Absorption and Antioxidant Effects of Quercetin from Onions, in Man

Absorption and antioxidant effects of quercetin from onions, in man

GT McAnlis1,2, J McEneny2, J Pearce1 and IS Young*2

1Department of Food Science, The Queen's University of Belfast, Newforge Lane, Belfast; and 2Department of Clinical Biochemistry, The Queen's University of Belfast, Royal Victoria Hospital, Belfast, Northern Ireland

Objective: To investigate the in vivo effects of quercetin following the ingestion of fried onions. Design: Five healthy volunteers, three males and two females aged between 25 and 39 y, ingested 225 g of fried onions after an overnight fast and peripheral venous blood was collected 0, 2, 4, 24 and 48 h after consumption. Quercetin in the plasma, total antioxidant capacity and susceptibility of low density lipoproteins (LDL) to oxidation were measured. Results: Following the onion meal, quercetin levels increased from baseline values (28.4Æ 1.9 ng=ml) to peak after 2 h (248.4Æ 103.9 ng=ml), decreasing to baseline again after 24 h (P > 0.05). This was accompanied by an increase in the total antioxidant activity of the plasma from baseline (1.70Æ 0.04 mmol=l trolox equivalents) to 1.75Æ 0.10 mmol=l trolox equivalents after 2 h and 1.76Æ 0.08 mmol=l trolox equivalents after 4 h. There was no signi®cant change in the susceptibility of the plasma or the isolated LDL to oxidation over the 48 h period after consumption of the fried onions. In view of these negative ®ndings, we isolated LDL and other lipoproteins from plasma at each time point. Quercetin was not detected in either LDL or VLDL, but was present in the HDL fraction, although this fraction also contains other proteins including albumin. Conclusions: Quercetin can be absorbed in humans from dietary sources to high enough concentrations to increase the overall antioxidant activity of the plasma. Quercetin, however, has a strong af®nity for protein and provides no direct protective effect during LDL oxidation. Sponsorship: This work was funded by the Department of Agriculture for Northern Ireland. Descriptors: ¯avonoids; low density lipoprotein; onions; quercetin

Introduction is tea and in the US and Northern Europe the main source is onions (Hertog et al, 1995). Quercetin occurs naturally in Flavonoids are a group of polyphenolic compounds which onions in the form of glucoside, with the main glucosides are widely distributed throughout the plant kingdom being quercetin-3,40-O-bis-b-D-glucoside and quercetin-40- (Kuhnau et al, 1976). They occur naturally in fruit and O-b-D-glucoside (Kiviranta et al, 1988). vegetables and are, therefore, an integral part of the human Several mechanisms may contribute to the protective diet (Hertog et al, 1992, 1993a). A number of epidemiol- role of ¯avonoids against cardiovascular disease, but ogical studies have shown an inverse correlation between recently much attention has been paid to their antioxidant dietary ¯avonoid intake and mortality from coronary heart properties. It is currently believed that oxidative modi®ca- disease (CHD) (Hertog et al, 1993b, 1995; Knekt et al, tion of low density lipoproteins (LDL) by free radicals is a 1996; Keli et al, 1996). The epidemiological evidence is, key early event in the pathogenesis of atherosclerosis however, not entirely consistent as a study involving (Steinberg et al, 1989). The rapid uptake of oxidatively American male health workers (Rimm et al, 1996) and modi®ed LDL via a scavenger receptor leads to the forma- the Caerphilly Heart Study (Hertog et al, 1997) found no tion of foam cells, and oxidised LDL also has a number of correlation between ¯avonoid intake and mortality from other atherogenic properties (Goldstein et al, 1979). A heart disease. number of mechanisms are likely to contribute to inhibition Dietary intake of ¯avonoids in Western Europe is of LDL oxidation by ¯avonoids. Flavonoids may directly estimated to be about 25 mg per day of ¯avonol (quercetin, scavenge some radical species by acting as chain-breaking kaempferol and myricetin) and ¯avone (luteolin and api- antioxidants (de-Whalley et al, 1990). In addition, they genin) aglycones (Hertog et al, 1993b). The main sources may recycle other chain-breaking antioxidants such as a- are tea, onions, apples and red wine (Hertog et al, 1993b); tocopherol by donating a hydrogen atom to the tocopheryl approximately 70% of our total ¯avonoid intake is querce- radical (Frankel et al, 1993). Transition metals such as iron tin (Hertog et al, 1993b). Major dietary sources of quercetin and copper are important pro-oxidants, and some ¯avo- show considerable geographical and cultural variation; in noids can chelate divalent metal ions, hence preventing free Italy the main source is red wine, in China the main source radical formation (de-Whalley et al, 1990). Extensive in vitro studies have found most of the main dietary ¯avonoids to be effective in protecting against the *Correspondence: Dr IS Young, Department of Clinical Biochemistry, The oxidative modi®cation of LDL (de-Whalley et al, 1990; Queen's University of Belfast, Royal Victoria Hospital, Belfast, Northern Ireland Laughton et al, 1991; Morel et al, 1993; Rankin et al, 1993; Received 3 May 1998; revised 13 August 1998; accepted 22 August 1998 Igarashi & Ohmuma, 1995; Fuhrman et al, 1995). The Absorption and antioxidant effects of quercetin GT McAnlis et al 93 ability of quercetin, and the quercetin glycosides, to protect isolated from plasma by rapid ultracentrifugation as LDL against oxidative modi®cation has been extensively described by McDowell et al (1995). HDL and VLDL studied, in every case showing a signi®cant protective were separated at the same time; the HDL appears as a effect (de-Whalley et al, 1990; McAnlis et al, 1997). It is clear band just above the plasma and the VLDL was therefore plausible that the consumption of ¯avonoid-rich removed by tube slicing (Beckman tube slicer) above the foods could reduce LDL oxidation and atherogenesis in LDL band. HDL protein concentration was standardised to vivo. However, the ef®ciency of the protection of LDL in 350 mg=l before HPLC analysis. In a separate study HDL vivo will depend on several factors, including the absorp- was isolated using the same methodology as above, but tion of the ¯avonoids and how they are distributed within centrifuging for 2 h. This reduced the albumin contamina- the plasma lipoprotein fractions. tion in the HDL. The concentration of albumin was The structure ± activity relationships and the absorption measured using radial immunodiffusion (RID). The purity and metabolism of the main dietary ¯avonoids have been of lipoprotein preparations was con®rmed by agarose gel extensively reviewed by Rice-Evans et al (1996). Only a electrophoresis using a Beckman Paragon system. small number of in vivo studies have been carried out to determine how effectively the ¯avonoids are absorbed. LDL oxidation Gugler et al (1975) found that after oral supplementation The LDL protein concentration was standardised to the quercetin aglycone was absorbed at < 1% ef®ciency. 50 mg=l and oxidation was catalysed by copper Nieder et al (1991) reported that ¯avonol glycosides from (2 mmol=l) at 37C. Conjugated diene formation was mon- Ginkgo biloba could be absorbed, but no details were given itored at l 234 nm. The lag phase prior to the onset of about quantities ingested and how ef®ciently they were rapid LDL oxidation is seen as a slope with a slow increase absorbed. More recently a sensitive technique for measur- in absorbance. The duration of the lag phase is indicative of ing ¯avonoids in plasma has been developed (Hollman et the resistance to oxidation of the LDL being examined. al, 1996). Plasma quercetin levels were found to reach Inter-assay c.v. was < 5% and intra-assay c.v. < 1%. maximum plasma concentrations in two subjects about 2.7 h after the ingestion of a fried onion breakfast (Hollman Susceptibility of plasma to oxidation et al, 1996). These ®ndings suggest that signi®cant ¯avo- The susceptibility of plasma to oxidation was measured noid absorption may occur following onion ingestion and using the method of Nyyssonen et al (1997). Plasma was point to a possible role for quercetin in protecting LDL diluted to a concentration of 0.67% in 0.02 mol=l phosphate against oxidative modi®cation. However, little is known buffered saline (PBS), pH 7.4. Tubes were gently inverted about how quercetin interacts with the plasma lipoproteins to ensure plasma had mixed well. Oxidation of the plasma and whether or not the quercetin is absorbed at great was initiated by the addition of 50 ml of 1 mmol=l CuCl2 enough levels to increase the antioxidant capacity of the into 1 ml pre-warmed (30C) plasma. The formation of plasma. In view of this, the aim of the present study was to conjugated dienes was followed by monitoring the change investigate the in vivo effects of quercetin on total anti- in absorbance at 234 nm at 30C using an Hitachi U2000 oxidant activity and susceptibility of LDL to oxidation spectrophotometer ®tted with a six-cell positioner. Inter- following the ingestion of fried onions. assay c.v. was < 10% and intra-assay c.v. was < 2.5%.

