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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1728


In vitro antioxidant capacity, bioavailability, and metabolism of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1728
Ex vivo antioxidant protection of human plasma and LDL after consumption of flavonoid-rich foods . . . . . . . . . . . . . . . . . 1731
Total antioxidant capacity of human plasma after consumption of flavonoid-rich foods . . . . . . . . . . . . . . . . . . . . . . . . . 1733
Plasma antioxidant capacity in humans after consumption of fruits or vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . 1733
Plasma antioxidant capacity in humans after tea consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736
Plasma antioxidant capacity in humans after consumption of wine or beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736
Plasma antioxidant capacity in humans after consumption of chocolate, cocoa products, or coffee . . . . . . . . . . . . . . . . . 1736
Factors affecting the total antioxidant capacity of plasma in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736
Increase in plasma urate after consumption of flavonoid-rich foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1737
Antioxidant effects of apples in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1738
Fructose-mediated urate production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1739
Other potential sources of endogenous urate or its derivatives: sucrose, sorbitol, lactate, and methylxanthines . . . . . . . . . . . . . 1740

⁎ Corresponding author. Fax: +1 541 737 5077.


E-mail address: balz.frei@oregonstate.edu (B. Frei).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2006.04.033
1728 S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1741
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1742
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1742

Flavonoids are a large family of polyphenolic compounds capita in the United States [14]. A serving of apple provides
synthesized by plants that have a common chemical structure. about 400 mg of total phenols (expressed as gallic acid
Polyphenolic compounds, or polyphenols, are polyhydroxy- equivalents) [15]. Pears and grapes can provide as much as
lated phytochemicals, of which the two main classes comprise 300 mg total phenols per serving, and a serving of cranberries,
flavonoids and phenolic acids. Flavonoids may be further cherries, or blueberries contains 200–400 mg [14]. Total
divided into several subclasses, i.e., flavones (and isoflavones), phenols in fruits, fruit juices or extracts, and vegetables is
flavanones, flavonols, flavanols (also called catechins), and strongly correlated with the total antioxidant capacity of these
anthocyanidins (Fig. 1). foods measured in vitro [15,16,19–21]. In fruits containing
Consumption of flavonoid-rich foods, in particular fruits and significant amounts of vitamin C, the in vitro antioxidant
vegetables, is associated with a lower incidence of heart disease, capacity often reflects both the polyphenol and the vitamin C
ischemic stroke, cancer, and other chronic diseases [1–5]. For content [22].
example, 7 of 12 epidemiological studies evaluating the risk of Vegetables such as spinach, broccoli, and onions also
coronary heart disease reported protective effects of dietary provide significant amounts of polyphenols in the human diet
flavonoids [6]. Additional studies also found inverse associa- [23] (Table 1). Other plant-derived foods and beverages further
tions between flavonoid intake and the risk of stroke [7,8] and contribute to the total daily intake of polyphenols in humans.
lung and colorectal cancers [7,9,10]. Because these chronic For example, tea, coffee, chocolate, wine, and beer have high in
diseases are associated with increased oxidative stress and vitro antioxidant capacity, which is almost exclusively due to
flavonoids are strong antioxidants in vitro, it has been suggested the presence of polyphenols. In black tea, the levels of
that dietary flavonoids exert health benefits through antioxidant polyphenols vary depending on the amount of leaves, brewing
mechanisms [11–13]. time, and brewing temperature and usually are between 150 and
Although there is conflicting evidence whether consumption 250 mg per 200-ml serving [24]. Because of its wide
of flavonoid-rich foods results in increased antioxidant protec- consumption, coffee has been recently claimed to be the main
tion of lipids and proteins in plasma and LDL, many studies source of polyphenols in the U.S. diet (J. Vinson, unpublished),
have found large, transient increases in the total antioxidant providing 150–180 mg per 200-ml serving [25]. In a Norwegian
capacity of plasma in humans. This observation led to the study of 2672 adults [26] coffee contributed 64% of the total
hypothesis that flavonoids, and polyphenols in general, play an daily intake of dietary antioxidants measured by the ferric-
important role as antioxidants in human blood and tissues. reducing antioxidant potential assay (see below).
However, accumulating evidence indicates that flavonoids are Red wine contains 200–500 mg total phenols per 200-ml
poorly bioavailable and reach only low, micromolar concentra- serving depending on type and varietal [27,28]. The total phenol
tions in human plasma, even after the intake of large amounts of content in white wine is considerably lower, about 40–60 mg
flavonoid-rich foods. In addition, most flavonoids are exten- per 200-ml serving [27]. Total phenol content in beer ranges
sively metabolized in vivo, which can affect their antioxidant from 50 to 100 mg in 200 ml [29]. Dark chocolate contains
activity. Based on an exhaustive review of these findings in the about 340 mg of total phenols per 40-g serving [25].
present article, we conclude that dietary flavonoids are unlikely Considering these amounts of total phenols per serving, a
to make a significant contribution to the antioxidant capacity of well-balanced diet with the recommended nine daily servings of
human plasma. The acute effects on total plasma antioxidant fruits and vegetables and moderate amounts of tea, coffee, wine,
capacity of consumption of flavonoid-rich foods, including beer, or chocolate can provide well over 1000 mg of total
fruits, vegetables, tea, wine, and chocolate, may instead be phenols per day (Table 1).
explained by changes in the concentration of the metabolic
antioxidant uric acid. In vitro antioxidant capacity, bioavailability, and
metabolism of flavonoids
Dietary antioxidant flavonoids
Flavonoids are strong antioxidants in vitro, mainly due to
The amounts of antioxidant flavonoids and polyphenols in their low redox potential and their capacity to donate several
plant-based foods of the human diet—in particular vegetables, electrons or hydrogen atoms [30,31]. For example, catechins
fruits, tea, and wine—are generally much greater than the have a redox potential of +0.53–0.57 V [31], which from a
amounts of other antioxidants in these foods, such as vitamins C thermodynamic standpoint enables them to protect urate
and E and carotenoids [14–18]. Fruits and fruit juices are (+0.59 V), but not ascorbate (+0.28 V), from oxidation by
among the best sources of polyphenols in the human diet peroxyl radicals (+1.06 V). Quercetin and (−)-epigallocatechin
because of their high content in most fruits (Table 1) and the gallate (EGCG) have even lower redox potentials (+0.33 and
relatively large serving sizes (100–200 g). Apples provide 0.43 V, respectively [32]), which places them close to ascorbate
approximately 22% of the total fruit phenols consumed per in the biological antioxidant network. Recent work has shown
S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 1729

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 Flavonoid levels in plasma (μM)
(mg/100 g edible portion or mg/100 ml) a
Flavonols Quercetin Vegetables: capers (316), celery (3.50), 0.65 (fried onions) [48]
Myricetin chives (21.7), onions (15.4), 0.74–7.60 (onions) [43,51]
Kaempferol red onions (38.8), dock leaves (120), fennel (84.4), 0.30 (apples) [49]
hot peppers (16.0–50.0), 2.10 (buckwheat tea) [43]
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)
Flavanols (–)-Epicatechin Fruits: apples (9.0), apricots (11.0), 1.00–1.80 (green tea) [55,61]
(+)-Catechin grapes (17.6), peaches (2.30), 0.09–0.34 (black tea) [54,52]
(–)-Epigallocatechin gallate nectarines (2.75), pears (3.43), 0.08–0.09 (red wine) [56,57]
plums (6.19), raisins (3.68), 0.26–4.77 (chocolate) [58,60]
raspberries (9.23), blackberries (18.7), 4.92–5.92 (cocoa) [59,60]
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)
Flavones Apigenin Vegetables: hot peppers (5.40), n/a
Luteolin celery hearts (22.6), fresh parsley (303)
Spices and herbs: oregano (4.50),
rosemary (4.00), dry parsley (13,506),
thyme (56.0)
Flavanones Naringenin Citrus fruits and juices: lemon (49.9), 5.99 (grapefruit juice) [72]
Hesperetin lemon juice (18.3), 0.06–0.64 (orange juice) [71,72]
Eriodictyol lime juice (11.5), orange (43.9),
orange juice (15.0), grapefruit (54.5),
tangerine juice (10.8)
Spices and herbs: peppermint (20.0)
Anthocyanidins Cyanidin Fruits: blackberries (100–400), 0.11 (black currant juice) [68]
Malvidin black currant (130–400), 0.12 (black currant concentrate) [69]
Delphinidin blueberries (25.0–500), black grape 0.10 (elderberry extract) [64]
Pelargonidin (30.0–750), elderberries (749), 0.01 (red wine) [70]
strawberries (15.0–75.0), cherries
(35.0–450), plums (0.20–25.0)
Others: red wine (20.0–35.0)
a
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 for anthocyanidins (Table 1) (reviewed in [34]). In addition, the
regenerating α-tocopherol in micellar solution [33]. half-lives of flavonoids in human plasma are short, usually in
Despite the strong antioxidant capacity of flavonoids in vitro, the range of a few hours. These factors severely limit the
their antioxidant efficacy in vivo is limited by several factors. capability of dietary flavonoids to act as antioxidants in plasma
First, the absorption of flavonoids in humans is low, in contrast in vivo, especially compared to other antioxidants present in
to other dietary antioxidants such as vitamins C and E. The plasma at high steady-state concentrations, e.g., 30–150 μM
maximal plasma concentrations of flavonoids in humans, ascorbate (vitamin C), 160–450 μM urate, or 15–40 μM α-
reached usually between 1 and 3 h after consumption of tocopherol (vitamin E) [35]. Chronic or long-term consumption
flavonoid-rich foods, are between 0.06 and 7.6 μM for of flavonoid-rich foods also does not result in accumulation of
flavonols, flavanols, and flavanones, and less than 0.15 μM significant amounts of flavonoids in plasma. For example,
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steady-state concentrations of quercetin in human plasma are human plasma after consumption of quercetin-rich foods
less than 1 μM [36]. Data on flavonoid concentrations in human range from 0.7 to 7.6 μM [43,48–51] (Table 1).
