8/11/2019 J. Nutr.-2000-Wang-2115S-9S

1/5

8/11/2019 J. Nutr.-2000-Wang-2115S-9S

2/5

levels increased from baseline values of 22 4 nmol/L tovalues of 257 66 nmol/L after 2 h. Importantly, this increasein epicatechin was associated with an increase in the antiox-idant capacity of the plasma and a decrease in plasma 2-thio-barbituric acid reactive substances (TBARS) (Rein et al.2000a). In the current work, we extend these observations byexamining dose-response changes in plasma epicatechin,plasma antioxidant capacity, plasma TBARS and 8-isopros-

tane after the acute consumption of different amounts of aprocyanidin-rich chocolate.

METHODS

Subjects. Twenty volunteers (14 women and 6 men, age range2056 years, weight 67 2.7 kg, body mass index 23.8 0.79 kg/m2)composed the subject pool from which subjects were randomly drawnto participate in each of four chocolate treatment groups. Ten to 12subjects were studied on four separate occasions, separated by 1-weekintervals. Subjects were nonsmokers and were free from any apparentdiseases. Volunteers signed informed consent before beginning thestudy, and all procedures were conducted in accordance with theguidelines of the Human Subjects Review Committee of the Univer-

sity of California, Davis.Chemicals. Chemicals were purchased from Sigma ChemicalCo. (St. Louis, MO) unless otherwise stated.

Study design. Participants were asked to refrain from takingvitamin supplements, nonsteroidal anti-inflammatory medicationsand foods rich in polyphenols for 24 h and to fast overnight for 12 hbefore each feeding trial. Blood was collected at baseline (0 h) and 2and 6 h after the first blood draw. Subjects were instructed toconsume the test foods (chocolate and bread, or bread only) within15 min of the first blood draw. All subjects were asked to consume anadditional 95 g of bread during the next 2 h.

Test foods provided 0, 27, 53 or 80 g of procyanidin-rich chocolatein the form of M&Ms Semi-Sweet Chocolate Mini Baking Bits madewith Cocoapro cocoa (Mars Incorporated, Hackettstown, NJ) andincluded a serving of 47 g of bread. The 27-g chocolate portion wasin the form of candy-coated M&Ms baking bits, which provided

0.732 MJ, 0.021 g caffeine and 0.18 g theobromine. Each 27-gchocolate portion contained 46 mg epicatechin and a total of 186 mgof procyanidins. The bread (bagel) provided 0.544 MJ, 0.75 g totalfat, 25 g carbohydrate and 4.5 g protein in a 47-g serving.

Collection of plasma samples. Eighteen milliliters of venousblood was drawn into Vacutainer tubes containing EDTA or sodiumheparin (Becton Dickinson, Franklin Lakes, NJ). Plasma was sepa-rated by low-speed centrifugation (1500 gat 4C for 10 min) andstored at 80C until analysis. For lipid oxidation determinations,aliquots of plasma samples were added with 4% w/v butylated hy-droxytoluene/ethanol (for a final ratio of 4:1) before freezing.

Assessment of epicatechin in plasma. Plasma concentration ofepicatechin was determined as described by Richelle et al. (1999),with some modifications. In brief, 200 l of heparinized humanplasma was vortexed with a solution of 0.2 g/ml ascorbic acid, 1mg/ml EDTA and 20 mol-glucuronidase suspension (2000 U ofglucuronidase activity and 80 U of sulfatase activity). After a 45-minincubation at 37C, 0.5 ml of acetonitrile was added, and the sampleswere vortexed and centrifuged at 10,000 g for 5 min. The super-natant fraction was combined with 50 mg of alumina and 1 ml of 50mmol/L Tris-HCl (pH 7.6), vortexed and centrifuged. After discard-ing the supernatant, the alumina was washed with 1 ml of 50 mmol/LTris-HCl (pH 7.6), followed by a final wash with 1 ml of methanol,and centrifuged as indicated. After discarding the methanol, residualmethanol was evaporated under nitrogen, and epicatechin was ex-tracted from the alumina with 250 l of 0.25 mol/L perchloric acid.The sample was centrifuged (10,000 g, 1 min, 4C), and thesupernatant fraction was filtered through a 0.2-m polyethersulfonemembrane (Nalgene).

