Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: Cause, consequence, or epiphenomenon?

Free Radical Biology & Medicine 41 (2006) 1727 – 1746 www.elsevier.com/locate/freeradbiomed

Review Article

Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: Cause, consequence, or epiphenomenon? Silvina B. Lotito, Balz Frei ⁎ Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331, USA Received 28 December 2005; revised 18 April 2006; accepted 20 April 2006 Available online 3 June 2006

Abstract Increased fruit and vegetable consumption is associated with a decreased incidence of cardiovascular diseases, cancer, and other chronic diseases. The beneficial health effects of fruits and vegetables have been attributed, in part, to antioxidant flavonoids present in these foods. Large, transient increases in the total antioxidant capacity of plasma have often been observed after the consumption of flavonoid-rich foods by humans. These observations led to the hypothesis that dietary flavonoids play a significant role as antioxidants in vivo, thereby reducing chronic disease risk. This notion, however, has been challenged recently by studies on the bioavailability of flavonoids, which indicate that they reach only very low concentrations in human plasma after the consumption of flavonoid-rich foods. In addition, most flavonoids are extensively metabolized in vivo, which can affect their antioxidant capacity. Furthermore, fruits and vegetables contain many macro- and micronutrients, in addition to flavonoids, that may directly or through their metabolism affect the total antioxidant capacity of plasma. In this article, we critically review the published research in this field with the goal to assess the contribution of dietary flavonoids to the total antioxidant capacity of plasma in humans. We conclude that the large increase in plasma total antioxidant capacity observed after the consumption of flavonoid-rich foods is not caused by the flavonoids themselves, but is likely the consequence of increased uric acid levels. © 2006 Elsevier Inc. All rights reserved. Keywords: Flavonoids; Polyphenols; Dietary antioxidants; Plasma antioxidant capacity; Uric acid

Contents Dietary antioxidant flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro antioxidant capacity, bioavailability, and metabolism of flavonoids . . . . . . . . . . . . . . . . . . Ex vivo antioxidant protection of human plasma and LDL after consumption of flavonoid-rich foods . . . . Total antioxidant capacity of human plasma after consumption of flavonoid-rich foods . . . . . . . . . . . . Plasma antioxidant capacity in humans after consumption of fruits or vegetables . . . . . . . . . . . . . Plasma antioxidant capacity in humans after tea consumption . . . . . . . . . . . . . . . . . . . . . . . Plasma antioxidant capacity in humans after consumption of wine or beer . . . . . . . . . . . . . . . . Plasma antioxidant capacity in humans after consumption of chocolate, cocoa products, or coffee . . . . Factors affecting the total antioxidant capacity of plasma in humans. . . . . . . . . . . . . . . . . . . . . . Increase in plasma urate after consumption of flavonoid-rich foods . . . . . . . . . . . . . . . . . . . . . . Antioxidant effects of apples in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fructose-mediated urate production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other potential sources of endogenous urate or its derivatives: sucrose, sorbitol, lactate, and methylxanthines

⁎ Corresponding author. Fax: +1 541 737 5077. E-mail address: [email protected] (B. Frei). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.04.033

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Flavonoids are a large family of polyphenolic compounds synthesized by plants that have a common chemical structure. Polyphenolic compounds, or polyphenols, are polyhydroxylated phytochemicals, of which the two main classes comprise flavonoids and phenolic acids. Flavonoids may be further divided into several subclasses, i.e., flavones (and isoflavones), flavanones, flavonols, flavanols (also called catechins), and anthocyanidins (Fig. 1). Consumption of flavonoid-rich foods, in particular fruits and vegetables, is associated with a lower incidence of heart disease, ischemic stroke, cancer, and other chronic diseases [1–5]. For example, 7 of 12 epidemiological studies evaluating the risk of coronary heart disease reported protective effects of dietary flavonoids [6]. Additional studies also found inverse associations between flavonoid intake and the risk of stroke [7,8] and lung and colorectal cancers [7,9,10]. Because these chronic diseases are associated with increased oxidative stress and flavonoids are strong antioxidants in vitro, it has been suggested that dietary flavonoids exert health benefits through antioxidant mechanisms [11–13]. Although there is conflicting evidence whether consumption of flavonoid-rich foods results in increased antioxidant protection of lipids and proteins in plasma and LDL, many studies have found large, transient increases in the total antioxidant capacity of plasma in humans. This observation led to the hypothesis that flavonoids, and polyphenols in general, play an important role as antioxidants in human blood and tissues. However, accumulating evidence indicates that flavonoids are poorly bioavailable and reach only low, micromolar concentrations in human plasma, even after the intake of large amounts of flavonoid-rich foods. In addition, most flavonoids are extensively metabolized in vivo, which can affect their antioxidant activity. Based on an exhaustive review of these findings in the present article, we conclude that dietary flavonoids are unlikely to make a significant contribution to the antioxidant capacity of human plasma. The acute effects on total plasma antioxidant capacity of consumption of flavonoid-rich foods, including fruits, vegetables, tea, wine, and chocolate, may instead be explained by changes in the concentration of the metabolic antioxidant uric acid.

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capita in the United States [14]. A serving of apple provides about 400 mg of total phenols (expressed as gallic acid equivalents) [15]. Pears and grapes can provide as much as 300 mg total phenols per serving, and a serving of cranberries, cherries, or blueberries contains 200–400 mg [14]. Total phenols in fruits, fruit juices or extracts, and vegetables is strongly correlated with the total antioxidant capacity of these foods measured in vitro [15,16,19–21]. In fruits containing significant amounts of vitamin C, the in vitro antioxidant capacity often reflects both the polyphenol and the vitamin C content [22]. Vegetables such as spinach, broccoli, and onions also provide significant amounts of polyphenols in the human diet [23] (Table 1). Other plant-derived foods and beverages further contribute to the total daily intake of polyphenols in humans. For example, tea, coffee, chocolate, wine, and beer have high in vitro antioxidant capacity, which is almost exclusively due to the presence of polyphenols. In black tea, the levels of polyphenols vary depending on the amount of leaves, brewing time, and brewing temperature and usually are between 150 and 250 mg per 200-ml serving [24]. Because of its wide consumption, coffee has been recently claimed to be the main source of polyphenols in the U.S. diet (J. Vinson, unpublished), providing 150–180 mg per 200-ml serving [25]. In a Norwegian study of 2672 adults [26] coffee contributed 64% of the total daily intake of dietary antioxidants measured by the ferricreducing antioxidant potential assay (see below). Red wine contains 200–500 mg total phenols per 200-ml serving depending on type and varietal [27,28]. The total phenol content in white wine is considerably lower, about 40–60 mg per 200-ml serving [27]. Total phenol content in beer ranges from 50 to 100 mg in 200 ml [29]. Dark chocolate contains about 340 mg of total phenols per 40-g serving [25]. Considering these amounts of total phenols per serving, a well-balanced diet with the recommended nine daily servings of fruits and vegetables and moderate amounts of tea, coffee, wine, beer, or chocolate can provide well over 1000 mg of total phenols per day (Table 1). In vitro antioxidant capacity, bioavailability, and metabolism of flavonoids

Dietary antioxidant flavonoids The amounts of antioxidant flavonoids and polyphenols in plant-based foods of the human diet—in particular vegetables, fruits, tea, and wine—are generally much greater than the amounts of other antioxidants in these foods, such as vitamins C and E and carotenoids [14–18]. Fruits and fruit juices are among the best sources of polyphenols in the human diet because of their high content in most fruits (Table 1) and the relatively large serving sizes (100–200 g). Apples provide approximately 22% of the total fruit phenols consumed per

Flavonoids are strong antioxidants in vitro, mainly due to their low redox potential and their capacity to donate several electrons or hydrogen atoms [30,31]. For example, catechins have a redox potential of +0.53–0.57 V [31], which from a thermodynamic standpoint enables them to protect urate (+0.59 V), but not ascorbate (+0.28 V), from oxidation by peroxyl radicals (+1.06 V). Quercetin and (−)-epigallocatechin gallate (EGCG) have even lower redox potentials (+0.33 and 0.43 V, respectively [32]), which places them close to ascorbate in the biological antioxidant network. Recent work has shown

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Fig. 1. Basic structures and examples of the main subclasses of dietary flavonoids.

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Table 1 Flavonoid content of selected foods and levels of flavonoids in human plasma after intake of flavonoid-rich foods Flavonoid subclass

Flavonoid

Flavonoid content of selected foods and beverages (mg/100 g edible portion or mg/100 ml) a

Flavonoid levels in plasma (μM)

Flavonols

Quercetin Myricetin Kaempferol

0.65 (fried onions) [48] 0.74–7.60 (onions) [43,51] 0.30 (apples) [49] 2.10 (buckwheat tea) [43]

Flavanols

(–)-Epicatechin (+)-Catechin (–)-Epigallocatechin gallate

Flavones

Apigenin Luteolin

Flavanones

Naringenin Hesperetin Eriodictyol

Anthocyanidins

Cyanidin Malvidin Delphinidin Pelargonidin

Vegetables: capers (316), celery (3.50), chives (21.7), onions (15.4), red onions (38.8), dock leaves (120), fennel (84.4), hot peppers (16.0–50.0), cherry tomatoes (2.30–20.3), spinach (4.86), sweet potato leaves (30.2), lettuce (1.20–9.40), celery (3.50), broccoli (raw, 9.37; cooked, 2.44), Hartwort leaves (38.9), kale (34.5) Cereal: buckwheat (23.1) Fruits: apples (4.42), apricots (2.55), grapes (2.54), plums (1.20), bilberries (4.13), blackberries (1.10), blueberries (3.93), cranberries (18.4), elderberries (42.0), currants (13.5), cherries (1.25), black currant juice (3.01) Spices and herbs: dill weed (55.0) Others: red wine (1.50), tea (green, 2.69; black, 2.07), cocoa powder (20.3) Fruits: apples (9.0), apricots (11.0), grapes (17.6), peaches (2.30), nectarines (2.75), pears (3.43), plums (6.19), raisins (3.68), raspberries (9.23), blackberries (18.7), blueberries (1.11), cranberries (4.20), cherries (11.7) Others: red wine (12.0), tea (green, 132; black, 33.0), chocolate (dark, 53.5; milk, 13.4) Vegetables: hot peppers (5.40), celery hearts (22.6), fresh parsley (303) Spices and herbs: oregano (4.50), rosemary (4.00), dry parsley (13,506), thyme (56.0) Citrus fruits and juices: lemon (49.9), lemon juice (18.3), lime juice (11.5), orange (43.9), orange juice (15.0), grapefruit (54.5), tangerine juice (10.8) Spices and herbs: peppermint (20.0) Fruits: blackberries (100–400), black currant (130–400), blueberries (25.0–500), black grape (30.0–750), elderberries (749), strawberries (15.0–75.0), cherries (35.0–450), plums (0.20–25.0) Others: red wine (20.0–35.0)

a

1.00–1.80 (green tea) [55,61] 0.09–0.34 (black tea) [54,52] 0.08–0.09 (red wine) [56,57] 0.26–4.77 (chocolate) [58,60] 4.92–5.92 (cocoa) [59,60]

n/a

5.99 (grapefruit juice) [72] 0.06–0.64 (orange juice) [71,72]

0.11 (black currant juice) [68] 0.12 (black currant concentrate) [69] 0.10 (elderberry extract) [64] 0.01 (red wine) [70]

Data recompiled from Manach et al. [46] and the USDA Database for Flavonoid Content of Selected Foods [18]. n/a, data not available.

that catechins may be even more effective than ascorbate in regenerating α-tocopherol in micellar solution [33]. Despite the strong antioxidant capacity of flavonoids in vitro, their antioxidant efficacy in vivo is limited by several factors. First, the absorption of flavonoids in humans is low, in contrast to other dietary antioxidants such as vitamins C and E. The maximal plasma concentrations of flavonoids in humans, reached usually between 1 and 3 h after consumption of flavonoid-rich foods, are between 0.06 and 7.6 μM for flavonols, flavanols, and flavanones, and less than 0.15 μM

for anthocyanidins (Table 1) (reviewed in [34]). In addition, the half-lives of flavonoids in human plasma are short, usually in the range of a few hours. These factors severely limit the capability of dietary flavonoids to act as antioxidants in plasma in vivo, especially compared to other antioxidants present in plasma at high steady-state concentrations, e.g., 30–150 μM ascorbate (vitamin C), 160–450 μM urate, or 15–40 μM αtocopherol (vitamin E) [35]. Chronic or long-term consumption of flavonoid-rich foods also does not result in accumulation of significant amounts of flavonoids in plasma. For example,