tissues are sparse, but it is unlikely that flavonoids make a In contrast to quercetin, catechins, which are the main
significant contribution to antioxidant defenses in cells and flavonoids in tea and chocolate, are not glycosidated in nature
tissues, where ascorbate and reduced glutathione are normally and can be absorbed directly into the bloodstream. After
found in millimolar concentrations. absorption, however, plasma levels of free catechins are low
In addition to being poorly absorbed, flavonoids are [52–61], and the catechins are mainly present as glucuronidated
extensively metabolized in intestine and liver. Flavonoids are and methylated metabolites. EGCG, the main catechin in tea, is
good substrates and inducers of phase II enzymes [37,38], found in human plasma in both its free and its methylated form
suggesting that they are recognized by the body as xenobiotic— [62,63]. Unlike quercetin and catechins, the anthocyanidins,
and thus potentially toxic—compounds, most likely due to their which are found in berries and wine, are absorbed intact as
polyhydroxylated structure and potent redox activity. It is glycosides [64,65]. It has been reported recently that methylated
tempting to speculate that the capacity of flavonoids to induce and glucuronidated conjugates of anthocyanidins can be
detoxifying enzymes is a major mechanism by which detected in blood [66,67]. Reported plasma levels of anthocya-
flavonoids protect against mutagens and carcinogens, i.e., act nidins from food sources are very low, i.e., in the nanomolar
as cancer chemopreventive agents. In addition, it is likely that range [64,68–70]. As mentioned above, the plasma concentra-
the low levels of flavonoids and their metabolites exert other tions of flavonols, flavanols, and flavanones from citrus fruits
biological effects, e.g., altered cell signaling and gene are also very low [71,72], i.e., in the nanomolar to low
expression, that contribute to their purported health benefits. micromolar range (Table 1).
Thus, flavonoids undergo extensive first-pass metabolism, The absorption of some flavonoids may be further restricted
and the chemical forms of flavonoids present in fruits and because they form high-molecular-weight polymers. Procyani-
vegetables (mainly glycosides, except for catechins and dins, which are polymers of catechins found in large amounts
proanthocyanidins, which are present as aglycones) are quite in fruits and cocoa products, are excellent antioxidants in vitro
different from the in vivo metabolites. In the intestinal mucosa because they contain many hydroxyl groups. The antioxidant
and liver, flavonoids undergo glucuronidation, methylation, and capacity of procyanidins depends on their oligomer chain
sulfation. This biotransformation greatly affects the physical length and the type of reactive oxygen species with which they
properties of flavonoids, making them more water soluble, and react. Thus, procyanidins and their free epicatechin units
also often affects their antioxidant activity. Some flavonoid exhibit about the same antioxidant efficacy toward aqueous
metabolites retain the antioxidant activity of their parent peroxyl radicals, whereas procyanidins of different degrees of
compounds, but in general the metabolites are less potent oligomerization differ significantly in their effectiveness in
antioxidants due to modification of their catechol and phenol protecting against lipid peroxidation induced by iron and
groups [39,40]. Furthermore, flavonoids are degraded by ascorbate [73]. Procyanidins often make a greater contribution
bacteria in the intestinal tract, and the resulting breakdown to the total phenol content, and thus total antioxidant capacity,
products may exert biological effects through antioxidant or of flavonoid-rich foods than their more bioavailable mono-
non-antioxidant mechanisms [41,42]. mers, (−)-epicatechin and (+)-catechin. Apples contain 80–
To illustrate the complexity and consequences of bio- 128 mg of procyanidins per 100 g wet wt, and hence
transformation of flavonoids, some representative examples procyanidins may contribute more than 80% to the total
will be discussed. Quercetin is one of the most studied antioxidant capacity of apples and apple juice or extracts [74].
polyphenolic flavonoids, mainly because it is widely The procyanidin content of chocolate can be as high as
consumed in the human diet and is a potent antioxidant in 1500 mg per 100 g [75] compared to about 400 mg of
vitro. Onions, apples, tea, and wine are the main sources of monomeric catechin per 100 g of dark chocolate. Thus,
quercetin in the human diet (Table 1). In these foods, procyanidins contribute substantially to the total phenol
quercetin is present in conjugated form, i.e., as quercetin content and in vitro antioxidant capacity of cocoa products.
glycosides. The nature of the sugar residues in the glycosides Recent studies have found extensive degradation of procyani-
influences the extent of absorption. For instance, quercetin dins by the intestinal bacterial microflora into more absorbable
glycosides from onions are more bioavailable than quercetin low-molecular-weight phenolic acids [42]. Whereas the
glycosides from apples [43]. Hollman and co-workers [44] bioavailability of quercetin, catechins, and anthocyanidins in
suggested that quercetin glycosides are transported into humans is very limited, the absorption of intact procyanidins
enterocytes by the sodium-dependent glucose transporter 1, seems even more restricted, or almost nil [59].
but other data indicate that previous deglycosidation in the
small intestine by human or bacterial enzymes is necessary Ex vivo antioxidant protection of human plasma and LDL
for absorption [45]. Once in the bloodstream, quercetin after consumption of flavonoid-rich foods
metabolites may be circulating for more than 10 h, which is
longer than many other flavonoids such as anthocyanidins When added in vitro to human plasma or isolated LDL,
and catechins (reviewed in [46]) and is likely due to flavonoids can prevent the oxidation of endogenous antiox-
enterohepatic recycling of quercetin metabolites [47]. idants, lipids, and proteins. For example, addition of catechins
Reported concentrations of total quercetin metabolites in to plasma protects α-tocopherol, β-carotene, and lipids from
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oxidation by aqueous peroxyl radicals [76,77]. Similarly, black extract daily for a week. Finally, Hodgson et al. [88] reported
tea extract added to plasma strongly inhibits peroxyl radical- that ex vivo oxidation of serum lipoproteins from human
induced lipid peroxidation [78]. Theaflavins in black tea, which subjects was slightly decreased after they acutely ingested black
are formed from partially oxidized catechins, and catechins in or green tea (Table 2).
