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Polyphenols Ins and Outs: Metabolism and Uptake from the Gut

Stephen Barnes, PhD
University of Alabama at Birmingham School of Medicine

The diet contains many different types of polyphenols derived from plants. The gastrointestinal tract is exposed to these compounds and their metabolites in concentrations substantially larger than in peripheral tissues.

Structure and Uptake of Polyphenols
Polyphenols exist in multiple forms, from the very hydrophobic, such as the methoxy flavonoids tangeritin and nobelitin, to the hydrophilic, such as glycosides of polyhydroxylated flavonoids. They exist as monomers, oligomers, and/or O- and C-glycosides. Examples of the latter include daidzein-7-O-glucoside and genistein-7-O-glucoside, both found in soy; and, daidzein-8-C-glucoside (puerarin), an isoflavone found in the kudzu root. The O-glycosides undergo hydrolysis in the gut by intestinal microflora and endogenous enzymes (bacterial glycosidases or lactose phlorizin hydrolase [1]) to aglycones.

It is possible that certain polyphenol glucosides are taken up by sodium-dependent glucose transporters, followed by hydrolysis in the enterocyte [2]. However, in the case of C-glucoside conjugates, they are resistant to hydrolysis and are rapidly transferred into the blood without any metabolism [3].

Within the enterocyte, polyphenol aglycones are glucuronidated and can pass through the enterocyte basolateral membrane and hence into the vascular system, or transferred back into the luminal compartment by P-glycoprotein and multi-drug resistance proteins (MDR1, MRP1 MDR2) [1,4]. For example, genistin, if absorbed, is converted to genistein in the intestinal cell. The polyphenol aglycones are further converted to sulfate ester derivatives, or ?-glucuronides. While these may be absorbed into the blood, a portion of the ?-glucuronide is prevented from entering the blood by an apical MRP-like protein and is transferred back into the intestinal lumen. This was demonstrated to be an important event in chrysin [5] and genistin bioavailability [6]. Overall uptake of an individual polyphenol is a complex interplay between the chemistry of polyphenol glycosides, metabolism of their aglycones and the rates of transport of each form [1].

Experimental Evidence of Uptake and Metabolism
The overall pathway of uptake and metabolism of polyphenols has been elucidated using a combined strategy of intact animals [7], isolated perfused intestine [8-12], and cultured Caco-2 cells [11-16].

We have used LC-mass spectrometry to study the efficiency of uptake of genistein, its sites of metabolism, its metabolic forms and routes of excretion (ADME) [7]. Using this technique we have further been able to study the influence of the glucoside moiety on ADME. In the first experiment, 14C-genistein was infused for 60 minutes into the upper duodenum of intact, anesthetized female rats fitted with a biliary cannula. After 60 minutes, infusion of physiological saline was initiated and continued throughout the rest of the experiment. After four hours, 70% of the 14C-radioactivity was recovered in bile, showing that genistein is efficiently taken up from the intestine into the blood, passed to the liver and secreted into the bile after duodenal administration. The collected bile was then administered into the mid-small intestine of a rat via a biliary cannula. After four hours, recovery of 14C-radioactivity was similar to, if somewhat slower, than recovery of genistein. These data are evidence of a quite efficient enterohepatic circulation for genistein [7].

We next looked at the biliary excretion of genistein infused in the portal vein and found it to be dose-dependent and transport limited. Genistein was excreted into bile as its ?-glucuronide. This prompted us to ask whether genistin, the O-glucoside of genistein and a major form of genistein found in soy foods, undergoes direct uptake in its glucosidic form. We found that genistin is present in bile after infusion into the portal and femoral veins, but when infused into the upper intestine of rats, no genistin is detected in bile [17]. Thus, genistin is not taken up in the intact form from the small intestine, but is hydrolyzed to its aglycone, genistein, prior to absorption. Genistein ??glucuronide again is the major metabolite of genistin, which is returned to the intestinal compartment via the bile.

Andlauer et al compared genistein vs. genistin uptake in a perfused small intestine model [8,9]. For genistein, they found that 46.4% of the genistein dose was absorbed in the first pass in the small intestine and 40.6% of the dose appeared in the vascular compartment [8]. There was extensive ?-glucuronidation in the gut wall, and the transport rate from the lumen to the vascular compartment was twice as high as the reverse rate. By comparison, they found that the uptake of genistin was slow (14.9% of the dose), and only 11.6% appeared on the vascular side as genistein glucuronide [9]. Genistein glucuronide also appeared on the luminal side (19.5% of dose), evidence of excretion from the intestinal cells.

The Caco-2 cell model is a colon cancer cell line that undergoes differentiation to behave like enterocytes and is widely used to study the intestinal transport of many substances, including flavonoids. It expresses multi-drug resistant proteins (MDR1, MRP2 and p-glycoprotein) and is useful in studying the net uptake of flavonoids and their bidirectional transport [11-16].