Methods HPLC analysis The HPLC system consisted of a TSP spectra system Chemicals (Thermo Separation Products) P2000 pump, a TSP A1000 All chemicals, unless otherwise stated, were purchased autoinjector and a TSP UV1000 detector. from Sigma Chemical Co., Poole, Dorset, UK and were > 95% pure. 1. Onions Study design Quercetin was determined quantitatively using a technique Five healthy volunteers, three males and two females aged developed by Hertog et al, 1992. Freeze-dried onion between 25 and 39 y, ingested 225 g of fried onions (Sains- powder (0.5 g) was hydrolysed with 1.2 M HCl for 2 h at bury's extra large onions, class II) after an overnight fast, 90C. The chromatography was carried out using an Ultra- and peripheral venous blood was collected 0, 2, 4, 24 and carb ODS 20 C18 (Phenomenex Ltd., UK) column 48 h after consumption. (4.66250 mm, 5 mm) protected by an Ultracarb ODS 20 C18 (Phenomenex Ltd., UK) guard column (4.6630 mm, Total antioxidant activity 5 mm). The mobile phase was ethanol ± water (70 ± 30) plus This was carried out according to a technique developed by 1% acetic acid, and peaks were detected at 375 nm; Miller et al (1996). The assay assesses the relative ability of calibration curves ranging from 5 ± 50 mg=ml were prepared antioxidants to scavenge the 2,20-azino-bis(3-ethylbenz- for quercetin. Inter-assay c.v. was < 5% and intra-assay thiazoline-6-sulphonic acid) (ABTS) radical cation in com- c.v. < 1%. parison with a standard amount of Trolox, a water soluble vitamin E analogue. The assay was automated using a 2. Quercetin in biological ¯uids COBAS FARA centrifugal analyser (Roche Products This assay was carried out according to a technique devel- Ltd., Welwyn Garden City, Herts., UK) (734 nm, 30C). oped by Hollman et al (1996). In a 4 ml vial, 1.0 ml of Inter-assay c.v. was < 3% and intra-assay c.v. was < 1%. methanol containing 2 g=l tert-butyl hydroxyquinone (TBHQ), 0.4 ml of 10 M HCl and 0.6 ml of plasma were Isolation of LDL=HDL and VLDL mixed. The vial was sealed tightly with a screw cap and Plasma was obtained from heparinised blood samples by septum and inserted into a preheated aluminium block at centrifugation at 1200 g for 10 min at 4C. LDL was 90C for 2 h. The vials were allowed to cool and 2.0 ml of Absorption and antioxidant effects of quercetin GT McAnlis et al 94 methanol containing 2 g=l TBHQ was added. The vials were then centrifuged at 1000 g for 15 min and 20 ml of the upper layer was injected onto the HPLC column. Chromatographic separations were carried out on an Inertsil ODS-2 (GL Sciences Inc., Tokyo, Japan) column (4.66150 mm, 5 mm) protected with an Ultracarb ODS (Phenomenex, Cheshire, UK) guard column. The main column was placed in a column oven at 30C. The HPLC system was the same as above with a ¯uorescence detector (Waters, UK) (Em 485 nm, Ex 422 nm) instead of a UV detector. The mobile phase consisted of aceton- itrile=methanol=(0.025 M) phosphate buffer pH 2.4, 10:38:52 (v=v=v), set at a ¯ow rate of 1 ml=min. The post-column ef¯uent was mixed with 0.4 ml=min 1.5 M aluminium nitrate in methanol containing 7.5% (v=v) acetic acid in a