You are on page 1of 6

Coffee drinking induces incorporation of phenolic acids into LDL and increases the resistance of LDL to ex vivo oxidation

in humans13
Fausta Natella, Mirella Nardini, Federica Belelli, and Cristina Scaccini
ABSTRACT Background: Epidemiologic and intervention studies indicate that both diet as a whole and single dietary components are involved in the risk of atherosclerosis. The resistance of LDL to oxidative modification is an ex vivo indicator of risk, which is modulated by dietary components. Coffee contains phenolic compounds with antioxidant activity. These molecules are found in plasma after the consumption of coffee, and it has been shown that, in vitro, they are able to decrease the susceptibility of LDL to oxidation. Objective: The aim of this study was to evaluate the effect of coffee consumption on the redox status of LDL as modulated by the possible incorporation of phenolic acids into LDL. Design: Ten healthy volunteers, after an overnight fast, drank 200 mL filtered coffee. Blood was drawn before and 30 and 60 min after drinking. Changes in LDL redox status were evaluated by the measure of LDL resistance to oxidative modification and the concentration of LDL(), a mildly modified, electronegative LDL subfraction. Chlorogenic and phenolic acids concentration in LDL were measured by electrochemical HPLC. Results: The resistance of LDL to oxidative modification increased significantly after coffee drinking, but the LDL() concentration did not increase. The concentration into LDL of conjugated forms of caffeic, p-coumaric, and ferulic acids increased significantly after coffee drinking. Conclusion: Drinking 200 mL (1 cup) coffee induces an increase in the resistance of LDL to oxidative modification, probably as a result of the incorporation of coffees phenolic acids into LDL. Am J Clin Nutr 2007;86:604 9. KEY WORDS mans INTRODUCTION Coffee, phenolic acids, LDL oxidation, hu-

In spite of the controversy about the role of cholesterol in the mechanisms underlying the pathogenesis of atherosclerosis, there is still consistent evidence that oxidatively modified LDL plays a key role (15). A large number of studies gave rise to the oxidative hypothesis of atherosclerosis, in which the oxidation of LDL represents the critical point. Oxidized LDL does have a wide range of atherogenic properties, from the formation of early lesions to plaque rupture (6), but the presence in vivo of circulating fully oxidized LDL is improbable. In fact, a complex defense system can rapidly remove oxidized LDL from the circulation.

Diet per se and single dietary components are involved in atherosclerotic risk, as evidenced by epidemiologic and intervention studies. Dietary patterns characterized by high fruit and vegetable intakes appear to be protective against the risk of cardiovascular disease (CVD) (7), and the susceptibility of LDL to oxidative modification can be influenced by dietary components. LDL(), a mildly modified, electronegative LDL subfraction found in vivo in human plasma, accounts in vitro for several biological proatherogenic events (8). LDL() is enriched with oxidized lipids and destabilized in the supramolecular structure of apolipoprotein B (9, 10). Its concentration can be modulated by extrinsic factors, such as a meal (11, 12). Coffee is among the most widely consumed beverages in the world. The relation between coffee consumption and CVD has been studied extensively. Several studies conducted in the past few years found a J-shaped association between coffee consumption and CVD risk (1316). Coffee contains several phenolic components (200 550 mg/cup; a cup equals 200 mL) that are endowed with antioxidant capacity. Chlorogenic acids are a family of esters formed between quinic acid and cinnamic acids such as caffeic, ferulic, and p-coumaric acids; of the chlorogenic acids, 5-caffeoylquinic acid is by far the most abundant isomer in coffee (Figure 1). With the use of 10 g powdered coffee per cup of brewed coffee, the concentration of 5-caffeoylquinic acid can range from 15 to 325 mg/200-mL cup. A value of 200 mg/cup has been reported for coffee brewed by drip-filtering (17). Chlorogenic acids are scarcely present in biological fluids, whereas their metabolites caffeic and ferulic acids reach micromolar concentrations (18 23). Caffeic acid possesses a high antioxidant capacity, it is absorbed and rapidly metabolized in rats and in humans (23, 24), and it is capable of inhibiting in vitro the oxidative modification of human LDL (25). The consumption of coffee increases the total plasma antioxidant capacity in humans (26), but the in vivo effect on LDL is not yet established. Filtered coffee did not seem to have any detectable short- or
1 From the National Research Institute for Food and Nutrition (INRAN), Rome, Italy. 2 Supported by a grant from the Institute for Scientific Information on Coffee and the Physiological Effects of Coffee Committee (both: La Tour-de-Peilz, Switzerland). 3 Reprints not available. Address correspondence to C Scaccini, INRAN, Via Ardeatina 546, 00178 Roma, Italy. E-mail: scaccini@inran.it. Received February 28, 2007. Accepted for publication April 20, 2007.

