respective plasma concentration obtained with the 17% fat diet (0.050 ± 0.013 µmol/L, n = 6) did not differ from the 3% fat diet value (P = 0.07) or the 32% fat diet value (P = 0.81). The values determined at 720 min after intake (c720) with the 17 and 32% fat diets (0.073 ± 0.016 µmol/L and 0.070 ± 0.015 µmol/L, respectively) were both significantly higher than the corresponding value attained after intake with the 3% fat diet (0.015 ± 0.015 µmol/L, P < 0.05) (Fig. 1). The fat content of the diet had a significant effect on total bioavailability (AUCtotal) of quercetin from the aglycone-containing diets (Table 2). Compared with the 3% fat diet, bioavailability was significantly increased with the fat-enriched diets, with no significant difference between the 17 and 32% fat diets.
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70 min (Table 2). When the flavonol glucoside was administered together with fat-enriched diets, the plasma concentrations of quercetin reached their peak levels earlier, which was significant only for the 32% fat diet. However, the cmax value of plasma quercetin was lowest after intake with 32% fat (Table 2). Similar to what was observed with the diets containing the aglycone, the elimination of quercetin was delayed with the fat-enriched diets (inset Fig. 1). Plasma concentrations after 480 min differed significantly among all 3 diets (0.053 ± 0.020 µmol/L in the 3% fat diet, 0.123 ± 0.020 µmol/L in the 17% fat diet, P < 0.05 vs. 3% fat diet, and 0.174 ± 0.020 µmol/L in the 32% fat diet, P < 0.05 vs. 3% and vs. 17% fat diet, n = 6). The c720 values from both the 17 and 32% fat diets (0.071 ± 0.015 µmol/L and 0.090 ± 0.020 µmol/L, respectively) were also significantly higher than the plasma concentration after intake with the 3% fat diet (0.026 ± 0.015 µmol/L, P < 0.05). Hence, the fat content of the diet had a significant effect on the AUCtotal from Q3G (Table 2). In the case of Q3G intake, the significantly increased AUCtotal values obtained with the fat-enriched diets were due solely to the delayed elimination of quercetin (inset Fig. 1). The amount of quercetin and methylated quercetin metabolites in the systemic circulation was also significantly influenced by the chemical form of quercetin administered. With quercetin aglycone, the mean total bioavailability from any of the diets was always significantly lower than from the respective diets containing the quercetin glucoside (P < 0.05) (Table 2). The fat content of the diet and the chemical form of the flavonol, however, did not have a significant interaction.
| DISCUSSION |
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In pig plasma, quercetin and its metabolites with a flavonol structure are present in substantial amounts only as conjugates (3,12). Therefore, we treated all plasma samples with ß-glucuronidase/sulfatase to release the flavonol aglycones. In all experiments, quercetin was found in plasma within 30 min after ingestion. This suggests that absorption had already occurred in the upper small intestine, irrespective of the diet and of the flavonol administered.
The increase in the dietary fat content from 3 to 17% crude fat enhanced the total bioavailability of quercetin (quercetin and its metabolites, isorhamnetin and tamarixetin) from the quercetin aglycone containing diet by
50%. This effect was already maximal because a further increase in dietary fat had no additional effect. It is generally believed that the rather lipophilic quercetin aglycone diffuses passively through the brush border membrane (BBM) of enterocytes (14,15). Azuma et al. (8) observed that coadministration of lipids (soybean oil or lecithin) and emulsifiers such as the bile constituent taurocholate was able to enhance and accelerate the intestinal absorption of quercetin in rats. In another rat study, the absorption of the flavanol catechin was enhanced when green tea catechins were administered as a phospholipid complex rather than as free catechins (16). Thus, it is likely that in the presence of dietary fat, quercetin was partly incorporated into mixed bile salt micelles in the lumen of the duodenum. This could have promoted its solubility and transport through the unstirred water layer and passive diffusion through the BBM.
