Chronic quercetin ingestion and exercise-inducedoxidative damage and inflammation

Steven R. McAnulty, Lisa S. McAnulty, David C. Nieman, John C. Quindry,Peter A. Hosick, Matthew H. Hudson, Laura Still, Dru A. Henson, Ginger L. Milne,Jason D. Morrow, Charles L. Dumke, Alan C. Utter, Nan T. Triplett, andAdrianna Dibarnardi

Abstract: Quercetin is a flavonoid compound that has been demonstrated to be a potent antioxidant in vitro. The objectiveof this study was to evaluate if quercetin ingestion would increase plasma antioxidant measures and attenuate increases inexercise-induced oxidative damage. Forty athletes were recruited and randomized to quercetin or placebo. Subjects con-sumed 1000 mg quercetin or placebo each day for 6 weeks before and during 3 d of cycling at 57% work maximum for3 h. Blood was collected before and immediately after exercise each day, and analyzed for F2-isoprostanes, nitrite, ferric-reducing ability of plasma, trolox equivalent antioxidant capacity, and C-reactive protein. Statistical analyses involved a2 (treatment) � 6 (times) repeated measures analysis of variance to test main effects. F2-isoprostanes, nitrite, ferric-reducing ability of plasma, trolox equivalent antioxidant capacity, and C-reactive protein were significantly elevated asa result of exercise, but no group effects were found. Despite previous data demonstrating potent antioxidant actionsof quercetin in vitro, this study indicates that this effect is absent in vivo and that chronic quercetin ingestion doesnot exert protection from exercise-induced oxidative stress and inflammation.

Key words: exercise, antioxidants, oxidative stress, inflammation, flavanoids, humans.

Resume : Evidence a l’appui, la quercetine est un flavonoıde au pouvoir antioxydant in vitro. Le but de cette etude est deverifier si l’ingestion de quercetine ameliore la capacite antioxydante du plasma et reduit l’ampleur des lesions dues al’oxydation suscitee par l’exercice physique. Quarante athletes sont repartis aleatoirement dans deux groupes, le groupe ex-perimental consommant un supplement de quercetine et le groupe placebo, n’en consommant pas. Six semaines avant les3 jours d’exercice sur bicyclette et durant ces memes 3 jours, les sujets consomment un supplement de quercetine ou unesubstance placebo a raison de 1000 mg par jour ; la seance d’exercice physique consiste en un effort realise a une intensitede 57% travail pour une duree maximale de 3 h. Avant et immediatement apres chacune des seances d’exercice, on pre-leve des echantillons de sang pour en analyser le contenu en F2-isoprostanes, en nitrite, en proteine C reactive et les capa-cites plasmatiques de reduction ferrique et d’antioxydation relative a celle du trolox. Pour tester statistiquement lesdifferences entre les groupes en des moments divers, on utilise une analyse de variance 2 � 6 avec mesures repetees. Chezles deux groupes, on observe une augmentation significative des concentrations de F2-isoprostanes, de nitrite, de proteineC reactive et des capacites plasmatiques de reduction ferrique et d’antioxydation relative a celle du trolox, mais on n’ob-serve pas de differences entre les deux groupes. Malgre la demonstration du pouvoir antioxydant in vitro de la quercetinedans des etudes anterieures, cette etude n’en fait pas l’observation in vivo ; de plus, l’apport d’un supplement de querce-tine sur une longue periode n’offre pas de protection contre le stress oxydant et l’inflammation suscites par l’exercice phy-sique.

Mots-cles : exercice physique, antioxydants, stress oxydatif, inflammation, flavonoıde, humains.

[Traduit par la Redaction]

Introduction

Polyphenols are abundant molecules in our diet, and thereis growing evidence linking polyphenols to the prevention of

degenerative diseases due to antioxidant, anti-inflammatory,cardioprotective, and anti-carcinogenic activities (Manach etal. 1998, 2005; Williamson and Manach 2005). Epidemio-logical observation studies have indicated that the intake of

Received 23 August 2007. Accepted 26 October 2007. Published on the NRC Research Press Web site at apnm.nrc.ca on 1 February2008.

S.R. McAnulty,1 D.C. Nieman, J.C. Quindry, P.A. Hosick, M.H. Hudson, C.L. Dumke, A.C. Utter, N.T. Triplett, andA. Dibarnardi. Department of Health, Leisure, and Exercise Science, Appalachian State University, Boone, NC 28608, USA.L.S. McAnulty and L. Still. Department of Family and Consumer Sciences, Appalachian State University, Boone, NC 28608, USA.D.A. Henson. Department of Biology, Appalachian State University, Boone, NC 28608, USA.G.L. Milne and J.D. Morrow. Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville,TN 37232, USA.

1Corresponding author (e-mail: mcanltysr@appstate.edu).

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flavonoids is inversely associated with subsequent coronaryheart disease (Hollman and Katan 1997). In support, Aviramand Fuhrman (2002) found that quercetin from red wine re-sulted in the enrichment of plasma LDL in healthy volun-teers, which was associated with a significant three-foldattenuation in copper-induced LDL oxidation. Quercetin hasalso shown to prevent chemically induced DNA damage inhuman lymphocytes, which suggests anticarcinogenic prop-erties (Wilms et al. 2005). Ackland et al. (2005) found thata combination of quercetin and kaempferol inhibited cancercell proliferation and suggested that the two compounds ex-erted a synergistic anti-proliferative effect.