Downloaded from www.ajcn.org by on January 18, 2009

604

Am J Clin Nutr 2007;86:604 9. Printed in USA. 2007 American Society for Nutrition

COFFEE INCREASES LDL RESISTANCE TO OXIDATION

605

FIGURE 1. Chemical structure of the principal phenolic compound present in coffee chlorogenic acid (5-caffeoylquinic acid). The oval highlights the caffeic acid structure.

long-term effect on lipid peroxidation (27) or on the susceptibility of LDL to oxidation (28) in healthy humans. A contrary result is reported in a supplementation study in which a 7-d standardized coffee consumption decreased both LDL susceptibility to oxidation and serum lipid concentrations in humans (29). The aims of this work were to evaluate tudy the incorporation of phenolic acids from coffee into human LDL and to evaluate the possible effect of coffee consumption on the redox status of LDL, as indicated by the modulation of LDL resistance to oxidative modification and by the LDL() concentrations.
SUBJECTS AND METHODS

spectrophotometer (model DU 70; Beckman Instruments) according to the method of Esterbauer et al (32). Susceptibility to oxidation was expressed as lag time and was calculated from the intercept of lines drawn through the linear portion of the lag and propagation phases for each samples. For the in vitro experiments, LDL from healthy subjects in a fasting condition was oxidized (as described above) in the presence of increasing concentrations of caffeic acid (0, 1, 10, and 100 nmol/L). For the measurement of LDL(), freshly isolated LDL was dialyzed in the dark for 18 h at 4 C in 5 mmol TrisHCl/L (pH 7.2) containing 10 mol EDTA/L. LDL() was separated from unmodified LDL by anion-exchange HPLC according to the method of Hodis et al (33). Phenolic acids in biological samples are routinely detected in our laboratory by HPLC with electrochemical detection (HPLCECD) (23, 34). The presence of phenolic acids into LDL was measured in untreated samples (free phenolic acids) and in samples subjected to alkaline hydrolysis (total phenolic acidsie, free plus bound forms). No treatment We added 200 ng m-coumaric acid to dialyzed LDL samples (0.5 mL each), acidified them to pH 3 with 1 N HCl, added 300 mg NaCl, and then conducted 3 extractions with ethyl acetate (4 volumes) by mixing in a vortex for 5 min. After each extraction, samples were centrifuged (3000 g, 10 min, room temperature) and supernatants were collected. The organic phase was dried under nitrogen flow. The residue was dissolved in 0.5 mL of water and mixed in a vortex for 5 min; then the pH was brought to pH 7 8 with 0.1 N NaOH, and the sample was passed through a 1-mL tube (Supelclean LC-SAX; Supelco, Bellefonte, PA) conditioned with 1 mL absolute methanol and 2 mL water. The tube was then washed with 1 mL water. Phenolic acid elution was obtained with 1 mL buffer containing 1 N acetic acid and MeOH at a 90:10 ratio. The eluant was immediately brought to pH 3 with 6 L of 4N NaOH and filtered, and an aliquot (100 L) was injected into the HPLC-ECD system. Alkaline hydrolysis treatment To dialyzed LDL samples (0.5 mL each) containing 200 ng m-coumaric acid were added, in the following order, 32.5 L H2O, 62.5 L 20% ascorbic acid, 25 L 0.5 mol EDTA/L, and 180 L of 8 N NaOH; then the samples were incubated at 30 C for 30 min. At the end of incubation, the pH was brought at 3.0 with 8 N HCl. After the addition of 600 mg NaCl, the samples were extracted 3 times with ethyl acetate (4 volumes) as reported above. The residue was dissolved in 0.5 mL of water, mixed in a vortex for 5 min, and then processed for solid-phase extraction as reported above. Treated and untreated samples were analyzed by HPLCECD as previously described (22). The concentration of phenolic acids is expressed as ng/mg protein. Free forms of phenolic acid in LDL were almost undetectable in our experimental conditions, so that all results showed represent the total concentration (free bound) of each single phenolic acid. The concentration of phenolic acid in coffee was measured as described previously (23). Statistical analysis Data are presented as means SDs or SEMs. Statistical analysis was carried out by using repeated-measures analysis of
Downloaded from www.ajcn.org by on January 18, 2009