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steady-state concentrations of quercetin in human plasma are less than 1 μM [36]. Data on flavonoid concentrations in human tissues are sparse, but it is unlikely that flavonoids make a significant contribution to antioxidant defenses in cells and tissues, where ascorbate and reduced glutathione are normally found in millimolar concentrations. In addition to being poorly absorbed, flavonoids are extensively metabolized in intestine and liver. Flavonoids are good substrates and inducers of phase II enzymes [37,38], suggesting that they are recognized by the body as xenobiotic— and thus potentially toxic—compounds, most likely due to their polyhydroxylated structure and potent redox activity. It is tempting to speculate that the capacity of flavonoids to induce detoxifying enzymes is a major mechanism by which flavonoids protect against mutagens and carcinogens, i.e., act as cancer chemopreventive agents. In addition, it is likely that the low levels of flavonoids and their metabolites exert other biological effects, e.g., altered cell signaling and gene expression, that contribute to their purported health benefits. Thus, flavonoids undergo extensive first-pass metabolism, and the chemical forms of flavonoids present in fruits and vegetables (mainly glycosides, except for catechins and proanthocyanidins, which are present as aglycones) are quite different from the in vivo metabolites. In the intestinal mucosa and liver, flavonoids undergo glucuronidation, methylation, and sulfation. This biotransformation greatly affects the physical properties of flavonoids, making them more water soluble, and also often affects their antioxidant activity. Some flavonoid metabolites retain the antioxidant activity of their parent compounds, but in general the metabolites are less potent antioxidants due to modification of their catechol and phenol groups [39,40]. Furthermore, flavonoids are degraded by bacteria in the intestinal tract, and the resulting breakdown products may exert biological effects through antioxidant or non-antioxidant mechanisms [41,42]. To illustrate the complexity and consequences of biotransformation of flavonoids, some representative examples will be discussed. Quercetin is one of the most studied polyphenolic flavonoids, mainly because it is widely consumed in the human diet and is a potent antioxidant in vitro. Onions, apples, tea, and wine are the main sources of quercetin in the human diet (Table 1). In these foods, quercetin is present in conjugated form, i.e., as quercetin glycosides. The nature of the sugar residues in the glycosides influences the extent of absorption. For instance, quercetin glycosides from onions are more bioavailable than quercetin glycosides from apples [43]. Hollman and co-workers [44] suggested that quercetin glycosides are transported into enterocytes by the sodium-dependent glucose transporter 1, but other data indicate that previous deglycosidation in the small intestine by human or bacterial enzymes is necessary for absorption [45]. Once in the bloodstream, quercetin metabolites may be circulating for more than 10 h, which is longer than many other flavonoids such as anthocyanidins and catechins (reviewed in [46]) and is likely due to enterohepatic recycling of quercetin metabolites [47]. Reported concentrations of total quercetin metabolites in

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human plasma after consumption of quercetin-rich foods range from 0.7 to 7.6 μM [43,48–51] (Table 1). In contrast to quercetin, catechins, which are the main flavonoids in tea and chocolate, are not glycosidated in nature and can be absorbed directly into the bloodstream. After absorption, however, plasma levels of free catechins are low [52–61], and the catechins are mainly present as glucuronidated and methylated metabolites. EGCG, the main catechin in tea, is found in human plasma in both its free and its methylated form [62,63]. Unlike quercetin and catechins, the anthocyanidins, which are found in berries and wine, are absorbed intact as glycosides [64,65]. It has been reported recently that methylated and glucuronidated conjugates of anthocyanidins can be detected in blood [66,67]. Reported plasma levels of anthocyanidins from food sources are very low, i.e., in the nanomolar range [64,68–70]. As mentioned above, the plasma concentrations of flavonols, flavanols, and flavanones from citrus fruits are also very low [71,72], i.e., in the nanomolar to low micromolar range (Table 1). The absorption of some flavonoids may be further restricted because they form high-molecular-weight polymers. Procyanidins, which are polymers of catechins found in large amounts in fruits and cocoa products, are excellent antioxidants in vitro because they contain many hydroxyl groups. The antioxidant capacity of procyanidins depends on their oligomer chain length and the type of reactive oxygen species with which they react. Thus, procyanidins and their free epicatechin units exhibit about the same antioxidant efficacy toward aqueous peroxyl radicals, whereas procyanidins of different degrees of oligomerization differ significantly in their effectiveness in protecting against lipid peroxidation induced by iron and ascorbate [73]. Procyanidins often make a greater contribution to the total phenol content, and thus total antioxidant capacity, of flavonoid-rich foods than their more bioavailable monomers, (−)-epicatechin and (+)-catechin. Apples contain 80– 128 mg of procyanidins per 100 g wet wt, and hence procyanidins may contribute more than 80% to the total antioxidant capacity of apples and apple juice or extracts [74]. The procyanidin content of chocolate can be as high as 1500 mg per 100 g [75] compared to about 400 mg of monomeric catechin per 100 g of dark chocolate. Thus, procyanidins contribute substantially to the total phenol content and in vitro antioxidant capacity of cocoa products. Recent studies have found extensive degradation of procyanidins by the intestinal bacterial microflora into more absorbable low-molecular-weight phenolic acids [42]. Whereas the bioavailability of quercetin, catechins, and anthocyanidins in humans is very limited, the absorption of intact procyanidins seems even more restricted, or almost nil [59]. Ex vivo antioxidant protection of human plasma and LDL after consumption of flavonoid-rich foods When added in vitro to human plasma or isolated LDL, flavonoids can prevent the oxidation of endogenous antioxidants, lipids, and proteins. For example, addition of catechins to plasma protects α-tocopherol, β-carotene, and lipids from

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oxidation by aqueous peroxyl radicals [76,77]. Similarly, black tea extract added to plasma strongly inhibits peroxyl radicalinduced lipid peroxidation [78]. Theaflavins in black tea, which are formed from partially oxidized catechins, and catechins in green tea inhibit copper-mediated LDL oxidation in vitro [79]. Zhu and co-workers [80] showed that green tea extracts regenerate α-tocopherol in human LDL. Red wine polyphenols bind to LDL and HDL and inhibit metal ion-dependent and -independent oxidation of the proteins and lipids in these lipoproteins [81]. Pearson and co-workers reported that extracts from apple skin or flesh inhibit copper-mediated LDL oxidation [82]. Apple extracts also effectively delay the oxidation of urate and α-tocopherol and the formation of lipid hydroperoxides in human plasma [15]. Another study showed that catechins, procyanidins, and procyanidin-rich extracts protect plasma components from oxidation [83]. Thus, there is ample and consistent evidence that in vitro addition of flavonoids or flavonoid-rich food extracts to human plasma or lipoproteins effectively protects their endogenous antioxidants, lipids, and proteins from oxidation. In contrast, studies of ex vivo oxidation of plasma and LDL obtained from humans before and after short- or long-term consumption of flavonoid-rich foods have yielded conflicting results (Table 2). Even studies using the same type of food have not found consistent results, indicating significant influences of experimental design, individual metabolism, and other uncontrolled variables such as potential oxidation artifacts introduced during preparation of LDL. For example, van het Hof and colleagues [84] observed that daily consumption of 900 ml of green or black tea by healthy individuals for 4 weeks did not increase the resistance of LDL to ex vivo oxidation. Cherubini et al. [78] and McAnlis et al. [85] also found no effect of acute or chronic consumption of black tea on plasma or LDL resistance to oxidation ex vivo. However, Nakagawa et al. [86] reported a reduction in plasma hydroperoxide levels in healthy subjects after acute ingestion of tea extract equivalent to 2 cups of tea, and Miura et al. [87] observed decreased oxidizability of LDL obtained from subjects who had ingested a green tea

extract daily for a week. Finally, Hodgson et al. [88] reported that ex vivo oxidation of serum lipoproteins from human subjects was slightly decreased after they acutely ingested black or green tea (Table 2). Several studies on flavonoid-rich beverages other than tea have been performed. Yukawa et al. [89] found decreased oxidizability of LDL isolated from 11 healthy male volunteers after they consumed coffee for 1 week. Results of studies on wine have been inconsistent. Whereas Chopra et al. [90] found that daily supplementation of male subjects for 2 weeks with either red wine extract (equivalent to 375 ml of wine) or quercetin resulted in significantly increased resistance of LDL to ex vivo oxidation, de Rijke et al. [91] did not observe such an increase after daily consumption of 550 ml of red wine for 4 weeks. In addition, Caccetta et al. [92] did not observe an effect on ex vivo serum or LDL oxidizability in healthy male nonsmokers after acute consumption of red wine (Table 2). Susceptibility of LDL or plasma to oxidation was also studied after the consumption of some vegetables, fruits, or fruit juices. McAnlis et al. [51] reported no significant changes in ex vivo oxidation of plasma or LDL isolated from volunteers before and after consumption of 225 g of fried onions. We reported that the resistance of plasma to oxidation in human volunteers did not increase after the consumption of five Red Delicious apples [15]. Marniemi et al. [93] observed an increase in ex vivo LDL resistance to oxidation after acute consumption of berries, but not after 8 weeks of daily consumption of berries. In contrast, O'Byrne et al. [94] showed that daily intake of Concord grape juice at 10 ml/kg body wt for 2 weeks was as effective as 400 IU of α-tocopherol daily in protecting LDL from ex vivo oxidation. Reduced LDL oxidizability was also observed in subjects who consumed 125 ml of concentrated grape juice daily for 7 days [95] (Table 2). Because chocolate and cocoa products are significant dietary sources of flavonoids, mainly (−)-epicatechin and procyanidins, many studies have evaluated their ex vivo antioxidant effects [96–99]. These studies have generally found consistent antioxidant protection of plasma and LDL, unlike the

Table 2 Resistance of human plasma and LDL to oxidation ex vivo after consumption of flavonoid-rich foods Flavonoid-rich food

Study reference

Characteristics of the study

Outcome on LDL or plasma oxidizability

Green and black tea Black tea

[84] [85]

Black tea Green tea extract Green tea extract Green and black tea

[78] [86] [87] [88]

900 ml (equivalent to 6 cups), daily for 4 weeks 600 ml 1.1% (w/v), acute consumption 3 × 300 ml 1.1 % (w/v), daily for 1 week 500 ml (equivalent to 6 cups), acute consumption Equivalent to 2 cups, acute consumption Twice/day (equivalent to 7–8 cups), daily for 1 week 400 ml 1.9 % (w/v), acute consumption

Red wine Red wine extract Red wine Onions Berries

[91] [90] [92] [51] [93]

Apples Grape juice Concord grape juice

[15] [95] [94]

No change No change No change No change ↓ Plasma hydroperoxides ↓ LDL oxidizability No change after green tea ↓ LDL oxidizability after black tea No change ↓ LDL oxidizability No change No change ↓ LDL diene conjugation No change No change in plasma oxidizability ↓ LDL oxidizability ↓ LDL oxidizability

550 ml, daily for 4 weeks Equivalent to 375 ml red wine, daily for 2 weeks 5 ml/kg body wt, acute consumption 225 g fried onions, acute consumption 240 g mixed berries, acute consumption 100 g mixed berries, daily for 8 weeks 1037 g (equivalent to 5 apples with peel), acute consumption 125 ml, daily for 1 week 10 ml/kg body wt, daily for 2 weeks

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conflicting data in Table 2 using fruits, wine, or tea. Only one study reported no change in susceptibility to oxidation of serum lipids or LDL after chocolate consumption in humans [100]. These consistent results may be due to the more uniform and lipophilic food matrix of cocoa products compared to fruits and vegetables and hence improved and more uniform flavonoid absorption. Nevertheless, plasma levels of flavonoids, in particular catechins, after consumption of chocolate or cocoa products are in the low micromolar range, similar to the levels observed after the consumption of other flavonoid-rich foods [58,59,101,102]. The effects of flavonoid-rich foods on lymphocyte DNA damage and 8-hydroxy-2′-deoxyguanosine formation have also been studied. Boyle et al. [103] found that consumption of fried onions, with or without fresh tomatoes, by six healthy female volunteers resulted in elevated flavonoid concentrations in plasma, which was associated with increased resistance of lymphocyte DNA to strand breakage ex vivo. Levels of urinary 8-hydroxy-2′-deoxyguanosine also decreased 4 h after ingestion of fried onions. Several factors may account for the contradictory results of the aforementioned studies on the effects of flavonoid-rich foods on ex vivo oxidation of plasma and LDL (Table 2). Differences in the absorption and metabolism of the various flavonoids may lead to the formation of metabolites with different antioxidant properties. The methods used to isolate LDL may also have contributed to the discrepant results, because flavonoids exhibit variable distribution and association with plasma constituents such as proteins and lipids. For example, quercetin exhibits greater affinity for albumin than LDL [51]. The standard isolation procedures for LDL will likely remove the water-soluble flavonoids that are not tightly bound to the lipoprotein particles. These flavonoids probably include EGCG from tea and glucuronide and sulfate metabolites of flavonoids, which tend to be more water-soluble than their parent compounds. Unfortunately, most of the studies that reported a significant decrease in the susceptibility of plasma or LDL to oxidation ex vivo after consumption of flavonoid-rich foods did not measure the levels of flavonoids in plasma or LDL. Total antioxidant capacity of human plasma after consumption of flavonoid-rich foods Several methods have been used to evaluate the antioxidant capacity of flavonoids and extracts of flavonoid-rich foods and that of human plasma before and after consumption of flavonoid-rich foods. These methods include the ferric-reducing antioxidant potential (FRAP) [104]; oxygen radical absorbance capacity (ORAC), without or with prior precipitation of plasma proteins with perchloric acid (ORAC-PCA) [105]; trolox equivalent antioxidant capacity (TEAC) [106]; and total radical-trapping antioxidant parameter (TRAP) [107] (Table 3). These assays have been extensively characterized, compared, and reviewed [108–111]. Although the assays vary in principle of detection, sensitivity, working pH, source of oxidants, and other assay conditions (Table 3), they all assess

