Effect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aortic endothelial cells

¹ From the Vascular Biology Laboratory, Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston.

² Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessary reflect the view of the US Department of Agriculture.

³ Supported by US Department of Agriculture agreement no. 58-1950-9-001. TK is a Visiting Scientist at the Vascular Biology Laboratory and is supported by the Noda Institute for Scientific Research, Japan.

⁴ Address reprint requests to M Meydani, Vascular Biology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail: mmeydani{at}hnrc.tufts.edu.

ABSTRACT  
Background: Flavonoids may exert their health benefit in cardiovascular disease by modulating monocyte adhesion in the inflammatory process of atherosclerosis. Most in vitro studies used forms of flavonoids present in food rather than forms that appear in plasma after ingestion.

Objectives: We tested the effects of plasma metabolites of (+)-catechin and quercetin on the modulation of monocyte adhesion to human aortic endothelial cells (HAEC) and on the production of reactive oxygen species (ROS).

Design: Plasma extracts of flavonoid metabolites were prepared after intragastric administration of pure compounds to rats. The plasma preparations contained sulfate or glucuronide conjugates or both and methylated forms. We measured adhesion of U937 monocytic cells to HAEC and the production of ROS in HAEC when cells were pretreated with either pure compounds or plasma extracts from control or treated rats. Adhesion assays were performed with HAEC stimulated with interleukin (IL)-1ß or U937 cells activated with phorbol myristyl acetate; ROS were measured after challenging HAEC with IL-1ß or hydrogen peroxide.

Results: Pretreatment of HAEC with (+)-catechin metabolites inhibited U937 cell adhesion to IL-1ß–stimulated cells, whereas pretreatment with intact (+)-catechin had no effect. Generation of ROS in hydrogen peroxide–stimulated HAEC was inhibited by (+)-catechin, its metabolites, and control plasma extract, whereas ROS generation in IL-1ß–stimulated HAEC was inhibited by (+)-catechin metabolites only. In contrast, quercetin inhibited U937 cell adhesion to IL-1ß–stimulated HAEC, whereas its metabolites were not effective.

Conclusions: Metabolic conversion of flavonoids such as (+)-catechin and quercetin modifies the flavonoids' biological activity. Metabolites of flavonoids, rather than their intact forms, may contribute to the reported effects of flavonoids on reducing the risk of cardiovascular disease.

Key Words: Flavonoid • metabolites • monocyte • endothelium • endothelial cell • reactive oxygen species • rats • cardiovascular disease

INTRODUCTION  
Flavonoids are found in a wide variety of plant products, such as fruit, vegetables, herbs, nuts, and tea (1, 2), and have been suggested to have a wide range of beneficial effects on human health, including protection from cardiovascular disease (CVD) and certain forms of cancer (3). The results of epidemiologic studies indicate an inverse relation between flavonoid intake and mortality from coronary artery disease and cancer (4, 5).

Evidence from in vitro and animal studies indicates that the high antioxidant activity of flavonoids (6, 7), their inhibition of some enzymes (8, 9), and their modulation of certain cell functions (10–13) may contribute to their beneficial effects. However, the biological effects of flavonoids have been attributed mainly to their antioxidant activities (6). Flavonoids may protect LDL from oxidation (14–16). Because oxidized LDL is implicated in the development of atherosclerosis (17), it is plausible that antioxidant activities contribute in part to the beneficial effects of flavonoids on the cardiovascular system. Chemotaxis and accumulation of leukocytes in the arterial wall are considered to be critical events in the inflammation associated with atherosclerosis (18, 19). Flavonoids were reported to inhibit adhesion of immune cells to endothelial cells (11, 12, 20, 21). However, most in vitro studies performed to date used forms of flavonoids present in foods, rather than the forms that appear in plasma after absorption and metabolism. Thus, the precise mechanism or mechanisms by which flavonoids exert positive effects have yet to be elucidated.