Samples (50 l) were chromatographed using a Hewlett Packard(Wilmington, DE) 1100 series system equipped with an ESA

(Chelmsford, MA) Coulochem II coulometric detector with a 5011analytical cell set as follows: guard cell 550 mV, conditioning cell

100 mV and analytical cell400 mV. Chromatography was carriedout using an Alltima C-18 column (5 m, 150 4.6 mm; Deerfield,IL) with a 0.5-m Frit precolumn filter (Upchurch, WA). Themobile phase was composed of two solvent solutions: solvent A, 40%methanol/60% 50 mmol/L sodium acetate, pH 5.8; and solvent B, 7%methanol/93% 100 mmol/L sodium acetate, pH 5.2.

Epicatechin separation was achieved with a flow rate of 1 ml/minand a gradient elution. For gradient elution, the composition of themobile phase was first set at 80% B, which was linearly decreased to

60% B by 1 min. This was followed by another linear decrease to 20%B by 3.5 min. The mobile phase composition was maintained at 20%B until 20 min, when B was linearly increased to 80% by 30 min.

Assessment of plasma TBARS. Plasma TBARS determinationwas conducted as previously described (Oteiza et al. 1997), withmodifications for using a plate reader. Briefly, 100 l of plasma, 200l of 3% sodium dodecyl sulfate, 800 l of 0.1 mol/L HCl, 100 l10% (wt/v) phosphotungstic acid and 400 l of 0.7% (wt/v) 2-thio-barbituric acid were combined and placed at 95C for 30 min.Samples were cooled on ice and mixed with 1 ml of 1-butanol. Aftercentrifugation (1800 g, 10 min, 4C), a 200-l aliquot of thebutanol phase was separated and analyzed spectrofluorometrically(excitation 515 nm and emission 555 nm) using a plate readerattachment (PerkinElmer Cetus, Norwalk, CT). TBARS are ex-pressed as malondialdehyde equivalents.

Assessment of plasma antioxidant capacity. Plasma samples(510 l) were assayed for their ability to inhibit the chemilumines-cence produced by a mixture of 3 ml of 5.4 mg/ml 2,2-azo-bis(amidi-nopropane) in 0.1 mmol/L phosphate-buffered saline, pH 7.4(GIBCO BRL, Life Technologies, Grand Island, NY) and 10l of 1mg/ml luminol. The chemiluminescence was measured in a liquidscintillation counter (Wallac 1410; Wallac Oy, Turku, Finland). Theplasma antioxidant capacity value was calculated as the lag timebefore an increase in the chemiluminescence was observed (Lissi etal. 1995). This lag time is proportional to the cumulative amount ofantioxidants present in the samples. A reference lag time was ob-tained by using a known amount of 6-hydroxy-2,5,7,8-tetramethoxy-chroman-2-carboxylic acid (Trolox; Aldrich Chemical Co, Milwau-kee WI).

Assessment of 8-isoprostane. 8-Isoprostane (8-epi-prostaglandinF2)was determined in plasma using 25 l of plasma added to 40 l

of the supplied enzyme immunoassay buffer from the 8-IsoprostaneEnzyme Immunoassay Kit (Cayman Chemical, Ann Arbor, MI.).Plates were read using a Shimadzu (Columbia, MD) spectrophotom-eter at 450 nm.

Statistical analysis. Results in the text and tables are expressedas means SEM. Changes between the baseline (0 h) and the 2- and6-h time points among treatment groups were examined using re-peated-measures ANOVA, with control for multiple measurementson the same subjects. Where appropriate, multiple comparisons weremade using Tukey-Kramer corrections.

Statistical significance was assessed at the 5% level. All statisticalanalyses were performed using SAS for Windows Release 7 (SASInstitute, Cary, NC).

RESULTS

Before the consumption of the test foods, 82% of thesubjects had baseline values of plasma epicatechin that werenondetectable (1 nmol/L). The subjects with detectablelevels of epicatechin in plasma showed values ranging from 5.5to 28.3 nmol/L.