Specifically, flavonoids such as quercetin have been re-ported to exhibit a wide range of biological activities relatedto their antioxidant capacity and are found at high concen-trations in onions, apples, red wine, broccoli, and tea (Aliaet al. 2006; Hannum 2004; Labuda et al. 2003). Most of thebeneficial health effects of flavonoids are attributed to anti-oxidant and chelating abilities. It is generally hypothesizedthat the antioxidant activities of flavonoids results in a de-crease of oxidative stress, which in turn reduces disease risk(Heim et al. 2002). Despite the epidemiological data andevidence obtained in vitro, the effects of polyphenolic com-pounds observed in vivo have been vastly more limited(Williamson and Manach 2005). Interestingly, human datasuggest that quercetin typically has small effects on plasmaantioxidant and oxidation biomarkers in vivo, although thiseffect is not consistent (Boyle et al. 2000). Primarily, thelikely reason for this lack of demonstrated antioxidant ef-fects in humans after oral ingestion is due to the extensiveconjugation and metabolism of flavanoids during absorptionfrom the gut, which forms vastly altered compounds fromthe original molecule (Spencer et al. 2004). Nonetheless, itremains unclear as to whether quercetin metabolites expressbiological activities at the cellular level (Williams et al.2004).

It is recognized that oxidative stress and damage occur inresponse to intense and long-duration exercise (McAnulty etal. 2003, 2005). However, the effects of exercise-inducedoxidative stress upon potential disease risks are not wellknown. Nonetheless, exercise functions as an acute modelin which to examine antioxidant countermeasures. Humanstudies with exercise and otherwise are virtually non-existent regarding the influence of quercetin ingestion onoxidative stress and inflammation in vivo (MacRae andMefferd 2006). Halliwell et al. (2005) suggest that it is dif-ficult to predict whether flavonoid metabolites in vivo aresufficient to exert systemic antioxidant effects until moreresearch is available. Therefore, given the potential impor-tance of quercetin in disease etiology and the paucity ofquercetin and human exercise studies, the purpose of thisstudy was to examine the effect of 1000 mg/d of oralquercetin for 6 weeks before and during 3 d of intense cy-cling upon oxidative damage and inflammation.

Materials and methods

SubjectsForty trained male cyclists were recruited as experimental

subjects through local and collegiate cycling clubs. Writteninformed consent was obtained from each subject, and the

experimental procedures were in accordance with the policystatements of the institutional review board of AppalachianState University.

Research designTwo to three weeks before the first test session subjects

reported to the ASU Human Performance Laboratory fororientation and measurement of body composition and cardi-orespiratory fitness. Body composition was assessed by hy-drostatic weighing using an electronic load cell system(Exertech, Dresbach, Minn.). VO2 max was determined usinga graded maximal protocol with the subjects using their ownbicycles on CompuTrainerTM Pro Model 8001 trainers(RaceMate, Seattle, Wash.). Oxygen uptake and ventilationwere measured using the MedGraphics CPX metabolic sys-tem (MedGraphics Corporation, St. Paul, Minn.). Heart ratewas measured using a chest heart rate monitor (Polar ElectroInc., Woodbury, N.Y.). Basic demographic and training datawere obtained through a questionnaire.

Subjects agreed to avoid the use of large-dose vitamin andmineral supplements (above 100% of recommended dietaryallowances), nutritional supplements, ergogenic aids, herbs,and medications known to affect antioxidant or oxidativestress function for 3 weeks before, during, and 2 weeks afterthe 3 d period of intensified exercise. During orientation, adietitian instructed the subjects to follow a diet moderate incarbohydrate (using a food list) during the 3 weeks beforeand during the 3 d test sessions. Subjects recorded food in-take in a 3 d food record before the first exercise test ses-sion. The food records were analyzed using a computerizeddietary assessment program (Food Processor, ESHAResearch, Salem, Ore.).

The cyclists were randomized under double blind proce-dures to quercetin (Q) (n = 20) or placebo (P) (n = 20)groups. Subjects received quercetin (1000 mg/d) or placebosupplements for 3 weeks before and during the 3 d period ofintensified exercise. Pure quercetin (QU995, QuercegenPharma, Newton, Mass.) was mixed in orange Tang powderwith powdered food coloring and two doses of 500 mg quer-cetin were taken by subjects before their first and last mealsof each day. Subjects returned empty vials to the study die-titian to verify compliance with the supplementation regi-men. The basis for the 1000 mg dose was obtained fromdata from a collaborating laboratory (unpublished) indicat-ing that rats given this equivalent dose (~14 mg quercetin /kg body mass = 1000 mg for reference 70 kg human) re-sponded by having significant increases in running perform-ance and changes in mitochondrial density.

Subjects came to the lab for 3 consecutive days followingthe 3 week quercetin or placebo supplementation period.Subjects cycled for 3 h at ~57% maximal workrate (Wmax).Subjects reported to the laboratory at 14:00 not having in-gested energy in any form after 12:30. Blood samples werecollected ~30 min before and ~15 min after exercise. Sub-jects ingested 0.5–1.0 L/h water during the 3 h cyclingbout, with no other beverages or food ingested during thetest sessions. During the test sessions, experimental subjectscycled using their own bicycles on CompuTrainerTM ProModel 8001 trainers (RaceMate) with the exercise load setat ~57% Wmax. Metabolic measurements were made every

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30 min of cycling using the MedGraphics CPX metabolicsystem to verify workload.

Blood collection and analysisBlood samples were drawn from an antecubital vein with

subjects in the supine position. A local clinical hematologylaboratory provided hemoglobin and hematocrit values thatwere used to evaluate changes in plasma volume shift ac-cording to the methodology of Dill and Costill (1974). Otherblood samples were collected and centrifuged in sodiumheparin or EDTA tubes, and the plasma aliquots were snapfrozen in liquid nitrogen and stored at –80 8C.

