brief communications

This opal-type structure is an example of 6. Sanders, J. V. & Darragh, P .J. Min. Rec. 2, 261–268 (1971). regarded as beneficial. Hyperuricaemia has a three-dimensional ‘photonic crystal’.Using 7. Philipse, A. P. J. Mat. Sci. Lett. 8, 1371–1373 (1989). been associated with stroke, cardiovascular ȕ ǃ ǂ Ꭷ 2ǁ 2Ȓ 8. Land, M. F. Progr. Biophys. Mol. Biol. 24, 75–106 (1972). 5–8 the formula max 2d 0.816 (n sin ) 9. Parker, A. R., McPhedran, R. C., McKenzie, D. R., Botten, L. C. and renal morbidity,and gout . as an approximation (for details, see & Nicorovici, N.-A. P. Nature 409, 36–37 (2001). Evidence is mounting regarding the www.icmm.csic.es/cefe/Infiltration/R6G/R 10.Nedelec, F. J., Surrey, T., Maggs, A. C. & Leibler, S. Nature 389, potential importance of the overall redox 6G_infill.htm),the wavelength of maximum 305–308 (1997). network in disease prevention.It is crucial to ȕ Ȓ 11.Blackwell, J. & Weih, M. A. in Chitin, Chitosan and Related reflectance, max, for an angle of incidence , Enzymes (ed. Zikakis, J. P.) 257–272 (Academic, New York, our understanding to elucidate the mecha- was calculated as 573 nm at Ȓǃ20ᑻ.The con- 1984). nisms by which TPAC is modulated, and the stant 0.816ǃ2/31/2 accounts for the spacing Competing financial interests: declared none. effect of food on this important parameter. between the close-packed planes in units of Barry Halliwell sphere diameter; n represents the average Department of Biochemistry, National University of refractive index in the system where point COMMUNICATIONS ARISING Singapore, 8 Medical Drive, MD7 #03-08, scatterers are arranged in planes, calculated Plasma antioxidants 117597 Singapore + as (ns nm)/2,where ns is the refractive index e-mail: [email protected] of the microspheres and is taken as 1.56, and Health benefits of eating 1. Halliwell, B. Nutr. Rev. 57, 104–111 (1999). 2. Serafini, M. et al. Nature 424, 1013 (2003). nm is the refractive index of the matrix (1.33) chocolate? 3. Rechner, A. R. et al. Free Radic. Res. 36, 1229–1241 (2002). — that is, the refractive index of the chitin- 4. Benzie, I. F. F. & Strain, J. J. Anal. Biochem. 239, 70–76 (1996). ous material and ‘water’ that make up the n assessing whether or not a compound or 5. Halliwell, B. & Gutteridge, J. M. C. Arch. Biochem. Biophys. 280, beetle’s exoskeleton7,8,respectively. food acts as an antioxidant in vivo,a con- 1–8 (1990). This is a good match with the measured ventional approach is to monitor biological 6. Weir, C. J. et al. Stroke 34, 1951–1956 (2003). I 7. Rott, K. T. et al. J. Am. Med. Assoc. 289, 2857–2860 (2003). ȕ max and indicates that the cause of the opti- markers of oxidative damage in response to 8. Johnson, R. J. et al. Hypertension 41, 1189–1190 (2003). cal effect is indeed similar to that of opal7. the intervention1. Another is to measure the Variations in Ȓ do,however,cause differences change in total plasma antioxidant capacity, ȕ Ȓ ᑻ 2 in max; the range of values of 0–70 equates as investigated by Serafini et al. in relation to ȕ to a range of max from 590 nm to 448 nm. the consumption of chocolate in the presence Nutrition The invariant colour (yellow–green) seen and absence of milk. The implications of the from the whole animal is a result of global authors’ finding that eating chocolate causes Milk and absorption of averaging of the different domains within an increase in total plasma antioxidant capa- dietary flavanols each scale and juxtaposed scales; the domain city, and the mechanism by which this is structure thus creates omnidirectional achieved, must also be considered — however, lavanol compounds in wine, cocoa colour,removing the iridescent effect. it should not be assumed that the effect is products and tea can exert a cardiopro- The first photonic crystal revealed as such necessarily beneficial. Ftective effect, for example by influenc- in an animal (in this case a polychaete)9 has I calculate that the maximum plasma epi- ing endothelial-cell function1, antithrombic a two-dimensional structure. By contrast, catechin concentration in the study of Serafini mechanisms2 and blood pressure3,4. Serafini the three-dimensional properties described et al. is about 1 ȖM. Epicatechin metabolites et al.5 claim that consuming dark chocolate, here allow for a relatively omnidirectional