postcolumn reaction coil (3.65 m Figure 2 Changes in the total antioxidant capacity of the plasma after 60.5 mm) placed in the column oven at 30C. Inter- ingesting 225 g fried onions. Mean of 5 observationsÆ s.d. assay c.v. was < 8% and intra-assay c.v. was < 1%. consumption of the fried onions (Figure 3). Similarly, the Incubation studies consumption of onions had no signi®cant effect on the Plasma was incubated with ethanolic additions of the main susceptibility of isolated LDL to copper oxidation in vitro dietary ¯avonoids at ®nal concentration of 1.0 mM for 4 h at (baseline lag time, 63.8Æ 2.8 min; 2 h after ingesting onions 37C. In a separate study, plasma was incubated with 63.4Æ 4.1 min). In view of these negative ®ndings, LDL quercetin (2.0 mM ®nal concentration) for 24 h at 4C. was isolated along with other lipoproteins from plasma at Studies were carried out according to the guidelines of each time point to determine their quercetin contents. the ethics committee of the Queen's University of Belfast. Quercetin was not detected in either LDL or VLDL at Results before and after ingestion of onions were compared any time point (Table 1). However, quercetin was present using paired t-tests in comparison to baseline. in the HDL fraction (Table 1). The HDL was puri®ed using a further ultracentrifugation process. The albumin concentration was reduced by > 75%, using ultracentrifugation for Results 2 h, as con®rmed by RID. There was no measurable Analysis of the quercetin in the onions showed that the total quercetin in the HDL fraction after puri®cation by ultra- intake of quercetin aglycone from 225 g fried onions was centrifugation for 2 h. 50.4Æ 3.8 mg. Following the onion meal, the plasma These results suggested that quercetin is not incorpo- quercetin concentrations increased from baseline values rated into lipoproteins in vivo in concentrations suf®cient to (28.4Æ 1.9 ng=ml) to peak after 2 h (248.4Æ 103.9 ng=ml), increase resistance to oxidation. In order to con®rm this, decreasing to baseline again after 24 h (Figure 1). This was plasma was incubated with various ¯avonoids prior to accompanied by an increase in the total antioxidant isolating the LDL. There was no signi®cant change in the activity of the plasma from baseline (1.70Æ 0.04 mmol=l susceptibility of LDL to oxidation between plasma incu- trolox equivalents) to 1.75Æ 0.10 mmol=l trolox equiva- bated for 4 h with ¯avonoids at 1.0 mM (quercetin= lents after 2 h and 1.76Æ 0.08 mmol=l trolox equivalents rutin=kaempferol and apigenin), and controls incubated (P < 0.05) 4 h after the ingestion of 225 g of fried onion with the same volume of ethanol (Table 2). There was no (Figure 2). signi®cant change in the susceptibility of LDL to oxidation Despite the increase in quercetin and total antioxidant between plasma incubated for 24 h with 2.0 mM quercetin capacity, there was no signi®cant change in the suscept- and the control incubated with the same volume of ethanol. ibility of the plasma to oxidation over the 48-h period after The lag times were 63.8Æ 4.1 min and 61.2Æ 0.2 min for LDL alone and LDL with 2.0 mM quercetin, respectively.