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either the radical-scavenging (hydrogen atom transfer) or -reducing (electron transfer) capacity of the compound or (biological) fluid under investigation. It should be noted, however, that all of these methods lack specificity. Thus, in a polyphenol-enriched plant extract, the antioxidant capacity measured by any of these methods reflects the content of all major reducing and antioxidant substances, including nonphenolic compounds such as vitamin C. Many studies have found highly significant correlations between the measured antioxidant capacity and the total phenol content of plant-derived extracts and beverages. For example, Proteggente et al. [22] studied a variety of fruits and vegetables and comparatively assessed the antioxidant capacity of aqueous and methanolic extracts using TEAC, FRAP, and ORAC. The values for each fruit and vegetable extract correlated well with its total phenolic content, as assessed by the Folin–Ciocalteau method, as well as the vitamin C content. Similar observations were made for tea, wine, and chocolate [112–114]. Whereas the observed antioxidant capacity of plant-derived extracts usually closely reflects their content of antioxidant polyphenols and flavonoids, the same is not true for human plasma, which contains large concentrations of antioxidant vitamins and metabolites such as vitamin C (30–150 μM), vitamin E (15– 40 μM), and urate (160–450 μM), but only very low concentrations of flavonoids (see Tables 1 and 3). Interestingly, and in striking contrast to the discrepant results of studies on the resistance of plasma or LDL to oxidation ex vivo (Table 2), virtually all studies on the total antioxidant capacity of human plasma before and after consumption of flavonoid-rich foods have found significant increases (Table 4). The extent of these increases in plasma antioxidant capacity, however, often greatly exceeded the increases in the plasma concentrations of flavonoids or total phenols. In the following sections, studies on plasma antioxidant capacity in humans after the intake of different types of flavonoid-rich foods will be discussed. Plasma antioxidant capacity in humans after consumption of fruits or vegetables Cao et al. [115] investigated the effects of a diet rich in fruit and vegetables on the antioxidant capacity of human plasma. Thirty-six healthy nonsmokers consumed a controlled diet containing 10 servings of fruit and vegetables each day for 15 days or the same diet with an additional 2 servings of broccoli each day on days 6–10. Both diets significantly increased the plasma total antioxidant capacity measured by ORAC. In another short-tem study, Cao et al. [116] investigated serum total antioxidant capacity in 8 elderly women after consumption of 240 g of strawberries, 294 g of spinach, or 300 ml of red wine compared to a control meal or a supplement of 1250 mg of vitamin C. The total antioxidant capacity of serum, assessed as area under the curve (AUC) for the time course of serum ORAC, TEAC, or FRAP, increased significantly during the 4-h period after consumption of strawberries, spinach, or wine (Table 4). The authors concluded that the antioxidant phenolic compounds in these

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Table 3 Methods used for the evaluation of plasma or serum total antioxidant capacity Method a

Principle

FRAP

Reduction of ferric ions to ferrous ions

ORAC

Based on peroxyl radical generation and oxidation of a fluorescent probe (β-phycoerythrin)

ORAC-PCA

Idem ORAC

TRAP (FL)

Based on peroxyl radical generation and oxidation of a fluorescent probe (β-phycoerythrin) Based on peroxyl radical generation and oxidation of luminol

TRAP (CL)

TEAC

Based on decolorization of preformed ABTS c radical cations

Assay conditions

Carried out at pH 3.6 Spectrophotometric (endpoint) Diluted plasma or serum Carried out at pH 7.4 Fluorescent (kinetics) Diluted plasma or serum Reaction followed to completion, results evaluated as area under the curve Idem ORAC Previous precipitation of plasma/serum proteins with perchloric acid (PCA) Carried out at pH 7.4 Fluorescent (kinetics) Diluted precipitated plasma or serum Results evaluated as lag times Carried out at pH 7.4 Chemiluminescent (kinetics) Undiluted plasma or serum Results evaluated as lag times Carried out at pH 7.4 Spectrophotometric (kinetics or endpoint) Results evaluated as area under the curve (kinetics) or % inhibition (endpoint)

Plasma or serum TAC b reference value (μM)

Contribution (%) to plasma or serum TAC

Ref.

Urate

Ascorbate

Bilirubin

Proteins

400–1000

60

15

5

10

[104]

1500–3100

7

1

<1

28

[105]

600–900

39

7

1

0

[105]

850–1300

20–60

2.5–5.3

n/a

n/a

[107]

250–400

60–90

10–20

n/a

n/a

[58]

19

3

1

28

[110]

1300–1600

a

Method: FRAP, ferric-reducing antioxidant potential or ferric-reducing ability of plasma; ORAC, oxygen radical absorbance capacity; TEAC, Trolox equivalent antioxidant capacity; TRAP (FL), total radical-trapping antioxidant parameter–fluorescence; TRAP (CL), total radical-trapping antioxidant parameter– chemiluminescence. b TAC, total antioxidant capacity; n/a = reliable data not available. c ABTS, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid).

foods were responsible for the increased serum antioxidant capacity, because it was not accounted for by the observed increases in the plasma concentrations of ascorbate (2–25%) and urate (10–30%). In another study [117], consumption of tomato products with olive oil for 1 week significantly raised the plasma antioxidant capacity measured as FRAP compared to consumption of tomato products with sunflower oil, despite similar plasma levels of lycopene after consumption of either tomato preparation. The change in FRAP after consumption of tomato products with olive oil was about 200 μM, i.e., about 20% of the baseline antioxidant capacity of human plasma. The tomato preparation also significantly increased serum LDL cholesterol levels and decreased triglycerides. The authors concluded that the type of oil may differentially affect the antioxidant capacity of plasma, although it is possible that the changes in FRAP were partly attributed to the polyphenolic constituents of olive oil. In a short-term study by Serafini et al. [118], six men and five women consumed 250 g of fresh lettuce, and blood was sampled before and 2, 3, and 6 h after consumption. Plasma antioxidant capacity measured as TRAP increased by 40–50%, which was associated with significantly increased plasma levels of quercetin, p-coumaric acid, and vitamin C (Table 4).

In a study of six men, Marniemi et al. [93] reported that TRAP of LDL was significantly increased by about 10% 5 h after consumption of 80 g of either bilberries, lingonberries, or black currants. Pedersen et al. [119] investigated the effect of blueberry or cranberry juice consumption on plasma phenolic content and antioxidant capacity. Nine healthy female subjects consumed 500 ml of blueberry juice, cranberry juice, or sucrose solution as control. Consumption of cranberry juice, but not blueberry juice, significantly increased the ability of plasma to reduce potassium nitrosodisulfonate and Fe(III)-2,4,6-tri(2-pyridyl)s-triazine (the indicator molecule for FRAP), reaching maximal levels 60 to 120 min after consumption. This increase in reducing capacity of plasma corresponded to a 30% increase in vitamin C levels and a small but significant increase in total phenols in plasma. The authors concluded that the observed increase in plasma antioxidant capacity after consumption of cranberry juice was mainly due to vitamin C, not phenolics. To evaluate the effects of wild blueberries on postprandial serum antioxidant capacity, a single-blinded crossover study was performed in a group of eight middle-aged male volunteers [120]. The subjects consumed a high-fat meal and a placebo

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Table 4 Plasma or serum total antioxidant capacity after consumption of flavonoid-rich foods Food

Study

Method a

Fruits and vegetables Strawberries, spinach, red wine

[115] [116]

Tomato with olive oil Berries Cranberry juice Lettuce Blueberries

[117] [93] [119] [118] [120]

Blueberries Grape juice

[121] [95]

ORAC ORAC FRAP TEAC FRAP TRAP (CL) LDL FRAP TRAP (FL) ORAC TEAC ORAC e FRAP

Concord grape juice Green and black tea Green tea Green and black tea Green tea Green tea Coffee Wine Wine Wine Wine Beer Wine, whiskey Chocolate Chocolate

[94] [123] [122] [54] [136] [133]

ORAC e TRAP (FL) FRAP FRAP TEAC TRAP

[126] [124] [125] [128] [129] [127] [58] [130]

TAC f FRAP TRAP (FL) TAC TRAP (FL) FRAP TRAP (CL) FRAP

Outcome b ↑ 5–15% ↑ 11–24% ↑ 7–24% ↑ 1–21% ↑ 20% ↑ 10% ↑ 40 μM d ↑ 40–50% ↑ 8.5% ↑ 4.5% ↑11–50% ↑ 8% (at 1 h) ↑ 11% (at day 8) ↑ 8% ↑ 40–48% ↑ 4% ↑ 3% ↑ 6–13% ↑ 5% (NS) ↑ 6% ↑ 11–18% ↑ 24% ↑ 14% ↑ 14% ↑ 17% ↑ 60–100 μM ↑ 31% ↑ 20%

Change in plasma antioxidants Ascorbate

Urate

Phe/Flav c

n/a ↑ 2–25%

n/a ↑ 10–30%

n/a n/a

n/a n/a ↑ 30% ↑ 10–11 μM n/a

n/a n/a n/a n/a n/a

n/a n/a ↑ 6 μg/ml ↑ 88 ng/ml n/a

n/a n/a

n/a n/a

↑ 13 ng/ml n/a

n/a n/a n/a No change n/a ↓ 3% No change n/a n/a n/a n/a n/a n/a No change n/a

n/a n/a n/a No change n/a ↑ 7% ↑ 5% n/a ↑ 23% n/a ↑ 12% ↑ 13% (NS) n/a ↑ 12% (NS) n/a

↑ 2.4 μg/ml n/a n/a n/a n/a n/a n/a n/a n/a ↑ 3 μg/ml n/a ↑ 18 ng/ml ↑ 2–2.5 μg/ml ↑ 257 nM ↑ 112.5 ng/ml g

NS, not statistically significant; n/a, data not available. a ORAC values refer to ORAC-PCA. b Expressed as % (when possible) or μM Trolox equivalents. c Phe/Flav, total phenolics or individual flavonoids. The maximal increases in phenolics or flavonoids are shown. d Expressed as Fe(II) equivalents. e Total ORAC. f Total antioxidant capacity (by chemiluminescence). g Calculated from area-under-the-curve data.

supplement followed 1 week later by the same high-fat meal supplemented with 100 g of freeze-dried wild blueberries. Consumption of the blueberry-supplemented meal was associated with a significant increase in serum antioxidant capacity 1 and 4 h postprandially. In a follow-up study, the same group [121] investigated the absorption of anthocyanins in humans after the consumption of a high-fat meal supplemented with freeze-dried blueberries. Of the 25 anthocyanins detected in the blueberries, 19 were also found in serum. The authors qualitatively, but not quantitatively, associated this increase in plasma anthocyanins with the increase in serum total antioxidant capacity. The antioxidant effects of red grape juice were studied in several trials. Day et al. [95] observed a significant increase in serum total antioxidant capacity in seven subjects 1 h after they consumed 125 ml of concentrated grape juice. Furthermore, after 8 days of daily grape juice consumption, serum total antioxidant capacity was increased compared to baseline and the susceptibility of isolated LDL to oxidation was decreased. O'Byrne et al. [94] compared the in vivo antioxidant efficacy of Concord grape juice with that of

vitamin E. Healthy subjects received daily for 2 weeks either 400 IU RRR-α-tocopherol or 10 ml/kg body wt of Concord grape juice. Plasma α-tocopherol concentrations increased by 92% in subjects who received α-tocopherol, and plasma total and conjugated phenols increased by 17 and 22%, respectively, in subjects who consumed grape juice. Both interventions significantly increased serum ORAC and LDL resistance to oxidation (Table 4). Vitamin C contained in fruits and vegetables can significantly affect plasma total antioxidant capacity assessed by FRAP or TRAP (CL) (Table 3). As indicated above, vitamin C is more readily absorbed than flavonoids and is present in human plasma at much higher concentrations (30– 150 μM) than flavonoids (nanomolar to low micromolar; Table 1). The contribution of ascorbate to FRAP can be as much as 20% [104] (Table 3). Therefore, it is important to distinguish between the antioxidant effects of ascorbate and flavonoids in plasma after consumption of vitamin C-containing, flavonoid-rich foods. Interestingly, ORAC is not sensitive to ascorbate, as indicated by the observation that treatment of plasma with ascorbate oxidase does not significantly affect the

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value of either ORAC or ORAC-PCA (S. Lotito and B. Frei, unpublished observations). In agreement with these observations, other researchers [108] have calculated that the contribution of ascorbate to plasma ORAC is only 1–7% (Table 3). Plasma antioxidant capacity in humans after tea consumption Benzie et al. [122] studied plasma and urine antioxidant capacity in healthy adults before and after ingestion of green tea or water as control and found about a 4% increase in plasma FRAP 40 min after drinking tea. Excretion of polyphenolic antioxidants peaked at 60–90 min, and a significant correlation was observed between urinary FRAP and total phenolics. Leenen et al. [54] investigated the effects of a single dose of black or green tea (2 g solids in 300 ml of water), with or without milk, or water as control on plasma antioxidant capacity in 21 healthy volunteers enrolled in a crossover study. Consumption of black tea significantly increased plasma antioxidant capacity, reaching maximal levels after about 60 min, and a larger increase was observed after consumption of green tea. Addition of milk did not affect the increase in plasma antioxidant capacity with either tea. In another similar study, two groups of 5 healthy adults drank 300 ml of either black tea or green tea, and the experiment was repeated on a separate day with the addition of 100 ml of whole milk to the tea [123]. The plasma antioxidant capacity measured as TRAP increased significantly after consumption of black or green tea and peaked after 30–50 min. Interestingly, the addition of milk completely inhibited the increase in plasma TRAP, in contrast to the observations made by Leenen et al. [54] (Table 4). Plasma antioxidant capacity in humans after consumption of wine or beer At least six studies investigated the effects of wine consumption on plasma antioxidant capacity in humans, and all of them found increased levels (Table 4). Day and Stansbie [124] reported a 24% increase in serum antioxidant capacity in 6 healthy men who consumed 250 ml of port wine, whereas ethanol ingestion as a control had no effect. Serafini et al. [125] found that plasma TRAP and polyphenol concentration significantly increased in 10 healthy subjects 50 min after consumption of alcohol-free red wine, but not after consumption of alcohol-free white wine or water. Similarly, Whitehead et al. [126] reported that mean serum antioxidant capacity, measured by chemiluminescence, in 9 subjects increased by 18 and 11%, respectively, 1 and 2 h after drinking 300 ml of red wine and 4 and 7%, respectively, after drinking an equivalent amount of white wine. In another study, 9 volunteers who consumed 100 ml of either red wine or malt whiskey showed a significant increase in plasma total phenols and FRAP within 30 min of consumption [127]. Maxwell and Thorpe [128] also found a significant mean increase of 14% in serum antioxidant capacity 60 min after ingestion of French Bordeaux red wine.