green tea inhibit copper-mediated LDL oxidation in vitro [79]. Several studies on flavonoid-rich beverages other than tea
Zhu and co-workers [80] showed that green tea extracts have been performed. Yukawa et al. [89] found decreased
regenerate α-tocopherol in human LDL. Red wine polyphenols oxidizability of LDL isolated from 11 healthy male volunteers
bind to LDL and HDL and inhibit metal ion-dependent and after they consumed coffee for 1 week. Results of studies on
-independent oxidation of the proteins and lipids in these wine have been inconsistent. Whereas Chopra et al. [90] found
lipoproteins [81]. Pearson and co-workers reported that extracts that daily supplementation of male subjects for 2 weeks with
from apple skin or flesh inhibit copper-mediated LDL oxidation either red wine extract (equivalent to 375 ml of wine) or
[82]. Apple extracts also effectively delay the oxidation of urate quercetin resulted in significantly increased resistance of LDL
and α-tocopherol and the formation of lipid hydroperoxides in to ex vivo oxidation, de Rijke et al. [91] did not observe such an
human plasma [15]. Another study showed that catechins, increase after daily consumption of 550 ml of red wine for
procyanidins, and procyanidin-rich extracts protect plasma 4 weeks. In addition, Caccetta et al. [92] did not observe an
components from oxidation [83]. Thus, there is ample and effect on ex vivo serum or LDL oxidizability in healthy male
consistent evidence that in vitro addition of flavonoids or nonsmokers after acute consumption of red wine (Table 2).
flavonoid-rich food extracts to human plasma or lipoproteins Susceptibility of LDL or plasma to oxidation was also
effectively protects their endogenous antioxidants, lipids, and studied after the consumption of some vegetables, fruits, or fruit
proteins from oxidation. juices. McAnlis et al. [51] reported no significant changes in ex
In contrast, studies of ex vivo oxidation of plasma and LDL vivo oxidation of plasma or LDL isolated from volunteers
obtained from humans before and after short- or long-term before and after consumption of 225 g of fried onions. We
consumption of flavonoid-rich foods have yielded conflicting reported that the resistance of plasma to oxidation in human
results (Table 2). Even studies using the same type of food have volunteers did not increase after the consumption of five Red
not found consistent results, indicating significant influences of Delicious apples [15]. Marniemi et al. [93] observed an increase
experimental design, individual metabolism, and other uncon- in ex vivo LDL resistance to oxidation after acute consumption
trolled variables such as potential oxidation artifacts introduced of berries, but not after 8 weeks of daily consumption of berries.
during preparation of LDL. For example, van het Hof and In contrast, O'Byrne et al. [94] showed that daily intake of
colleagues [84] observed that daily consumption of 900 ml of Concord grape juice at 10 ml/kg body wt for 2 weeks was as
green or black tea by healthy individuals for 4 weeks did not effective as 400 IU of α-tocopherol daily in protecting LDL
increase the resistance of LDL to ex vivo oxidation. Cherubini from ex vivo oxidation. Reduced LDL oxidizability was also
et al. [78] and McAnlis et al. [85] also found no effect of acute observed in subjects who consumed 125 ml of concentrated
or chronic consumption of black tea on plasma or LDL grape juice daily for 7 days [95] (Table 2).
resistance to oxidation ex vivo. However, Nakagawa et al. [86] Because chocolate and cocoa products are significant dietary
reported a reduction in plasma hydroperoxide levels in healthy sources of flavonoids, mainly (−)-epicatechin and procyanidins,
subjects after acute ingestion of tea extract equivalent to 2 cups many studies have evaluated their ex vivo antioxidant effects
of tea, and Miura et al. [87] observed decreased oxidizability of [96–99]. These studies have generally found consistent
LDL obtained from subjects who had ingested a green tea 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 [84] 900 ml (equivalent to 6 cups), daily for 4 weeks No change
Black tea [85] 600 ml 1.1% (w/v), acute consumption No change
3 × 300 ml 1.1 % (w/v), daily for 1 week No change
Black tea [78] 500 ml (equivalent to 6 cups), acute consumption No change
Green tea extract [86] Equivalent to 2 cups, acute consumption ↓ Plasma hydroperoxides
Green tea extract [87] Twice/day (equivalent to 7–8 cups), daily for 1 week ↓ LDL oxidizability
Green and black tea [88] 400 ml 1.9 % (w/v), acute consumption No change after green tea
↓ LDL oxidizability after black tea
Red wine [91] 550 ml, daily for 4 weeks No change
Red wine extract [90] Equivalent to 375 ml red wine, daily for 2 weeks ↓ LDL oxidizability
Red wine [92] 5 ml/kg body wt, acute consumption No change
Onions [51] 225 g fried onions, acute consumption No change
Berries [93] 240 g mixed berries, acute consumption ↓ LDL diene conjugation
100 g mixed berries, daily for 8 weeks No change
Apples [15] 1037 g (equivalent to 5 apples with peel), acute consumption No change in plasma oxidizability
Grape juice [95] 125 ml, daily for 1 week ↓ LDL oxidizability
Concord grape juice [94] 10 ml/kg body wt, daily for 2 weeks ↓ LDL oxidizability
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conflicting data in Table 2 using fruits, wine, or tea. Only one either the radical-scavenging (hydrogen atom transfer) or
study reported no change in susceptibility to oxidation of serum -reducing (electron transfer) capacity of the compound or
lipids or LDL after chocolate consumption in humans [100]. (biological) fluid under investigation.
These consistent results may be due to the more uniform and It should be noted, however, that all of these methods lack
lipophilic food matrix of cocoa products compared to fruits and specificity. Thus, in a polyphenol-enriched plant extract, the
vegetables and hence improved and more uniform flavonoid antioxidant capacity measured by any of these methods reflects
absorption. Nevertheless, plasma levels of flavonoids, in the content of all major reducing and antioxidant substances,
particular catechins, after consumption of chocolate or cocoa including nonphenolic compounds such as vitamin C. Many
products are in the low micromolar range, similar to the levels studies have found highly significant correlations between the
observed after the consumption of other flavonoid-rich foods measured antioxidant capacity and the total phenol content of
[58,59,101,102]. plant-derived extracts and beverages. For example, Proteggente
The effects of flavonoid-rich foods on lymphocyte DNA et al. [22] studied a variety of fruits and vegetables and
damage and 8-hydroxy-2′-deoxyguanosine formation have also comparatively assessed the antioxidant capacity of aqueous and
been studied. Boyle et al. [103] found that consumption of fried methanolic extracts using TEAC, FRAP, and ORAC. The
onions, with or without fresh tomatoes, by six healthy female values for each fruit and vegetable extract correlated well with
volunteers resulted in elevated flavonoid concentrations in its total phenolic content, as assessed by the Folin–Ciocalteau
plasma, which was associated with increased resistance of method, as well as the vitamin C content. Similar observations
lymphocyte DNA to strand breakage ex vivo. Levels of urinary were made for tea, wine, and chocolate [112–114]. Whereas the
8-hydroxy-2′-deoxyguanosine also decreased 4 h after inges- observed antioxidant capacity of plant-derived extracts usually
tion of fried onions. closely reflects their content of antioxidant polyphenols and
Several factors may account for the contradictory results of flavonoids, the same is not true for human plasma, which
the aforementioned studies on the effects of flavonoid-rich contains large concentrations of antioxidant vitamins and
foods on ex vivo oxidation of plasma and LDL (Table 2). metabolites such as vitamin C (30–150 μM), vitamin E (15–
Differences in the absorption and metabolism of the various 40 μM), and urate (160–450 μM), but only very low
flavonoids may lead to the formation of metabolites with concentrations of flavonoids (see Tables 1 and 3).
different antioxidant properties. The methods used to isolate Interestingly, and in striking contrast to the discrepant results
LDL may also have contributed to the discrepant results, of studies on the resistance of plasma or LDL to oxidation ex
because flavonoids exhibit variable distribution and association vivo (Table 2), virtually all studies on the total antioxidant
with plasma constituents such as proteins and lipids. For capacity of human plasma before and after consumption of
example, quercetin exhibits greater affinity for albumin than flavonoid-rich foods have found significant increases (Table 4).