Plasma quercetin analysisPlasma aliquots were spiked with 3.5 mmol/L of diosmetin

for use as an internal standard and acidified (to pH 4.9) with0.1 v/v of acetic acid, 0.58 mol/L. The samples were treatedfor 30 min at 37 8C with 5 � 106 U/L b-glucuronidase and2.5 � 105 U/L sulfatase. The reactions were stopped by addi-tion of 2.85 mL of acetone, and the resulting mixtures werecentrifuged. After centrifugation, a 20 mL aliquot of super-natant was injected in the high-performance liquid chroma-tography (HPLC) system for analysis. The HPLC systemused consisted of an autosampler (Kontron 360), an ultravio-let detector (set at 370 nm), and a software system for datarecording and processing. The system was fitted with a5 mm C-18 Hypersil-based deactivated silica analytical col-umn (150 34.6 mm; Life Sciences International, Cergy,France). The mobile phase consisted of water and H3PO4(99.5:0.5, solvent A) and acetonitrile (solvent B).

F2-isoprostanesPlasma F2-isoprostanes were determined using gas chro-

matography mass spectrometry (GC-MS) according to themethodology of Morrow and Roberts (1999). Briefly, freeF2-isoprostanes were extracted from 1 mL of plasma. Oneto 5 pmol of deuterated [2H4] PGF2� internal standard wasadded and the mixture was vortexed. This mixture was thenadded to a C18 Sep Pak column, followed by silica solid-phase extractions. F2-isoprostanes were converted intopentafluorobenzyl esters, subjected to thin-layer chromatog-raphy, and converted to trimethylsilyl ether derivatives.Samples were then analyzed by a negative ion chemical ion-ization GC-MS using a Nermag R10-10C mass spectrometerinterfaced with an Agilent computer system.

Ferric-reducing antioxidant potentialTotal plasma antioxidant potential was determined by the

ferric-reducing ability of plasma (FRAP) assay according tothe methodology of Benzie and Strain (1996). The basis ofthis assay is that water-soluble reducing agents (antioxi-dants) in the plasma will reduce ferric ions to ferrous ions,which then react with an added chromogen. Samples andstandards were analyzed in duplicate and expressed as ascor-bate equivalents based on a physiological ascorbate standardcurve (0–1000 mmol). Intra- and inter-assay coefficients ofvariation were less than 5% and 7%, respectively.

Trolox equivalent antioxidant capacityAntioxidant activity was determined in EDTA-treated

plasma samples using the trolox equivalent antioxidant ca-pacity (TEAC) method as described by Villano et al.

(2005). Briefly, a free radical producing enzymatic systemwas created using the horseradish peroxidase enzyme and2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS). This radical is produced by a reaction between1.5 mmol/L ABTS, 15 mmol/L hydrogen peroxide, and0.25 mmol/L peroxidase in 50 mmol/L glycine–HCl buffer(pH 4.5). One hundred microlitres of sample were placed inindividual cuvettes, and 2 mL of ABTS was then added.After 2 min incubation, the wavelength of the solution wasmeasured at 414 nm. All samples were tested in duplicate.The decrease in absorbency was quantified using a linear re-gression model of an 8 point standard curve (0–200 mmol/L)and expressed as Trolox equivalents. Intra- and inter-assaycoefficients of variation were less than 3% and 4%, respec-tively.

C-reactive protein (CRP)CRP was determined using an enzyme-linked immuno-

sorbent assay (ELISA) kit (catalog No. 1000) obtained fromAlpha Diagnostics (San Antonio, Tex.). Intra- and inter-assay coefficients of variation were less than 3% and 5%,respectively.

Plasma nitriteTotal plasma nitrate and nitrite were determined by spec-

trophotometric analysis using a nitric oxide non-enzymaticassay (Oxis International, Portland, Ore.). This assay isbased upon reaction of nitrite with Greiss reagent. Sampleswere pre-reacted with cadmium to convert any nitrate intonitrite. Plasma samples were diluted 3.8 times and read on anon-protein-binding microtiter plate at 540 nm. Concentra-tion was determined by linear regression of a standard curve(0–100 mmol/L). Intra- and inter-assay coefficients of varia-tion were less than 6% and 8%, respectively.

Statistical analysisData are expressed as means ± SEM and analyzed using a

2 (treatment) � 6 (times) repeated measures analysis of var-iance (ANOVA). Subject descriptive data in Table 1 werecompared between groups using Student’s t tests. Pearsonproduct–moment correlations were used to test the relation-ship between changes in measured outcomes. All statisticalanalyses were accomplished using Instat version 1.01 (SanDiego, Calif.) and SPSS version 13.0 (Chicago, Ill.).

ResultsSubject characteristics for the 40 cyclists randomized to

quercetin and placebo groups are summarized in Table 1.No significant differences were found between groups forage, body composition, training history, or maximal per-formance measures. Subjects in the quercetin and placebogroups came into the study averaging approximately 1.5 ±0.2 and 1.6 ± 0.2 h cycling per training bout. Thus, the 3 dintensified exercise period (9 h total exercise) representednearly a doubling of their normal exercise workload. Threeday food records before the exercise period revealed no sig-nificant group differences in energy or macronutrient intake(data not shown), and for all subjects combined, energy in-take was 2684 ± 117 kcal/d with carbohydrate representing56.6% ± 2.5%, protein 16.2% ± 0.8%, and fat 27.2% ±1.8%.