After the consumption of the test foods, plasma epicatechinlevels were higher than baseline values for the three testgroups that consumed the procyanidin-rich chocolate. At the2-h point, the plasma epicatechin concentrations averaged133, 258 and 355 nmol/L for the subjects who consumed 27,53 and 80 g of the procyanidin-rich chocolate, respectively(Table 1). There were no significant changes in plasma epi-catechin levels in the subjects who consumed bread only. In

the procyanidin-rich chocolate groups, the average decrease inepicatechin concentration between the 2- and 6-h point was

SUPPLEMENT2116S

bygueston

February20,2012

jn.nutrition.org

Do

wnloadedfrom

http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/

8/11/2019 J. Nutr.-2000-Wang-2115S-9S

3/5

81, 77 and 74% for the 27-, 53- and 80-g groups, respectively.It can be noted that the 6-h epicatechin concentrations in the53- and 80-g procyanidin-rich chocolate groups were still

markedly higher than the baseline concentrations (P 0.001), and they were higher than plasma epicatechinconcentrations in the subjects who consumed bread only (P 0.0001).

With respect to plasma antioxidant capacity, there was adecrease in the time of inhibition produced by plasma in thesubjects who consumed the nonchocolate-containing foodfrom 489 88 s (0 h) to 401 70 s (2 h) and 350 69 s (6h). These decreases were ameliorated in the subjects whoconsumed procyanidin-rich chocolate. Although there wereno trends toward increased plasma antioxidant capacity 2 hafter the consumption of chocolate, 6 h after the consumptionof chocolate, there was a trend for an increase in the time ofinhibition with an increase in the amount of procyanidin-richchocolate ingested (Fig. 1). Subjects who consumed 27 g hadan increase in total antioxidant capacity of 78 s at 2 h and adecrease of 38 s at 6 h compared with baseline values. Subjectswho consumed 53 g had a decrease of 57 s at 2 h and anincrease of 138 s at 6 h compared with baseline values. Finally,subjects who consumed 80 g of chocolate had increases of 36and 253 s at 2 and 6 h compared with baseline values, respec-tively.

With respect to lipid oxidation, there was an increase inplasma TBARS that was dependent on the time after whichsubjects consumed bread only (0.63 0.12, 0.77 0.11 and

0.83

0.11mol/L at the 0-, 2- and 6-h time points, respec-tively). There was a trend for the increase in plasma TBARSto be reversed, in a dose-dependent manner, in the subjectswho consumed procyanidin-rich chocolate (Fig. 2). A secondmarker of lipid oxidation, plasma 8-isoprostane, was also mea-sured in the subjects. Plasma 8-isoprostane values for thesubjects who consumed the different amounts of the procya-nidin-rich chocolate were not statistically different at the 2-and 6-h time points (Table 2).

DISCUSSION

In this study, we confirm previous observations that inhealthy adult human subjects, epicatechin is rapidly absorbedafter the consumption of procyanidin-rich chocolate (Rein et

al. 1999 and 2000). Importantly, the data support the theorythat in healthy adults, a linear relationship exists betweenconsumption of procyanidin-rich chocolate and plasma pro-cyanidin concentration. In addition, we have extended thisobservation by showing dose-dependent increases in plasmaepicatechin that are associated with increases in plasma anti-oxidant capacity and reductions in plasma lipid oxidation.

The highest plasma epicatechin concentration observed in

TABLE 1

Concentration of epicatechin in plasma of subjects fed different amounts of procyanidin-rich chocolate

Epicatechin

Procyanidin-rich chocolate foodconsumed,g 0 27 53 80

Time,h nmol/L0 1 1 (9)1 2 2 (13)1 4 2 (13)1 4 3 (10)1

2 19 14 (9)1 133 27 (13)3 258 29 (13)3 355 49 (10)3

6 1 1 (9)1 26 8 (13)2 66 8 (13)2 103 16 (10)2

Area under the curve,mol h L1 0 (9) 0.5 (13) 1.0 (13) 1.5 (10)

Values are expressed as means SEM. The area under the curve for epicatechin versus time was calculated from the means of the different timepoints. Values in parenthesesindicate number of subjects. Means within a column not sharing a common superscript numberare significantly differentat P 0.05.

FIGURE 1 Antioxidant capacity of plasma at baseline and 2 and6 h after the consumption of procyanidin-rich chocolate. Values are

expressed as changes in lag time(s) before chemiluminescence is ob-served. A weak trend (P 0.5302) was observed for increasing anti-oxidant capacity with increasing amounts of procyanidin-rich chocolate

consumed. Variability is represented as SEM. Please refer to Table 1 forthe number of subjects in each group.