Flavonoids are partly absorbed from the gastrointestinal tract in animals (22–26) and humans (27–31). Recent studies showed that metabolites such as glucuronide or sulfate conjugates and methylated conjugates accumulate in plasma, whereas the unconjugated compounds are detected in very low concentrations (25, 32, 33). Conjugated metabolites have been suggested to play greater roles in the biological activity of flavonoids than do their parent compounds (31, 34–36). Nevertheless, little is known about the biological activities of the metabolites of flavonoids that appear in the blood. Therefore, in the present study, we prepared plasma metabolites of (+)-catechin and quercetin from rats after intragastric administration and investigated the effects of these metabolites on the modulation of monocyte adhesion to human aortic endothelial cells (HAEC).

MATERIALS AND METHODS  
Materials
(+)-Catechin and quercetin (3,3',4,5,7-pentahydroxyflavone) sulfatase (type H-5, which contains ß-glucuronidase) were purchased from Sigma Chemical Co (St Louis). Isorhamnetin (3,3',4',5-tetrahydroxy-7-methoxyflavone) was purchased from Funakoshi Co (Tokyo). Methylcatechin was prepared by enzymatic synthesis according to the method of Manach et al (32). All other chemicals were of analytic grade and solvents were of HPLC grade. In all cell culture experiments, endotoxin-screened distilled water was used (Gibco, Grand Island, NY).

Animals and oral administration of (+)-catechin and quercetin
The protocol was reviewed and approved by the Animal Care and Use Committee of the US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University. Seven-week-old male Wistar rats (Charles River, Wilmington, MA) were housed in an air-conditioned room (23 ± 1°C and 55 ± 5% humidity) under a 12-h dark-light cycle. Rats were fed a polyphenol-free semipurified diet (23) and water ad libitum for 1 wk. All animals were then deprived of food for 16–18 h before intragastric administration of flavonoid preparations. (+)-Catechin was dissolved in water at a concentration of 25 g/L and quercetin was dissolved in propylene glycol at a concentration of 12 g/L. (+)-Catechin and quercetin were administered at doses of 250 and 120 mg/kg body wt, respectively. Two milliliters of water was administered to the control rats. Previous studies showed that the maximal plasma concentrations of these flavonoids appear within 1 h of intragastric administration (22, 25, 30, 37–40). Three rats from each group were anesthetized with pentobarbital 1 h after flavonoid administration and blood was drawn from the abdominal vena cava into heparin-treated tubes. Plasma samples were separated by centrifugation at 1000 x g for 20 min at 4°C, pooled, and stored at -80°C until extraction.

Preparation of (+)-catechin and quercetin metabolites from rat plasma
Ten milliliters pooled plasma obtained from control rats or rats administered (+)-catechin or quercetin was mixed with 40 mL acetone containing 1% acetic acid. The mixture was shaken continuously at 4°C for 2 h and then centrifuged for 30 min at 5000 x g and 4°C. The supernatant fluid was separated from the precipitate and evaporated in a rotary evaporator. The remaining water phase was further lyophilized. The residue was washed 2 times with 10 mL chloroform, dried under nitrogen gas, and dissolved in 1 mL dimethylsulfoxide (DMSO):water (1:9, by vol). The plasma extracts were stored at -80°C.

HPLC analysis of plasma metabolites of (+)-catechin and quercetin
The plasma extract (10 µL) was mixed with 190 µL acetate buffer containing 0.2 mol acetate/L (pH 5.0), sulfatase (25 U), and ß-glucuronidase (500 U) and incubated at 37°C in a shaking water bath for 1 h to hydrolyze the conjugated metabolites into their free forms. The hydrolysate was mixed with 800 µL methanol:phosphoric acid (100:5, by vol). This mixture was mixed by vortex for 1 min, sonicated for 30 s, and centrifuged for 5 min at 5000 x g and 4°C. An aliquot (50 µL) was mixed with 50 µL of each respective solvent of mobile phase and 20 µL was injected into the HPLC instrument (Waters 600E; Millipore, Milford, MA) equipped with a TSK-gel octadecylsilane 80 Ts QA column (4.6 x 150 mm; TOSOH, Tokyo) and an electrochemical detector (Bioanalytical Systems, West Lafatette, IN) with an applied potential of 950 mV. The mobile phase was composed of acetonitrile:ethyl acetate:phosphoric acid (0.5%; 12:2:86, by vol) for the analysis of (+)-catechin metabolites and methanol:water:acetic acid (48:50:2, by vol) for the analysis of quercetin metabolites. The column was eluted at a flow rate of 0.9 mL/min. Peaks were matched with standards on the basis of their retention times. In this system, the lowest detectable amounts of (+)-catechin and quercetin were estimated to be 0.5 and 0.2 pmol, respectively, at a signal-to-noise ratio of 5.