Ghiselli et al. [129] studied the effects of drinking beer, dealcoholized beer, or ethanol on total antioxidant capacity and the profile of selected phenolic acids, including caffeic, sinapic, syringic, and vanillic acids, in the plasma of 14 healthy subjects. Beer consumption caused a significant increase in plasma antioxidant capacity after 1 h, returning to baseline after 2 h, and also increased plasma levels of all measured phenolic acids. Plasma antioxidant capacity in humans after consumption of chocolate, cocoa products, or coffee Several studies investigated the effects of chocolate consumption on plasma antioxidant capacity (Table 4). The absorption of (−)-epicatechin and plasma antioxidant capacity measured as TRAP were evaluated in 17 healthy volunteers who consumed 80 g of procyanidin-rich, semisweet chocolate [58]. Two hours after chocolate ingestion, a 12-fold increase in plasma (−)-epicatechin was observed, from 22 to 257 nM, and a significant 31% increase in plasma total antioxidant capacity, corresponding to about 90 μM trolox equivalents. Both plasma (−)-epicatechin and total antioxidant capacity returned to baseline 4 h later. This study is a particularly striking example of the large discrepancy between the increase in plasma antioxidant capacity and plasma flavonoid concentration [58]. In a crossover study, 12 healthy volunteers consumed 100 g of dark chocolate, 100 g of dark chocolate with 200 ml of fullfat milk, or 200 g of milk chocolate [130]. Plasma FRAP significantly increased by 20% 1 h after ingestion of dark chocolate, but did not increase after consumption of dark chocolate with milk or milk chocolate. The authors speculated that milk may interfere with the absorption of antioxidants from chocolate and may, therefore, negate the potential health benefits of eating moderate amounts of dark chocolate. This notion, however, has been questioned recently by other investigators, who did not find such an effect of adding milk to chocolate on plasma antioxidant capacity [131]. Coffee is widely consumed in the Western world, but only few studies have assessed the antioxidant effects of coffee consumption in humans. Coffee is particularly rich in phenolic acids such as caffeic, ferulic, chlorogenic, and p-coumaric acids. Nardini et al. [132] showed that coffee consumption in 10 healthy male volunteers acutely increased total plasma caffeic acid concentration, present mainly as glucuronate and sulfate metabolites, peaking 1 h after coffee consumption. Natella et al. [133] reported that plasma antioxidant capacity measured as TRAP significantly increased after drinking 200 ml of coffee. Factors affecting the total antioxidant capacity of plasma in humans Studies assessing the total antioxidant capacity of plasma, serum, or other biological samples often do not take into account possible postprandial or diurnal variations that are not directly related to the intake of dietary antioxidants. Plasma antioxidant capacity may be significantly affected by nonantioxidant dietary constituents that affect uptake, tissue mobilization, or metabolism of endogenous or exogenous

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antioxidants. Food intake per se may cause variations in the plasma concentrations of ascorbate and urate, the major contributors to plasma total antioxidant capacity (Table 3). For example, in a short-term study, Cao and Prior [134] fed eight elderly women a diet with negligible amounts of antioxidants and found that plasma ascorbate and urate varied during the trial. In particular, plasma total antioxidant capacity measured as FRAP and TEAC increased 30 min after breakfast consumption, which was paralleled by significant changes in plasma urate. Interestingly, after this initial increase, FRAP, TEAC, and urate decreased over time, reaching a minimum at 4 h. In contrast, the postprandial increase in ORAC could not be explained entirely by variations in urate, suggesting that other unknown factors contributed. Interestingly, whereas urate and ascorbate are known to contribute about 39 and 7%, respectively, of the ORAC-PCA value of normal human plasma, over 50% could not be assigned to any specific antioxidant [108] (Table 3). Mazza et al. [121] studied the absorption of anthocyanins in humans after the consumption of a high-fat meal with a freezedried blueberry powder containing 25 different anthocyanins. As discussed above, 19 of the 25 anthocyanins were detected in human serum, and the authors associated the increase in anthocyanins with the increase in serum antioxidant capacity. However, ORAC of serum or acetone-precipitated serum timedependently increased not only after consumption of the high-fat meal supplemented with blueberries, but also after the placebosupplemented high-fat meal used as control. In both experiments, the increase in ORAC was closely paralleled by the increase in triglycerides. Hence, this study [121] does not provide evidence that the increase in serum ORAC is directly linked to anthocyanins, but suggests an effect of the high-fat meal itself. In a short-term study with human volunteers, we observed a decrease in FRAP and urate 6 h after consumption of plain bagels [135], in agreement with the data of Cao and Prior [134]. These findings indicate that plasma urate levels are affected by food intake, most likely due to changes in the levels of ATP and inorganic phosphate (see below). In contrast, in that same study (for study design, see [135]), we observed a large, significant increase in plasma ORAC-PCA after the consumption of either flavonoid-rich Red Delicious apples or plain bagels in amounts matched by their carbohydrate content (S. Lotito and B. Frei, unpublished observations). The increase in ORAC-PCA was similar after the intake of either food, indicating that the increase in plasma antioxidant capacity was independent of the antioxidant and flavonoid content of the foods consumed, which was negligible for the bagels. Interestingly, consumption of a fructose solution also acutely increased plasma ORACPCA, which was proportional to the amount of carbohydrates consumed. These observations suggest a strong postprandial effect of carbohydrates on plasma antioxidant capacity, regardless of the amount of dietary antioxidants. Taken together, the above data emphasize the need to consider the effect of food intake per se, including fats and carbohydrates, on plasma total antioxidant capacity in humans. Hence, studies that assessed the effects of flavonoid-rich foods on plasma antioxidant capacity (Table 4) may have to be

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reevaluated, and future studies should control for postprandial effects of non-antioxidant dietary constituents. Increase in plasma urate after consumption of flavonoid-rich foods As discussed above, the consumption of flavonoid-rich foods is almost always associated with substantial increases in plasma or serum total antioxidant capacity (Table 4). Even when the kinetics of the appearance of flavonoids in plasma and the increase in plasma antioxidant capacity are closely related, the extent of the increase in plasma antioxidant capacity usually far exceeds the plasma concentrations of flavonoids and their metabolites, which are in the nanomolar to low micromolar range (Tables 1 and 4). For example, Serafini et al. [118] showed that after consumption of lettuce, plasma TRAP increased by 40–50% from a baseline value of about 800 μM, i.e., TRAP increased by about 320–400 μM trolox equivalents (Table 4). This large increase in plasma total antioxidant capacity could not be explained by the small increases in plasma ascorbate of about 10 μM, in phenolics of about 90 μg/L, in caffeic acid of 29 μg/L (0.16 μM), in coumaric acid of 39 μg/L (0.24 μM), and in quercetin of 20 μg/L (0.07 μM) [118]. As mentioned above, consumption of tomato products with olive oil increased plasma FRAP by as much as 200 μM [117], again far exceeding maximally achievable flavonoid concentrations in plasma, and Rein et al. [58] reported an increase in plasma TRAP of about 90 μM after consumption of chocolate, in contrast to an increase in plasma (−)-epicatechin of only about 257 nM. After consumption of 100 g of wild blueberries, plasma ORACPCA increased more than 8% over baseline, corresponding to about 50 μM trolox equivalents [120], and increases of 40 μM in plasma antioxidant capacity were observed 1 h after drinking grape juice [95]. Furthermore, large increases in plasma FRAP of 20%, corresponding to 80–200 μM, were reported after chocolate consumption [130]. Serafini et al. [125] observed increases of 14% or 159 μM in plasma TRAP in 10 volunteers within 1 h after consuming 300 ml of alcohol-free red wine, and Duthie et al. [127] found an increase in FRAP of 60–100 μM in 9 healthy males within 30 min after drinking 100 ml of red wine (Table 4). Finally, tea consumption has been reported to dosedependently increase plasma antioxidant capacity by 200– 600 μM trolox equivalents [136]. How can these large increases in plasma total antioxidant capacity after consumption of flavonoid-rich foods and beverages be reconciled with the small increases in plasma concentrations of flavonoids? One possible explanation is that only a small fraction of the flavonoids in plasma is measured and that the total amount of flavonoids is greatly underestimated. However, this is an unlikely explanation, as all flavonoids detected in human plasma thus far are found in concentrations that are orders of magnitude lower than the observed increases in plasma antioxidant capacity. Furthermore, the plasma polyphenols and flavonoids that are putatively responsible for the increased antioxidant capacity of plasma as assessed, e.g., by FRAP or ORAC, should also protect plasma constituents from oxidation ex vivo as assessed by other assays.

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However, the evidence in support of this notion is weak and inconsistent, with 7 of 14 studies showing no effect on ex vivo oxidation of plasma (Table 2). It has also been proposed that dietary flavonoids exert synergistic effects, resulting in unusually large increases in plasma antioxidant capacity. However, the chemical mechanisms for such synergistic interactions have not been explained nor have such interactions been observed in plasma to which flavonoid-rich extracts were added in vitro. Interestingly, the physiological antioxidant urate is a major contributor to plasma total antioxidant capacity (Table 3). This is not surprising given the high concentration of urate in human plasma (160–450 μM) and its powerful reducing and free radical scavenging activities. For instance, urate may contribute as much as 60% to plasma FRAP, 60–90% to plasma TRAP, and about 40% to plasma ORAC-PCA (Table 3). Ascorbate also makes a significant contribution, e.g., about 15% to FRAP and up to 20% of TRAP (CL). Considering the magnitude of these contributions to total antioxidant capacity, it is important to assess the changes in the plasma concentrations of urate and ascorbate after consumption of flavonoid-rich foods, which will allow for the determination of the contributions of dietary flavonoids. Whereas an increase in plasma ascorbate can be expected after consumption of flavonoid-rich foods that also contain vitamin C, such as many fruits and vegetables, the increase in plasma urate observed in numerous studies is surprising (Table 4). Flavonoid-rich foods usually do not contain urate or its precursors, such as inosine. Nevertheless, Cao et al. [116] found significant increases in plasma urate after consumption of strawberries, spinach, or red wine that corresponded to significant increases in plasma ORAC-PCA, FRAP, and TEAC. For example, FRAP increased by 21, 53, and 21 μM trolox equivalents during the first 2 h after the intake of strawberries, spinach, or red wine, respectively, compared to 8 μM after the intake of a control meal. Plasma urate levels also increased significantly after the intake of each food. Based on AUC measurements, the increase in plasma urate after consumption of spinach was much greater than that after consumption of strawberry, which was greater than that after wine, and these increases were similar to the increases in plasma FRAP. Strawberry consumption produced the highest increase in plasma ascorbate, but ascorbate did not appreciably affect plasma antioxidant capacity. Thus, it can be inferred that urate significantly affected plasma FRAP in this study. Other studies have found that wine increases serum urate. Day and Stansbie [124] observed an increase of 23% in serum urate concentration, corresponding to a mean increase of 81 μM, in 6 healthy men 30 min after drinking port wine. This increase in urate was associated with a 24% increase in total antioxidant capacity, corresponding to a mean increase of 109 μM trolox equivalents (Table 4). Both urate and plasma antioxidant capacity declined slowly thereafter, with a highly significant correlation between the two measures. The authors concluded that the acute increase in serum antioxidant capacity after the ingestion of port wine may be attributed to the increase in serum urate, not wine-derived flavonoids. Maxwell and Thorpe [128]