are likely to be present at lower concentrations but not milk chocolate or dark chocolate optical effect, which is important to the and to have reduced antioxidant activity together with milk, increases the anti- behaviour of this weevil because it appears compared with that of epicatechin itself, oxidant capacity of human plasma, and sug- to be strongly coloured from every direction because of blocking of radical-scavenging gest that interaction between milk proteins in situ. This could be useful for interspecific hydroxyl groups by conjugation3.However, and chocolate flavonoids inhibits the in vivo colour or pattern recognition. the total plasma antioxidant capacity (TPAC) antioxidant activity of chocolate and the Opal is notoriously difficult to manu- measured by Serafini et al.2 rose by up to 18%. absorption of epicatechin into the blood- facture in solid form. However, transmission When measured by the ferric-reducing stream. This inference could have implica- electron micrographs of the weevil’s opal-like antioxidant-potential (FRAP) used by the tions beyond chocolate consumption if structure reveal repeating patterns of light and authors, TPAC is usually 0.6–1.6 mM (ref. 4), dairy products do indeed counteract the dark areas within each microsphere, provid- of which 18% would be 108–288 ȖM.The rise putative health benefits of dietary flavanols. ing a clue to their molecular structure (which in TPAC is therefore so large that it is unlikely The results of Serafini et al.5 are open to may be revealed by X-ray diffraction analysis) to be due in significant part to the antioxidant a different interpretation if the biological and production. These microspheres must action of epicatechin and its metabolites,or of availability and subsequent activity of any be constructed by molecular self-assembly, a other phenolics in chocolate. compound depends on the varying nutri- process that could potentially be reproduced10 The FRAP activity of human plasma is tional and biophysical properties of the by the synthetic-opal industry. After all, the mainly attributable to ascorbate, Ȋ-toco- matrix in which it is ingested (that is, caloric self-assembly technique of the abalone has pherol, bilirubin and urate4. Given normal background, lipid/water content, viscosity, been successfully copied in the manufacture plasma levels of these substances5,an increase density, extent of mastication). To compare of a nanocomposite coating that is analogous in urate concentration is most likely to the absorption of epicatechin for chocolate to the nacre of its shell11. account for the results of Serafini et al., ingested in the presence and absence of Andrew R. Parker, Victoria L. Welch, because urate is present in plasma at much milk,it is necessary to control for the compo- Dominique Driver, Natalia Martini higher levels than those of other antioxidants, sition of the matrix in which the flavanols Department of Zoology, University of Oxford, and chocolate is unlikely to contain much are delivered. Oxford OX1 3PS, UK ascorbate. Although Ȋ-tocopherol may be Compared with values obtained after e-mail: [email protected] present in chocolate, even a high intake of consumption of dark chocolate alone (100 g, 1. Schultz, T. D. Bull. Ent. Soc. Am. 32, 142–146 (1986). this vitamin can increase its concentration in of which about 30 g is lipid), Serafini et al. 2. Parker, A. R., McKenzie, D. R. & Large, M. C. J. J. Exp. Biol. 201, plasma by only a few micromolar at most5. find a lower plasma antioxidant capacity, as 1307–1313 (1998). The mechanism and consequences of well as a reduction in the area under the 3. Parker, A. R. J. Opt. A 2, 15–28 (2000). 4. Sanders, J. V. Nature 204, 1151–1153 (1964). increased plasma urate levels following con- curve of a plot of plasma antioxidant activity 5. Ajgaonkar, M., Zhang, Y., Grebel, H. & Brown, R. A. J. Opt. Soc. sumption of chocolate would be interesting for epicatechin against time, in the case of Am. B 19, 1391–1395 (2002). to investigate, but should not necessarily be milk chocolate (200 g, of which about 60 g is