Cell culture
HAEC were obtained from Clonetics (San Diego) and cells from passages 4–7 were used in this study. The HAEC were cultured in MCDB-131 medium (Sigma) supplemented with 2% fetal bovine serum (FBS) (Gibco), 10 µg human epidermal growth factor/L (Clonetics), 9 mg bovine brain extract/L (Clonetics), 0.5 mg hydrocortisone/L (Clonetics), 1 x 10⁵ U penicillin/L (Gibco), 100 mg streptomycin/L (Gibco), and 1.25 mg amphotericin B/L (Sigma). The cells were seeded in culture flasks or plates coated with 2% gelatin (Sigma) and allowed to grow to confluence before experimental treatment. The U937 human monocytic cell line (American Type Culture Collection, Rockville, MD) was used for the adhesion assay. U937 cells have been used as a model for blood-borne monocytes in endothelial cell adhesion experiments (41). This cell line exhibits many characteristics of monocytes, is readily available, and can be used to prepare a virtually unlimited number of relatively uniform cells. U937 cells were maintained in RPMI-1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 2 mmol L-glutamine/L (Life Technologies), 1 x 10⁵ U penicillin/L, and 100 mg streptomycin/L.

Fluorescent labeling of cells
U937 cells were fluorescently labeled with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR) for the quantitative adhesion assay (42). Nonfluorescent BCECF-AM is lipophilic and is cleaved by intracellular esterase and becomes a highly charged fluorescent BCECF that is retained by viable cells. The BCECF-AM was prepared as a 1-g/L stock in DMSO and was stored at -80°C. The U937 cells were fluorescently labeled by incubating the cells (1 x 10⁷ cells/5 mL) with 5 µmol BCECF-AM/L in RPMI-1640 medium for 30 min at 37°C and 5% CO₂. After the cells were labeled with BCECF-AM, they were washed 3 times with 1% FBS–phosphate buffered saline (PBS) to remove excess dye. Finally, cells were resuspended in MCDB-131 medium at a density of 5 x 10⁸ cells/L.

U937 cell adhesion to HAEC
HAEC were cultured to confluence in 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) and were treated at 37°C with pure flavonoid or plasma extract for 20 h. In some experiments, U937 cells were incubated with pure flavonoid or plasma extract in a 25-mL culture flask (Becton Dickinson Labware) for 20 h, activated with phorbol myristyl acetate (PMA; 100 µg/L) for 2 h, and then fluorescently labeled. After incubation with flavonoids, HAEC were washed with PBS and stimulated with 10 µg recombinant human IL-1ß/L (Endogen, Woburn, MA) for 6 h. BCECF-labeled U937 cells (2.5 x 10⁵ cells/well) were incubated with HAEC for 30 min at 37°C. After incubation, nonadherent cells were removed by washing each well 3 times with 1% FBS-PBS. The attached cells were lysed with 0.5 mL of 50-µmol/L tris buffer (pH 7.6) containing 0.1% sodium dodecyl sulfate. The fluorescence intensity of each well was measured with a Cytofluor (PerSeptive Biosystems, Framingham, MA) fluorescence multiwell plate reader set at excitation and emission wavelengths of 485 and 530 nm, respectively. With each set of experiments, a separate plate containing known numbers of labeled cells was prepared for determination of a standard curve of fluorescence units per cell.