Subjects Ten healthy volunteers (5 M, 5 F) aged 24 35 y who were moderate coffee drinkers (2 4 cups/d) were recruited. Subjects acted as their own controls, and they were instructed to avoid coffee and food and other beverages that are rich in phenolic acids for 2 d before the experiment. After an overnight (10 12-h) fast, a venous blood sample was taken at time 0. Immediately after the first blood collection, subjects drank 200 mL (1 cup) of freshly prepared American-style coffee. Filtered coffee was prepared by using a commercial automatic brewing machine and 60 g roasted and ground coffee/L water. Further blood collections were made 30 and 60 min after coffee consumption. All subjects gave written informed consent. The Ethics Committee of the National Institute for Food and Nutrition Research approved all procedures. Methods Venous blood samples were collected into evacuated tubes containing EDTA. LDL (d 1.019 1.063 g/mL) was isolated from plasma immediately after blood centrifugation by the use of sequential ultracentrifugation in salt solutions, according to the method of Havel et al (30), by using a bench-top ultracentrifuge (model T-100; Beckman Instruments, Irvine, CA) with a T-100.3 rotor (Beckman Instruments). Protein was measured by the method of Lowry et al (31) with the use of bovine serum albumin as a standard. For oxidation experiments, freshly isolated LDL was dialyzed in the dark for 18 h at 4 C against 2 L of 0.01 mol phosphate-buffered saline/L and 0.15 mol NaCl/L at a pH of 7.4. Dialyzed LDL (50 g protein/mL) was incubated in phosphate-buffered saline at 37 C for 3 h in the presence of 2.5 mol CuCl2/L. The kinetics of conjugated diene formation was followed by continuous monitoring of the change in the 234-nm absorbance, conducted with the use of a

606

NATELLA ET AL

TABLE 1 Phenolic acids in 1 cup (200 mL) of brewed coffee before and after hydrolysis1 5-Caffeoylquinic acid 88.4 1.7 ND Caffeic acid mg/cup Nonhydrolyzed coffee Hydrolyzed coffee
1

p-Coumaric acid

Ferulic acid

ND 214.0 13.4

ND 4.7 0.4

ND 34.9 1.9

All values are x SEM of 4 independent experiments. ND, not determined.

variance, which was followed by Tukeys test for multiple comparisons. Analyses were performed by using KALEIDAGRAPH software (version 3.6; Synergy Software, Reading, PA). P values 0.05 were considered statistically significant.

Coffee consumption, susceptibility of LDL to oxidative modification, and the proportion of LDL() The consumption of 200 mL of coffee significantly influenced the resistance of LDL to Cu(II)-catalyzed oxidative modification, extending the lag phase of conjugate diene formation. Coffee consumption induced a significant increase in the lag phase from 55.6 8.6 (time 0) to 61.6 10.7 min (time 30 min) and to 66.8 17.0 min (time 60 min) (Figure 2). In contrast, the proportion of LDL() was not significantly affected by coffee consumption. At time 0, LDL() accounted for 4.7 2.4% of all LDL, and the proportion did not change significantly after coffee consumption (4.6 2.8% and 4.5 2.8% at time 30 and time 60, respectively) (Figure 3). Coffee consumption and the incorporation of hydroxycinnamic acids into LDL Here we show for the first time that phenolic acids from food are incorporated into LDL in humans. After LDL was subjected to alkaline hydrolysis, a statistically significant increase in caffeic, p-coumaric, and ferulic acids was observed (Table 2). The maximum incorporation peak occurred at 60 min after coffee consumption for caffeic acid and at 30 min after coffee consumption for p-coumaric and ferulic acids. Phenolic acids were present in LDL mainly as bound forms. In fact, after coffee consumption, free phenolic acids were undetectable (p-coumaric acid) or present at the most as traces (caffeic and ferulic acids) in nonhydrolyzed samples (in these experimental conditions, the detection limit in the injected volume was 200, 30, and 50 pg for chlorogenic acid, p-coumaric acid, and ferulic and caffeic acids, respectively. Moreover, the absence in nonhydrolyzed plasma

RESULTS

Phenolic acids in coffee Phenolic acids in filtered coffee are almost exclusively present as chlorogenic acids, and free forms are undetectable. In the present sample, the principal form of chlorogenic acid in coffee, 5-caffeoylquinic acid, reached a concentration of 1.25 mmol/L. After hydrolysis, caffeic acid is most often the main phenolic acid in coffee, with a concentration of 6 mmol/L, whereas ferulic acid has a concentration of 1 mmol/L. The reason that the concentration in coffee of caffeic acid apparently exceeds that of chlorogenic acid is that 5-caffeoylquinic acid is the most representative but not the only bound form of caffeic acid in coffee. In the present experiment, a cup of coffee corresponded to an intake of 200, 5, and 35 mg of caffeic, p-coumaric, and ferulic acid, respectively (Table 1).