FIGURE 2 Plasma TBARS at baseline and 2 and 6 h after the

consumption of procyanidin-rich chocolate. Values are expressed asmalondialdehyde equivalents (mol/L). A weak trend (P 0.3451) wasobserved for decreasing plasma TBARS with increasing amounts of

procyanidin-rich chocolate consumed. Variability is represented as SEM.Please refer to Table 1 for the number of subjects in each group.

CHOCOLATE, EPICATECHIN DOSE-RESPONSE AND ANTIOXIDANTS 2117S

bygueston

February20,2012

jn.nutrition.org

Do

wnloadedfrom

http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/

8/11/2019 J. Nutr.-2000-Wang-2115S-9S

4/5

this study was 562 nmol/L, which occurred 2 h after consump-tion in a subject who consumed 80 g of the procyanidin-richchocolate. The average plasma epicatechin concentration at

the 2-h point in this group was 355 nmol/L. This concentra-tion is similar to that reported by Richelle et al. (1999),although it is higher than the average value of 285 nmol/Lachieved in a previous study by our group (Rein et al. 2000a).In contrast to the present study, in the study by Rein et al.(2000a), subjects did not consume any additional food (bread)until 4 h after the chocolate test meal. Therefore, we suggestthat the difference in results between our two studies is due tosubtle changes in the experimental methods that were used.This can be shown by examining the change in plasma epi-catechin concentration between the 2- and 6-h time pointsafter chocolate consumption. In the present study, this differ-ence was 263 nmol/L, whereas in the study of Rein et al.(2000a), this difference was 104 nmol/L. The estimated area

under the curve of epicatechin concentration versus time issimilar between the two studies (1480 versus 1540 nmol h1

L1), suggesting that although maximum absorption time wasdelayed with chocolate consumption alone, the total epicat-echin absorbed after the consumption of 80 g of procyanidin-rich chocolate was unchanged between the two studies.

The influence of the bread on the bioavailability of epicat-echin from chocolate must be determined in future studies.When green tea and black tea were consumed with semi-skimmed milk, the addition of milk did not significantly affectthe blood catechin levels for black tea with milk (van het Hofet al. 1998). In the current study, we can estimate the amountof epicatechin present in the plasma, extrapolated to a 6-hpostconsumption time, to 1.6, 1.5 and 1.4% of the total

epicatechin consumed in 80, 53 and 27 g of procyanidin-richchocolate. These percentages can be compared with a plasmacatechin absorption of 330mol h1 L1 and a recovery of0.24% after subjects consumed 126 mol of ()-catechin inreconstituted red wine (Bell et al. 2000). Collectively, the datafrom the above studies suggest that catechins from chocolateare more bioavailable than catechins from wine.

Similar to plasma epicatechin kinetics, there were alsodifferences in chocolate-induced changes in plasma antioxi-dant capacity between our previous work (Rein et al. 2000a)and the current study. In the former study, plasma antioxidantcapacity peaked at 2 h after chocolate consumption, whereasin the present study, plasma antioxidant capacity showed afurther increase at the 6-h time point from that of the 2-h time

point. This response is consistent with the different pattern ofepicatechin absorption that was observed in the two studies.

It has been suggested that the measurement of plasmaconcentrations of F2-isoprostanes can provide an additionalbiomarker for oxidative stress in vivo (Pratico, 1999, Robertsand Morrow, 2000). In addition, because of their function asrenal and pulmonary vasoconstrictors, the assessment of F2-isoprostanes may provide insight into the effects of specificfood products on vascular health (Morrow and Roberts 1997,Morrow et al. 1999, Practico, 1999). In the current study, we

could not document a dose-related change between chocolateconsumption and plasma F2-isoprostane concentrations.

Given the results of the current study, as well as the findingsof others, we suggest that the daily consumption of procyani-din-rich foods, such as chocolate, can result in improvementsin the antioxidant potential of plasma. A potential mechanismfor such improvement would be a sparing capacity that in-volved other antioxidant molecules such as ascorbate, -to-copherols and carotenes (Lotito and Fraga 1998 and 1999 and2000, Salah et al. 1995).