Discussion The average daily intake of dietary ¯avonoids compares favourably to the other main dietary antioxidants such as a- tocopherol and b-carotene (Hertog et al, 1993). This would

Table 1 Quercetin present in plasma and the main lipoprotein fractions. Values expressed as mean of ®ve observationsÆ s.d. HDL was standardised to 250mg=l protein, LDL and VLDL were standardised to 50mg=l prior to quercetin analysis

Time (h) Plasma (ng=ml) HDL (ng=ml) LDL (ng=ml) VLDL (ng=ml)

0 28.4Æ 2.1 69.3Æ 0.0 < 20.0 < 20.0 2 248.4Æ 116.2 79.7Æ 62.5 < 20.0 < 20.0 4 166.3Æ 113.1 117.4Æ 78.0 < 20.0 < 20.0 24 40.3Æ 14.7 21.9Æ 0.0 < 20.0 < 20.0 Figure 1 Plasma quercetin levels after ingesting 225 g fried onions, 48 31.8Æ 6.5 < 20.0 < 20.0 < 20.0 equivelant to 50.4Æ 3.8 mg quercetin. Mean of 5 observationsÆ s.d. Absorption and antioxidant effects of quercetin GT McAnlis et al 95 The total antioxidant activity of the plasma reached a peak 2 h after ingestion corresponding to the peak in plasma quercetin (Figure 2). A number of studies have reported increases in antioxidant capacity after the ingestion of ¯avonoid rich foods (Sera®ni et al, 1996; Whitehead et al, 1995) and have suggested this may also lead to a protective effect against LDL oxidation, thereby showing the progression of atherosclerosis. The peak concentration of plasma quercetin (0.73Æ 0.30 mM), when compared to in vitro studies, should be high enough to protect the LDL from oxidative modi®cation (McAnlis et al, 1997). How- ever, after isolating and oxidising the LDL there was no signi®cant prolongation of lag time. HPLC analysis of the main lipid fractions con®rmed that no quercetin could be detected in the LDL or VLDL fractions. Quercetin appeared to be present in the protein-rich HDL fraction (Table 1), probably due to the high af®nity of ¯avonoids for Figure 3 Changes in total plasma oxidation lag times after ingesting 225 g fried oninos, equivelant to 50.4Æ 3.8 mg quercetin. Mean of 5 proteins (Rice-Evans et al, 1996). The albumin was observationsÆ s.d. reduced in the HDL fraction by further ultracentrifugation after which the quercetin concentrations were no longer high enough to be measurable, suggesting the majority of Table 2 Changes in LDL lag time after incubating with ¯avonoids for 4h at 37C. Mean of three observationsÆ s.d. the quercetin was bound to the albumin protein and not present within the HDL. Flavonoid Concentration (mM) Lag time (min) The majority of in vitro studies until recently have used Quercetin 0 67.3Æ 3.4 isolated LDL to which a metal or non-metal catalyst is 1.0 63.3Æ 1.9 added and the resistance to oxidation is measured. Oxida- Rutin 0 54.1Æ 4.2 tion of the isolated LDL excludes the effect of antioxidants 1.0 48.8Æ 1.8 present in the aqueous phase in vivo, the ¯avonoids are Apigenin 0 50.7Æ 0.9 1.0 51.5Æ 2.3 known to be sparingly soluble in both the aqueous and lipid Kaempferol 0 56.9Æ 2.7 phases depending on their chemical structure (Kuhnau, 1.0 60.6Æ 2.7 1976; Rice-Evans et al, 1996). Using the method of Nyyssonen et al (1997) to oxidise LDL within whole plasma should give a better overall view of the oxidation in vivo. However, again there was no signi®cant difference suggest that they may have more signi®cant nutritional in the susceptibility of the LDL, within the whole plasma, bene®ts than has been previously recognised. However, to oxidation (Figure 3). there is still little information about the absorption, dis- It is therefore clear from this in