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Plasma volume shift did not differ between groups duringthe 3 exercise bouts and averaged less than 1% due to inges-tion of 0.5–1.0 L/h water during exercise (data not shown).Figure 1 indicates that after 3 weeks of supplementation,plasma quercetin levels for the quercetin and placebo groupswere 304 ± 71 and 47.7 ± 8.7 mg/L, respectively (p =0.002). This demonstrates that quercetin supplementationwas effective in raising free and conjugated quercetin me-tabolites. Breaking the variables into individual treatmentsindicates that the average pre- to post-exercise increase forF2-isoprostanes was 130% in Q and 113% in P, FRAP in-creased 4% in Q and 7% in P, TEAC increased 11% in Qand 9% in P, nitrite increased 19% in Q and 16% in P, andCRP increased 13% in Q and decreased 13% in P. As re-ported in the Figure legends, sample numbers (n) do not al-ways equal 20 because of specimen losses incurred duringprocessing. Overall, F2-isoprostanes, FRAP, TEAC, nitrite,and CRP were significantly elevated as a result of exercise,but no significant treatment–time interaction effects werefound (Figs. 2, 3, 4, 5, and 6). Significant but modest corre-lations were found between Q ((F2-isoprostanes and FRAP,r = 0.293, p = 0.001), (F2-isoprostanes and TEAC, r =0.284, p = 0.002), (F2-isoprostanes and CRP, r = 0.490, p £0.001), (FRAP and CRP, r = 0.231, p = 0.012), (CRP andnitrite, r = 0.279, p = 0.012), and (TEAC and CRP, r =0.243, p = 0.010)) and P )(F2-isoprostanes and TEAC, r =0.309, p = 0.001), (F2-isoprostanes and CRP, r = 0.211, p =0.023), and (TEAC and CRP, r = 0.214, p = 0.020)).

Discussion

The findings from this study indicate that quercetin sup-plementation increases circulating plasma values of conju-gated forms of quercetin. However, in contrast to ourhypothesis, the increase in plasma quercetin metabolites didnot affect oxidative stress, inflammation, or plasma antioxi-dant capacity. This finding is surprising and counter to awealth of previous investigations demonstrating the efficacyof quercetin as an in vitro antioxidant. In this regard, doseissues may be relevant as average dietary intake of quercetinfor humans appears to range from 5 to 40 mg/d (Manach etal. 1997). Human intervention studies in vivo with quercetinare few but Williamson and Manach reviewed human stud-ies using dosages of between 21 and 1000 mg of quercetin,all of which exhibited at least some beneficial effects,although not with markers used in the present study.Williamson and Manach also indicated that lower doses ofquercetin are more methylated than larger doses. Therefore,it is possible that larger doses may allow higher blood val-ues of unconjugated quercetin, but this was not seen in ourinvestigation. Our data could be interpreted to imply thatquercetin ingestion results in circulating quercetin metabo-

lites that must exert beneficial effects through mechanismsbeyond those related to strict antioxidant function.

The plasma antioxidant capacity increased significantly asa result of exercise, but not due to quercetin ingestion, asthere were not any significant treatment–time interactions.This increase in plasma antioxidant capacity most likely rep-resents release of urate and ascorbate into the blood duringexercise (Aguilo et al. 2005; Yanai and Morimoto 2004).Our results indicate that the antioxidant capacity of querce-tin and blood plasma is not additive. This may be partly dueto interactions between quercetin and plasma proteins thatinhibit antioxidant activity. In contrast, the antioxidant ca-pacity of a-tocopherol, which also binds to protein, is notaffected by the interaction. This suggests that the antioxidantcapacity of quercetin may be important, but the bloodplasma environment where antioxidant functions may negatea net change in antioxidant capacity (Arts et al. 2001).

Other plausible rationale for a lack of change in antioxi-dant capacity could be that (i) exogenous supplementationof quercetin at the current dosages may not significantly al-ter the redox status of blood plasma or (ii) the antioxidantfunction of quercetin in vivo may be preserved despite con-jugation, but the conjugated metabolites do not exhibit anti-oxidant activities detectable within the confines of theantioxidant assays we employed (FRAP and TEAC). Quer-cetin exhibits powerful antioxidant activity in vitro due tothe presence of a 3,4 B-ring hydroxyl group configuration.During metabolism in humans, substitution of the 3,4 B-ring hydroxyl groups with methyl or glycosyl groups mayhave abolished a large amount of the free radical scavengingability of quercetin detectable by the FRAP and TEAC as-says. Nonetheless, one study found that quercetin metabo-lites exhibit some antioxidant capacity despite extensivemetabolism. Also, other relevant mechanisms of antioxidantactivity such as lipophilicity and membrane partitioning maynot have been abolished . Regardless, these results confirmthat the total antioxidant capacity of blood plasma is com-plex and highly regulated.

The effect of antioxidants is often executed in complexbiological mixtures where various interactions may takeplace. The TEAC assay derives the antioxidant capacity ofa compound by measuring spectrophotometrically the disap-pearance of the stable ABTS radical by free radical scaveng-ing. The ABTS radical is introduced within the assay incontrast to the FRAP assay. Also, the TEAC assay measuresthe contribution of plasma proteins, whereas FRAP does notmeasure considerable amounts of plasma proteins (Cao andPrior 1998). This may be an advantage of the FRAP assayin that only the contribution of other plasma antioxidants,such as quercetin, can be assessed. The FRAP assay dependsupon the reduction of a ferric–tripyridyltriazine complex toa ferrous–tripyridyltriazine complex by an electron- orhydrogen-donating antioxidant.