LDL [51]. The standard isolation procedures for LDL will likely The extent of these increases in plasma antioxidant capacity,
remove the water-soluble flavonoids that are not tightly bound however, often greatly exceeded the increases in the plasma
to the lipoprotein particles. These flavonoids probably include concentrations of flavonoids or total phenols. In the following
EGCG from tea and glucuronide and sulfate metabolites of sections, studies on plasma antioxidant capacity in humans after
flavonoids, which tend to be more water-soluble than their the intake of different types of flavonoid-rich foods will be
parent compounds. Unfortunately, most of the studies that discussed.
reported a significant decrease in the susceptibility of plasma or
LDL to oxidation ex vivo after consumption of flavonoid-rich Plasma antioxidant capacity in humans after consumption of
foods did not measure the levels of flavonoids in plasma or fruits or vegetables
LDL.
Cao et al. [115] investigated the effects of a diet rich in fruit
Total antioxidant capacity of human plasma after and vegetables on the antioxidant capacity of human plasma.
consumption of flavonoid-rich foods Thirty-six healthy nonsmokers consumed a controlled diet
containing 10 servings of fruit and vegetables each day for
Several methods have been used to evaluate the antioxidant 15 days or the same diet with an additional 2 servings of
capacity of flavonoids and extracts of flavonoid-rich foods and broccoli each day on days 6–10. Both diets significantly
that of human plasma before and after consumption of increased the plasma total antioxidant capacity measured by
flavonoid-rich foods. These methods include the ferric-reducing ORAC. In another short-tem study, Cao et al. [116]
antioxidant potential (FRAP) [104]; oxygen radical absorbance investigated serum total antioxidant capacity in 8 elderly
capacity (ORAC), without or with prior precipitation of plasma women after consumption of 240 g of strawberries, 294 g of
proteins with perchloric acid (ORAC-PCA) [105]; trolox spinach, or 300 ml of red wine compared to a control meal or
equivalent antioxidant capacity (TEAC) [106]; and total a supplement of 1250 mg of vitamin C. The total antioxidant
radical-trapping antioxidant parameter (TRAP) [107] (Table capacity of serum, assessed as area under the curve (AUC) for
3). These assays have been extensively characterized, com- the time course of serum ORAC, TEAC, or FRAP, increased
pared, and reviewed [108–111]. Although the assays vary in significantly during the 4-h period after consumption of
principle of detection, sensitivity, working pH, source of strawberries, spinach, or wine (Table 4). The authors
oxidants, and other assay conditions (Table 3), they all assess concluded that the antioxidant phenolic compounds in these
1734 S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

Table 3
Methods used for the evaluation of plasma or serum total antioxidant capacity
Method a Principle Assay conditions Plasma or serum Contribution (%) to plasma or serum TAC Ref.
TAC b reference
Urate Ascorbate Bilirubin Proteins
value (μM)
FRAP Reduction of ferric ions Carried out at pH 3.6 400–1000 60 15 5 10 [104]
to ferrous ions Spectrophotometric (endpoint)
Diluted plasma or serum
ORAC Based on peroxyl radical Carried out at pH 7.4 1500–3100 7 1 <1 28 [105]
generation and oxidation Fluorescent (kinetics)
of a fluorescent probe Diluted plasma or serum
(β-phycoerythrin) Reaction followed to completion,
results evaluated as area under
the curve
ORAC-PCA Idem ORAC Idem ORAC 600–900 39 7 1 0 [105]
Previous precipitation of
plasma/serum proteins with
perchloric acid (PCA)
TRAP (FL) Based on peroxyl radical Carried out at pH 7.4 850–1300 20–60 2.5–5.3 n/a n/a [107]
generation and Fluorescent (kinetics)
oxidation of a Diluted precipitated
fluorescent probe plasma or serum
(β-phycoerythrin) Results evaluated as lag times
TRAP (CL) Based on peroxyl radical Carried out at pH 7.4 250–400 60–90 10–20 n/a n/a [58]
generation and oxidation Chemiluminescent (kinetics)
of luminol Undiluted plasma or serum
Results evaluated as lag times
TEAC Based on decolorization Carried out at pH 7.4 1300–1600 19 3 1 28 [110]
of preformed ABTS c Spectrophotometric
radical cations (kinetics or endpoint)
Results evaluated as area under
the curve (kinetics) or %
inhibition (endpoint)
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 In a study of six men, Marniemi et al. [93] reported that
capacity, because it was not accounted for by the observed TRAP of LDL was significantly increased by about 10%
increases in the plasma concentrations of ascorbate (2–25%) 5 h after consumption of 80 g of either bilberries,
and urate (10–30%). lingonberries, or black currants. Pedersen et al. [119]
In another study [117], consumption of tomato products with investigated the effect of blueberry or cranberry juice
olive oil for 1 week significantly raised the plasma antioxidant consumption on plasma phenolic content and antioxidant
capacity measured as FRAP compared to consumption of capacity. Nine healthy female subjects consumed 500 ml of
tomato products with sunflower oil, despite similar plasma blueberry juice, cranberry juice, or sucrose solution as
levels of lycopene after consumption of either tomato prepara- control. Consumption of cranberry juice, but not blueberry
tion. The change in FRAP after consumption of tomato products juice, significantly increased the ability of plasma to reduce
with olive oil was about 200 μM, i.e., about 20% of the baseline potassium nitrosodisulfonate and Fe(III)-2,4,6-tri(2-pyridyl)-
antioxidant capacity of human plasma. The tomato preparation s-triazine (the indicator molecule for FRAP), reaching
also significantly increased serum LDL cholesterol levels and maximal levels 60 to 120 min after consumption. This
decreased triglycerides. The authors concluded that the type of increase in reducing capacity of plasma corresponded to a
oil may differentially affect the antioxidant capacity of plasma, 30% increase in vitamin C levels and a small but significant
although it is possible that the changes in FRAP were partly increase in total phenols in plasma. The authors concluded
attributed to the polyphenolic constituents of olive oil. In a that the observed increase in plasma antioxidant capacity
short-term study by Serafini et al. [118], six men and five after consumption of cranberry juice was mainly due to
women consumed 250 g of fresh lettuce, and blood was vitamin C, not phenolics.