Interestingly, an enhancing effect of dietary fat on total bioavailability of quercetin was also found after administration of Q3G-containing diets. At first glance, this was an unexpected observation, because the more hydrophilic glucoside of quercetin should not directly interact with the absorption of lipids. In agreement with this finding, Azuma et al. (17) recently observed an enhancement of quercetin bioavailability from quercetin glucosides in onions by administration with at least 4.6% fat. In the present study, the effect of dietary fat on quercetin bioavailability from Q3G was due exclusively to a delayed elimination of quercetin from plasma. In contrast, the addition of fat to the quercetin aglyconecontaining diets also enhanced the absorption of the flavonol. Several studies pointed to the involvement of ß-glycosidases such as the BBM enzyme, lactase-phlorizin hydrolase (LPH, EC 3.2.1.62), in the intestinal absorption of Q3G (1820). According to those studies, Q3G is hydrolyzed by LPH in the small intestine and the liberated aglycone diffuses passively across the BBM into the enterocyte. Because the liberation of quercetin from Q3G by LPH occurs adjacent to the BBM, enhanced solubility and diffusion through the unstirred water layer as promoted by the formation of micelles cannot be expected in this case. However, an enhancing effect of dietary fat on the reabsorption of quercetin during enterohepatic circulation can be expected after the administration of both Q3G and quercetin aglycone. Stimulation of biliary secretion by dietary fat intensifies excretion of quercetin glucuronides and/or sulfates with the bile (21). After microbial deconjugation of the excreted conjugates in the lower small and large intestine, quercetin could be partially reabsorbed (22). This could explain the higher plasma concentrations of quercetin several hours after ingestion with the fat-enriched diets.
The earlier appearance of maximal quercetin plasma concentrations after ingestion with fat could be explained by a partial incorporation of the quercetin conjugates formed in the enterocyte into chylomicrons with consecutive export into the peripheral blood via lymph. Thus, part of the quercetin would bypass the liver and, consequently, increase the plasma concentration. However, because no data are available concerning the incorporation of quercetin conjugates into lipoproteins during absorption, this remains speculative at present.
In our previous study, administration of Q3G together with meat enhanced total quercetin bioavailability by 140% (3), whereas the addition of fat in the present study enhanced bioavailability of Q3G by only
30%. This suggests that other dietary factors in addition to fat influence the bioavailability of quercetin. It is conceivable that the lack of carbohydrates in the meat could have favored carrier-mediated uptake of Q3G by the intestinal sodium-dependent glucose transporter (SGLT1) (3). Unspecific binding to food components such as soy protein could have limited the absorption of quercetin from the test meal used compared with the meat meal (23). In this regard, it is important to note that the absolute amount of protein in the meat meal was only
10% of that in the standard meal used (3).
In the present study, all test meals were isoenergetic to avoid differences in the rate of stomach emptying, which in turn would influence absorption kinetics. Hence, the test meals differed inevitably in protein (32, 23, and 16 g crude protein) and carbohydrate content (100, 68, and 48 g nonfiber carbohydrates) in the 3, 17, and 32% fat test meals, respectively. These differences in protein and carbohydrate content were rather small, however, compared with the relative differences in fat content (7, 28, and 43 g crude fat in the 3, 17, and 32% fat test meals, respectively). Thus, in our opinion, it is most likely that the differences in the fat content were responsible for the observed effects on quercetin bioavailability.
Regardless of these latter considerations, Q3G was always more bioavailable than quercetin aglycone with each of the different diets. This observation agrees with our previous study using pigs (3), and is also known from studies of humans (7) and rats (24). Most authors explain the higher bioavailability of Q3G compared with the aglycone by the occurrence of higher local quercetin concentrations adjacent to the BBM due to the Q3G-hydrolyzing activity of LPH (see above). In addition, transport of Q3G by SGLT1 could also contribute to the higher bioavailability of quercetin from Q3G compared with quercetin aglycone. Several studies showed an interaction of Q3G with SGLT1 (5,25,26). A recent study demonstrated transport of a quercetin glucoside by SGLT1 (27). In experiments with pig small intestine BBM vesicles, we also found evidence for transport of Q3G by SGLT1 (28).
In summary, we showed that the dietary fat content influences the bioavailability of quercetin. Bioavailability from both quercetin aglycone and quercetin-3-O-glucoside is enhanced in a diet enriched with fat compared with a low-fat diet. This could be explained by an improved solubility and an accelerated absorption of the lipophilic quercetin aglycone via lipid micelles and a prolonged enterohepatic circulation.
| FOOTNOTES |
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2 Supported by grant no. WO 763/22 and Graduiertenkolleg 820 of the Deutsche Forschungsgemeinschaft (DFG). ![]()
4 Abbreviations used: AUC, area under the curve; BBM, brush border membrane; BW, body weight; c480, c720, plasma concentration at 480 or 720 min after ingestion of test meal, respectively; cmax, maximal plasma concentration; LPH, lactase-phlorizin hydrolase; LSM, least-squares means; Q3G, quercetin-3-O-glucoside; SGLT1, sodium-dependent glucose transporter; tmax, time at maximal plasma concentration. ![]()
Manuscript received 5 February 2004. Initial review completed 8 March 2004. Revision accepted 26 March 2004.
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