Table 1. Subject characteristics (mean ± SEM; n = 20).

Age (y)Body mass(kg) % Body fat

Training volume(km�week–1)

VO2 max

(mL�kg–1�min–1)Maximumpower (W)

HRmax

(beats�min–1)Quercetin 26.1±1.8 74.7±0.2 13.8±1.2 242±27 53.2±1.2 314±9 188±1Placebo 29.1±2.4 74.2±1.4 11.5±0.6 270±29 54.7±1.1 320±69 190±2p value 0.321 0.846 0.094 0.493 0.365 0.554 0.328

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We did not observe any effects of quercetin upon the oxi-dative damage markers used in this study. No differenceswere observed between quercetin and placebo in the patternof change in F2-isoprostanes and nitrite. However, F2-isoprostanes were significantly increased by exercise,which indicates that the subjects underwent significant in-creases in oxidative damage. It would certainly be ex-pected that despite the lack of effect of quercetin uponplasma F2-isoprostanes or antioxidant potential, that the in-crease in antioxidant potential would have diminished orprevented the rise in F2-isoprostanes. Since this was notthe case, the most obvious conclusions one would draware that a disconnect exists between the antioxidant assaysused and the ability to detect changes in antioxidant ca-pacity, that the antioxidant supplement is altered after in-gestion with the resulting metabolites unable to exertantioxidant activity, or that F2-isoprostanes are produced ina mechanism or compartment that is unaffected by the

antioxidant supplement or increase in antioxidant capacity.Specifically, one of the main strengths of this investigationis that F2-isoprostanes measured via GS-MS are widelyregarded as a precise and accurate marker of oxidativestress in vivo. The development of methods to quantifyF2-isoprostanes (prostaglandin-like compounds derived fromthe free radical catalyzed peroxidation of arachidonicacid) has provided an accurate assessment of oxidant stressin vivo (Basu and Helmersson 2005; Morrow 2005).F2-isoprostanes are typically increased during exercise, butthis rise can be attenuated by antioxidant supplementation(Mastaloudis et al. 2004).

Plasma nitrite was used as a biomarker for nitric oxidedegradation and was increased as a result of exercise, butwas unaffected by quercetin supplementation. This impliesthat the heavy exercise caused significant changes in nitricoxide turnover, changes in production, or both. In one ofthe few human studies to examine blood nitrite in an exer-

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cise application, Vassalle et al. (2002) found that athleteshad significantly higher baseline values of plasma nitrateand nitrite compared with controls. Nitrite has been foundto cause nitrosative stress leading to nitration of aromaticcompounds (Pannala et al. 1997). The nitric oxide synthase(NOS) isoforms are differentially expressed and regulatedand, in turn, regulate several important physiological func-tions including vascular tone, inflammation, and cancer.Quercetin administration in vitro has been found to inhibitNOS and reduce nitrite formation (Hirota et al. 2005;Martinez-Florez et al. 2005). However, we did not observethis effect of quercetin. This again suggests that quercetinmetabolites must function differently from unconjugatedquercetin.

Elevation of CRP levels in blood is recognized as amarker of inflammation and is one of the cardiac diseaserisk factors, as well as the acute exercise response in healthy

individuals. Administration of polyphenolic compounds hasbeen found to reduce inflammatory cytokine (IL-1b and IL-6)induced CRP expression in Hep3B cells. Quercetin and re-sveratrol suppressed cytokine-induced CRP expression in adose-dependent manner (Kaur et al. 2007). Garcia-Mediavilla et al. (2007) found that quercetin exhibitedanti-inflammatory effects and subsequent reduction of CRPin Chang liver cells via mechanisms likely to involveblockade of NF-kb activation. Similar to the other markersused in our study, no effect of quercetin was found in vivoin reducing CRP and the inflammatory response of theheavy exercise.

In agreement with our findings, Beatty et al. (2000) foundthat 14 d of either a low- or high-flavonol (predominantlyquercetin) diet in 36 healthy human subjects did not resultin differences in oxidative DNA damage. Interestingly, inone of the few exercise studies available, MacRae and

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Fig. 3. Plasma ferric-reducing ability of plasma (FRAP) over time in quercetin (n = 19) and placebo (n = 18) groups before and after 3 d ofcycling for 3 h at ~57% Wmax. Main effects were time (p = 0.008) and treatment � time interaction (p = 0.521). Values are means ± SEM.

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Fig. 4. Plasma trolox equivalent antioxidant capacity (TEAC) over time in quercetin (n = 18) and placebo (n = 19) groups before and after3 d of cycling for 3 h at ~57% Wmax. Main effects were time (p £ 0.001) and treatment � time interaction (p = 0.606). Values are means ±SEM.

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Mefferd (2006) found that 6 weeks of quercetin supplemen-tation improved 30 km time-trial performance by 3.1% in 11male cyclists, and speculated that the performance increasewith quercetin use may have been related to an attenuationof oxidative stress and muscle IL-6 release. Unfortunately,no oxidative stress indicators were measured. Phillips et al.(2003) found that a mix of antioxidants containing quercetinattenuated IL-6 and CRP after eccentric exercise in un-trained males. As stated previously, we did not observe aquercetin effect upon CRP. In this case, it could be inter-preted that the mode and duration of exercise were differentfrom the cycling exercise used in our study, but this has notbeen evaluated. It should also be emphasized that our sub-ject population consisted of highly trained athletes who areknown to possess up-regulated antioxidant defenses (Powerset al. 1999). Although it remains to be determined, the pres-ence of highly elevated antioxidant defense systems mayhave masked any beneficial effects of quercetin administra-tion. It would be interesting to repeat our study using un-trained individuals and examine whether the outcomeswould be similar.