vivo study that quercetin tribution, metabolism and excretion of ¯avonoids in can be absorbed and increase the total antioxidant capacity humans. Kuhnau (1976) concluded that only ¯avonoid of the plasma without having a signi®cant protective effect aglycones could pass through the gut wall, because the on LDL oxidation. In part, this is because quercetin is not naturally occurring glycoside were too large. Hydrolysis signi®cantly incorporated into the LDL particle. This would was thought to occur in the colon as a result of the action of explain the ®nding of a number of recent in vivo studies micro-organisms which at the same time degrade the which have concluded that the ingestion of tea, a beverage ¯avonoids, giving a poor absorption ef®ciency (Kuhnau, rich in ¯avonoids, does not protect the LDL against 1976). Gugler et al (1975) concluded < 1% of quercetin oxidation in vivo (van het Hof et al, 1997; McAnlis et aglycone was absorbed after supplementing subjects with al, 1998; Princen et al, 1998). The ¯avonoids may be 4 g of quercetin. However, more recently, using more absorbed from the tea into the plasma, as shown by Lee accurate analysis techniques, Hollman et al (1995) demon- et al (1995), but become bound to the plasma proteins strated that 24% of orally administered quercetin aglycone leaving them unable to protect the LDL particle against was absorbed. In a further study they found the quercetin oxidation. glucosides present in onions were shown to be absorbed The antioxidant effects of ¯avonoids, especially querce- even more ef®ciently than the aglycone (Hollman et al, tin, have been studied extensively in vitro (de-Whalley et 1996). al, 1990; McAnlis et al, 1997). Most of the LDL studies The object of this study was to assess the biological have used puri®ed and isolated LDL to which ¯avonoids effects and distribution within the plasma of quercetin after are added before oxidation is initiated by a metal or non- ingesting onions. The amount of quercetin present in the metal catalyst (de-Whalley et al, 1990). Under these con- onion meal was 50.4Æ 3.8 mg, signi®cantly greater than the ditions, ¯avonoids may be acting in the aqueous phase average daily intake of 25 mg=d (Hertog et al, 1993). The without incorporation into the LDL particle. As the results quercetin concentration in plasma peaked 2 h after inges- from our in vivo study suggested that quercetin does not tion at 248.4Æ 103.9 ng=ml (Figure 1), corresponding to a accumulate within the LDL particle at all, in vitro incuba- plasma quercetin concentration of 0.73Æ 0.30 mM, and then tion studies were carried out to assess ¯avonoid uptake by decreased, returning to baseline levels 24 h after ingestion. LDL. In the ®rst study plasma was incubated with the main These results are comparable to those of Hollman et al dietary ¯avonoids (1.0 mM) for 4 h at 37C. In the second (1996) in a similar study where two subjects consumed study the experiment was repeated using only quercetin 215 g of fried onions. (20.0 mM) and incubating for 24 h instead of 4 h. There was Absorption and antioxidant effects of quercetin GT McAnlis et al 96 still no reduction in the susceptibility of the LDL to van het Hof K, de Boer HSM, Wiseman SA, Lien N, Weststrate JA & oxidation despite the concentrations being signi®cantly Tijburg LBM (1997): Consumption of green or black tea does not increase resistance of low-density lipoprotein to oxidation in humans higher than the peak in vivo concentrations ( 