sampled before and 2, 3, and 6 h after consumption. Plasma To evaluate the effects of wild blueberries on postprandial
antioxidant capacity measured as TRAP increased by 40–50%, serum antioxidant capacity, a single-blinded crossover study
which was associated with significantly increased plasma levels was performed in a group of eight middle-aged male volunteers
of quercetin, p-coumaric acid, and vitamin C (Table 4). [120]. The subjects consumed a high-fat meal and a placebo
S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 1735

Table 4
Plasma or serum total antioxidant capacity after consumption of flavonoid-rich foods
Food Study Method a Outcome b Change in plasma antioxidants
Ascorbate Urate Phe/Flav c
Fruits and vegetables [115] ORAC ↑ 5–15% n/a n/a n/a
Strawberries, spinach, red wine [116] ORAC ↑ 11–24% ↑ 2–25% ↑ 10–30% n/a
FRAP ↑ 7–24%
TEAC ↑ 1–21%
Tomato with olive oil [117] FRAP ↑ 20% n/a n/a n/a
Berries [93] TRAP (CL) LDL ↑ 10% n/a n/a n/a
Cranberry juice [119] FRAP ↑ 40 μM d ↑ 30% n/a ↑ 6 μg/ml
Lettuce [118] TRAP (FL) ↑ 40–50% ↑ 10–11 μM n/a ↑ 88 ng/ml
Blueberries [120] ORAC ↑ 8.5% n/a n/a n/a
TEAC ↑ 4.5%
Blueberries [121] ORAC e ↑11–50% n/a n/a ↑ 13 ng/ml
Grape juice [95] FRAP ↑ 8% (at 1 h) n/a n/a n/a
↑ 11% (at day 8)
Concord grape juice [94] ORAC e ↑ 8% n/a n/a ↑ 2.4 μg/ml
Green and black tea [123] TRAP (FL) ↑ 40–48% n/a n/a n/a
Green tea [122] FRAP ↑ 4% n/a n/a n/a
Green and black tea [54] FRAP ↑ 3% No change No change n/a
Green tea [136] TEAC ↑ 6–13% n/a n/a n/a
Green tea [133] TRAP ↑ 5% (NS) ↓ 3% ↑ 7% n/a
Coffee ↑ 6% No change ↑ 5% n/a
Wine [126] TAC f ↑ 11–18% n/a n/a n/a
Wine [124] FRAP ↑ 24% n/a ↑ 23% n/a
Wine [125] TRAP (FL) ↑ 14% n/a n/a ↑ 3 μg/ml
Wine [128] TAC ↑ 14% n/a ↑ 12% n/a
Beer [129] TRAP (FL) ↑ 17% n/a ↑ 13% (NS) ↑ 18 ng/ml
Wine, whiskey [127] FRAP ↑ 60–100 μM n/a n/a ↑ 2–2.5 μg/ml
Chocolate [58] TRAP (CL) ↑ 31% No change ↑ 12% (NS) ↑ 257 nM
Chocolate [130] FRAP ↑ 20% n/a n/a ↑ 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 vitamin E. Healthy subjects received daily for 2 weeks
supplemented with 100 g of freeze-dried wild blueberries. either 400 IU RRR-α-tocopherol or 10 ml/kg body wt of
Consumption of the blueberry-supplemented meal was asso- Concord grape juice. Plasma α-tocopherol concentrations
ciated with a significant increase in serum antioxidant capacity increased by 92% in subjects who received α-tocopherol,
1 and 4 h postprandially. In a follow-up study, the same group and plasma total and conjugated phenols increased by 17
[121] investigated the absorption of anthocyanins in humans and 22%, respectively, in subjects who consumed grape
after the consumption of a high-fat meal supplemented with juice. Both interventions significantly increased serum
freeze-dried blueberries. Of the 25 anthocyanins detected in the ORAC and LDL resistance to oxidation (Table 4).
blueberries, 19 were also found in serum. The authors Vitamin C contained in fruits and vegetables can
qualitatively, but not quantitatively, associated this increase in significantly affect plasma total antioxidant capacity assessed
plasma anthocyanins with the increase in serum total antiox- by FRAP or TRAP (CL) (Table 3). As indicated above,
idant capacity. vitamin C is more readily absorbed than flavonoids and is
The antioxidant effects of red grape juice were studied in present in human plasma at much higher concentrations (30–
several trials. Day et al. [95] observed a significant increase 150 μM) than flavonoids (nanomolar to low micromolar;
in serum total antioxidant capacity in seven subjects 1 h Table 1). The contribution of ascorbate to FRAP can be as
after they consumed 125 ml of concentrated grape juice. much as 20% [104] (Table 3). Therefore, it is important to
Furthermore, after 8 days of daily grape juice consumption, distinguish between the antioxidant effects of ascorbate and
serum total antioxidant capacity was increased compared to flavonoids in plasma after consumption of vitamin C-contain-
baseline and the susceptibility of isolated LDL to oxidation ing, flavonoid-rich foods. Interestingly, ORAC is not sensitive
was decreased. O'Byrne et al. [94] compared the in vivo to ascorbate, as indicated by the observation that treatment of
antioxidant efficacy of Concord grape juice with that of plasma with ascorbate oxidase does not significantly affect the
1736 S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

value of either ORAC or ORAC-PCA (S. Lotito and B. Frei, Ghiselli et al. [129] studied the effects of drinking beer,
unpublished observations). In agreement with these observa- dealcoholized beer, or ethanol on total antioxidant capacity and
tions, other researchers [108] have calculated that the the profile of selected phenolic acids, including caffeic, sinapic,
contribution of ascorbate to plasma ORAC is only 1–7% syringic, and vanillic acids, in the plasma of 14 healthy subjects.
(Table 3). Beer consumption caused a significant increase in plasma
antioxidant capacity after 1 h, returning to baseline after 2 h, and
Plasma antioxidant capacity in humans after tea consumption also increased plasma levels of all measured phenolic acids.

Benzie et al. [122] studied plasma and urine antioxidant Plasma antioxidant capacity in humans after consumption of
capacity in healthy adults before and after ingestion of green chocolate, cocoa products, or coffee
tea or water as control and found about a 4% increase in
plasma FRAP 40 min after drinking tea. Excretion of Several studies investigated the effects of chocolate
polyphenolic antioxidants peaked at 60–90 min, and a consumption on plasma antioxidant capacity (Table 4). The
significant correlation was observed between urinary FRAP absorption of (−)-epicatechin and plasma antioxidant capacity
and total phenolics. Leenen et al. [54] investigated the effects measured as TRAP were evaluated in 17 healthy volunteers
of a single dose of black or green tea (2 g solids in 300 ml of who consumed 80 g of procyanidin-rich, semisweet chocolate
water), with or without milk, or water as control on plasma [58]. Two hours after chocolate ingestion, a 12-fold increase in
antioxidant capacity in 21 healthy volunteers enrolled in a plasma (−)-epicatechin was observed, from 22 to 257 nM, and a
crossover study. Consumption of black tea significantly significant 31% increase in plasma total antioxidant capacity,
increased plasma antioxidant capacity, reaching maximal corresponding to about 90 μM trolox equivalents. Both plasma
levels after about 60 min, and a larger increase was observed (−)-epicatechin and total antioxidant capacity returned to
after consumption of green tea. Addition of milk did not affect baseline 4 h later. This study is a particularly striking example
the increase in plasma antioxidant capacity with either tea. In of the large discrepancy between the increase in plasma
another similar study, two groups of 5 healthy adults drank antioxidant capacity and plasma flavonoid concentration [58].
300 ml of either black tea or green tea, and the experiment In a crossover study, 12 healthy volunteers consumed 100 g
was repeated on a separate day with the addition of 100 ml of of dark chocolate, 100 g of dark chocolate with 200 ml of full-
whole milk to the tea [123]. The plasma antioxidant capacity fat milk, or 200 g of milk chocolate [130]. Plasma FRAP
measured as TRAP increased significantly after consumption significantly increased by 20% 1 h after ingestion of dark
of black or green tea and peaked after 30–50 min. Interest- chocolate, but did not increase after consumption of dark
ingly, the addition of milk completely inhibited the increase in chocolate with milk or milk chocolate. The authors speculated
plasma TRAP, in contrast to the observations made by Leenen that milk may interfere with the absorption of antioxidants from
et al. [54] (Table 4). chocolate and may, therefore, negate the potential health
benefits of eating moderate amounts of dark chocolate. This
Plasma antioxidant capacity in humans after consumption of notion, however, has been questioned recently by other
wine or beer investigators, who did not find such an effect of adding milk
to chocolate on plasma antioxidant capacity [131].
