Dietary flavonoids with a catechol structure increase α-tocopherol in rats and protect the vitamin from oxidation in vitro
2006; Elsevier BV; Volume: 47; Issue: 12 Linguagem: Inglês
10.1194/jlr.m600291-jlr200
ISSN1539-7262
AutoresJan Frank, Alicja Z. Budek, Torbjörn Lundh, Robert S. Parker, Joy E. Swanson, Cátia F. Lourenço, Bruno Gago, João Laranjinha, Bengt Vessby, Afaf Kamal‐Eldin,
Tópico(s)Free Radicals and Antioxidants
ResumoTo identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (−)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of α-tocopherol (α-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of α-T, the capacities of the flavonoids to i) protect α-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce α-tocopheroxyl radicals (α-T · ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected α-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of α-T · . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the α-T · by the flavonoids. To identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (−)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of α-tocopherol (α-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of α-T, the capacities of the flavonoids to i) protect α-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce α-tocopheroxyl radicals (α-T · ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected α-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of α-T · . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the α-T · by the flavonoids. ERRATAJournal of Lipid ResearchVol. 48Issue 9PreviewIn the article "Dietary flavonoids with a catechol structure increase α-tocopherol in rats and protect the vitamin from oxidation in vitro" by Frank et al., published in the December 2006 issue of the Journal of Lipid Research (Volume 47, pages 2718–2725), the text giving the concentrations of vitamins contained in the vitamin premix that was used to make the rat diets contained errors. In the Materials and Methods section, under the heading "Experimental animals and diets" (page 2719, right column, second paragraph, lines 5–10), the corrected text should read as follows: Full-Text PDF Open Access Of the eight natural substances exerting vitamin E activity (α-, β-, δ-, and γ-tocopherols and α-, β-, δ-, and γ-tocotrienols), α-tocopherol (α-T) has traditionally been regarded as the most important vitamer because it exerts the highest biological activity of all vitamers when assessed in animal model systems (1Brigelius-Flohé R. Traber M.G. Vitamin E: function and metabolism.FASEB J. 1999; 13: 1145-1155Crossref PubMed Scopus (1263) Google Scholar). All forms of vitamin E are equally well absorbed in the small intestine. From the intestinal mucosal cells, vitamin E enters the circulation via the lymphatic system incorporated into lipoproteins and is eventually transported to the liver, from which α-T is preferentially secreted into the blood (1Brigelius-Flohé R. Traber M.G. Vitamin E: function and metabolism.FASEB J. 1999; 13: 1145-1155Crossref PubMed Scopus (1263) Google Scholar). Tocopherol-ω-hydroxylase catalyzes the initial step in the degradation of vitamin E to its water-soluble carboxyethyl hydroxychroman urinary metabolites and has a higher catalytic activity toward the non-α-vitamers (2Sontag T.J. Parker R.S. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status.J. Biol. Chem. 2002; 277: 25290-25296Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar); thus, it may be responsible for the several times higher concentrations of α-T compared with the other vitamers in blood and most other tissues. α-T, the major lipid-soluble antioxidant in blood and tissues (3Burton G.W. Joyce A. Ingold K.U. First proof that vitamin E is major lipid-soluble, chain-breaking antioxidant in human blood plasma.Lancet. 