It should also be pointed out that although excessivestates of oxidative stress are likely detrimental, it is alsoknown that oxidative stress from exercise coincides with po-tential beneficial changes in cell signaling and gene expres-sion. Increased gene expression leads to production ofprotective proteins and antioxidant enzymes (Johnson 2002;Jokelainen et al. 2001; McArdle et al. 2001; Powers et al.1993). As an example of the beneficial expression of protec-tive proteins, Thompson et al. (2005) found an increase inthe antioxidant enzyme heme oxygenase 1 (HO-1) protein2 h after exercise. These changes likely coincide with acti-vation of the transcription factor known as nuclear factorkappa beta (NFkb). NFkb is redox sensitive and Cuevas etal. (2005) concluded that even short-term anaerobic exerciseinduced oxidative stress and gave rise to activation of NFkb.

In summary, despite the well-established potential ofquercetin as an in vitro antioxidant, we did not observe abenefit in highly trained subjects exposed to 3 consecutivedays of prolonged endurance exercise. Quercetin ingestiondid not significantly alter antioxidant capacity or prevent ox-idative damage from heavy exercise. Lastly, since circulat-

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Fig. 5. Plasma nitrite over time in quercetin (n = 20) and placebo (n = 20) groups before and after 3 d of cycling for 3 h at ~57% Wmax.Main effects were time (p = 0.025) and treatment � time interaction (p = 0.171). Values are means ± SEM.

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Fig. 6. Plasma C-reactive protein (CRP) over time in quercetin (n = 19) and placebo (n = 19) groups before and after 3 d of cycling for 3 hat ~57% Wmax. Main effects were time (p £ 0.001) and treatment � time interaction (p = 0.179). Values are means ± SEM.

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ing plasma quercetin exists as metabolites, the beneficial ef-fects of quercetin may likely exist via mechanisms related togene expression rather than those related to strict antioxidantfunction. To our knowledge, this is the first study to solelyexamine the effects of quercetin supplementation with exer-cise in humans.

AcknowledgementsSupported by a grant from DARPA and the US Army Re-

search Office, award number W911NF-06-0014, and Na-tional Institutes of Health Grants (to J.D. Morrow) DK-48831, GM-15431, CA-77839, and RR00095. J.D. Morrowis the recipient of a Burroughs Wellcome Fund ClinicalScientist Award in Translational Research. Fischer/NycomLaboratory.

ReferencesAckland, M.L., van de Waarsenburg, S., and Jones, R. 2005. Syner-

gistic antiproliferative action of the flavonols quercetin andkaempferol in cultured human cancer cell lines. In Vivo, 19:69–76. PMID:15796157.

Aguilo, A., Tauler, P., Fuentespina, E., Tur, J.A., Cordova, A., andPons, A. 2005. Antioxidant response to oxidative stress inducedby exhaustive exercise. Physiol. Behav. 84: 1–7. doi:10.1016/j.physbeh.2004.07.034. PMID:15642600.

Alia, M., Ramos, S., Mateos, R., Granado-Serrano, A.B., Bravo, L.,and Goya, L. 2006. Quercetin protects human hepatoma HepG2against oxidative stress induced by tert-butyl hydroperoxide.Toxicol. Appl. Pharmacol. 212: 110–118. doi:10.1016/j.taap.2005.07.014. PMID:16126241.

Arts, M.J., Haenen, G.R., Voss, H.P., and Bast, A. 2001. Maskingof antioxidant capacity by the interaction of flavonoids with pro-tein. Food Chem. Toxicol. 39: 787–791. doi:10.1016/S0278-6915(01)00020-5. PMID:11434985.

Aviram, M., and Fuhrman, B. 2002. Wine flavonoids protectagainst LDL oxidation and atherosclerosis. Ann. N. Y. Acad.Sci. 957: 146–161. PMID:12074969.

Basu, S., and Helmersson, J. 2005. Factors regulating isoprostaneformation in vivo. Antioxid. Redox Signal. 7: 221–235. doi:10.1089/ars.2005.7.221. PMID:15650410.

Beatty, E.R., O’Reilly, J.D., England, T.G., McAnlis, G.T., Young,I.S., Geissler, C.A., Sanders, T.A., and Wiseman, H. 2000. Ef-fect of dietary quercetin on oxidative DNA damage in healthyhuman subjects. Br. J. Nutr. 84: 919–925. PMID:11177210.

Benzie, I.F.F., and Strain, J.J. 1996. The ferric reducing ability ofplasma (FRAP) as a measure of ‘‘antioxidant power’’: TheFRAP assay. Anal. Biochem. 239: 70–76. doi:10.1006/abio.1996.0292. PMID:8660627.

Boyle, S.P., Dobson, V.L., Duthie, S.J., Hinselwood, D.C., Kyle,J.A., and Collins, A.R. 2000. Bioavailability and efficiency ofrutin as an antioxidant: a human supplementation study. Eur. J.Clin. Nutr. 54: 774–782. doi:10.1038/sj.ejcn.1601090. PMID:11083486.

Cao, G., and Prior, R.L. 1998. Comparison of different analyticalmethods for assessing total antioxidant capacity of humanserum. Clin. Chem. 44: 1309–1315. PMID:9625058.