also reported an increase in serum urate levels by 12% or 39 μM in 10 healthy subjects 1 h after drinking French Bordeaux, which was paralleled by a significant increase in antioxidant capacity by 14% or 66 μM trolox equivalents. Natella et al. [137] observed increases of 25% or 72 μM in urate 1 h after ingestion of a meal with red wine, compared to a postprandial increase of 8% or 23 μM after ingestion of a meal with ethanol instead of wine. Another study [92] also found an increase in average plasma urate levels of about 40 μM, or 12% from baseline, after the intake of regular or dealcoholized red wine. Many studies have assessed the antioxidant capacity of plasma after drinking tea, but data on levels of plasma urate in these studies are scarce. Natella and colleagues [133] found significant increases in plasma urate of 18 and 24 μM, or 5 and 7%, 1 h after drinking coffee or green tea, respectively, which was paralleled by increases of 6 and 5% in total antioxidant capacity (Table 4). Thus, several studies indicate that the consumption of flavonoid-rich foods may increase plasma urate, although the underlying mechanism(s) was not elucidated. Because plasma concentrations of urate are much greater than those of flavonoids, it is possible that changes in urate levels account for the relatively large increases in plasma total antioxidant capacity after consumption of flavonoid-rich foods. Antioxidant effects of apples in vitro and in vivo Apples are one of the main sources of flavonoids in the Western diet [22,138,139] and contain as much as 2 g of total phenols per kilogram wet weight, or about 400 mg per apple [15]. The main classes of polyphenols in apples are flavonoids, including quercetin, (−)-epicatechin, (+)-catechin, procyanidins, and anthocyanidins (Fig. 1); dihydrochalcones such as phloretin and phloridzin; and other phenolic compounds such as chlorogenic acid. In an in vitro study, we examined the antioxidant capacity of individual apple polyphenols and apple extracts by the FRAP and ORAC assays. Aqueous extracts of whole Red Delicious apples containing 176 ± 3 mg total phenols per 100 g of apple exhibited FRAP and ORAC values of 1421 ± 45 and 1508 ± 44 μmol trolox equivalents per 100 g of apple, respectively. However, individual polyphenols accounted for only 14–18% of total FRAP and ORAC [74], suggesting that a large proportion of the in vitro antioxidant capacity was due to other compounds, most likely highmolecular-weight procyanidins that are found in amounts of about 130 mg per 100 g apple. Addition of Red Delicious apple extract to human plasma in vitro dose-dependently increased plasma FRAP [74], but not ORAC-PCA (S. Lotito and B. Frei, unpublished observation). For example, plasma FRAP increased from 454 ± 9 to 486 ± 15 (+7%) and 532 ± 12 μM (+17%), respectively, after the addition of 7 or 14 μg total apple phenols per milliliter of plasma. These increases in plasma FRAP are comparable to those observed in humans after the consumption of flavonoid-rich foods (Table 4). Furthermore, in vitro addition of apple phenols significantly increased the resistance of plasma to oxidation. Although apple phenols could not protect plasma ascorbate from oxidation by aqueous peroxyl radicals, the half-life of

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endogenous urate and α-tocopherol and the lag time preceding detectable lipid peroxidation were all significantly increased [15]. Our in vitro observations led us to conclude that Red Delicious apple extracts exhibit high polyphenol content and in vitro antioxidant capacity. Apple polyphenols also significantly increased the resistance of human plasma to oxidation [15] when added in amounts that increased plasma FRAP by 7–17% [74]. To investigate the in vivo relevance of these in vitro observations, we conducted a study in which six healthy volunteers ate five Red Delicious apples, providing 1825 mg of total phenols. Plasma was obtained immediately before and up to 6 h after apple consumption and assessed for resistance to oxidation and total antioxidant capacity. In a second sitting, the same subjects consumed 260 g of plain bagels as a flavonoidfree control and 750 ml of water, matching the carbohydrate content and mass of the five apples. In contrast to the in vitro data, no significant increase in the resistance of plasma to aqueous peroxyl radical-mediated oxidation ex vivo was observed after apple consumption [15]. However, plasma antioxidant capacity measured as FRAP increased significantly 1 h after apple consumption by about 60 μM trolox equivalents (Fig. 2A) [135]. The apples provided about 60 mg of vitamin C, and hence plasma ascorbate increased slightly after apple consumption. However, removal of ascorbate from plasma with ascorbate oxidase did not affect the increase in antioxidant capacity. These data indicate that vitamin C from apples did not make a significant contribution to the change in plasma antioxidant capacity in our study. Consumption of bagels resulted in a time-dependent decrease in plasma FRAP (Fig. 2A), suggesting an effect of food intake per se, in agreement with previous reports [121,134]. Surprisingly, plasma urate levels increased in all subjects by about 35% 1 h after apple consumption and paralleled the increase in plasma FRAP (Fig. 2). The contribution of urate to the total antioxidant capacity was confirmed by preincubation of plasma with uricase, which completely abolished the changes in FRAP after apple consumption [74]. Both FRAP and urate returned to baseline levels 6 h after apple consumption (Fig. 2). These data strongly suggest that transient increases in urate, and not apple polyphenols, are responsible for the increase in plasma antioxidant capacity after apple consumption. Fructose-mediated urate production The rapid and large increases in both plasma urate and antioxidant capacity after apple consumption suggested that the active component(s) is present in apples in relatively large amounts and is easily absorbed. However, apples do not contain urate or its dietary precursors, inosine or other purines. On the other hand, fructose has been known for more than 30 years to increase plasma urate levels consequent to its rapid metabolism by fructokinase [140–142]. Fructose metabolism in this manner leads to a transient decrease in hepatic ATP and inorganic phosphate, which are important inhibitors of 5′-nucleotidase and AMP deaminase, respectively, and thus increased degradation of AMP to uric acid (Fig. 3) [141–143].

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Fig. 2. Changes in plasma antioxidant capacity and urate after consumption of apples, fructose, or bagels. (A) Changes in antioxidant capacity of plasma at the indicated times after food consumption, expressed as ferric-reducing antioxidant potential with ascorbate oxidase treatment (FRAPAO). (B) Changes in plasma urate concentrations in the same subjects. Values are mean changes ± SEM relative to baseline (n = 6 subjects). For experimental details, see Ref. [135]. The p values shown are for time-dependent changes using repeated-measures analysis of variance; *significantly different from time 0 h, using Tukey– Kramer post hoc analysis.

Fructose is found in most fruits. The fructose content of apples is about 6–8 g per 100-g serving [144,145]. Pears and grapes also contain fructose in amounts of about 5–9 g per 100 g. Cherries, blackberries, and blueberries contain 5–7 g, 2– 4 g, and about 4 g of fructose per 100-g serving, respectively. The fructose content is particularly high in dried fruits such as dried prunes (12 g per 100 g) and raisins (34 g per 100 g). Honey also is a well-known source of fructose (about 40 g per 100 g) [17]. Considering the role of fructose metabolism in urate production, we hypothesized that fructose in apples caused the increase in plasma urate—and thus antioxidant capacity—in human subjects after apple consumption. This hypothesis was tested in the same six volunteers enrolled in our study [135]. The subjects drank a solution of 64 g of fructose in 1000 ml of water, matching the fructose content and mass of the five apples. Plasma FRAP increased significantly by about 40 μM 1 h after fructose consumption, and the time-dependent changes in plasma antioxidant capacity were comparable to those observed after apple consumption (Fig. 2A) [135]. In addition, plasma urate levels significantly increased in all subjects after fructose intake (Fig. 2B), as had been seen after apple consumption, whereas plasma ascorbate levels remained unchanged. The increase in plasma urate after apple consumption was strongly

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Fig. 3. Purine nucleotide degradation resulting from fructose catabolism. Rapid conversion of fructose to fructose 1-phosphate catalyzed by fructokinase results in decreased ATP and inorganic phosphate (Pi) levels. Intracellular Pi has a regulatory effect on urate production in the liver by inhibiting AMP deaminase, the enzyme controlling purine breakdown. In addition, ATP levels have a regulatory effect on 5′-nucleotidase. Thus, fructose induces acute depletion of ATP and Pi and causes increased activity of the enzymes involved in the degradation of purine nucleotides to urate. AMP, adenosine monophosphate; IMP, inosine monophosphate; PNP, purine nucleoside phosphorylase.

correlated with the increase in plasma urate after fructose consumption. These observations indicate that the plasma antioxidant capacity after apple consumption increased because of the increase in urate, which was due to the effect of applecontained fructose on adenine nucleotide degradation (Fig. 3). The hyperuricemic effect of fructose administered by intravenous infusion has been observed above a threshold of 0.5 g of fructose per kilogram body weight per hour, with doses of 1.0 and 1.5 g/kg/h significantly increasing serum urate by about 50 and 80 μM, respectively [142]. In addition, a study of six subjects undergoing biliary surgery showed that intravenous infusion of 50 g of fructose (0.67– 0.83 g/kg body wt) within 30 min reduced hepatic ATP levels by about 50% and hepatic total adenosine phosphates and inorganic phosphate by about 35%, which was paralleled by a 64 μM increase in mean serum urate [143]. Average plasma urate levels in our study increased by 31 μM 1 h after an oral dose of fructose of 0.83 g/kg body wt. Thus, our data are in close qualitative and quantitative agreement with other studies on the hyperuricemic effect of fructose in humans [140–143]. Other potential sources of endogenous urate or its derivatives: sucrose, sorbitol, lactate, and methylxanthines Other carbohydrates in fruit also could influence urate production and plasma antioxidant capacity. Sucrose, which like fructose is present in high amounts in fruits, can undergo hydrolysis in vivo to yield equal amounts of fructose and glucose before absorption. Sucrose can range from 2.5 to 5.0 g per 100 g

of apple [144]. In contrast to fructose, glucose does not have a direct effect on plasma urate [143], but may facilitate the absorption of fructose [145]. Solyst et al. [146] demonstrated that a high sucrose intake (2 g/kg body wt) in healthy volunteers produced significant rises in both serum fructose and urate and a decrease in serum phosphate. Serum urate increased by 24 (+8%), 36 (+11%), and 18 μM (+6%) 0.5, 1, and 2 h, respectively, after the sucrose load. These results are in good agreement with our data (Fig. 2) for an oral fructose dose of 0.83 g/kg body wt, considering that the amount of sucrose used by Solyst et al. is equivalent to a fructose dose of 1 g/kg body wt. In another study [147], a sucrose dose of 1 g/kg body wt had no effect on serum urate, emphasizing that doses greater than 0.5 g fructose/kg body wt are needed to produce a measurable effect on serum urate [142]. Taken together, these observations indicate that fructose from sucrose can increase serum or plasma levels of urate. Sorbitol is another carbohydrate found in fruits. Sorbitol is converted to fructose during metabolism in the liver and produces biochemical effects similar to those of fructose on hepatic adenosine phosphate levels in humans [143]. In apples, sorbitol ranges from 0.5 to 1.0 g per 100 g fresh wt [144]. Cherries contain even higher amounts of sorbitol (1.4–2.1 g/ 100 g), and in dried prunes the sorbitol content can be as high as 12 g per 100-g serving [17]. Thus, sorbitol in fruits, in addition to fructose and sucrose, likely contributes to increased plasma urate and antioxidant capacity. Future studies on the in vivo antioxidant effects of flavonoid-rich foods need to consider fructose, sucrose, and sorbitol content, which may significantly affect plasma urate concentrations. Several studies have reported increases in plasma urate after intake of red wine (Table 4). In addition to the sugar in wine, Caccetta et al. [92] suggested that the observed increases in plasma urate could be due to the metabolic effect of lactate. Indeed, it has been shown that lactate can increase urate levels. For example, Burch and Kurke [148] reported that infusion of lactate in healthy volunteers reduced the renal clearance of urate by 70–80%, resulting in an increase of 8–18% in serum urate. Tea, coffee, and chocolate are rich sources of methylxanthines, including caffeine, theobromine, and theophylline (Fig. 4). Green and black tea, respectively, contain 8–40 and 35– 60 mg of caffeine per 200-ml serving, and coffee contains 60– 110 mg of caffeine per 200 ml [149]. Chocolate is a particularly rich source of theobromine (486 and 205 mg per 100 g dark or milk chocolate, respectively) and also contains caffeine (62 and 20 mg per 100 g dark and milk chocolate, respectively) [17]. It is well known that both caffeine and theobromine are readily bioavailable, in contrast to flavonoids, and can be metabolized to methyl derivatives of uric acid (Fig. 4). Caffeine metabolites, such as 1-methylxanthine and 1-methyl uric acid, exhibit in vitro antioxidant activity in the ORAC assay and prevent LDL oxidation comparable to the effects of urate, trolox, and ascorbate [150]. In addition, we found that the methyl derivatives of uric acid exhibit a reducing activity similar to that of uric acid (Fig. 5). High levels of methylxanthines in tea, coffee, and chocolate, and their rapid absorption and metabolism

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Fig. 4. Structures of methylxanthines (caffeine, theobromine, and theophylline), uric acid, and uric acid methyl derivatives (1-methyl uric acid, 1,3-dimethyl uric acid, 1,7-dimethyl uric acid, and 3,7-dimethyl uric acid).

to methyl uric acid derivatives, could significantly increase plasma total antioxidant capacity. Conclusions Some of the health benefits of fruits and vegetables have been attributed to their content of polyphenols and flavonoids. However, the specific mechanism(s) by which these compounds affect human health remains unclear, despite extensive research

Fig. 5. Reducing activity of uric acid and its methyl derivatives. The reducing activity was measured as FRAP according to Benzie et al. [104] and is expressed as mM trolox equivalents/mM test compound.