1982; 320: 327Abstract Scopus (274) Google Scholar), can scavenge reactive species and therefore is thought to have an important function in the prevention of degenerative diseases (4Brigelius-Flohé R. Kelly F.J. Salonen J.T. Neuzil J. Zingg J.M. Azzi A. The European perspective on vitamin E: current knowledge and future research.Am. J. Clin. Nutr. 2002; 76: 703-716Crossref PubMed Scopus (513) Google Scholar). High blood concentrations of vitamin E, for instance, have been associated with a reduced risk of heart disease and cancer (5Gey K.F. 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Med. 1996; 239: 111-117Crossref PubMed Scopus (140) Google Scholar). Hence, increasing vitamin E levels in blood and tissues may be helpful in the prevention of these conditions. Yet, the intake of high doses of supplemental vitamin E has been associated with increases in all-cause mortality (9Miller 3rd, E.R. Pastor-Barriuso R. Dalal D. Riemersma R.A. Appel L.J. Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality.Ann. Intern. Med. 2005; 142: 37-46Crossref PubMed Scopus (2156) Google Scholar) and heart failure (10Lonn E. Bosch J. Yusuf S. Sheridan P. Pogue J. Arnold J.M. Ross C. Arnold A. Sleight P. Probstfield J. et al.Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial.J. Am. Med. Assoc. 2005; 293: 1338-1347Crossref PubMed Scopus (1072) Google Scholar). These highly controversial findings, however, are currently a matter of debate (11Hathcock J.N. Azzi A. Blumberg J. Bray T. Dickinson A. Frei B. Jialal I. Johnston C.S. Kelly F.J. Kraemer K. et al.Vitamins E and C are safe across a broad range of intakes.Am. J. Clin. Nutr. 2005; 81: 736-745Crossref PubMed Scopus (254) Google Scholar) and are awaiting clarification. Nevertheless, the identification of alternative strategies to improve vitamin E status, preferably by simple means such as dietary intervention, may prove important for optimal nutrition. Plant foods and beverages contain a great number of potentially bioactive compounds acting, similarly to α-T, as strong antioxidants in vitro, among which the flavonoids are the most abundant. Flavonoids, including the flavonol quercetin (Q) and the flavanols (−)-epicatechin (EC) and (+)-catechin (C), are ubiquitously distributed in plant foods (12Kühnau J. The flavonoids. A class of semi-essential food components: their role in human nutrition.World Rev. Nutr. Diet. 1976; 24: 117-191Crossref PubMed Google Scholar, 13Scalbert A. Williamson G. Dietary intake and bioavailability of polyphenols.J. Nutr. 2000; 130: 2073-2085Crossref PubMed Google Scholar). In humans, the daily consumption of flavonols and flavanols has been estimated to be up to 30 and 124 mg, respectively (14Arts I.C. Hollman P.C. Feskens E.J. Bueno De Mesquita H.B. Kromhout D. Catechin intake might explain the inverse relation between tea consumption and ischemic heart disease: the Zutphen Elderly Study.Am. J. Clin. Nutr. 2001; 74: 227-232Crossref PubMed Scopus (309) Google Scholar, 15Beecher G.R. Overview of dietary flavonoids: nomenclature, occurrence and intake.J. Nutr. 2003; 133: 3248-3254Crossref Google Scholar). Flavonoids in general, and Q, EC, and C in particular, have a number of reported functions in vitro and in vivo that are hypothesized to promote health. In vitro, they have the ability, inter alia, to scavenge reactive oxygen and nitrogen species, chelate metal ions, inhibit redox-sensitive transcription factors (e.g., nuclear factor-κB), inhibit the expression of free radical-generating enzymes (e.g., inducible nitric oxide synthase) (16Frei B. Higdon J.V. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies.J. Nutr. 2003; 133: 3275-3284Crossref PubMed Google Scholar), and, thus, function as strong antioxidants (17Rice-Evans C.A. Miller N.J. Bolwell P.G. Bramley P.M. Pridham J.B. The relative antioxidant activities of plant-derived polyphenolic flavonoids.Free Radic. Res. 1995; 22: 375-383Crossref PubMed Scopus (1908) Google Scholar). Furthermore, previous studies support the notion of a regeneration of α-T from its α-tocopheroxyl radical (α-T · ) at water-lipid interfaces by dietary phenols (18Laranjinha J. Vieira O. Madeira V. Almeida L. Two related phenolic antioxidants with opposite effects on vitamin E content in low density lipoproteins oxidized by ferrylmyoglobin: consumption vs regeneration.Arch. Biochem. Biophys. 1995; 323: 373-381Crossref PubMed Scopus (179) Google Scholar) in a way reminiscent of that of vitamin C (19Packer J.E. Slater T.F. Willson R.L. Direct observation of a free radical interaction between vitamin E and vitamin C.Nature. 1979; 278: 737-738Crossref PubMed Scopus (1201) Google Scholar). Accordingly, Q, EC, and C have been shown to reduce chemically generated α-T · in SDS micelles and organic solutions (20Pedrielli P. Skibsted L.H. Antioxidant synergy and regeneration effect of quercetin, (−)-epicatechin, and (+)-catechin on α-tocopherol in homogeneous solutions of peroxidating methyl linoleate.J. Agric. Food Chem. 2002; 50: 7138-7144Crossref PubMed Scopus (141) Google Scholar, 21Zhou B. Wu L.M. Yang L. Liu Z.L. Evidence for alpha-tocopherol regeneration reaction of green tea polyphenols in SDS micelles.Free Radic. Biol. Med. 2005; 38: 78-84Crossref PubMed Scopus (155) Google Scholar). In agreement with ex vivo findings that C preserves α-T in human plasma (22Lotito S.B. Fraga C.G. Catechins delay lipid oxidation and alpha-tocopherol and beta-carotene depletion following ascorbate depletion in human plasma.Proc. Soc. Exp. Biol. Med. 2000; 225: 32-38Crossref PubMed Google Scholar), we previously reported that it increases α-T levels in rats (23Frank J. Lundh T. Parker R.S. Swanson J.E. Vessby B. Kamal-Eldin A. Dietary (+)-catechin and BHT markedly increase α-tocopherol concentrations in rats by a tocopherol-ω-hydroxylase-independent mechanism.J. Nutr. 2003; 133: 3195-3199Crossref PubMed Scopus (28) Google Scholar). Despite the great number of in vitro studies published on the effects of Q, EC, and C, the literature on their effects in vivo is limited. Therefore, we studied the effects of these major dietary flavonoids on the concentration of vitamin E in rats. In a search for potential mechanisms of polyphenol-vitamin E interactions, we determined the protection of vitamin E in human LDL, the reduction of α-T · in micellar solutions, and the effects of these flavonoids on the activity of vitamin E-metabolizing enzymes in hepatocyte cultures. This experimental strategy allows the direct comparison of the efficacy of these three related catechol-type flavonoids, which differ only marginally in the spatial configuration of their chemical structures, to interact with vitamin E in a range of in vivo and in vitro model systems. To study the interactions of dietary catechols with vitamin E, the project was divided into an in vivo part, to establish potential physiological effects, and several in vitro studies (using human LDL, SDS micelles, and HepG2 cells as model systems), to investigate the mechanisms underlying the observed in vivo effects. Thirty-two male, 21 d old Sprague-Dawley rats with a mean body weight of 63 g (B&K Universal AB, Sollentuna, Sweden) were used for this study. The rats were housed individually in Macrolon IV cages (Ehret GmbH and Co., Emmendingen, Germany) with aspen wood bedding (Beekay bedding; B&K Universal AB) in a conditioned room at 23°C and 50% relative humidity with 12 h of light (7:00 AM to 7:00 PM) and 12 h of darkness. Each cage was equipped with a water bottle with metal lid, a feed container attached to a stainless-steel plate to avoid overthrowing and spilling, two black plastic tubes that the rats used for resting and hiding, and a table tennis ball for