At least six studies investigated the effects of wine Coffee is widely consumed in the Western world, but only
consumption on plasma antioxidant capacity in humans, and few studies have assessed the antioxidant effects of coffee
all of them found increased levels (Table 4). Day and consumption in humans. Coffee is particularly rich in phenolic
Stansbie [124] reported a 24% increase in serum antioxidant acids such as caffeic, ferulic, chlorogenic, and p-coumaric
capacity in 6 healthy men who consumed 250 ml of port acids. Nardini et al. [132] showed that coffee consumption in 10
wine, whereas ethanol ingestion as a control had no effect. healthy male volunteers acutely increased total plasma caffeic
Serafini et al. [125] found that plasma TRAP and polyphenol acid concentration, present mainly as glucuronate and sulfate
concentration significantly increased in 10 healthy subjects metabolites, peaking 1 h after coffee consumption. Natella et al.
50 min after consumption of alcohol-free red wine, but not [133] reported that plasma antioxidant capacity measured as
after consumption of alcohol-free white wine or water. TRAP significantly increased after drinking 200 ml of coffee.
Similarly, Whitehead et al. [126] reported that mean serum
antioxidant capacity, measured by chemiluminescence, in 9 Factors affecting the total antioxidant capacity of plasma in
subjects increased by 18 and 11%, respectively, 1 and 2 h humans
after drinking 300 ml of red wine and 4 and 7%, respectively,
after drinking an equivalent amount of white wine. In another Studies assessing the total antioxidant capacity of plasma,
study, 9 volunteers who consumed 100 ml of either red wine serum, or other biological samples often do not take into
or malt whiskey showed a significant increase in plasma total account possible postprandial or diurnal variations that are not
phenols and FRAP within 30 min of consumption [127]. directly related to the intake of dietary antioxidants. Plasma
Maxwell and Thorpe [128] also found a significant mean antioxidant capacity may be significantly affected by non-
increase of 14% in serum antioxidant capacity 60 min after antioxidant dietary constituents that affect uptake, tissue
ingestion of French Bordeaux red wine. mobilization, or metabolism of endogenous or exogenous
S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 1737

antioxidants. Food intake per se may cause variations in the reevaluated, and future studies should control for postprandial
plasma concentrations of ascorbate and urate, the major effects of non-antioxidant dietary constituents.
contributors to plasma total antioxidant capacity (Table 3).
For example, in a short-term study, Cao and Prior [134] fed Increase in plasma urate after consumption of
eight elderly women a diet with negligible amounts of flavonoid-rich foods
antioxidants and found that plasma ascorbate and urate varied
during the trial. In particular, plasma total antioxidant capacity As discussed above, the consumption of flavonoid-rich foods
measured as FRAP and TEAC increased 30 min after breakfast is almost always associated with substantial increases in plasma
consumption, which was paralleled by significant changes in or serum total antioxidant capacity (Table 4). Even when the
plasma urate. Interestingly, after this initial increase, FRAP, kinetics of the appearance of flavonoids in plasma and the
TEAC, and urate decreased over time, reaching a minimum at increase in plasma antioxidant capacity are closely related, the
4 h. In contrast, the postprandial increase in ORAC could not be extent of the increase in plasma antioxidant capacity usually far
explained entirely by variations in urate, suggesting that other exceeds the plasma concentrations of flavonoids and their
unknown factors contributed. Interestingly, whereas urate and metabolites, which are in the nanomolar to low micromolar
ascorbate are known to contribute about 39 and 7%, range (Tables 1 and 4). For example, Serafini et al. [118] showed
respectively, of the ORAC-PCA value of normal human plasma, that after consumption of lettuce, plasma TRAP increased by
over 50% could not be assigned to any specific antioxidant 40–50% from a baseline value of about 800 μM, i.e., TRAP
[108] (Table 3). increased by about 320–400 μM trolox equivalents (Table 4).
Mazza et al. [121] studied the absorption of anthocyanins in This large increase in plasma total antioxidant capacity could not
humans after the consumption of a high-fat meal with a freeze- be explained by the small increases in plasma ascorbate of about
dried blueberry powder containing 25 different anthocyanins. As 10 μM, in phenolics of about 90 μg/L, in caffeic acid of 29 μg/L
discussed above, 19 of the 25 anthocyanins were detected in (0.16 μM), in coumaric acid of 39 μg/L (0.24 μM), and in
human serum, and the authors associated the increase in quercetin of 20 μg/L (0.07 μM) [118]. As mentioned above,
anthocyanins with the increase in serum antioxidant capacity. consumption of tomato products with olive oil increased plasma
However, ORAC of serum or acetone-precipitated serum time- FRAP by as much as 200 μM [117], again far exceeding
dependently increased not only after consumption of the high-fat maximally achievable flavonoid concentrations in plasma, and
meal supplemented with blueberries, but also after the placebo- Rein et al. [58] reported an increase in plasma TRAP of about
supplemented high-fat meal used as control. In both experiments, 90 μM after consumption of chocolate, in contrast to an increase
the increase in ORAC was closely paralleled by the increase in in plasma (−)-epicatechin of only about 257 nM. After
triglycerides. Hence, this study [121] does not provide evidence consumption of 100 g of wild blueberries, plasma ORAC-
that the increase in serum ORAC is directly linked to PCA increased more than 8% over baseline, corresponding to
anthocyanins, but suggests an effect of the high-fat meal itself. about 50 μM trolox equivalents [120], and increases of 40 μM in
In a short-term study with human volunteers, we observed a plasma antioxidant capacity were observed 1 h after drinking
decrease in FRAP and urate 6 h after consumption of plain grape juice [95]. Furthermore, large increases in plasma FRAP
bagels [135], in agreement with the data of Cao and Prior [134]. of 20%, corresponding to 80–200 μM, were reported after
These findings indicate that plasma urate levels are affected by chocolate consumption [130]. Serafini et al. [125] observed
food intake, most likely due to changes in the levels of ATP and increases of 14% or 159 μM in plasma TRAP in 10 volunteers
inorganic phosphate (see below). In contrast, in that same study within 1 h after consuming 300 ml of alcohol-free red wine, and
(for study design, see [135]), we observed a large, significant Duthie et al. [127] found an increase in FRAP of 60–100 μM in 9
increase in plasma ORAC-PCA after the consumption of either healthy males within 30 min after drinking 100 ml of red wine
flavonoid-rich Red Delicious apples or plain bagels in amounts (Table 4). Finally, tea consumption has been reported to dose-
matched by their carbohydrate content (S. Lotito and B. Frei, dependently increase plasma antioxidant capacity by 200–
unpublished observations). The increase in ORAC-PCA was 600 μM trolox equivalents [136].