NATURE | VOL 426 | 18/25 DECEMBER 2003 | www.nature.com/nature © 2003 Nature Publishing Group 787 brief communications lipid) and for dark chocolate consumed with not influence the cocoa-mediated increase in after 1 h and in plasma (–)epicatechin for 4 h milk (100 g plus 200 ml milk, corresponding plasma antioxidant capacity (mean AUCmilk, after ingestion (as measured by the area under DŽ DŽ to more than 30 g lipid). These differences 1,028.7 40.5; mean AUCwater,1,005.6 35.7 the curve in Fig.1a of ref.1).In a pilot study,we may therefore reflect matrix-dependent nmol trolox equivalents mlǁ1 hǁ1; t-test, also monitored plasma TAC for 6 h (results not effects on flavanol absorption. PDŽ0.369). Furthermore, the consumption shown) and found no increase following the A mechanistic link between the antioxi- of a milk-containing cocoa beverage resulted consumption of either milk chocolate or dark dant properties of flavonoids in vitro and their in a 25–30% reduction in platelet-mediated chocolate with milk. biological activity in vivo is not properly estab- haemostasis (R.R.H. and C.L.K., unpub- The study described by Schroeter et al. lished,partly because factors such as biological lished results), a functional marker that is differs in key respects from ours. The authors transformation and tissue/plasma concentra- related to cardiovascular health. used a chocolate beverage, rather than solid tions are often not considered6.Measurements Our findings show that the presence of chocolate. The amount of milk consumed of plasma antioxidant capacity ideally need to milk in cocoa products does not counteract with the beverage is not stated but,on the basis be complemented with markers of cardiovas- the absorption and biological activity of of the lipid content (3% as opposed to 30%),it cular function to assess the biological effect of monomeric flavanols from cocoa products, is substantially less than in our study, which flavanols on cardiovascular health in vivo. nor does it affect plasma antioxidant capa- could explain why they did not observe an We tested the possible inhibition by milk city.In the context of potential health benefits inhibition of plasma TAC by milk. Another of the absorption of chocolate flavanols in mediated by dietary flavanols, food-matrix- factor could be that Schroeter et al. measured 12 healthy, non-smoking volunteers whose dependent, temporal effects on absorption plasma TAC by using the TRAP-luminol blood-lipid profiles were normal and who must be taken into account when comparing assay, which is unlike the FRAP assay that we were not taking any dietary supplement (the different food formulations and processing used in that it measures radical-scavenging study was approved by the Internal Review methods in terms of the biological activity of activity rather than reducing power, and as Board of the University of California, Davis, their constituent compounds. such determines a different feature of TAC2. and informed consent was obtained from all Hagen Schroeter*, Roberta R. Holt*, We were careful to distinguish between subjects). Participants consumed a cocoa Timothy J. Orozco*, Harold H. Schmitz†, TAC and (–)epicatechin absorption because beverage (containing 0.66 g cocoa solids, the Carl L. Keen*‡ the aim of our investigation was to evaluate equivalent of 5.44 mg monomeric flavanols Departments of *Nutrition and ‡Internal Medicine, the effect of food associations on plasma TAC, and 15.65 mg proanthocyanidins, per kg University of California, Davis, and not to identify the compounds responsi- body weight) prepared with either whole California 95616, USA ble for the effect.We agree that the increase in milk (3.25% lipid) or water (supplemented e-mail: [email protected] plasma TAC cannot be explained solely on the with carbohydrate and 3.25% lipid as a †Analytical and Applied Sciences, Masterfoods USA, basis of increased (–)epicatechin levels.Other control). Both beverages delivered an equal Hackettstown, New Jersey 07840, USA chocolate polyphenols, such as procyanidins amount of fluid (4.8 g per kg body weight). 1. Heiss, C. et al. J. Am. Med. Assoc. 290, 1030–1031 (2003). or their in vivo metabolites, could also be The area under the curve (AUC) in plots 2. Holt, R. R., Schramm, D. D., Keen, C. L., Lazarus, S. A. & involved in increasing FRAP values.However, Schmitz, H. H. J. Am. Med. Assoc. 