Measurement of the intracellular generation of reactive oxygen species
The determination of intracellular reactive oxygen species (ROS) was based on the oxidation of 2',7'-dichlorodihydrofluorescein (DCHF) by intracellular peroxides, forming the fluorescent compound 2',7'-dichlorofluorescein (DCF), which was measured by a Cytofluor (PerSeptive Biosystems) fluorescence multiwell plate reader. HAEC were cultured to confluence in 24-well plates (Becton Dickinson Labware) and treated at 37°C with pure flavonoid or plasma extract for 20 h. All treatments contained the same amount of DMSO (0.1%). After incubation, the cells were washed 3 times with PBS and then incubated in Hank's Balanced Salt Solution (HBSS) containing 50 µmol DCHF diacetate/L (Molecular Probes) for an additional 30 min at 37°C. The cells were washed and maintained in HBSS. After the addition of IL-1ß (10 µg/L) or hydrogen peroxide (20 µmol/L), fluorescence was monitored for 45 min at excitation and emission wavelengths of 485 and 530 nm, respectively. Data are presented as the percentage increase in DCF fluorescence compared with that in unstimulated cells.

Statistical analysis
Data were analyzed by using the SYSTAT statistical package (version 9.0; SPSS Inc, Chicago). The overall treatment effect was determined by analysis of variance followed by Fisher's least-significant-difference post hoc test. Significance was set at P < 0.05.

RESULTS  
Metabolites of (+)-catechin and quercetin in plasma
Concentrations of the metabolites in the plasma extracts were 10 times the original plasma concentrations. As shown in Figure 1, enzymatic treatment released (+)-catechin and methylcatechin from their conjugated forms. Trace amounts of conjugated (+)-catechin were detected in the plasma of untreated rats (Figure 1A) and trace amounts of free (+)-catechin (0.017 mmol/L) and free methylcatechin (0.012 mmol/L) were detected in the unhydrolyzed plasma of treated rats (Figure 1B). The concentrations of the conjugated metabolites of (+)-catechin and metylcatechin in the plasma of treated rats were 0.617 and 0.121 mmol/L, respectively.

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FIGURE 1. . HPLC chromatogram of plasma extracts from control rats with enzymatic hydrolysis (A), from (+)-catechin–treated rats without enzymatic hydrolysis (B), and from (+)-catechin–treated rats with enzymatic hydrolysis (C). Extracts were prepared from rat plasma obtained 1 h after treatment. Preparation and analytic conditions are described in the Methods.

 
Shown in Figure 2 are the chromatographic profiles of plasma from control and quercetin-administered rats. Trace amounts of conjugated quercetin and conjugated isorhamnetin were present in the plasma of untreated rats (Figure 2A). Free quercetin was detected in unhydrolyzed plasma (0.05 mmol/L) but free isorhamnetin was not (Figure 2B). The concentrations of the conjugated metabolites of quercetin and isorhamnetin in the plasma of treated rats were 0.24 and 0.041 mmol/L, respectively.

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FIGURE 2. . HPLC chromatogram of plasma extracts from control rats with enzymatic hydrolysis (A), from quercetin-treated rats without enzymatic hydrolysis (B), and from quercetin-treated rats with enzymatic hydrolysis (C). Extracts were prepared from rat plasma obtained 1 h after treatment. Preparation and analytic conditions are described in the Methods.

 
Effect of (+)-catechin and its plasma metabolites on U937 cell adhesion to HAEC
HAEC were incubated in culture medium containing 100-fold dilutions of plasma extracts from control or (+)-catechin–administered rats. The medium contained 0.2 µmol (+)-catechin/L, 0.1 µmol methylcatechin/L, and 7.3 µmol/L of their conjugated forms. As shown in Figure 3A, pretreatment of HAEC with plasma extract from (+)-catechin–administered rats significantly reduced U937 cell adhesion to IL-1ß–stimulated HAEC. In contrast, pretreatment of HAEC with intact (+)-catechin at a concentration of 10 µmol/L or with plasma extract from control rats had no significant effect on U937 cell adhesion to IL-1ß–stimulated HAEC. When U937 cells were pretreated with plasma extract from (+)-catechin-administered rats for 20 h followed by PMA (100 µg/L) activation for 2 h, U937 cell adhesion to IL-1ß–stimulated HAEC was also inhibited (Figure 3B). Pretreatment of U937 with intact (+)-catechin at a concentration of 10 µmol/L or with plasma extract from control rats had no significant effect on U937 cell adhesion to IL-1ß–stimulated HAEC.