playing. The rats had free access to feed and water throughout the experiment, which was carried out in accordance with the guidelines of and approved by the Ethical Committee for Animal Experiments in the Uppsala region. The basal diet was prepared from (all values in g/kg diet): maize starch, 528; casein (vitamin-free), 200; rapeseed oil, 100; sucrose, 80; cellulose powder, 40; mineral and trace element premix (Lactamin, Lidköping, Sweden), 40; vitamin premix (vitamin E-free; Lactamin), 10; and cholesterol, 2. The composition of the vitamin premix was as follows (mg/kg diet): retinol, 23.8; cholecalciferol, 3.0; thiamin, 4.0; riboflavin, 14.8; pyridoxine, 6.2; calcium pantothenate, 24.6; niacin, 40.0; cobalamin, 20.0; menadione, 3.1;biotin, 15.0; ascorbic acid, 1,429.0; inositol, 30.0; choline chloride, 2,000.0; folic acid, 0.5; and corn starch, 6,385.9. All vitamin E in the diet originated from the rapeseed oil (Izegem), which was obtained from a local grocery store. Tocopherol and tocotrienol concentrations in the oil were as follows (μg/g): α-T, 212; γ-T, 345; and δ-T, 8; α-, γ-, and δ-tocotrienols were present at concentrations of <5 μg/g. The mineral and trace element premix contained (mg/kg diet): KH2PO4, 13,653.2; CaCO3, 14,365.3; KCl, 996.8; NaCl, 7,189.6; MgSO4 × 1 H2O, 2,023.6; FeC6H5O7 × 5 H2O, 1,333.2; MnO, 109.7; Cu2C6H4O7 × 2.5 H2O, 25.2; Zn3(C6H5O7)2 × 2 H2O, 14.0; CoCl2 × 6 H2O, 0.8; KAI(SO4)2 × 2 H2O, 3.2; NaF, 10.0; KIO3, 3.6; Na2B4O7 × 10 H2O, 0.8; Na2SeO3, 2.7; Na2MoO4 × 2 H2O, 0.4; and corn starch, 267.9. Cholesterol and the phenolic compounds EC (Chemical Absract Service No. 490-46-0), (+)-catechin (CAS No. 154-23-4), and Q dihydrate (CAS No. 6151-25-3) were added to the basal diet at concentrations of 2 g/kg and purchased from Sigma Chemical Co. (St. Louis, MO). The 32 rats were divided into groups of eight animals with similar mean body weights and fed their respective diets for 30 days. Body weights were measured weekly. At the end of the experiment, the rats were food-deprived for 12 h before intraperitoneal injection of an overdose of sodium pentobarbital and euthanized by exsanguination. Blood samples were withdrawn from the heart, collected in tubes containing EDTA as anticoagulant, and centrifuged (1,000 g, 10 min), and the blood plasma was transferred to test tubes with screw caps and stored at −20°C until analyzed. Liver tissues were excised, weighed, and stored in 2-propanol at −80°C until analyzed. For the extraction of plasma tocopherol, blood plasma (500 μl) was mixed with ethanol containing 0.005% butylated hydroxytoluene (500 μl) and extracted with hexane (2 ml) after manual shaking for 3 min. The lipids from livers were extracted according to the method developed by Hara and Radin (24Hara A. Radin N.S. Lipid extraction of tissues with a low-toxicity solvent.Anal. Biochem. 1978; 90: 420-426Crossref PubMed Scopus (2094) Google Scholar) as described previously (25Frank J. Kamal-Eldin A. Razdan A. Lundh T. Vessby B. The dietary hydroxycinnamate caffeic acid and its conjugate chlorogenic acid increase vitamin E and cholesterol concentrations in Sprague-Dawley rats.J. Agric. Food Chem. 2003; 51: 2526-2531Crossref PubMed Scopus (29) Google Scholar). Briefly, the liver tissue was homogenized in hexane/2-propanol (3:2, v/v) and centrifuged, and the lipid extract was collected in a separatory funnel. The extract was washed with aqueous sodium sulfate, the supernatant was evaporated, and the lipids were weighed and dissolved in 10 ml of hexane. The extract was stored at −20°C until analyzed. Plasma and liver concentrations of tocopherols, cholesterol, and triacylglycerols were determined by standard methods as described previously (25Frank J. Kamal-Eldin A. Razdan A. Lundh T. Vessby B. The dietary hydroxycinnamate caffeic acid and its conjugate chlorogenic acid increase vitamin E and cholesterol concentrations in Sprague-Dawley rats.J. Agric. Food Chem. 2003; 51: 2526-2531Crossref PubMed Scopus (29) Google Scholar). LDLs were isolated from plasma of healthy volunteers by density gradient ultracentrifugation as described previously (26Vieira O.V. Laranjinha J.A. Madeira V.M. Almeida L.M. Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants.J. Lipid Res. 1996; 37: 2715-2721Abstract Full Text PDF PubMed Google Scholar). Briefly, plasma was centrifuged for 3 h at 290,000 g and 15°C, and the LDL fraction was collected and washed by ultrafiltration. Protein concentrations were measured according to the method of Lowry et al. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using BSA as the standard. Incubation of LDL with 4 μM α-T in the absence (control) or presence of 2 μM EC, C, or Q was carried out at 37°C in a closed glass vessel protected from light under a stream of N2. LDL particles were exposed to a constant flux of peroxyl radicals [generated from thermal decomposition of 2,2′-azobis(2-methylpropionamidine) dihydrochloride]. Aliquots were removed at 0, 5, 10, 20, and 30 min, and oxidation was immediately stopped by the addition of butylated hydroxytoluene. α-T concentrations in LDL were determined by HPLC as described previously (28Laranjinha J. Cadenas E. Redox cycles of caffeic acid, α-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation.IUBMB Life. 1999; 48: 57-65Crossref PubMed Google Scholar). Experiments were performed in duplicate. In a second experiment, human plasma was incubated with 50 μM EC or C before LDL isolation, and then the isolated LDLs were exposed to a constant flux of peroxyl radicals identical to the treatment of control samples described above. A micellar solution of SDS was prepared in 50 mM phosphate buffer (pH 7.4). α-T (in ethanol) was dispersed in SDS micelles to a final concentration of 2 mM. α-T · was generated by exposure of the micelles to ultraviolet (UV) irradiation for 3 min. Immediately upon terminating the UV irradiation, EC, C, or Q was added to the micelles at concentrations of 0, 5, 10, 25, 50, and 100 μM. Micellar solutions were then transferred to bottom-sealed Pasteur pipettes and immediately inserted into the electron paramagnetic resonance cavity of a Bruker EMX electron paramagnetic resonance spectrometer, and spectra were recorded using the following instrument settings: microwave frequency, 9.8 GHz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 2 G; time constant, 0.65 s. The time between deactivation of the UV irradiation and recording of the electron paramagnetic resonance spectra was 2 min for all samples. α-T · concentrations are given as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide equivalents. All measurements were performed in triplicate. The effect of EC, C, and Q on tocopherol-ω-hydroxylase activity was evaluated in a hepatocyte cell culture assay. HepG2 cells (subclone C3A; American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% FBS under conditions recommended by the supplier and used at 3–5 days after confluence. Test compounds, in ethanol stock solutions, were first added drop-wise to FBS, which was then diluted 10-fold with DMEM for a final concentration of 20 μM. Cells were preincubated with medium containing the test compounds for 4 h, after which the medium was changed to one containing 10 μM δ-T and 20 μM EC, C, or Q, or 1 μM sesamin. Sesamin (Cayman Chemical, Ann Arbor, MI) was used as a positive control. After 48 h of incubation, the concentrations of 3′- and 5′-δ-carboxychromanol metabolites in the medium were determined by gas chromatography-mass spectrometry of their trimethylsilyl ethers, using d9-α-3′-carboxychromanol as the internal standard, as described previously (29Parker R.S. Sontag T.J. Swanson J.E. Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin.Biochem. Biophys. Res. Commun. 2000; 277: 531-534Crossref PubMed Scopus (195) Google Scholar). Total cell protein was determined by dye binding (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA). Experiments were replicated three times, and representative results are shown. δ-T was used as a substrate for assays of tocopherol-ω-hydroxylase activity because it is a substantially better substrate than α-T and therefore offers greater sensitivity in assays of enzyme activity and inhibition. Concentrations of cell-associated δ-T were quantified by gas chromatography-mass spectrometry as described previously (29Parker R.S. Sontag T.J. Swanson J.E. Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin.Biochem. Biophys. Res. Commun. 2000; 277: 531-534Crossref PubMed Scopus (195) Google Scholar). Statistical comparisons were made using the statistical software StatView (version 4.51; Abacus Concepts, Inc., Berkeley, CA) by way of an ANOVA with the Bonferroni-Dunn posthoc test, and effects were considered significant at 5% (P < 0.0083). During the 30 days of this experiment, the rats generally ate all of the feed provided, amounting to a total consumption of 380 g per rat and a mean intake of Q, EC, or C of 25 mg/day. Animal performance, assessed by measuring feed consumption, weight gain, total body weight, and total and relative liver weight, was not affected by any of the test compounds (Table 1 ).TABLE 1Body weights, liver weights, and liver lipid contents of rats fed control or flavonoid-supplemented (2 g/kg) diets for 4 weeksVariableControlQuercetin(−)-Epicatechin(+)-CatechinBody weight (g)221 ± 7.4217 ± 11.2221 ± 6.9220 ± 4.8Liver weight (g)10.2 ± 1.310.3 ± 1.110.8 ± 0.810.2 ± 0.9Relative liver weight (g/100 g body weight)4.6 ± 0.54.7 ± 0.34.9 ± 0.34.6 ± 0.4Liver lipids (mg/g fresh weight)102 ± 1192 ± 1597 ± 14103 ± 26Values represent means ± SD (n = 8). No statistically significant differences were observed. Open table in a new tab Values represent means ± SD (n = 8). No statistically significant differences were observed. Rats fed Q, EC, or C had substantially increased concentrations of α-T in their blood plasma and liver (P < 0.0002) (Table 2 ), whereas γ-T concentrations were unaltered. In EC-fed rats, however, plasma and liver γ-T concentrations were slightly, although not significantly, increased. Consequently, the ratio of γ-T to α-T was significantly reduced in plasma (P < 0.0001) and liver (P < 0.0003) by Q and C but unaltered by EC. Consumption of Q, EC, or C did not change the plasma concentrations of triacylglycerols, total cholesterol, HDL-cholesterol, or VLDL+LDL-cholesterol, or the total cholesterol content of the liver (Table 3 ).TABLE 2Plasma and liver tocopherol concentrations of rats fed control or flavonoid-supplemented (2 g/kg) diets for 4 weeksVariableControlQuercetin(−)-Epicatechin(+)-CatechinPlasma (μmol/l)n = 7n = 8n = 8n = 8α-T6.5 ± 0.914.3 ± 2.8aDifferent from control at P < 0.0001.11.0 ± 1.6bDifferent from control at P < 0.001.14.3 ± 2.1aDifferent from control at P < 0.0001.γ-T0.7 ± 0.20.8 ± 0.31.1 ± 0.30.6 ± 0.2α-T + γ-T7.2 ± 1.115.1 ± 3.0aDifferent from control at P < 0.0001.12.0 ± 2.0bDifferent from control at P < 0.001.14.9 ± 2.2aDifferent from control at P < 0.0001.γ-T/α-T0.10 ± 0.030.05 ± 0.01aDifferent from control at P < 0.0001.0.10 ± 0.020.04 ± 0.01aDifferent from control at P < 0.0001.Liver (nmol/g)n = 8n = 8n = 8n = 8α-T13.1 ± 2.835.4 ± 2.4aDifferent from control at P < 0.0001.23.5 ± 2.3aDifferent from control at P < 0.0001.36.8 ± 4.2aDifferent from control at P < 0.0001.γ-T2.0 ± 0.92.1 ± 0.72.9 ± 0.72.0 ± 0.6α-T + γ-T15.1 ± 3.237.8 ± 2.1aDifferent from control at P < 0.0001.26.4 ± 2.7aDifferent from control at P < 0.0001.38.8 ± 4.6aDifferent from control at P < 0.0001.