Whereas some polyphenols are readily absorbed by passive diffusion and facilitated transport in the colon, their glucuronidated metabolites formed in the enterocyte may alter the bioavailability of other xenobiotics. However, not all polyphenols can be absorbed. For example, oligomeric proanthocyanidins cannot pass through the enterocytes. Nonetheless, fractionated high molecular weight proanthocyanidins have effects at peripheral sites, which raises the question of whether they are in fact absorbed, or whether they alter bacterial flora of the gut and hence the metabolism of physiologic metabolites such as steroids. Industry-funded investigation of proanthocyanidin metabolism has yielded two interesting findings re digestive health: (1) proanthocyanidins prevent ethanol-induced gastric injury; and (2) proanthocyanidin fractions appear to reduce fecal odor.

The Kudzu: An Unexpected Source of Flavonoids
The kudzu vine, pueraria lobato, was presented to the U.S. government in 1876 by Japan to celebrate the first century of independence. It was used to prevent soil erosion in the Southern states during the Great Depression, but since it has no known adversaries, it has overgrown the areas where it was planted. Today it has overwhelmed trees all over the South and is considered a weed by the USDA.

Interestingly, the kudzu is a rich source of isoflavones. Its root, Radix Puerariae, is sold as a dietary supplement for hangovers, cardiovascular disease, angina, and as an anti-pyretic. Kudzu root isoflavones are mostly C-glycosides of daidzein as opposed to the O-glucosides in soy. They are the principal isoflavones in over-the-counter preparations used by peri- and postmenopausal women as alternatives to equine estrogens [18]. Studies of the metabolism of kudzu isoflavones found that the C-glycosides are excreted rapidly and are largely recovered in urine in the unmetabolized form [3]. However, puerarin was found in the brain of an untreated rat [3], and in another study had a favorable effect on glucose tolerance [19]. Therefore, it is apparent that the C-glycosides are absorbed intact from the intestine without hydrolysis, and can enter peripheral tissue and potentially have biologic effects.

Conclusions
Taken altogether, these studies reveal that polyphenols are important players in the lumen of the gastrointestinal tract. Some polyphenols are readily absorbed and glucuronidated in the intestinal cell, and their metabolites may affect the availability of other compounds. The colon is also a site of absorption of polyphenols and their metabolites, but even unabsorbed polyphenols may play a role in gut homeostasis.

References
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  2. Walle, T. Flavonoids and isoflavones (phytoestrogens): absorption, metabolism, and bioactivity. Free Radical Biology and Medicine 36: 829-837, 2004.
  3. Prasain, J.K., Jones, K., Brissie, N., Moore, D.R. II, Wyss, J.M., and Barnes, S. Identification of puerarin and its metabolites in rats by liquid chromatography-tandem mass spectrometry. Journal of Agricultural Food Chemistry 52: 3708-3712, 2004.
  4. Walgren, R.A., Karnaky, K.J. Jr., Lindenmayer, G.E., and Walle, T. Efflux of dietary flavonoid quercetin 4'-B-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2. Journal of Pharmacology and Experimental Therapeutics 294: 830-836, 2000.
  5. Walle, U.K., Galijatovic, A., and Walle, T. Transport of the flavonoid chrysin and its conjugated metabolites by the human intestinal cell line Caco-2. Biochemical Pharmacology 58:431-438, 1999.
  6. Walle, U.K., French, K.L., Walgren, R.A., and Walle, T. Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2. Research Communications in Molecular Pathology & Pharmacology 103: 45-56, 1999.
  7. Sfakianos, J., L. Coward, M. Kirk and S. Barnes. Intestinal uptake and biliary excretion of the isoflavone genistein in the rat. Journal of Nutrition 127: 1260-1268, 1997.
  8. Andlauer, W., Kolb, J., Stehle, P., and Furst, P. Absorption and metabolism of genistein in isolated rat small intestine. Journal of Nutrition 130: 843-846, 2000.
  9. Andlauer, W., Kolb, J., and Furst, P. Absorption and metabolism of genistin in the isolated rat small intestine. FEBS Letters 475: 127-130, 2000.
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  14. Murota, K., Shimizu, S., Miyamoto, S., Izumi, T., Obata, A., Kikuchi, M., and Terao, J. Unique uptake and transport of isoflavone aglycones by human intestinal caco-2 cells: comparison of isoflavonoids and flavonoids. Journal of Nutrition 132: 1956-1961, 2002.
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  16. Chen, J., Lin, H., and Hu, M. Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model. Cancer Chemotherapy & Pharmacology 55: 159-169, 2005.
  17. Barnes, S., Xu, J., Smith. M., and Kirk, M. Lack of evidence for the intestinal absorption of isoflavone B-glucosides in the intact rat. Presented at the 221st National Meeting of the American Chemical Society, San Diego, CA, 2001.
  18. Prasain, J.K., Jones, K., Kirk, M., Wilson, L., Smith-Johnson, M., Weaver, C.M., Barnes, S. Identification and quantitation of isoflavonoids in Kudzu dietary supplements by HPLC and electrospray ionization tandem mass spectrometry. Journal of Agricultural Food Chemistry, 51: 4213-4218, 2003.
  19. Meezan, E., Meezan, E.M., Jones, K., Moore II, D.R., Barnes, S., and Prasain, J.K. Contrasting effects of the isoflavones glucosides puerarin and daidzein on glucose metabolism in mice. Journal of Agricultural and Food Chemistry, accepted for publication.



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