287, 2212–2213 (2002). of epicatechin plasma concentration against 3. Corder, R. et al. Nature 414, 863–864 (2001). whatever the components responsible, they 7 time (where plasma epicatechin was extracted 4. Taubert, D., Berkels, R., Roesen, R. & Klaus, W. J. Am. Med. too are likely to be affected by milk, causing a and analysed by high-performance liquid Assoc. 290, 1029–1030 (2003). reduction in vivoin the antioxidant properties chromatography8) shows that there is no 5. Serafini, M. et al. Nature 424, 1013 (2003). of dark chocolate. 6. Spencer, J. P., Schroeter, H., Rechner, A. R. & Rice-Evans, C. significant difference (analysis of variance Antioxid. Redox Signal 3, 1023–1039 (2001). We measured urate concentrations in (ANOVA), Pǃ0.499) in epicatechin concen- 7. Abd El Mohsen, M. M. et al. Free Rad. Biol. Med. 33, 1693–1702 plasma under similar conditions (results not tration in plasma after the consumption of a (2002). shown) and the results are commensurate milk-containing (mean AUC, 3,074.8DŽ536.7 8. Holt, R. R. et al. Am. J. Clin. Nutr. 76, 798–804 (2002). with an involvement of urate in the redox ǁ1 ǁ1 9. Lissi, E., Salim-Hanna, M., Pascual, C. & del Castillo, M. D. Free ng ml h ) or water-based (mean AUC, Rad. Biol. Med. 18, 153–158 (1995). network. However, as noted by Halliwell, the ǁ1 ǁ1 2,580.5DŽ655.4 ng ml h ) cocoa beverage Competing financial interests: declared; see online version for situation is not straightforward and we did under isocaloric and isolipidaemic conditions. details. not therefore speculate on the contribution Bearing in mind that measurements of of urate in this context. plasma antioxidant capacity must be inter- TAC measurement is increasingly being preted with caution in evaluating flavonoid- Serafini et al. reply — Our results indicate used to monitor redox status in vivo as well as mediated biological effects, we used the total that there is an increase in total antioxidant for epidemiological purposes3. Understand- antioxidant potential (TRAP) assay9 as an capacity (TAC) and (–)epicatechin content ing the factors that regulate plasma TAC will independent test of the results of Serafini of plasma in people who have consumed help to clarify the interplay between plant- et al.5, who measured the ferric-reducing dark chocolate, and that these effects are derived foods, plasma redox status and antioxidant potential (FRAP).We found that reduced by the presence of milk1. Halliwell oxidative disease; the effect of food asso- consumption of a cocoa-containing beverage and Schroeter et al. raise issues that are cen- ciations on TAC in vivo is just one, albeit resulted in a statistically significant, milk- tral to the debate over the fate and potential complicated,variable. independent increase in plasma antioxidant protective effects of dietary antioxidants. Mauro Serafini*, Alan Crozier† capacity,as shown by comparing the baseline Schroeter et al. suggest that a matrix effect *Antioxidant Research Laboratory, Unit of Human value (mean, 234.2DŽ12.47 nmol trolox (vit- in our study may have delayed the absorption Nutrition, National Institute for Food and amin E equivalent) per ml) with the maxi- of antioxidants when chocolate was consumed Nutrition Research, 00178 Rome, Italy mum values for the milk-containing (mean with milk, and that this went unnoticed e-mail: [email protected] ǁ maximum, 291.4DŽ8.6 nmol trolox ml 1; because our experiments were terminated †Plant Products and Human Nutrition Group, ANOVA, P<0.001) and water-containing after 4 h. However, this is unlikely for several Division of Biochemistry and Molecular Biology, (mean maximum, 283.72DŽ21.6 nmol trolox reasons. All of the participants in our trials Institute of Biomedical and Life Sciences, University ǁ ml 1;ANOVA,P<0.001) drinks. fasted overnight for 12 h and,after supplemen- of Glasgow, Glasgow G12 8QQ, UK Results based on AUC values for plasma tation with chocolate/milk, no other product 1. Serafini, M. et al. Nature 424, 1013 (2003). antioxidant capacity plotted against time, was consumed for the duration of the experi- 2. Del Rio, D., Serafini, M. & Pellegrini, N. Nutr. Met. Card. Dis. 12, 343–351 (2002). which take into account temporal differences ment. Individuals who ate dark chocolate 3. Serafini, M., Bellocco, R., Wolk, A. & Ekström, A. M. in absorption, also indicate that milk does showed a significant increase in plasma TAC Gastroenterology 123, 985–991 (2002).

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