similar after the intake of either food, indicating that the How can these large increases in plasma total antioxidant
increase in plasma antioxidant capacity was independent of the capacity after consumption of flavonoid-rich foods and
antioxidant and flavonoid content of the foods consumed, beverages be reconciled with the small increases in plasma
which was negligible for the bagels. Interestingly, consumption concentrations of flavonoids? One possible explanation is that
of a fructose solution also acutely increased plasma ORAC- only a small fraction of the flavonoids in plasma is measured
PCA, which was proportional to the amount of carbohydrates and that the total amount of flavonoids is greatly under-
consumed. These observations suggest a strong postprandial estimated. However, this is an unlikely explanation, as all
effect of carbohydrates on plasma antioxidant capacity, flavonoids detected in human plasma thus far are found in
regardless of the amount of dietary antioxidants. concentrations that are orders of magnitude lower than the
Taken together, the above data emphasize the need to observed increases in plasma antioxidant capacity. Furthermore,
consider the effect of food intake per se, including fats and the plasma polyphenols and flavonoids that are putatively
carbohydrates, on plasma total antioxidant capacity in humans. responsible for the increased antioxidant capacity of plasma as
Hence, studies that assessed the effects of flavonoid-rich foods assessed, e.g., by FRAP or ORAC, should also protect plasma
on plasma antioxidant capacity (Table 4) may have to be 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 also reported an increase in serum urate levels by 12% or 39 μM
inconsistent, with 7 of 14 studies showing no effect on ex vivo in 10 healthy subjects 1 h after drinking French Bordeaux, which
oxidation of plasma (Table 2). It has also been proposed that was paralleled by a significant increase in antioxidant capacity
dietary flavonoids exert synergistic effects, resulting in by 14% or 66 μM trolox equivalents. Natella et al. [137]
unusually large increases in plasma antioxidant capacity. observed increases of 25% or 72 μM in urate 1 h after ingestion
However, the chemical mechanisms for such synergistic of a meal with red wine, compared to a postprandial increase of
interactions have not been explained nor have such interactions 8% or 23 μM after ingestion of a meal with ethanol instead of
been observed in plasma to which flavonoid-rich extracts were wine. Another study [92] also found an increase in average
added in vitro. plasma urate levels of about 40 μM, or 12% from baseline, after
Interestingly, the physiological antioxidant urate is a major the intake of regular or dealcoholized red wine. Many studies
contributor to plasma total antioxidant capacity (Table 3). This is have assessed the antioxidant capacity of plasma after drinking
not surprising given the high concentration of urate in human tea, but data on levels of plasma urate in these studies are scarce.
plasma (160–450 μM) and its powerful reducing and free radical Natella and colleagues [133] found significant increases in
scavenging activities. For instance, urate may contribute as plasma urate of 18 and 24 μM, or 5 and 7%, 1 h after drinking
much as 60% to plasma FRAP, 60–90% to plasma TRAP, and coffee or green tea, respectively, which was paralleled by
about 40% to plasma ORAC-PCA (Table 3). Ascorbate also increases of 6 and 5% in total antioxidant capacity (Table 4).
makes a significant contribution, e.g., about 15% to FRAP and Thus, several studies indicate that the consumption of
up to 20% of TRAP (CL). Considering the magnitude of these flavonoid-rich foods may increase plasma urate, although the
contributions to total antioxidant capacity, it is important to underlying mechanism(s) was not elucidated. Because plasma
assess the changes in the plasma concentrations of urate and concentrations of urate are much greater than those of
ascorbate after consumption of flavonoid-rich foods, which will flavonoids, it is possible that changes in urate levels account
allow for the determination of the contributions of dietary for the relatively large increases in plasma total antioxidant
flavonoids. capacity after consumption of flavonoid-rich foods.
Whereas an increase in plasma ascorbate can be expected
after consumption of flavonoid-rich foods that also contain Antioxidant effects of apples in vitro and in vivo
vitamin C, such as many fruits and vegetables, the increase in
plasma urate observed in numerous studies is surprising (Table Apples are one of the main sources of flavonoids in the
4). Flavonoid-rich foods usually do not contain urate or its Western diet [22,138,139] and contain as much as 2 g of total
precursors, such as inosine. Nevertheless, Cao et al. [116] found phenols per kilogram wet weight, or about 400 mg per apple
significant increases in plasma urate after consumption of [15]. The main classes of polyphenols in apples are flavonoids,
strawberries, spinach, or red wine that corresponded to including quercetin, (−)-epicatechin, (+)-catechin, procyani-
significant increases in plasma ORAC-PCA, FRAP, and dins, and anthocyanidins (Fig. 1); dihydrochalcones such as
TEAC. For example, FRAP increased by 21, 53, and 21 μM phloretin and phloridzin; and other phenolic compounds such as
trolox equivalents during the first 2 h after the intake of chlorogenic acid. In an in vitro study, we examined the
strawberries, spinach, or red wine, respectively, compared to antioxidant capacity of individual apple polyphenols and apple
8 μM after the intake of a control meal. Plasma urate levels also extracts by the FRAP and ORAC assays. Aqueous extracts of
increased significantly after the intake of each food. Based on whole Red Delicious apples containing 176 ± 3 mg total
AUC measurements, the increase in plasma urate after phenols per 100 g of apple exhibited FRAP and ORAC values
consumption of spinach was much greater than that after of 1421 ± 45 and 1508 ± 44 μmol trolox equivalents per 100 g
consumption of strawberry, which was greater than that after of apple, respectively. However, individual polyphenols
wine, and these increases were similar to the increases in plasma accounted for only 14–18% of total FRAP and ORAC [74],
FRAP. Strawberry consumption produced the highest increase in suggesting that a large proportion of the in vitro antioxidant
plasma ascorbate, but ascorbate did not appreciably affect capacity was due to other compounds, most likely high-
plasma antioxidant capacity. Thus, it can be inferred that urate molecular-weight procyanidins that are found in amounts of
significantly affected plasma FRAP in this study. about 130 mg per 100 g apple. Addition of Red Delicious apple
Other studies have found that wine increases serum urate. extract to human plasma in vitro dose-dependently increased
Day and Stansbie [124] observed an increase of 23% in serum plasma FRAP [74], but not ORAC-PCA (S. Lotito and B. Frei,
urate concentration, corresponding to a mean increase of 81 μM, unpublished observation). For example, plasma FRAP
in 6 healthy men 30 min after drinking port wine. This increase increased from 454 ± 9 to 486 ± 15 (+7%) and 532 ± 12 μM
in urate was associated with a 24% increase in total antioxidant (+17%), respectively, after the addition of 7 or 14 μg total apple
capacity, corresponding to a mean increase of 109 μM trolox phenols per milliliter of plasma. These increases in plasma
equivalents (Table 4). Both urate and plasma antioxidant FRAP are comparable to those observed in humans after the
capacity declined slowly thereafter, with a highly significant consumption of flavonoid-rich foods (Table 4).
correlation between the two measures. The authors concluded Furthermore, in vitro addition of apple phenols signifi-
that the acute increase in serum antioxidant capacity after the cantly increased the resistance of plasma to oxidation.
ingestion of port wine may be attributed to the increase in serum Although apple phenols could not protect plasma ascorbate
urate, not wine-derived flavonoids. Maxwell and Thorpe [128] from oxidation by aqueous peroxyl radicals, the half-life of
S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 1739

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 sig-
nificantly 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 flavonoid-
free 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 Fig. 2. Changes in plasma antioxidant capacity and urate after consumption of
ascorbate oxidase did not affect the increase in antioxidant apples, fructose, or bagels. (A) Changes in antioxidant capacity of plasma at the
indicated times after food consumption, expressed as ferric-reducing antioxidant
capacity. These data indicate that vitamin C from apples did not potential with ascorbate oxidase treatment (FRAPAO). (B) Changes in plasma
make a significant contribution to the change in plasma urate concentrations in the same subjects. Values are mean changes ± SEM
antioxidant capacity in our study. Consumption of bagels relative to baseline (n = 6 subjects). For experimental details, see Ref. [135]. The
resulted in a time-dependent decrease in plasma FRAP (Fig. p values shown are for time-dependent changes using repeated-measures
2A), suggesting an effect of food intake per se, in agreement analysis of variance; *significantly different from time 0 h, using Tukey–
Kramer post hoc analysis.
with previous reports [121,134].