conducted in this area in recent years. Most of that research has focused on the antioxidant properties of flavonoids, which are well characterized and well established in vitro. However, the in vitro data often conflict with results obtained from in vivo studies on the antioxidant capacity of plasma or the resistance of plasma and lipoproteins to oxidation ex vivo after the consumption of flavonoid-rich foods by human subjects. These inconsistencies between the in vitro and the in vivo data are likely explained by the limited bioavailability of dietary flavonoids and their extensive metabolism in humans. Based on the data reviewed in this article, it seems highly unlikely that flavonoids can make a significant contribution to antioxidant protection of plasma and other extracellular fluids in vivo. It should be noted that the efficacy of a compound to act as an antioxidant in vivo cannot be estimated solely from its effect on the total antioxidant capacity of plasma. The contribution of flavonoids to plasma antioxidant protection in vivo—measured by the methods discussed in this review—is expected to be small, because of the low plasma concentrations of flavonoids, their effective and extensive biotransformation, and the large contribution of other physiological antioxidants such as urate and ascorbate to plasma total antioxidant capacity. This does not preclude the possibility that flavonoids may accumulate in tissues where they might exert local antioxidant effects or that very low concentrations of flavonoids may modulate cell signaling, gene regulation, angiogenesis, and other biological processes by non-antioxidant mechanisms, which may explain the purported health benefits of flavonoids. Foods rich in flavonoids contain other substances that may affect plasma total antioxidant capacity, either directly, e.g., by providing vitamin C, or indirectly, e.g., by stimulating

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endogenous production of urate. Although urate is a known physiological antioxidant, its biologic functions and potential health benefits are unclear. Ames and co-workers [151] pointed out that during primate evolution, the increase in urate levels due to loss of uricase coincided with a large increase in life span. Remarkably, more than 90% of urate is reabsorbed in the kidneys, suggesting that urate has an important physiological function [152]. Excessive urate causes gout, and urate has also been reported to be an independent risk factor for cardio- and cerebrovascular diseases, but these data are controversial [153,154]. Under certain experimental conditions, urate can act as a pro-oxidant in vitro, although this effect was not observed at physiological concentrations of urate [155,156]. In contrast, urate may protect against multiple sclerosis and possibly other inflammatory conditions due to its capacity to inhibit peroxynitrite-mediated nitration reactions and leakage of the blood–brain barrier [157–159]. Whether plasma urate is a marker of ischemia–reperfusion events or has a physiological function per se remains unknown. The data reviewed here and our own observations [135] show that plasma urate levels readily and transiently increase after consumption of fructose-containing foods, resulting in increased plasma total antioxidant capacity. Other dietary constituents such as sucrose, sorbitol, and lactate may also increase endogenous production of urate and, hence, plasma antioxidant capacity. In addition, readily absorbable methylxanthines in flavonoid-rich foods, upon metabolism to methyl derivatives of uric acid, may contribute to increased plasma antioxidant capacity. These observations emphasize the need to include proper controls of food, carbohydrate, and methylxanthine intake in studies of the in vivo antioxidant effects of flavonoid-rich foods to rule out postprandial effects on plasma antioxidant capacity not related to flavonoids. Therefore, the consumption of flavonoid-rich foods is the cause of the increased plasma total antioxidant capacity, which is not just an epiphenomenon. However, the flavonoids make only a small, if any, contribution to the increased plasma antioxidant capacity, which seems to be largely the consequence of urate production stimulated by fructose and other components of flavonoid-rich foods. Acknowledgments The work in the authors' laboratory is supported by a grant from the Washington Tree Fruit Research Commission (Wenatchee, WA, USA), NIH Grants P01 AT002034 and T32 AT002688 from the National Center for Complementary and Alternative Medicine, and American Heart Association Grant 03254862. The authors are indebted to Stephen Lawson from the Linus Pauling Institute, Oregon State University, for carefully editing the manuscript. References [1] Verlangieri, A. J.; Kapeghian, J. C.; el-Dean, S.; Bush, M. Fruit and vegetable consumption and cardiovascular mortality. Med. Hypotheses 16:7–15; 1985.

[2] Joshipura, K. J.; Ascherio, A.; Manson, J. E.; Stampfer, M. J.; Rimm, E. B.; Speizer, F. E.; Hennekens, C. H.; Spiegelman, D.; Willett, W. C. Fruit and vegetable intake in relation to risk of ischemic stroke. JAMA 282:1233–1239; 1999. [3] Riboli, E.; Norat, T. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am. J. Clin. Nutr. 78:559S–569S; 2003. [4] Bosetti, C.; Spertini, L.; Parpinel, M.; Gnagnarella, P.; Lagiou, P.; Negri, E.; Franceschi, S.; Montella, M.; Peterson, J.; Dwyer, J.; Giacosa, A.; La Vecchia, C. Flavonoids and breast cancer risk in Italy. Cancer Epidemiol. Biomarkers Prev. 14:805–808; 2005. [5] Peterson, J.; Lagiou, P.; Samoli, E.; Lagiou, A.; Katsouyanni, K.; La Vecchia, C.; Dwyer, J.; Trichopoulos, D. Flavonoid intake and breast cancer risk: a case-control study in Greece. Br. J. Cancer 89:1255–1259; 2003. [6] Arts, I. C.; Hollman, P. C. Polyphenols and disease risk in epidemiologic studies. Am. J. Clin. Nutr. 81:317S–325S; 2005. [7] Knekt, P.; Kumpulainen, J.; Jarvinen, R.; Rissanen, H.; Heliovaara, M.; Reunanen, A.; Hakulinen, T.; Aromaa, A. Flavonoid intake and risk of chronic diseases. Am. J. Clin. Nutr. 76:560–568; 2002. [8] Keli, S. O.; Hertog, M. G.; Feskens, E. J.; Kromhout, D. Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen study. Arch. Intern. Med. 156:637–642; 1996. [9] Hirvonen, T.; Virtamo, J.; Korhonen, P.; Albanes, D.; Pietinen, P. Flavonol and flavone intake and the risk of cancer in male smokers (Finland). Cancer Causes Control 12:789–796; 2001. [10] Arts, I. C.; Jacobs Jr., D. R.; Gross, M.; Harnack, L. J.; Folsom, A. R. Dietary catechins and cancer incidence among postmenopausal women: the Iowa Women's Health Study (United States). Cancer Causes Control 13:373–382; 2002. [11] Aviram, M.; Fuhrman, B. Wine flavonoids protect against LDL oxidation and atherosclerosis. Ann. N. Y. Acad. Sci. 957:146–161; 2002. [12] Rietveld, A.; Wiseman, S. Antioxidant effects of tea: evidence from human clinical trials. J. Nutr. 133:3285S–3292S; 2003. [13] Serafini, M.; Laranjinha, J. A.; Almeida, L. M.; Maiani, G. Inhibition of human LDL lipid peroxidation by phenol-rich beverages and their impact on plasma total antioxidant capacity in humans. J. Nutr. Biochem. 11:585–590; 2000. [14] Vinson, J. A.; Su, X.; Zubik, L.; Bose, P. Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem. 49:5315–5321; 2001. [15] Lotito, S. B.; Frei, B. Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects. Free Radic. Biol. Med. 36:201–211; 2004. [16] Aaby, K.; Skrede, G.; Wrolstad, R. E. Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa). J. Agric. Food Chem. 53:4032–4040; 2005. [17] Nutrient Data Laboratory; Beltsville Human Nutrition Research Center, USDA. Nutrient database for standard reference, release 18. USDA Database; 2005. Available at http://www.nal.usda.gov/fnic/foodcomp. [18] Nutrient Data Laboratory; Beltsville Human Nutrition Research Center, USDA. Flavonoid content of selected foods. USDA Database; 2003. Available at http://www.nal.usda.gov/fnic/foodcomp. [19] Moyer, R. A.; Hummer, K. E.; Finn, C. E.; Frei, B.; Wrolstad, R. E. Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes. J. Agric. Food Chem. 50:519–525; 2002. [20] Kuti, J. O.; Konuru, H. B. Antioxidant capacity and phenolic content in leaf extracts of tree spinach (Cnidoscolus spp.). J. Agric. Food Chem. 52:117–121; 2004. [21] Karakaya, S.; El, S. N.; Tas, A. A. Antioxidant activity of some foods containing phenolic compounds. Int. J. Food. Sci. Nutr. 52:501–508; 2001. [22] Proteggente, A. R.; Pannala, A. S.; Paganga, G.; Van Buren, L.; Wagner, E.; Wiseman, S.; Van De Put, F.; Dacombe, C.; Rice-Evans, C. A. The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin C composition. Free Radic. Res. 36:217–233; 2002. [23] Chu, Y. F.; Sun, J.; Wu, X.; Liu, R. H. Antioxidant and antiproliferative

S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

[24]

[25] [26]

[27]

[28]

[29]

[30] [31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

activities of common vegetables. J. Agric. Food Chem. 50:6910–6916; 2002. Astill, C.; Birch, M. R.; Dacombe, C.; Humphrey, P. G.; Martin, P. T. Factors affecting the caffeine and polyphenol contents of black and green tea infusions. J. Agric. Food Chem. 49:5340–5347; 2001. Scalbert, A.; Williamson, G. Dietary intake and bioavailability of polyphenols. J. Nutr. 130:2073S–2085S; 2000. Svilaas, A.; Sakhi, A. K.; Andersen, L. F.; Svilaas, T.; Strom, E. C.; Jacobs Jr., D. R.; Ose, L.; Blomhoff, R. Intakes of antioxidants in coffee, wine, and vegetables are correlated with plasma carotenoids in humans. J. Nutr. 134:562–567; 2004. Lotito, S. B.; Renart, M. L.; Fraga, C. G. Assessing the antioxidant capacity in the hydrophilic and lipophilic domains: study of a sample of Argentine wines. Ann. N. Y. Acad. Sci. 957:284–287; 2002. Actis-Goretta, L.; Mackenzie, G. G.; Oteiza, P. I.; Fraga, C. G. Comparative study on the antioxidant capacity of wines and other plant-derived beverages. Ann. N. Y. Acad. Sci. 957:279–283; 2002. Rivero, D.; Perez-Magarino, S.; Gonzalez-Sanjose, M. L.; Valls-Belles, V.; Codoner, P.; Muniz, P. Inhibition of induced DNA oxidative damage by beers: correlation with the content of polyphenols and melanoidins. J. Agric. Food Chem. 53:3637–3642; 2005. Bors, W.; Saran, M. Radical scavenging by flavonoid antioxidants. Free Radic. Res. Commun. 2:289–294; 1987. Bors, W.; Michel, C.; Stettmaier, K. Structure–activity relationships governing antioxidant capacities of plant polyphenols. Methods Enzymol. 335:166–180; 2001. Jovanovic, S. V.; Simic, M. G. Antioxidants in nutrition. Ann. N. Y. Acad. Sci. 899:326–334; 2000. Mukai, K.; Mitani, S.; Ohara, K.; Nagaoka, S. Structure–activity relationship of the tocopherol-regeneration reaction by catechins. Free Radic. Biol. Med. 38:1243–1256; 2005. Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Bioavailability and bioefficacy of polyphenols in humans: I. Review of 97 bioavailability studies. Am. J. Clin. Nutr. 81:230S–242S; 2005. Stocker, R.; Frei, B. Endogenous antioxidant defences in human blood plasma. In: Sies, H., ed. Oxidative stress: oxidants and antioxidants. New York: Academic Press; 1991:213–243. Moon, J. H.; Nakata, R.; Oshima, S.; Inakuma, T.; Terao, J. Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279: R461–R467; 2000. Kong, A. N.; Owuor, E.; Yu, R.; Hebbar, V.; Chen, C.; Hu, R.; Mandlekar, S. Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metab. Rev. 33:255–271; 2001. Walle, U. K.; Walle, T. Induction of human UDP-glucuronosyltransferase UGT1A1 by flavonoids—Structural requirements. Drug Metab. Dispos. 30:564–569; 2002. Natsume, M.; Osakabe, N.; Yasuda, A.; Baba, S.; Tokunaga, T.; Kondo, K.; Osawa, T.; Terao, J. In vitro antioxidative activity of (−)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic. Res. 38:1341–1348; 2004. Janisch, K. M.; Williamson, G.; Needs, P.; Plumb, G. W. Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin. Free Radic. Res. 38:877–884; 2004. Ward, N. C.; Croft, K. D.; Puddey, I. B.; Hodgson, J. M. Supplementation with grape seed polyphenols results in increased urinary excretion of 3-hydroxyphenylpropionic acid, an important metabolite of proanthocyanidins in humans. J. Agric. Food Chem. 52:5545–5549; 2004. Rios, L. Y.; Gonthier, M. P.; Remesy, C.; Mila, I.; Lapierre, C.; Lazarus, S. A.; Williamson, G.; Scalbert, A. Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects. Am. J. Clin. Nutr. 77:912–918; 2003. Graefe, E. U.; Wittig, J.; Mueller, S.; Riethling, A. K.; Uehleke, B.; Drewelow, B.; Pforte, H.; Jacobasch, G.; Derendorf, H.; Veit, M. Pharmacokinetics and bioavailability of quercetin glycosides in humans. J. Clin. Pharmacol. 41:492–499; 2001. Hollman, P. C.; de Vries, J. H.; van Leeuwen, S. D.; Mengelers, M. J.;