The physiological relevance of such an increase in plasmaepicatechin after the consumption of procyanidin-rich choc-olate can be shown by the results presented in this study andin Rein et al. (2000a) on modifying select oxidative stress

variables. In addition, it can be speculated that changes inoxidative stress may contribute to the suppressive effects ofprocyanidins on platelet activation (Rein et al. 2000b). Insummary, the results obtained to date demonstrate an in vivophysiological effect of chocolate consumption on several bio-logical variables associated with vascular pathologies, includ-ing cardiovascular disease.

ACKNOWLEDGMENT

We would like to thank Janet Peerson for her assistance in thestatistical analysis of these data.

LITERATURE CITED

Arteel, G. E. & Sies, H. (1999) Protection against peroxynitrite by cocoapolyphenol oligomers. FEBS Lett. 462: 167170.

Adamson, G. E., Lazarus, S. A., Mitchell, A. E., Prior, R. L., Cao, G., Jacobs, P. H.,Kremers, B. G., Hammerstone, J. F., Rucker, R. B., Ritter, K. A. & Schmitz,H. H. (1999) HPLC method for the quantification of procyanidins in cocoaand chocolate samples and correlation to total antioxidant capacity. J Agric.Food Chem. 47: 41844188.

Bearden, M. M., Pearson, D. A., Rein, D., Chevaux, K. A., Carpenter, D. R., Keen,C. L. & Schmitz, H. H. (2000) Potential cardiovascular health benefits ofprocyanidins present in chocolate and cocoa. ACS Symposium Series,Chemistry and Health Benefits of Caffeinated Beverages.

Bell, J. R., Donovan, J. L., Wong, R., Waterhouse, A. L., German, J. B., Walzem,R. L., & Kasim-Karakas, S. E. (2000) ()-Catechin in human plasma afteringestion of a single serving of reconstituted red wine. Am J Clin Nutr. 71:103108.

Bravo, L. (1998) Polyphenols: Chemistry, dietary sources, metabolism, andnutritional significance. Nutr. Rev. 56: 317333.

de Groot, H. & Rauen, U. (1998) Tissue injury by reactive oxygen species andthe protective effects of flavonoids. Fundam. Clin. Pharmacol. 12: 249255.

Hammerstone, J. F., Lazarus, S. A., Mitchell, A. E., Rucker, R. & Schmitz, H. H.(1999) Identification of procyanidins in cocoa (Theobroma cacao) and choc-olate using high performance liquid chromatography mass spectrometry. J.Agric. Food Chem. 47: 490 496.

Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B. & Kromhout, D.(1993) Dietary antioxidant flavonoids and risk of coronary heart disease: TheZutphen Elderly Study. Lancet. 342: 10071011.

Hertog, M. G., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F.,Giampaoli, S., Jansen, A., Menotti, A., Nedelijovic, S., Pekkarinen, M., Simic,B. S., Toshima, H., Feskens, E.J.M., Hollman, P.C.H. & Katan, M. B. (1995)Flavonoid intake and long-term risk of coronary heart disease and cancer inthe Seven Countries Study. Arch. Intern. Med. 155: 381386.

Kinsella, J. E., Frankel, E., German, J. B. & Kanner, J. (1993) Possible mech-anisms for the protective role of antioxidants in wine and plant foods. FoodTechnol. 47: 8589.

Kondo, K., Hirano, R., Matsumoto, A., Igarashi, O. & Itakura, H. (1996) Inhibi-tion of LDL oxidation by cocoa. Lancet. 348: 1514.

Lissi, E., Salim-Hanna, M., Pascual, C. & del Castillo, M. D. (1995) Evaluationof total antioxidant potential (TRAP) and total antioxidant reactivity from

TABLE 2

8-Isoprostane in plasma of subjects fed different amounts

of procyanidin-rich chocolate

8-Isoprostane

Procyanidin-richchocolate foodconsumed,g 0 27 53 80

Time,h mol/L0 48 5 (7) 39 8 (8) 52 7 (10) 50 7 (7)2 44 9 (7) 51 8 (8) 52 7 (10) 57 9 (7)6 58 7 (7) 42 5 (8) 49 4 (10) 42 9 (7)

Values are expressed as means SEM. Values in parenthesesindicate number of subjects. There were no significant differencesacross treatments or time.

SUPPLEMENT2118S

bygueston

February20,2012

jn.nutrition.org

Do

wnloadedfrom

http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/

8/11/2019 J. Nutr.-2000-Wang-2115S-9S

5/5

luminol-enhanced chemiluminescence measurements. Free Radic. Biol. Med.18: 153158.