0.7 mM). .Am. J. Clin. Nut. 66, 1125 ± 1132. Both of these incubation studies suggest ¯avonoids are not Igarashi K & Ohmuma M (1995): Effect of Isohamneti, Rhamnetin, and taken up signi®cantly into the LDL particle. Although it Quercetin on the concentrations of cholesterol and lipoperoxide in the remains possible that ¯avonoid metabolites have a higher serum and liver and on the blood and liver antioxidative enzyme af®nity for the LDL particle, the present in vivo studies did activities in rats. Biosci. Biotech. Biochem. 59, 595 ± 601. Keli SO, Hertog MGL, Feskens EJM & Kromhout D (1996): Dietary not provide any evidence for this. ¯avonoids, antioxidant vitamins, and incidence of stroke. Arch. Int. Med. 156, 637 ± 642. Kiviranta J, Huovinen K & Hiltunen R (1988): Variation of phenolic Conclusion substances in onion. Acta Pharm. Fenn. 97, 67 ± 72. It is clear that ¯avonoids can be absorbed in humans from Knekt P, Jarvinen R, Reunanen A & Maatela J (1996): Flavonoid intake and coronary mortality in Finland: a cohort study. Br. Med. J. 312, dietary sources to high enough concentrations to increase the 478 ± 481. overall antioxidant activity of the plasma. The ¯avonoids, Kuhnau J (1976): The Flavonoids: A class of semi-essential food compo- however, have a strong af®nity for protein and provide no nents; their role in human nutrition. World Rev. Nutr. Diet 24, 117 ± direct protective effect during LDL oxidation, although they 191. may still exert an indirect antioxidant effect as a result of Laughton MJ, Evans PJ, Moroney MA, Hoult JRS & Halliwell B (1991): Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by incorporation into vascular cells or by protecting endogenous ¯avonoids and phenolic dietary additives. Biochem. Pharmacol. 42, antioxidants. Further research using labelled ¯avonoids in 1673 ± 1681. vivo would help to clarify how the ¯avonoids are absorbed, Lee MJ, Wang ZY, Li H, Sun Y, Gobbo S, Balentine DA & Yang CS distributed and metabolised. (1995): Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol. Biomarkers and Prevention 4, 393 ± 399. McAnlis GT, McEneny J, Pearce J & Young IY (1997): The effect of various dietary ¯avonoids on the susceptibility of low density lipoproteins to oxidation in vitro using both metallic and non-metallic oxidising agents. Biochem. Soc. Trans. 25, 142S. References McAnlis GT, McEneny J, Pearce J & Young IY (1998): Black tea consumption does not protect low density lipoprotein from oxidative de-Whalley C, Rankin SM, Hoult JRS, Jessup W & Leake DS (1990): Flavonoids inhibit the oxidative modi®cation of low density lipopro- modi®cation. Eur. J. Clin. Nut. 52, 202 ± 206. teins by macrophages. Biochem. Pharmacol. 39, 1743 ± 1750. McDowell IFW, McEneny J & Trimble ER (1995): A rapid method for Frankel EN, Kanner J, German JB, Parks E & Kinsella JE (1993): measurement of the susceptibility to oxidation of LDL. Clin. Biochem. Inhibition of oxidation of human low-density lipoprotein by phenolic 32, 167 ± 174. substances in red wine. Lancet 341, 454 ± 457. Miller NJ & Rice-Evans CA (1996): Spectrophotometric determination of Fuhrman B, Lavy A & Aviram M (1995): Consumption of red wine with antioxidant activity. Redox Report. 