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

1743

Katan, M. B. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am. J. Clin. Nutr. 62:1276–1282; 1995. Nemeth, K.; Plumb, G. W.; Berrin, J. G.; Juge, N.; Jacob, R.; Naim, H. Y.; Williamson, G.; Swallow, D. M.; Kroon, P. A. Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans. Eur. J. Nutr. 42:29–42; 2003. Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jimenez, L. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79:727–747; 2004. Arts, I. C.; Sesink, A. L.; Faassen-Peters, M.; Hollman, P. C. The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides. Br. J. Nutr. 91:841–847; 2004. Hollman, P. C.; van der Gaag, M.; Mengelers, M. J.; van Trijp, J. M.; de Vries, J. H.; Katan, M. B. Absorption and disposition kinetics of the dietary antioxidant quercetin in man. Free Radic. Biol. Med. 21:703–707; 1996. Hollman, P. C.; van Trijp, J. M.; Buysman, M. N.; van der Gaag, M. S.; Mengelers, M. J.; de Vries, J. H.; Katan, M. B. Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Lett. 418:152–156; 1997. Aziz, A. A.; Edwards, C. A.; Lean, M. E.; Crozier, A. Absorption and excretion of conjugated flavonols, including quercetin-4′-O-betaglucoside and isorhamnetin-4′-O-beta-glucoside by human volunteers after the consumption of onions. Free Radic. Res. 29:257–269; 1998. McAnlis, G. T.; McEneny, J.; Pearce, J.; Young, I. S. Absorption and antioxidant effects of quercetin from onions, in man. Eur. J. Clin. Nutr. 53:92–96; 1999. Widlansky, M. E.; Duffy, S. J.; Hamburg, N. M.; Gokce, N.; Warden, B. A.; Wiseman, S.; Keaney Jr., J. F.; Frei, B.; Vita, J. A. Effects of black tea consumption on plasma catechins and markers of oxidative stress and inflammation in patients with coronary artery disease. Free Radic. Biol. Med. 38:499–506; 2005. van het Hof, K. H.; Kivits, G. A.; Weststrate, J. A.; Tijburg, L. B. Bioavailability of catechins from tea: the effect of milk. Eur. J. Clin. Nutr. 52:356–359; 1998. Leenen, R.; Roodenburg, A. J.; Tijburg, L. B.; Wiseman, S. A. A single dose of tea with or without milk increases plasma antioxidant activity in humans. Eur. J. Clin. Nutr. 54:87–92; 2000. van het Hof, K. H.; Wiseman, S. A.; Yang, C. S.; Tijburg, L. B. Plasma and lipoprotein levels of tea catechins following repeated tea consumption. Proc. Soc. Exp. Biol. Med. 220:203–209; 1999. Donovan, J. L.; Bell, J. R.; Kasim-Karakas, S.; German, J. B.; Walzem, R. L.; Hansen, R. J.; Waterhouse, A. L. Catechin is present as metabolites in human plasma after consumption of red wine. J. Nutr. 129:1662–1668; 1999. Bell, J. R.; Donovan, J. L.; Wong, R.; Waterhouse, A. L.; German, J. B.; Walzem, R. L.; Kasim-Karakas, S. E. (+)-Catechin in human plasma after ingestion of a single serving of reconstituted red wine. Am. J. Clin. Nutr. 71:103–108; 2000. Rein, D.; Lotito, S.; Holt, R. R.; Keen, C. L.; Schmitz, H. H.; Fraga, C. G. Epicatechin in human plasma: in vivo determination and effect of chocolate consumption on plasma oxidation status. J. Nutr. 130:2109S–2114S; 2000. Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm, D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L. Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 76:798–804; 2002. Baba, S.; Osakabe, N.; Yasuda, A.; Natsume, M.; Takizawa, T.; Nakamura, T.; Terao, J. Bioavailability of (−)-epicatechin upon intake of chocolate and cocoa in human volunteers. Free Radic. Res. 33:635–641; 2000. Pietta, P. G.; Simonetti, P.; Gardana, C.; Brusamolino, A.; Morazzoni, P.; Bombardelli, E. Catechin metabolites after intake of green tea infusions. Biofactors 8:111–118; 1998. Meng, X.; Sang, S.; Zhu, N.; Lu, H.; Sheng, S.; Lee, M. J.; Ho, C. T.; Yang, C. S. Identification and characterization of methylated and ring-

1744

[63]

[64]

[65]

[66]

[67] [68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76] [77]

[78]

[79]

[80]

[81]

S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 fission metabolites of tea catechins formed in humans, mice, and rats. Chem. Res. Toxicol. 15:1042–1050; 2002. Lee, M. J.; Maliakal, P.; Chen, L.; Meng, X.; Bondoc, F. Y.; Prabhu, S.; Lambert, G.; Mohr, S.; Yang, C. S. Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol. Biomarkers Prev. 11:1025–1032; 2002. Cao, G.; Muccitelli, H. U.; Sanchez-Moreno, C.; Prior, R. L. Anthocyanins are absorbed in glycated forms in elderly women: a pharmacokinetic study. Am. J. Clin. Nutr. 73:920–926; 2001. McGhie, T. K.; Ainge, G. D.; Barnett, L. E.; Cooney, J. M.; Jensen, D. J. Anthocyanin glycosides from berry fruit are absorbed and excreted unmetabolized by both humans and rats. J. Agric. Food Chem. 51:4539–4548; 2003. Felgines, C.; Talavera, S.; Gonthier, M. P.; Texier, O.; Scalbert, A.; Lamaison, J. L.; Remesy, C. Strawberry anthocyanins are recovered in urine as glucuro- and sulfoconjugates in humans. J. Nutr. 133:1296–1301; 2003. Kay, C. D.; Mazza, G.; Holub, B. J.; Wang, J. Anthocyanin metabolites in human urine and serum. Br. J. Nutr. 91:933–942; 2004. Nielsen, I. L.; Dragsted, L. O.; Ravn-Haren, G.; Freese, R.; Rasmussen, S. E. Absorption and excretion of black currant anthocyanins in humans and watanabe heritable hyperlipidemic rabbits. J. Agric. Food Chem. 51:2813–2820; 2003. Matsumoto, H.; Inaba, H.; Kishi, M.; Tominaga, S.; Hirayama, M.; Tsuda, T. Orally administered delphinidin 3-rutinoside and cyanidin 3rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms. J. Agric. Food Chem. 49:1546–1551; 2001. Bub, A.; Watzl, B.; Heeb, D.; Rechkemmer, G.; Briviba, K. Malvidin-3glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and red grape juice. Eur. J. Nutr. 40:113–120; 2001. Manach, C.; Morand, C.; Gil-Izquierdo, A.; Bouteloup-Demange, C.; Remesy, C. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 57:235–242; 2003. Erlund, I.; Meririnne, E.; Alfthan, G.; Aro, A. Plasma kinetics and urinary excretion of the flavanones naringenin and hesperetin in humans after ingestion of orange juice and grapefruit juice. J. Nutr. 131:235–241; 2001. Lotito, S. B.; Actis-Goretta, L.; Renart, M. L.; Caligiuri, M.; Rein, D.; Schmitz, H. H.; Steinberg, F. M.; Keen, C. L.; Fraga, C. G. Influence of oligomer chain length on the antioxidant activity of procyanidins. Biochem. Biophys. Res. Commun. 276:945–951; 2000. Lotito, S. B.; Frei, B. Apples and plasma antioxidant capacity in humans: the roles of fructose and urateIn: Puntarulo, S., Boveris, A., eds. Proceedings of the XII Biennial Meeting of the Society for Free Radical Research International, Medimond International Proceedings Bologna: Monduzzi; 2004:41–48. Nutrient Data Laboratory; Beltsville Human Nutrition Research Center, USDA. Proanthocyanidin content of selected foods. USDA Database; 2004. Available at http://www.nal.usda.gov/fnic/foodcomp. Lotito, S. B.; Fraga, C. G. (+)-Catechin prevents human plasma oxidation. Free Radic. Biol. Med. 24:435–441; 1998. Lotito, S. B.; Fraga, C. G. Catechins delay lipid oxidation and alphatocopherol and beta-carotene depletion following ascorbate depletion in human plasma. Proc. Soc. Exp. Biol. Med. 225:32–38; 2000. Cherubini, A.; Beal, M. F.; Frei, B. Black tea increases the resistance of human plasma to lipid peroxidation in vitro, but not ex vivo. Free Radic. Biol. Med. 27:381–387; 1999. Leung, L. K.; Su, Y.; Chen, R.; Zhang, Z.; Huang, Y.; Chen, Z. Y. Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J. Nutr. 131:2248–2251; 2001. Zhu, Q. Y.; Huang, Y.; Tsang, D.; Chen, Z. Y. Regeneration of alphatocopherol in human low-density lipoprotein by green tea catechin. J. Agric. Food Chem. 47:2020–2025; 1999. Ivanov, V.; Carr, A. C.; Frei, B. Red wine antioxidants bind to human lipoproteins and protect them from metal ion-dependent and -independent oxidation. J. Agric. Food Chem. 49:4442–4449; 2001.

[82] Pearson, D. A.; Tan, C. H.; German, J. B.; Davis, P. A.; Gershwin, M. E. Apple juice inhibits human low density lipoprotein oxidation. Life Sci. 64:1913–1920; 1999. [83] Shafiee, M.; Carbonneau, M. A.; Urban, N.; Descomps, B.; Leger, C. L. Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production: a comparison to other extracts or compounds. Free Radic. Res. 37:573–584; 2003. [84] van het Hof, K. H.; de Boer, H. S.; Wiseman, S. A.; Lien, N.; Westrate, J. A.; Tijburg, L. B. Consumption of green or black tea does not increase resistance of low-density lipoprotein to oxidation in humans. Am. J. Clin. Nutr. 66:1125–1132; 1997. [85] McAnlis, G. T.; McEneny, J.; Pearce, J.; Young, I. S. Black tea consumption does not protect low density lipoprotein from oxidative modification. Eur. J. Clin. Nutr. 52:202–206; 1998. [86] Nakagawa, K.; Ninomiya, M.; Okubo, T.; Aoi, N.; Juneja, L. R.; Kim, M.; Yamanaka, K.; Miyazawa, T. Tea catechin supplementation increases antioxidant capacity and prevents phospholipid hydroperoxidation in plasma of humans. J. Agric. Food Chem. 47:3967–3973; 1999. [87] Miura, Y.; Chiba, T.; Miura, S.; Tomita, I.; Umegaki, K.; Ikeda, M.; Tomita, T. Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans. J. Nutr. Biochem. 11:216–222; 2000. [88] Hodgson, J. M.; Burke, V.; Puddey, I. B. Acute effects of tea on fasting and postprandial vascular function and blood pressure in humans. J. Hypertens. 23:47–54; 2005. [89] Yukawa, G. S.; Mune, M.; Otani, H.; Tone, Y.; Liang, X. M.; Iwahashi, H.; Sakamoto, W. Effects of coffee consumption on oxidative susceptibility of low-density lipoproteins and serum lipid levels in humans. Biochemistry (Moscow) 69:70–74; 2004. [90] Chopra, M.; Fitzsimons, P. E.; Strain, J. J.; Thurnham, D. I.; Howard, A. N. Nonalcoholic red wine extract and quercetin inhibit LDL oxidation without affecting plasma antioxidant vitamin and carotenoid concentrations. Clin. Chem. 46:1162–1170; 2000. [91] de Rijke, Y. B.; Demacker, P. N.; Assen, N. A.; Sloots, L. M.; Katan, M. B.; Stalenhoef, A. F. Red wine consumption does not affect oxidizability of low-density lipoproteins in volunteers. Am. J. Clin. Nutr. 63:329–334; 1996. [92] Caccetta, R. A.; Croft, K. D.; Beilin, L. J.; Puddey, I. B. Ingestion of red wine significantly increases plasma phenolic acid concentrations but does not acutely affect ex vivo lipoprotein oxidizability. Am. J. Clin. Nutr. 71:67–74; 2000. [93] Marniemi, J.; Hakala, P.; Maki, J.; Ahotupa, M. Partial resistance of low density lipoprotein to oxidation in vivo after increased intake of berries. Nutr. Metab. Cardiovasc. Dis. 10:331–337; 2000. [94] O'Byrne, D. J.; Devaraj, S.; Grundy, S. M.; Jialal, I. Comparison of the antioxidant effects of Concord grape juice flavonoids alpha-tocopherol on markers of oxidative stress in healthy adults. Am. J. Clin. Nutr. 76:1367–1374; 2002. [95] Day, A. P.; Kemp, H. J.; Bolton, C.; Hartog, M.; Stansbie, D. Effect of concentrated red grape juice consumption on serum antioxidant capacity and low-density lipoprotein oxidation. Ann. Nutr. Metab. 41:353–357; 1997. [96] Hirano, R.; Osakabe, N.; Iwamoto, A.; Matsumoto, A.; Natsume, M.; Takizawa, T.; Igarashi, O.; Itakura, H.; Kondo, K. Antioxidant effects of polyphenols in chocolate on low-density lipoprotein both in vitro and ex vivo. J. Nutr. Sci. Vitaminol. (Tokyo) 46:199–204; 2000. [97] Osakabe, N.; Baba, S.; Yasuda, A.; Iwamoto, T.; Kamiyama, M.; Takizawa, T.; Itakura, H.; Kondo, K. Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers. Free Radic. Res. 34:93–99; 2001. [98] Wan, Y.; Vinson, J. A.; Etherton, T. D.; Proch, J.; Lazarus, S. A.; KrisEtherton, P. M. Effects of cocoa powder and dark chocolate on LDL oxidative susceptibility and prostaglandin concentrations in humans. Am. J. Clin. Nutr. 74:596–602; 2001. [99] Mathur, S.; Devaraj, S.; Grundy, S. M.; Jialal, I. Cocoa products decrease low density lipoprotein oxidative susceptibility but do not affect biomarkers of inflammation in humans. J. Nutr. 132:3663–3667; 2002.