Lotito, S. B. & Fraga, C. G. (1998) ( )-Catechin prevents human plasmaoxidation. Free Radic. Biol. Med. 24: 435441.

Lotito, S. B. & Fraga, C. G. (1999) Catechins as antioxidant: Mechanismspreventing human plasma oxidation and activity in red wines. Biofactors 10:125130.

Lotito, S. B. & Fraga, C. G. (2000) Catechins delay lipid oxidation and alpha-tocopherol and beta-carotene depletion following ascorbate depletion in hu-man plasma. Proc. Soc. Exp. Biol.

Morrow, J. D., Chen, Y., Brame, C. J., Yang, J., Zackert, W. E., Awad, J. A. &Roberts, L. J. (1999) The isoprostanes: Unique prostaglandin-like prod-ucts of free-radical-initiated lipid peroxidation. Drug Metab. Rev. 31: 117139.

Morrow, J. D. & Roberts, L. J. (1997) The isoprostanes: Unique bioactiveproducts of lipid peroxidation. Prog. Lipid Res. 36: 121.

Oteiza, P. I., Uchitel, O. D., Carrasquedo, F., Dubrovski, A. L., Roma, J. C. &Fraga, C. G. (1997) Evaluation of antioxidants, protein, and lipid oxidationproducts in blood from sporadic amyotrophic lateral sclerosis patients. Neu-rochem. Res. 22: 535539.

Pratico, D. (1999) F2-isoprostanes: Sensitive and specific non-invasive indicesof lipid peroxidation in vivo. Atherosclerosis. 147: 110.

Renaud, S. & de Lorgeril, M. (1992) Wine, alcohol, platelets, and the Frenchparadox for coronary heart disease. Lancet. 339: 15231526.

Rein, D., Lotito, S., Holt, R. R., Keen, C. L., Schmitz, H. H. & Fraga, C. G. (2000a)Epicatechin in human plasma: In vivo determination and effect of chocolateconsumption on plasma antioxidant capacity. J. Nutr. 130: 2109S2114S.

Rein, D., Paglieroni, T. G., Pearson, D. A., Wun, T., Schmitz, H. H., Gosselin, R. &Keen, C. L. (2000b) Cocoa and wine polyphenols modulate platelet acti-vation and function. Am. J. Clin. Nutr.

Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M. & Pridham, J. B.(1995) The relative antioxidant activities of plant-derived polyphenolic fla-vonoids. Free Radic. Res. 22: 375383.

Richelle, M., Tavazzi, I., Enslen, M. & Offord, E. A. (1999) Plasma kinetics inman of epicatechin from black chocolate. Eur. J. Clin. Nutr. 53: 2226.

Roberts, L. J., & Morrow, J. D. (2000) Measurement of F2-isoprostanes as anindex of oxidative stress in vivo. Free Radic. Biol. Med. 28: 505513.

Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, G. P. & Rice-Evans, C.(1995) Polyphenolic flavanols as scavengers of aqueous phase radicals andas chain-breaking antioxidants. Arch. Biochem. Biophys. 322: 339346.

Sirving, O. K., Hertog, M. G. L., Feskens, E. J. M. & Kromhout, D. (1996)

Dietary flavonoids, antioxidant vitamins, and incidence of stroke. Arch. Intern.Med. 154: 637642.

Steinmetz, K. A. & Potter, J. D. (1991) Vegetables, fruit, and cancer. I. Epide-miology. Cancer Causes Control. 5: 325357.

Van het hof, K. H., Kivits, G. A., Westrate, J. A. & Tijburg, L. B. (1998) Bio-availability of catechins from tea: The effect of milk. Eur. J. Clin. Nutr. 52(5):356359.

Waterhouse, A. L., Shirley, J. R. & Donovan, J. L. (1996) Antioxidants in

chocolate. Lancet. 348: 834.Wiseman, H. (1999) The bioavailability of non-nutrient plant factors: Dietary

flavonoids and phyto-oestrogens. Proc. Nutr. Soc. 58: 139146.

CHOCOLATE, EPICATECHIN DOSE-RESPONSE AND ANTIOXIDANTS 2119S

bygueston

February20,2012

jn.nutrition.org

Do

wnloadedfrom

http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/http://jn.nutrition.org/