Surprisingly, plasma urate levels increased in all subjects by
about 35% 1 h after apple consumption and paralleled the Fructose is found in most fruits. The fructose content of
increase in plasma FRAP (Fig. 2). The contribution of urate to apples is about 6–8 g per 100-g serving [144,145]. Pears and
the total antioxidant capacity was confirmed by preincubation grapes also contain fructose in amounts of about 5–9 g per
of plasma with uricase, which completely abolished the changes 100 g. Cherries, blackberries, and blueberries contain 5–7 g, 2–
in FRAP after apple consumption [74]. Both FRAP and urate 4 g, and about 4 g of fructose per 100-g serving, respectively.
returned to baseline levels 6 h after apple consumption (Fig. 2). The fructose content is particularly high in dried fruits such as
These data strongly suggest that transient increases in urate, and dried prunes (12 g per 100 g) and raisins (34 g per 100 g).
not apple polyphenols, are responsible for the increase in Honey also is a well-known source of fructose (about 40 g per
plasma antioxidant capacity after apple consumption. 100 g) [17].
Considering the role of fructose metabolism in urate
Fructose-mediated urate production production, we hypothesized that fructose in apples caused the
increase in plasma urate—and thus antioxidant capacity—in
The rapid and large increases in both plasma urate and human subjects after apple consumption. This hypothesis was
antioxidant capacity after apple consumption suggested that the tested in the same six volunteers enrolled in our study [135]. The
active component(s) is present in apples in relatively large subjects drank a solution of 64 g of fructose in 1000 ml of water,
amounts and is easily absorbed. However, apples do not contain matching the fructose content and mass of the five apples.
urate or its dietary precursors, inosine or other purines. On the Plasma FRAP increased significantly by about 40 μM 1 h after
other hand, fructose has been known for more than 30 years to fructose consumption, and the time-dependent changes in
increase plasma urate levels consequent to its rapid metabolism plasma antioxidant capacity were comparable to those observed
by fructokinase [140–142]. Fructose metabolism in this manner after apple consumption (Fig. 2A) [135]. In addition, plasma
leads to a transient decrease in hepatic ATP and inorganic urate levels significantly increased in all subjects after fructose
phosphate, which are important inhibitors of 5′-nucleotidase intake (Fig. 2B), as had been seen after apple consumption,
and AMP deaminase, respectively, and thus increased degrada- whereas plasma ascorbate levels remained unchanged. The
tion of AMP to uric acid (Fig. 3) [141–143]. increase in plasma urate after apple consumption was strongly
1740 S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

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
Fig. 3. Purine nucleotide degradation resulting from fructose catabolism. Rapid converted to fructose during metabolism in the liver and
conversion of fructose to fructose 1-phosphate catalyzed by fructokinase results produces biochemical effects similar to those of fructose on
in decreased ATP and inorganic phosphate (Pi) levels. Intracellular Pi has a
regulatory effect on urate production in the liver by inhibiting AMP deaminase,
hepatic adenosine phosphate levels in humans [143]. In apples,
the enzyme controlling purine breakdown. In addition, ATP levels have a sorbitol ranges from 0.5 to 1.0 g per 100 g fresh wt [144].
regulatory effect on 5′-nucleotidase. Thus, fructose induces acute depletion of Cherries contain even higher amounts of sorbitol (1.4–2.1 g/
ATP and Pi and causes increased activity of the enzymes involved in the 100 g), and in dried prunes the sorbitol content can be as high as
degradation of purine nucleotides to urate. AMP, adenosine monophosphate; 12 g per 100-g serving [17]. Thus, sorbitol in fruits, in addition
IMP, inosine monophosphate; PNP, purine nucleoside phosphorylase.
to fructose and sucrose, likely contributes to increased plasma
urate and antioxidant capacity. Future studies on the in vivo
correlated with the increase in plasma urate after fructose antioxidant effects of flavonoid-rich foods need to consider
consumption. These observations indicate that the plasma fructose, sucrose, and sorbitol content, which may significantly
antioxidant capacity after apple consumption increased because affect plasma urate concentrations.
of the increase in urate, which was due to the effect of apple- Several studies have reported increases in plasma urate
contained fructose on adenine nucleotide degradation (Fig. 3). after intake of red wine (Table 4). In addition to the sugar in
The hyperuricemic effect of fructose administered by wine, Caccetta et al. [92] suggested that the observed
intravenous infusion has been observed above a threshold of increases in plasma urate could be due to the metabolic
0.5 g of fructose per kilogram body weight per hour, with effect of lactate. Indeed, it has been shown that lactate can
doses of 1.0 and 1.5 g/kg/h significantly increasing serum increase urate levels. For example, Burch and Kurke [148]
urate by about 50 and 80 μM, respectively [142]. In reported that infusion of lactate in healthy volunteers reduced
addition, a study of six subjects undergoing biliary surgery the renal clearance of urate by 70–80%, resulting in an
showed that intravenous infusion of 50 g of fructose (0.67– increase of 8–18% in serum urate.
0.83 g/kg body wt) within 30 min reduced hepatic ATP Tea, coffee, and chocolate are rich sources of methyl-
levels by about 50% and hepatic total adenosine phosphates xanthines, including caffeine, theobromine, and theophylline
and inorganic phosphate by about 35%, which was paralleled (Fig. 4). Green and black tea, respectively, contain 8–40 and 35–
by a 64 μM increase in mean serum urate [143]. Average 60 mg of caffeine per 200-ml serving, and coffee contains 60–
plasma urate levels in our study increased by 31 μM 1 h 110 mg of caffeine per 200 ml [149]. Chocolate is a particularly
after an oral dose of fructose of 0.83 g/kg body wt. Thus, rich source of theobromine (486 and 205 mg per 100 g dark or
our data are in close qualitative and quantitative agreement milk chocolate, respectively) and also contains caffeine (62 and
with other studies on the hyperuricemic effect of fructose in 20 mg per 100 g dark and milk chocolate, respectively) [17]. It is
humans [140–143]. well known that both caffeine and theobromine are readily
bioavailable, in contrast to flavonoids, and can be metabolized to
Other potential sources of endogenous urate or its methyl derivatives of uric acid (Fig. 4). Caffeine metabolites,
derivatives: sucrose, sorbitol, lactate, and methylxanthines such as 1-methylxanthine and 1-methyl uric acid, exhibit in vitro
antioxidant activity in the ORAC assay and prevent LDL
Other carbohydrates in fruit also could influence urate oxidation comparable to the effects of urate, trolox, and
production and plasma antioxidant capacity. Sucrose, which ascorbate [150]. In addition, we found that the methyl
like fructose is present in high amounts in fruits, can undergo derivatives of uric acid exhibit a reducing activity similar to
hydrolysis in vivo to yield equal amounts of fructose and glucose that of uric acid (Fig. 5). High levels of methylxanthines in tea,
before absorption. Sucrose can range from 2.5 to 5.0 g per 100 g coffee, and chocolate, and their rapid absorption and metabolism
S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746 1741

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 conducted in this area in recent years. Most of that research has
plasma total antioxidant capacity. focused on the antioxidant properties of flavonoids, which are
well characterized and well established in vitro. However, the in
Conclusions vitro data often conflict with results obtained from in vivo
studies on the antioxidant capacity of plasma or the resistance of
Some of the health benefits of fruits and vegetables have plasma and lipoproteins to oxidation ex vivo after the
been attributed to their content of polyphenols and flavonoids. consumption of flavonoid-rich foods by human subjects.
However, the specific mechanism(s) by which these compounds These inconsistencies between the in vitro and the in vivo
affect human health remains unclear, despite extensive research 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.
Fig. 5. Reducing activity of uric acid and its methyl derivatives. The reducing Foods rich in flavonoids contain other substances that may
activity was measured as FRAP according to Benzie et al. [104] and is expressed affect plasma total antioxidant capacity, either directly, e.g., by
as mM trolox equivalents/mM test compound. providing vitamin C, or indirectly, e.g., by stimulating
1742 S.B. Lotito, B. Frei / Free Radical Biology & Medicine 41 (2006) 1727–1746

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