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FIGURE 3. . Effect of (+)-catechin metabolites on U937 cell adhesion to human aortic endothelial cells (HAEC). HAEC (A) or U937 cells (B) were incubated without catechin or plasma extracts (control), with the plasma extract from control rats (plasma control), with 10 µmol (+)-catechin /L, or with the plasma extract from (+)-catechin–treated rats for 20 h. HAEC were stimulated with interleukin 1ß (IL-1ß; 10 µg/L) for 6 h and U937 cells were stimulated with phorbol myristyl acetate (PMA; 100 µg/L) for 2 h. The adhesion assay was performed as described in the Methods. Data are the mean (±SD) of 3 experiments, each performed in quadruplicate. ^*Significantly different from the other treatments, P < 0.05.

 
Effect of (+)-catechin and its plasma metabolites on ROS production in HAEC
The intracellular concentration of ROS in HAEC, as assessed by DCHF oxidation, was increased by hydrogen peroxide and IL-1ß treatments. Pretreatment of HAEC with plasma extract from control rats, (+)-catechin (10 µmol/L), or plasma extract from (+)-catechin–treated rats [containing (+)-catechin metabolites] for 20 h significantly inhibited the increase of intracellular ROS when cells were stimulated with hydrogen peroxide (Figure 4A). In contrast, when cells were challenged with IL-1ß, the generation of intracellular ROS in HAEC was significantly reduced only when HAEC were pretreated with plasma extract of (+)-catechin–treated rats (Figure 4B). Pretreatment of HAEC with plasma extract from control rats and intact (+)-catechin at a concentration of 10 µmol/L had no effect on ROS generation by IL-1ß stimulation.

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FIGURE 4. . Effect of (+)-catechin metabolites on the generation of reactive oxygen species in human aortic endothelial cells (HAEC). HAEC were incubated without catechin or plasma extracts (control), with the plasma extract from control rats (plasma control), with 10 µmol (+)-catechin/L, or with the extract from (+)-catechin–treated rats for 20 h, loaded with 50 µmol 2',7'-dichlorodihydrofluorescein diacetate/L for 30 min, and stimulated by 20 µmol H₂O₂/L (A) or 10 µg interleukin 1ß/L (B). 2',7'-Dichlorofluorescein (DCF) fluorescence was monitored 45 min after stimulation. Data are presented as the percentage increase in DCF fluorescence compared with unstimulated cells and are the mean (±SD) of 2 experiments, each performed in quadruplicate. ^*,**Significantly different from control: ^*P < 0.05, ^**P < 0.01.

 
Effect of quercetin and its plasma metabolites on U937 cell adhesion to HAEC
Quercetin inhibits immune cell–endothelial cell interaction (12, 13, 20). As shown in Figure 5A, pretreatment of HAEC with quercetin reduced U937 cell adhesion to IL-1ß–stimulated HAEC dose-dependently. In contrast, when HAEC were pretreated with plasma extract of quercetin-treated rats—in which free quercetin (0.05 µmol/L), glucuronide, or sulfate conjugates of quercetin and isorhamnetin (2.9 µmol/L) were present in the medium—U937 cell adhesion to IL-1ß–stimulated HAEC was not significantly reduced. When U937 cells were pretreated with 1 or 10 µmol quercetin/L for 20 h followed by PMA activation (100 µg/L) for 6 h, U937 cell adhesion to IL-1ß–stimulated HAEC was significantly inhibited (Figure 5B). However, pretreatment of U937 cells with plasma extract from quercetin-treated rats had no significant effect on PMA-activated U937 cell adhesion to IL-1ß-stimulated HAEC. In these experiments, plasma extract from control rats had no significant effect on U937 cell adhesion to HAEC.