Cuevas, M.J., Almar, M., Garcia-Glez, J.C., Garcia-Lopez, D.,De Paz, J.A., Alvear-Ordenes, I., and Gonzalez-Gallego, J.2005. Changes in oxidative stress markers and NF-kappaB acti-vation induced by sprint exercise. Free Radic. Res. 39: 431–439.doi:10.1080/10715760500072149. PMID:16028368.

Dill, D.B., and Costill, D.L. 1974. Calculation of percentage

changes in volumes of blood, plasma, and red cells in dehydra-tion. J. Appl. Physiol. 37: 247–248. PMID:4850854.

Garcia-Mediavilla, V., Crespo, I., Collado, P.S., Esteller, A.,Sanchez-Campos, S., Tunon, M.J., and Gonzalez-Gallego, J.2007. The anti-inflammatory flavones quercetin and kaempferolcause inhibition of inducible nitric oxide synthase, cyclooxygen-ase-2 and reactive C-protein, and down-regulation of the nuclearfactor kappaB pathway in Chang Liver cells. Eur. J. Pharmacol.557: 221–229. doi:10.1016/j.ejphar.2006.11.014. PMID:17184768.

Halliwell, B., Rafter, J., and Jenner, A. 2005. Health promotion byflavonoids, tocopherols, tocotrienols, and other phenols: director indirect effects? Antioxidant or not? Am. J. Clin. Nutr. 81:268S–276S. PMID:15640490.

Hannum, S.M. 2004. Potential impact of strawberries on humanhealth: a review of the science. Crit. Rev. Food Sci. Nutr. 44:1–17. doi:10.1080/10408690490263756. PMID:15077879.

Heim, K.E., Tagliaferro, A.R., and Bobilya, D.J. 2002. Flavonoidantioxidants: chemistry, metabolism and structure-activity rela-tionships. J. Nutr. Biochem. 13: 572–584. doi:10.1016/S0955-2863(02)00208-5. PMID:12550068.

Hirota, S., Takahama, U., Ly, T.N., and Yamauchi, R. 2005.Quercetin-dependent inhibition of nitration induced by peroxi-dase/H2O2/nitrite systems in human saliva and characterizationof an oxidation product of quercetin formed during the inhibi-tion. J. Agric. Food Chem. 53: 3265–3272. doi:10.1021/jf0404389. PMID:15853358.

Hollman, P.C., and Katan, M.B. 1997. Absorption, metabolism andhealth effects of dietary flavonoids in man. Biomed. Pharmac-other. 51: 305–310. doi:10.1016/S0753-3322(97)88045-6.PMID:9436520.

Johnson, P. 2002. Antioxidant enzyme expression in health and dis-ease: effects of exercise and hypertension. Comp. Biochem.Physiol. C Pharmacol. Toxicol. 133: 493–505. doi:10.1016/S1532-0456(02)00120-5.

Jokelainen, K., Reinke, L.A., and Nanji, A.A. 2001. Nf-kappab ac-tivation is associated with free radical generation and endotoxe-mia and precedes pathological liver injury in experimentalalcoholic liver disease. Cytokine, 16: 36–39. doi:10.1006/cyto.2001.0930. PMID:11669585.

Kaur, G., Rao, L.V., Agrawal, A., and Pendurthi, U.R. 2007. Effectof wine phenolics on cytokine-induced C-reactive protein ex-pression. J. Thromb. Haemost. 5: 1309–1317. doi:10.1111/j.1538-7836.2007.02527.x. PMID:17388968.

Labuda, J., Buckova, M., Heilerova, L., Silhar, S., and Stepanek, I.2003. Evaluation of the redox properties and anti/pro-oxidant ef-fects of selected flavonoids by means of a DNA-based electro-chemical biosensor. Anal. Bioanal. Chem. 376: 168–173.PMID:12712310.

MacRae, H.S., and Mefferd, K.M. 2006. Dietary antioxidant sup-plementation combined with quercetin improves cycling timetrial performance. Int. J. Sport Nutr. Exerc. Metab. 16: 405–419. PMID:17136942.

Manach, C., Morand, C., Demigne, C., Texier, O., Regerat, F., andRemesy, C. 1997. Bioavailability of rutin and quercetin in rats.FEBS Lett. 409: 12–16. doi:10.1016/S0014-5793(97)00467-5.PMID:9199494.

Manach, C., Morand, C., Crespy, V., Demigne, C., Texier, O.,Regerat, F., and Remesy, C. 1998. Quercetin is recovered in hu-man plasma as conjugated derivatives which retain antioxidantproperties. FEBS Lett. 426: 331–336. doi:10.1016/S0014-5793(98)00367-6. PMID:9600261.

Manach, C., Williamson, G., Morand, C., Scalbert, A., andRemesy, C. 2005. Bioavailability and bioefficacy of polyphenols

McAnulty et al. 261

# 2008 NRC Canada

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

nive

rsity

of

Reg

ina

on 0

5/02

/13

For

pers

onal

use

onl

y.

in humans. I. Review of 97 bioavailability studies. Am. J. Clin.Nutr. 81: 230S–242S. PMID:15640486.

Martinez-Florez, S., Gutierrez-Fernandez, B., Sanchez-Campos, S.,Gonzalez-Gallego, J., and Tunon, M.J. 2005. Quercetin attenu-ates nuclear factor-kappaB activation and nitric oxide productionin interleukin-1beta-activated rat hepatocytes. J. Nutr. 135:1359–1365. PMID:15930438.