S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 [100] Mursu, J.; Voutilainen, S.; Nurmi, T.; Rissanen, T. H.; Virtanen, J. K.; Kaikkonen, J.; Nyyssonen, K.; Salonen, J. T. Dark chocolate consumption increases HDL cholesterol concentration and chocolate fatty acids may inhibit lipid peroxidation in healthy humans. Free Radic. Biol. Med. 37:1351–1359; 2004. [101] Engler, M. B.; Engler, M. M.; Chen, C. Y.; Malloy, M. J.; Browne, A.; Chiu, E. Y.; Kwak, H. K.; Milbury, P.; Paul, S. M.; Blumberg, J.; MietusSnyder, M. L. Flavonoid-rich dark chocolate improves endothelial function and increases plasma epicatechin concentrations in healthy adults. J. Am. Coll. Nutr. 23:197–204; 2004. [102] Wiswedel, I.; Hirsch, D.; Kropf, S.; Gruening, M.; Pfister, E.; Schewe, T.; Sies, H. Flavanol-rich cocoa drink lowers plasma F(2)-isoprostane concentrations in humans. Free Radic. Biol. Med. 37:411–421; 2004. [103] Boyle, S. P.; Dobson, V. L.; Duthie, S. J.; Kyle, J. A.; Collins, A. R. Absorption and DNA protective effects of flavonoid glycosides from an onion meal. Eur. J. Nutr. 39:213–223; 2000. [104] Benzie, I. F.; Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239:70–76; 1996. [105] Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic. Biol. Med. 14:303–311; 1993. [106] Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; RiceEvans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26:1231–1237; 1999. [107] Ghiselli, A.; Serafini, M.; Maiani, G.; Azzini, E.; Ferro-Luzzi, A. A fluorescence-based method for measuring total plasma antioxidant capability. Free Radic. Biol. Med. 18:29–36; 1995. [108] Cao, G.; Prior, R. L. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin. Chem. 44:1309–1315; 1998. [109] Prior, R. L.; Cao, G. In vivo total antioxidant capacity: comparison of different analytical methods. Free Radic. Biol. Med. 27:1173–1181; 1999. [110] Rice-Evans, C. A. Measurement of total antioxidant activity as a marker of antioxidant status in vivo: procedures and limitations. Free Radic. Res. 33:S59–S66 (Suppl.); 2000. [111] Ghiselli, A.; Serafini, M.; Natella, F.; Scaccini, C. Total antioxidant capacity as a tool to assess redox status: critical view and experimental data. Free Radic. Biol. Med. 29:1106–1114; 2000. [112] Chandra, S.; De Mejia Gonzalez, E. Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas. J. Agric. Food Chem. 52:3583–3589; 2004. [113] Pazourek, J.; Gajdosova, D.; Spanila, M.; Farkova, M.; Novotna, K.; Havel, J. Analysis of polyphenols in wines: correlation between total polyphenolic content and antioxidant potential from photometric measurements. Prediction of cultivars and vintage from capillary zone electrophoresis fingerprints using artificial neural network. J. Chromatogr. A 1081:48–54; 2005. [114] Lee, K. W.; Kim, Y. J.; Lee, H. J.; Lee, C. Y. Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine. J. Agric. Food Chem. 51:7292–7295; 2003. [115] Cao, G.; Booth, S. L.; Sadowski, J. A.; Prior, R. L. Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. Am. J. Clin. Nutr. 68:1081–1087; 1998. [116] Cao, G.; Russell, R. M.; Lischner, N.; Prior, R. L. Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. J. Nutr. 128:2383–2390; 1998. [117] Lee, A.; Thurnham, D. I.; Chopra, M. Consumption of tomato products with olive oil but not sunflower oil increases the antioxidant activity of plasma. Free Radic. Biol. Med. 29:1051–1055; 2000. [118] Serafini, M.; Bugianesi, R.; Salucci, M.; Azzini, E.; Raguzzini, A.; Maiani, G. Effect of acute ingestion of fresh and stored lettuce (Lactuca sativa) on plasma total antioxidant capacity and antioxidant levels in human subjects. Br.J. Nutr. 88:615–623; 2002. [119] Pedersen, C. B.; Kyle, J.; Jenkinson, A. M.; Gardner, P. T.; McPhail,

[120]

[121]

[122]

[123] [124] [125] [126]

[127]

[128] [129]

[130] [131]

[132]

[133]

[134] [135]

[136] [137]

[138]

[139]

[140] [141]

[142] [143]

1745

D. B.; Duthie, G. G. Effects of blueberry and cranberry juice consumption on the plasma antioxidant capacity of healthy female volunteers. Eur. J. Clin. Nutr. 54:405–408; 2000. Kay, C. D.; Holub, B. J. The effect of wild blueberry (Vaccinium angustifolium) consumption on postprandial serum antioxidant status in human subjects. Br.J. Nutr. 88:389–398; 2002. Mazza, G.; Kay, C. D.; Cottrell, T.; Holub, B. J. Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects. J. Agric. Food Chem. 50:7731–7737; 2002. Benzie, I. F.; Szeto, Y. T.; Strain, J. J.; Tomlinson, B. Consumption of green tea causes rapid increase in plasma antioxidant power in humans. Nutr. Cancer 34:83–87; 1999. Serafini, M.; Ghiselli, A.; Ferro-Luzzi, A. In vivo antioxidant effect of green and black tea in man. Eur. J. Clin. Nutr. 50:28–32; 1996. Day, A.; Stansbie, D. Cardioprotective effect of red wine may be mediated by urate. Clin. Chem. 41:1319–1320; 1995. Serafini, M.; Maiani, G.; Ferro-Luzzi, A. Alcohol-free red wine enhances plasma antioxidant capacity in humans. J. Nutr. 128:1003–1007; 1998. Whitehead, T. P.; Robinson, D.; Allaway, S.; Syms, J.; Hale, A. Effect of red wine ingestion on the antioxidant capacity of serum. Clin. Chem. 41:32–35; 1995. Duthie, G. G.; Pedersen, M. W.; Gardner, P. T.; Morrice, P. C.; Jenkinson, A. M.; McPhail, D. B.; Steele, G. M. The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. Eur. J. Clin. Nutr. 52:733–736; 1998. Maxwell, S.; Thorpe, G. Impact of red wine on antioxidant status in vivo. Eur. J. Heart 21:1482–1483; 2000. Ghiselli, A.; Natella, F.; Guidi, A.; Montanari, L.; Fantozzi, P.; Scaccini, C. Beer increases plasma antioxidant capacity in humans. J. Nutr. Biochem. 11:76–80; 2000. Serafini, M.; Bugianesi, R.; Maiani, G.; Valtuena, S.; De Santis, S.; Crozier, A. Plasma antioxidants from chocolate. Nature 424:1013; 2003. Schroeter, H.; Holt, R. R.; Orozco, T. J.; Schmitz, H. H.; Keen, C. L. Nutrition: milk and absorption of dietary flavanols. Nature 426:787–788; discussion 788; 2003. Nardini, M.; Cirillo, E.; Natella, F.; Scaccini, C. Absorption of phenolic acids in humans after coffee consumption. J. Agric. Food Chem. 50:5735–5741; 2002. Natella, F.; Nardini, M.; Giannetti, I.; Dattilo, C.; Scaccini, C. Coffee drinking influences plasma antioxidant capacity in humans. J. Agric. Food Chem. 50:6211–6216; 2002. Cao, G.; Prior, R. L. Postprandial increases in serum antioxidant capacity in older women. J. Appl. Physiol. 89:877–883; 2000. Lotito, S. B.; Frei, B. The increase in human plasma antioxidant capacity after apple consumption is due to the metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids. Free Radic. Biol. Med. 37:251–258; 2004. Sung, H.; Nah, J.; Chun, S.; Park, H.; Yang, S. E.; Min, W. K. In vivo antioxidant effect of green tea. Eur. J. Clin. Nutr. 54:527–529; 2000. Natella, F.; Ghiselli, A.; Guidi, A.; Ursini, F.; Scaccini, C. Red wine mitigates the postprandial increase of LDL susceptibility to oxidation. Free Radic. Biol. Med. 30:1036–1044; 2001. Hertog, M. G.; Feskens, E. J.; Hollman, P. C.; Katan, M. B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342:1007–1011; 1993. Hertog, M. G.; Kromhout, D.; Aravanis, C.; Blackburn, H.; Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.; Nedeljkovic, S., et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the Seven Countries Study. Arch. Intern. Med. 155:381–386; 1995. Perheentupa, J.; Raivio, K. Fructose-induced hyperuricaemia. Lancet 2:528–531; 1967. Maenpaa, P. H.; Raivio, K. O.; Kekomaki, M. P. Liver adenine nucleotides: fructose-induced depletion and its effect on protein synthesis. Science 161:1253–1254; 1968. Heuckenkamp, P. U.; Zollner, N. Fructose-induced hyperuricaemia. Lancet 1:808–809; 1971. Bode, J. C.; Zelder, O.; Rumpelt, H. J.; Wittkamp, U. Depletion of liver adenosine phosphates and metabolic effects of intravenous infusion of

1746

[144]

[145]

[146]

[147]

[148] [149]

[150]

[151]

S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 fructose or sorbitol in man and in the rat. Eur. J. Clin. Invest. 3:436–441; 1973. Wrolstad, R. E.; Shallenberger, R. S. Free sugars and sorbitol in fruits—A complication from the literature. J. Assoc. Off. Anal. Chem. 64:91–103; 1981. Rumessen, J. J. Fructose and related food carbohydrates: sources, intake, absorption, and clinical implications. Scand. J. Gastroenterol. 27:819–828; 1992. Solyst, J. T.; Michaelis, O. E. t.; Reiser, S.; Ellwood, K. C.; Prather, E. S. Effect of dietary sucrose in humans on blood uric acid, phosphorus, fructose, and lactic acid responses to a sucrose load. Nutr. Metab. 24:182–188; 1980. Kelsay, J. L.; Behall, K. M.; Moser, P. B.; Prather, E. S. The effect of kind of carbohydrate in the diet and use of oral contraceptives on metabolism of young women. I. Blood and urinary lactate, uric acid, and phosphorus. Am. J. Clin. Nutr. 30:2016–2022; 1977. Burch, R. E.; Kurke, N. The effect of lactate infusion on serum uric acid. Proc. Soc. Exp. Biol. Med. 127:17–20; 1968. Higdon, J. Tea. Linus Pauling Institute, Micronutrient Information Center; 2005. Available at http://lpi.oregonstate.edu/infocenter/phytochemicals/ tea/index.html. Lee, C. Antioxidant ability of caffeine and its metabolites based on the study of oxygen radical absorbing capacity and inhibition of LDL peroxidation. Clin. Chim. Acta 295:141–154; 2000. Ames, B. N.; Cathcart, R.; Schwiers, E.; Hochstein, P. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci. USA 78:6858–6862; 1981.

[152] Kirschbaum, B. Renal regulation of plasma total antioxidant capacity. Med. Hypotheses 56:625–629; 2001. [153] Alderman, M. H. Serum uric acid as a cardiovascular risk factor for heart disease. Curr. Hypertens. Rep. 3:184–189; 2001. [154] Landmesser, U.; Drexler, H. Toward understanding of extracellular superoxide dismutase regulation in atherosclerosis: a novel role of uric acid? Arterioscler. Thromb. Vasc. Biol. 22: 1367–1368; 2002. [155] Filipe, P.; Haigle, J.; Freitas, J.; Fernandes, A.; Maziere, J. C.; Maziere, C.; Santus, R.; Morliere, P. Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation. Eur. J. Biochem. 269:5474–5483; 2002. [156] Patterson, R. A.; Horsley, E. T.; Leake, D. S. Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid. J. Lipid Res. 44:512–521; 2003. [157] Hooper, D. C.; Spitsin, S.; Kean, R. B.; Champion, J. M.; Dickson, G. M.; Chaudhry,I.;Koprowski,H.Uricacid,anaturalscavengerof peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis. Proc. Natl. Acad. Sci. USA 95:675–680; 1998. [158] Hooper, D. C.; Scott, G. S.; Zborek, A.; Mikheeva, T.; Kean, R. B.; Koprowski, H.; Spitsin, S. V. Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. FASEB J. 14:691–698; 2000. [159] Scott, G. S.; Spitsin, S. V.; Kean, R. B.; Mikheeva, T.; Koprowski, H.; Hooper, D. C. Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors. Proc. Natl. Acad. Sci. USA 99:16303–16308; 2002.