Kaeko Murota

Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism.

Quercetin is a typical flavonoid ubiquitously present in fruits and vegetables, and its antioxidant effect is implied to be helpful for human health. The bioavailability of quercetin glycosides should be clarified, because dietary quercetin is mostly present as its glycoside form. Although quercetin glycosides are subject to deglycosidation by enterobacteria for the absorption at large intestine, small intestine acts as an effective absorption site for glucose-bound glycosides (quercertin glucosides). This is because small intestinal cells possess a glucoside-hydrolyzing activity and their glucose transport system is capable of participating in the glucoside absorption. A study using a cultured cell model for intestinal absorption explains that the hydrolysis of the glucosides accelerates their absorption in the small intestine. Small intestine is also recognized as the site for metabolic conversion of quercetin and other flavonoids as it possesses enzymatic activity of glucuronidation and sulfation. Modulation of the intestinal absorption and metabolism may be beneficial for regulating the biological effects of dietary quercetin.

Macrophage as a Target of Quercetin Glucuronides in Human Atherosclerotic Arteries IMPLICATION IN THE ANTI-ATHEROSCLEROTIC MECHANISM OF DIETARY FLAVONOIDS

Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases.

Quercetin appears in the lymph of unanesthetized rats as its phase II metabolites after administered into the stomach

Quercetin is a major flavonoid in plant foods and potentially has beneficial effects on disease prevention. The present work demonstrated that quercetin was transported into the lymph after being metabolized in the gastrointestinal mucosa of rats. Glucuronide/sulfate and methylated conjugates of quercetin appeared in the lymph, but not quercetin aglycone. The highest lymphatic concentration was found at as rapid as 30 min after administration, suggesting gastric absorption, whereas the mucosal glucuronidation activity was significantly higher in the duodenum and jejunum than in the stomach. This is the first report to show the lymphatic flavonoid transport pathway from the gastrointestinal tract.

(−)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: Implication in the anti-atherosclerotic actions of tea catechins

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (-)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.

Effect of Quercetin and Its Conjugated Metabolite on the Hydrogen Peroxide-induced Intracellular Production of Reactive Oxygen Species in Mouse Fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-β-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.

Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes.

Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO(2) and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPARα activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPARα activity is a novel target of PPARα agonist for decreasing circulating levels of lipids under postprandial conditions.

Prenylation Enhances Quercetin Uptake and Reduces Efflux in Caco-2 Cells and Enhances Tissue Accumulation in Mice Fed Long Term

Prenyl flavonoids are widely distributed in plant foods and have attracted appreciable attention in relation to their potential benefits for human health. Prenylation may enhance the biological functions of flavonoids by introducing hydrophobic properties in their basic structures. Previously, we found that 8-prenyl naringenin exerted a greater preventive effect on muscle atrophy than nonprenylated naringenin in a mouse model. Here, we aimed to estimate the effect of prenylation on the bioavailability of dietary quercetin (Q). The cellular uptake of 8-prenyl quercetin (PQ) and Q in Caco-2 cells and C2C12 myotube cells was examined. Prenylation significantly enhanced the cellular uptake by increasing the lipophilicity in both cell types. In Caco-2 cells, efflux of PQ to the basolateral side was <15% of that of Q, suggesting that prenylation attenuates transport from the intestine to the circulation. After intragastric administration of PQ or Q to mice or rats, the area under the concentration-time curve for PQ in plasma and lymph was 52.5% and 37.5% lower than that of Q, respectively. PQ and its O-methylated form (MePQ) accumulated at much higher amounts than Q and O-methylated Q in the liver (Q: 3400%; MePQ: 7570%) and kidney (Q: 385%; MePQ: 736%) of mice after 18 d of feeding. These data suggest that prenylation enhances the accumulation of Q in tissues during long-term feeding, even though prenylation per se lowers its intestinal absorption from the diet.

Phospholipid hydroperoxides are detoxified by phospholipase A2 and GSH peroxidase in rat gastric mucosa

The aim of this study was to determine the metabolic fate of phospholipid hydroperoxides (PLOOH) in rat gastric mucosa. Here we report evidence concering the mechanism for PLOOH detoxification in gastric mucosa homogenate. Analysis by the TLC blot technique showed that the gastric mucosa has the highest potential to eliminate 1-palmitoyl-2-linoleoyl-phosphatidylcholine hydroperoxides (PL-PtdChoOOH) compared with the intestinal mucosa and liver. Major products detected after incubation with gastric mucosa were the partially reduced linoleic acid hydroperoxides (LAOOH) and lysophosphatidylcholine, indicating the involvement of phospholipase A2 (PLA2) in the elimination pathway. Using unilamellar vesicles, we demonstrated that gastric mucosal PLA2 does not distinguish between PLOOH and intact phospholipids. Although gastric mucosal PLA2 activity efficiently eliminated excess amounts of PLOOH, the complete reduction of LAOOH was dependent on the supply of exogenous GSH. In a separate experiment, administration of egg yolk PtdChoOOH to rats for 6 d significantly elevated GSH peroxidase (GPx) activity in the gastric mucosa. We concluded that excess amounts of PLOOH are efficiently eliminated through the hydrolysis by PLA2, and the subsequent reduction of FA hydroperoxide by GPx is the critical step for complete detoxification of oxidized phospholipids in the stomach.

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Background: The intracellular carrier protein(s) for monoacylglycerols (MGs) is unknown. Results: Using chromatography and NMR and fluorescence spectroscopy, we show that liver fatty acid-binding protein (LFABP) is a binding protein for MG and promotes rapid MG transfer to membranes. Conclusion: LFABP binds MG in vitro and in liver cytosol. Significance: LFABP may transport MG, a metabolic intermediate and signaling molecule, in liver and intestinal cytosol. Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.

Influence of fatty acid patterns on the intestinal absorption pathway of quercetin in thoracic lymph duct-cannulated rats

Since it is known that dietary fats improve the bioavailability of the flavonol quercetin, we purposed to investigate whether this effect is due to increased lymphatic transport of quercetin. In rats with implanted catheters in the thoracic lymph duct, we administered quercetin into the duodenum with TAG emulsions containing either long-chain fatty acids (LCT) or medium-chain fatty acids (MCT). Controls received quercetin together with a glucose solution. LCT administration increased the lymphatic output of quercetin (19.1 (SEM 1.2) nmol/8 h) as well as the lymph-independent bioavailability of the flavonol, determined as area under the plasma concentration curve (1091 (SEM 142) microM x min). Compared with glucose administration, MCT neither increased the lymphatic output (12.3 (SEM 1.5) nmol/8 h) nor the bioavailability of quercetin (772 (SEM 99) microM x min) significantly (glucose group: 9.8 (SEM 1.5) nmol/8 h and 513 (SEM 55) microM x min, respectively). Because LCT are released within chylomicrons into the intestinal lymph while MCT are mainly released into the portal blood, we conclude from the present results that dietary fats that are mainly composed of LCT improve quercetin bioavailability by increasing its transport via the lymph, thereby circumventing hepatic first-pass metabolism of the flavonol. In addition, LCT could enhance quercetin absorption by improving its solubility in the intestinal tract.