Mastaloudis, A., Morrow, J.D., Hopkins, D.W., Devaraj, S., andTraber, M.G. 2004. Antioxidant supplementation preventsexercise-induced lipid peroxidation, but not inflammation, inultramarathon runners. Free Radic. Biol. Med. 36: 1329–1341.doi:10.1016/j.freeradbiomed.2004.02.069. PMID:15110397.

McAnulty, S.R., McAnulty, L.S., Nieman, D.C., Morrow, J.D.,Utter, A.C., Henson, D.A., Dumke, C.L., and Vinci, D.M. 2003.Influence of carbohydrate ingestion on oxidative stress andplasma antioxidant potential following a 3 h run. Free Radic.Res. 37: 835–840. doi:10.1080/1071576031000136559. PMID:14567443.

McAnulty, S.R., McAnulty, L.S., Nieman, D.C., Morrow, J.D.,Shooter, L.A., Holmes, S., Heward, C., and Henson, D.A. 2005.Effect of alpha-tocopherol supplementation on plasma homocys-teine and oxidative stress in highly trained athletes before andafter exhaustive exercise. J. Nutr. Biochem. 16: 530–537.doi:10.1016/j.jnutbio.2005.02.001. PMID:16115541.

McArdle, A., Pattwell, D., Vasilaki, A., Griffiths, R.D., andJackson, M.J. 2001. Contractile activity-induced oxidative stress:cellular origin and adaptive responses. Am. J. Physiol. Cell Phy-siol. 280: C621–C627. PMID:11171582.

Morrow, J.D. 2005. Quantification of isoprostanes as indices ofoxidant stress and the risk of atherosclerosis in humans. Arter-ioscler. Thromb. Vasc. Biol. 25: 279–286. doi:10.1161/01.ATV.0000152605.64964.c0. PMID:15591226.

Morrow, J.D., and Roberts, L.J. 1999. Mass spectrometric quantifi-cation of F2-isoprostanes in biological fluids and tissues as mea-sure of oxidant stress. Methods Enzymol. 300: 3–12. PMID:9919502.

Pannala, A.S., Rice-Evans, C.A., Halliwell, B., and Singh, S. 1997.Inhibition of peroxynitrite-mediated tyrosine nitration by cate-chin polyphenols. Biochem. Biophys. Res. Commun. 232: 164–168. doi:10.1006/bbrc.1997.6254. PMID:9125123.

Phillips, T., Childs, A.C., Dreon, D.M., Phinney, S., andLeeuwenburgh, C. 2003. A dietary supplement attenuates IL-6and CRP after eccentric exercise in untrained males. Med. Sci.

Sports Exerc. 35: 2032–2037. doi:10.1249/01.MSS.0000099112.32342.10. PMID:14652498.

Powers, S.K., Criswell, D., Lawler, J., Martin, D., Lieu, F.K., Ji,L.L., and Herb, R.A. 1993. Rigorous exercise training increasessuperoxide dismutase activity in ventricular myocardium. Am. J.Physiol. 265: H2094–H2098. PMID:8285249.

Powers, S.K., Ji, L.L., and Leeuwenburgh, C. 1999. Exercisetraining-induced alterations in skeletal muscle antioxidant capa-city: a brief review. Med. Sci. Sports Exerc. 31: 987–997.doi:10.1097/00005768-199907000-00011. PMID:10416560.

Spencer, J.P., Abd-el-Mohsen, M.M., and Rice-Evans, C. 2004.Cellular uptake and metabolism of flavonoids and their metabo-lites: implications for their bioactivity. Arch. Biochem. Biophys.423: 148–161. doi:10.1016/j.abb.2003.11.010. PMID:14989269.

Thompson, D., Basu-Modak, S., Gordon, M., Poore, S.,Markovitch, D., and Tyrrell, R.M. 2005. Exercise-inducedexpression of heme oxygenase-1 in human lymphocytes. FreeRadic. Res. 39: 63–69. doi:10.1080/10715760400022327.PMID:15875813.

Vassalle, C., Lubrano, V., L’Abbate, A., and Clerico, A. 2002. De-termination of nitrite plus nitrate and malondialdehyde in humanplasma: analytical performance and the effect of smoking andexercise. Clin. Chem. Lab. Med. 40: 802–809. doi:10.1515/CCLM.2002.139. PMID:12392309.

Villano, D., Fernadez-Panchon, M.S., Troncosa, A.M., and Garcia-Parilla, M.C. 2005. Comparison of antioxidant activity of winephenolic compounds and metabolites in vitro. Anal. Chim.Acta, 538: 391–398. doi:10.1016/j.aca.2005.02.016.

Williams, R.J., Spencer, J.P., and Rice-Evans, C. 2004. Flavonoids:antioxidants or signalling molecules? Free Radic. Biol. Med. 36:838–849. doi:10.1016/j.freeradbiomed.2004.01.001. PMID:15019969.

Williamson, G., and Manach, C. 2005. Bioavailability and bioeffi-cacy of polyphenols in humans. II. Review of 93 interventionstudies. Am. J. Clin. Nutr. 81: 243S–255S. PMID:15640487.

Wilms, L.C., Hollman, P.C., Boots, A.W., and Kleinjans, J.C.2005. Protection by quercetin and quercetin-rich fruit juiceagainst induction of oxidative DNA damage and formation ofBPDE-DNA adducts in human lymphocytes. Mutat. Res. 582:155–162. PMID:15781220.

Yanai, H., and Morimoto, M. 2004. Effect of ascorbate on serumlipids and urate metabolism during exhaustive training. Clin.Sci. (Lond.), 106: 107–109. PMID:12927020.

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