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FIGURE 5. . Effect of quercetin metabolites on U937 cell adhesion to human aortic endothelial cells (HAEC). HAEC (A) and U937 cells (B) were incubated without catechin or plasma extracts (control) or with 1 or 10 µmol quercetin/L or the plasma extract from quercetin-treated rats for 20 h. HAEC were stimulated with interleukin 1ß (IL-1ß; 10 µg/L) for 6 h and U937 cells were stimulated with phorbol myristyl acetate (PMA; 100 µg/L) for 2 h. The adhesion assay was performed as described in the Methods. Data are the mean (±SD) of 3 experiments, each performed in quadruplicate. ^*,**Significantly different from the other treatments: ^*P < 0.05, ^**P < 0.01.

 

DISCUSSION  
Consumption of flavonoids is reported to be associated with a lowered risk of CVD mortality (4, 5). Despite mounting evidence for the biological activity of flavonoids—including antioxidant activity (6, 7), enzyme inhibition (8, 9, 43), and modulation of cell functions (10–13)—the precise mechanism or mechanisms by which flavonoids exert their beneficial effects are not well understood. This is partly because most in vitro studies to date used the flavonoid forms present in foods, rather than the forms that appear in plasma after absorption and metabolism. Therefore, in the present study, we investigated the metabolites of (+)-catechin and quercetin as they appeared in rat plasma after intragastric administration of pure compounds.

(+)-Catechin and quercetin are commonly found in food products (44) and their absorption, metabolism, and antioxidant activity have been studied in vivo (25, 27, 29, 32–36). (+)-Catechin and quercetin are detected as their sulfate or glucuronide conjugates in plasma (25, 27, 31, 32, 35). Administration of quercetin was shown to increase plasma antioxidant capacity in rats (25, 36). In vitro antioxidant activity was also reported for glucuronide conjugates of (+)-catechin (33) and quercetin (34), indicating that (+)-catechin and quercetin might act as antioxidants in biological systems even after metabolic conversion and conjugation.

In the present study, HAEC were treated with (+)-catechin metabolites at a concentration of 7.6 µmol/L, which was determined to be the total concentration of metabolites in the HPLC analysis (Figure 1). Although the physiologic concentration of (+)-catechin or quercetin in humans is not known, Hollman et al (45) reported a plasma quercetin concentration of 0.6 µmol/L after ingestion of 150 g fried onions containing the equivalent of 64 mg quercetin. Donovan et al (31) reported that the concentration of total conjugated forms was 91 ± 14 nmol/L in human plasma 1 h after consumption of 120 mL red wine. Richelle et al (46) reported concentrations of total epicatechin metabolites in human plasma of 0.38 and 0.7 µmol/L after consumption of 40 and 80 g chocolate, respectively. Lee et al (30) reported that glucuronide- and sulfate-conjugated metabolites of flavan-3-ols in plasma ranged from 0.50 to 0.99 µmol/L 1 h after consumption of 1.2 g decaffeinated green tea in warm water. In these studies, although the consumption of flavonoids was restricted in subjects before testing, previous daily food intake might have contributed to the total amount of flavonoid metabolites measured. Thus, the amount of (+)-catechin metabolites used in our study appears to approximate the amount normally found in plasma.

In our in vitro study, supplementing HAEC or U937 cells with pure (+)-catechin or with the extract of control plasma had no significant effect on U937 cell adhesion to HAEC, whereas the plasma extract of (+)-catechin–administered rats, which contained the metabolites of (+)-catechin, inhibited this process. Furthermore, supplementing HAEC with (+)-catechin metabolites decreased IL-1ß–induced generation of ROS. These results indicate that some metabolites of (+)-catechin, which might be formed from (+)-catechin by metabolic conversion during absorption and metabolism, are more biologically active than their parent compounds and inhibit the activation of HAEC by IL-1ß or of U937 cells by PMA, resulting in reduced U937 cell adhesion. Surprisingly, pretreatment of HAEC with (+)-catechin metabolites had no significant effect on HAEC expression of adhesion molecules such as intracellular adhesion molecule 1, vascular adhesion molecule 1, and E-selectin (data not shown) that are known to be involved in leukocyte adhesion to HAEC. Although not tested in this study, other adhesion molecules, including P-selectin (47), vascular monocyte adhesion-associated proteins that mediate endothelial adhesion to immune cells (48), and connecting segment 1 fibronectin, which is a ligand for very late-acting antigen-4 expressed on monocyte (49), might have been involved in inhibiting monocyte adhesion to endothelial cells by (+)-catechin metabolites. Inhibition of expression of adhesion molecules via decreased activation of nuclear factor B by other antioxidants such as vitamin E has been reported (50). Further study is needed to clarify the mechanism of inhibition of monocyte adhesion to HAEC mediated by (+)-catechin metabolites.