2, 161 ± 171. meals reduces the susceptibility of human plasma and low-density Morel I, Lescoat G, Cogrel P & Cillard J (1993): Antioxidant and lipoprotein to lipid peroxidation. Am. Soc. Clin. Nutr. 61, 549 ± 554. iron-chelating activities of the ¯avonoids catechin, quercetin and Goldstein JL, Ho YK, Basu SK & Brown MS (1979): Binding site on diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. macrophages that mediates uptake and degradation of acetylated low 45, 13 ± 19. density lipoprotein, producing massive cholesterol deposition. Proc. Nieder M (1991): Pharmakokinetik der Ginkgo-¯avonole im plasma. Natl. Acad. Sci. 76, 333 ± 337. Munch Med Wochenschr 133, S61 ± S62. Gugler R, Leschik M & Dengler HJ (1975): Disposition of quercetin in man Nyyssonen K, Porkkala-Sarataho E, Kaikkonen J & Salonen JT (1997): after single oral and intravenous doses. E. J. Clin. Pharm. 9, 229 ± 234. Ascorbate and urate are the strongest determinants of plasma antiox- Hertog MGL, Hollman PCH & Katan MB (1992): Content of potentially idative capacity and serum lipid resistance to oxidation in Finnish men. anticarcinogenic ¯avonoids of 28 vegetables and 9 fruits commonly Atherosclerosis 130, 223 ± 233. consumed in The Netherlands. J. Agric. Food Chem. 40, 2379 ± 2383. Princen HMG, van Duyvenvoorde W, Buytenhek R, Blonk C, Tijburg Hertog MGL, Hollman PCH & Putte B (1993a): Content of potentially LBM, Langius JAE, Meinders AE & Pijl H (1998): No effect of anticarcinogenic ¯avonoids of tea infusions, wines, and fruit juices. J. consumption of green and black tea on plasma lipid and antioxidant Agric. Food Chem. 41, 1242 ± 1246. levels and on LDL oxidation in smokers. Arterioscl. Thromb. Vasc. Hertog MGL, Feskens EJM, Hollman PCH, Katan MB & Kromhout D Biol. 18, 833 ± 841. (1993b): Dietary antioxidant ¯avonoids and risk of coronary heart Rankin SM, De Whalley CV, Hoult JRS, Jessup W, Wilkins GM, Collard J disease: the zutphen elderly study. Lancet 342, 1007 ± 1011. & Leake DS (1993): The modi®cation of low density lipoprotein by the Hertog MGL, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza ¯avonoids myricetin and gossypetin. Biochem. Pharmacol. 45, 67 ± 75. F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, Pekkarinen M, Rice-Evans CA, Miller NJ, Paganga G (1996): Structure ± antioxidant Simic BS, Toshima H, Feskens EJM, Hollman PCH & Katan MB activity relationships of ¯avonoids and phenolic acids. Ree Rad. Biol. (1995): Flavonoid intake and long-term risk of coronary-heart-disease Med. 20, 933 ± 956. and cancer in the 7 countries study. Arch. Int. Med. 155, 381 ± 386. Rimm EB, Katan MB, Ascerio A, Stampfer MJ & Willet WC (1996): Hertog MGL, Sweetnam PM, Fehily AM, Elwood PC & Kromhout D (1997): Relation between intake of ¯avonoids and risk of coronary heart disease Antioxidant ¯avonoids and ischemic heart disease in a Welsh population in male health professionals. Ann. Intern. Med. 125, 384 ± 389. of men: the Caerphilly Study. Am. J. Clin. Nutr. 65, 1489 ± 1494. Sera®ni M, Ghiselli A & Ferro-Luzzi A (1996): In vivo antioxidant effect Hollman PCH, Vries JHM, Leeuwen SD, Mengelers MJB & Katan MB of green and black tea in man. Eur. J. Clin. Nut. 50, 28 ± 32. (1995): Absorption of dietary quercetin glycosides and quercetin in Steinberg D, Parthasarathy S, Carew TE, Khoo JC & Witztum JL (1989): healthy ileostomy volunteers. Am. J. Clin. Nut. 62, 1276 ± 1282. Beyond cholesterol. New England J. Med. 320, 915 ± 924. Hollman PCH, Vantrijp JMP & Buysman MNCP (1996): Fluorescence Whitehead TP, Thorpe GHG & Maxwell SRJ (1992): Enhanced chemilu- detection of ¯avonols in HPLC by postcolumn chelation with alumi- minescent assay for antioxidant capacity in biological ¯uids. Anal. num. Anal. Chem. 68, 3511 ± 3515. Chem. 266, 265 ± 277.