γ-T/α-T0.16 ± 0.080.07 ± 0.02bDifferent from control at P < 0.001.0.12 ± 0.030.05 ± 0.01aDifferent from control at P < 0.0001.α-T, α-tocopherol. Values represent means ± SD.a Different from control at P < 0.0001.b Different from control at P < 0.001. Open table in a new tab TABLE 3Plasma and liver cholesterol concentrations of rats fed control or flavonoid-supplemented (2 g/kg) diets for 4 weeksVariableControlQuercetin(−)-Epicatechin(+)-CatechinPlasma (mmol/l)n = 7n = 8n = 8n = 8Total cholesterol1.7 ± 0.41.7 ± 0.41.9 ± 0.31.6 ± 0.5HDL-cholesterol0.7 ± 0.10.7 ± 0.10.9 ± 0.20.7 ± 0.2VLDL+LDL-cholesterol0.5 ± 0.40.6 ± 0.40.6 ± 0.20.6 ± 0.5HDL-cholesterol/total cholesterol0.5 ± 0.20.4 ± 0.10.5 ± 0.10.4 ± 0.2Triacylglycerol1.1 ± 0.41.0 ± 0.30.9 ± 0.30.8 ± 0.2Livern = 8n = 8n = 8n = 8Total cholesterol (μmol/g)49.3 ± 8.841.6 ± 3.847.3 ± 8.345.2 ± 11.0Total cholesterol in liver lipids (%)18.8 ± 4.017.7 ± 2.219.0 ± 3.217.5 ± 3.7Values represent means ± SD. No statistically significant differences were observed. Open table in a new tab α-T, α-tocopherol. Values represent means ± SD. Values represent means ± SD. No statistically significant differences were observed. The tested flavonoids markedly protected α-T from oxidative degradation in human LDL challenged with a constant flux of peroxyl radicals in the order EC > C > Q (Fig. 1A ). Consistent with this observation, LDLs isolated from plasma preincubated with 50 μM EC or C, although devoid of detectable amounts of the flavanols, were protected from free radical-induced oxidative degradation of α-T (Fig. 1B). In SDS micelles, incubation with the flavonoids dose-dependently (0–100 μM) reduced the concentrations of α-T · generated by UV irradiation (Fig. 2 ).Fig. 1.α-Tocopherol concentrations (percentage of control) over time upon exposure to a constant flux of peroxyl radicals in LDL incubated with flavonoids (2 μM; A) and LDL isolated from human plasma preincubated with flavonoids (50 μM; B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2.Concentrations of α-tocopheroxyl radicals in micellar solutions of sodium dodecyl sulfate upon incubation with increasing concentrations of the antioxidants ascorbate, quercetin, (+)-catechin, and (−)-epicatechin. Error bars represent SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) None of the test compounds affected tocopherol-ω-hydroxylase activity in HepG2 cultures. The concentrations of δ-T metabolites (sum of 3′- and 5′-δ-carboxychromanols) in the 48 h cell culture medium were (means ± SD, n = 3) 171 ± 19, 173 ± 10, 200 ± 20, 198 ± 16, and 53 ± 3 nmol/l for control, EC, C, Q, and sesamin, respectively. Sesamin, which was used as a positive control to verify the functioning of the assay, inhibited tocopherol-ω-hydroxylase activity as expected. The uptake of δ-T into HepG2 cells was not affected by the test compounds (data not shown). To identify dietary phenolic compounds capable of improving vitamin E status in vivo, we chose the structurally related catechol-type flavonoids Q, EC, and C because of their abundance in the diet (13Scalbert A. Williamson G. Dietary intake and bioavailability of polyphenols.J. Nutr. 2000; 130: 2073-2085Crossref PubMed Google Scholar), their documented bioavailability in rats and humans (30Manach C. Texier O. Morand C. Crespy V. Regerat F. Demigne C. Remesy C. Comparison of the bioavailability of quercetin and catechin in rats.Free Radic. Biol. Med. 1999; 27: 1259-1266Crossref PubMed Scopus (134) Google Scholar), their reported sparing of α-T in various in vitro systems (20Pedrielli P. Skibsted L.H. Antioxidant synergy and regeneration effect of quercetin, (−)-epicatechin, and (+)-catechin on α-tocopherol in homogeneous solutions of peroxidating methyl linoleate.J. Agric. Food Chem. 2002; 50: 7138-7144Crossref PubMed Scopus (141) Goo
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