ROS are known to play an important role in the regulation of cell adhesion (51). For example, exposure of endothelial cells to IL-1ß induces leukocyte adhesion and enhances adhesion molecule expression, both of which are known to be mediated by cellular generation of ROS (52, 53). (+)-Catechin metabolites might scavenge ROS because of their antioxidant activity (33). Indeed, we found that hydrogen peroxide–induced ROS generation was significantly reduced by pretreatment of HAEC with (+)-catechin metabolites as well as with (+)-catechin and with plasma extract from control rats. However, only (+)-catechin metabolites reduced IL-1ß–induced ROS generation in HAEC, suggesting that some (+)-catechin metabolites may act not only as ROS scavengers but also as inhibitors of IL-1ß activation. Because intact (+)-catechin and plasma extract of control rats had an inhibitory effect against hydrogen peroxide–induced ROS generation but not against IL-1ß–induced ROS generation, metabolic conversion of (+)-catechin may produce an active compound to modulate U937 cell adhesion to IL-1ß–stimulated endothelial cells.

Quercetin was reported to be a potent antioxidant (54, 55) and to inhibit enzyme activity (8, 9, 43), inflammatory processes (20), and adhesion molecule expression (13). The present study also showed that pretreatment of HAEC or U937 cells with quercetin significantly inhibited U937 cell adhesion. Interestingly, when either U937 or HAEC were pretreated with the plasma extract of quercetin-treated rats, which contained quercetin metabolites, the inhibitory effect on U937 cell adhesion was not observed. In these experiments, the concentration of quercetin showing a significant inhibitory effect on PMA-activated U937 cell adhesion to HAEC was 1 µmol/L, whereas quercetin metabolites at a total concentration of 3.0 µmol/L showed no effect. These results indicate that the inhibitory effect of quercetin on U937 cell adhesion to HAEC was abolished or significantly lowered by metabolic conversion during absorption and metabolism. Previous studies found no intact quercetin in rat plasma after oral quercetin administration (25). In our study also, quercetin was detected at very low concentrations in rat plasma extract. Detecting intact forms required concentrating the plasma extract from quercetin-treated rats 10-fold. These results indicate that the biological activity observed in vitro with intact quercetin is different from that of the metabolites that appear in vivo, such as in plasma.

Because quercetin metabolites such as glucuronide or sulfate conjugates possess differential hydrophilicity than does intact quercetin, their cellular uptake might be different. Boulton et al (56) reported that when quercetin was added to a culture of the human hepatocarcinoma cell line HepG₂, although cellular uptake and metabolism through methylation had occurred, a large fraction of added quercetin was chemically degraded and the degradation products may have been biologically active. Plasma quercetin metabolites may have different stability against the oxidative degradation by cells, in which the products may be involved in the modulation of cell functions.

In conclusion, this study showed for the first time that (+)-catechin and quercetin exert differential effects on monocyte– endothelial cell interaction after metabolic conversion to their conjugated forms. Because (+)-catechin metabolites had an inhibitory effect on monocyte adhesion to IL-1ß–stimulated endothelial cells, these metabolites may be responsible for the beneficial effects of flavonoid-rich foods, including red wine, on cardiovascular disease risk.

ACKNOWLEDGMENTS  
We thank Hong Wang for technical assistance.

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