Downloaded from www.ajcn.org by on January 18, 2009

FIGURE 2. The effect of coffee consumption on the susceptibility of LDL to oxidative modification. Values are mean SD, n 10. LDL, separated at time 0 and 30 and 60 min after coffee consumption, was oxidized in phosphate-buffered saline at 37 C with 2.5 mol Cu(II)/L. The kinetics of conjugated diene formation was monitored after the absorbance at 234 nm. Lag time, expressed in minutes, was calculated from the intercept of lines drawn through the linear portion of the lag and propagation phases of conjugated diene formation. Differences were analyzed by repeated-measures ANOVA, followed by Tukeys test. *,**Significantly different from time 0: * P 0.05, **P 0.01.

FIGURE 3. The effect of coffee consumption on LDL() concentrations. Values are mean SD, n 10. LDL(), expressed as the percentage of total LDL, was measured before and 30 and 60 min after coffee consumption. Differences were analyzed by repeated-measures ANOVA, followed by Tukeys test.

COFFEE INCREASES LDL RESISTANCE TO OXIDATION


TABLE 2 Total phenolic acid content in LDL1 Caffeic acid p-Coumaric acid Ferulic acid Isoferulic acid

607

DISCUSSION

pmol/mg protein time 0 time 30 min time 60 min


1

17.8 6.1 31.1 8.32 36.1 7.22

1.8 1.2 9.7 3.6 9.1 1.82

1.5 1.5 21.6 6.12 12.9 2.12

ND TR TR

All values are x SEM; n 10. ND, not determined; TR, traces. Values represent the total amount of each phenolic acid measured after alkaline hydrolysis (free bound forms). Differences were analyzed by repeated-measure ANOVA, followed by Tukey test. 2 Significantly different from time 0, P 0.01.

samples of 5-caffeoylquinic acid, the most abundant phenolic in coffee, indicated an extensive metabolism of coffee chlorogenic acids in humans. In vitro experiment To test whether caffeic acid modulates LDL oxidation at the concentration observed in LDL after coffee consumption, we conducted an in vitro experiment using caffeic acid concentrations from 1 to 100 nmol/L. These concentrations were calculated on the basis of the concentration of caffeic acid measured in LDL after coffee consumption (36.1 pmol/mg protein) that corresponds to a concentration of 2 nmol/L in the in vitro experiments (in which the protein concentration was 50 g/mL). As shown in Figure 4, even at nanomolar concentrations, caffeic acid is able to limit the in vitro oxidation of LDL.

FIGURE 4. Dose-response effect of caffeic acid on in vitro LDL oxidation. Values are mean SD, n 3. LDL was oxidized, in the absence or presence of caffeic acid (from 1 to 100 nmol/L), in phosphate-buffered saline at 37 C with 2.5 mol Cu(II)/L. The kinetics of conjugated diene formation was monitored after the absorbance at 234 nm. Lag time, expressed in minutes, was calculated from the intercept of lines drawn through the linear portion of the lag and propagation phases of conjugated diene formation. Differences were analyzed by repeated-measures ANOVA, followed by Tukeys test. *Significantly different from control subjects, P 0.05.

This study provides novel evidence that specific phenolic acids are absorbed into the bloodstream and incorporated into LDL after coffee consumption. In addition, the ex vivo oxidation of LDL was significantly influenced by acute consumption of coffee, whereas the proportion of LDL() remained unchanged. Combined evidence indicates that phenolic acids from coffee have extensive antioxidant activity in in vitro systems (25, 35). In contrast, an effect of caffeine (the other principal bioactive component of coffee) on LDL resistance to oxidative modification has been excluded by several in vitro (36 38) and ex vivo (39) studies. However, the in vitro antioxidant capacity of an antioxidant cannot be directly transposed to an in vivo efficacy, because both absorption and metabolism must be considered. The first evidence of the capacity of coffee to transfer in vivo its in vitro antioxidant potential was offered by the demonstration that coffee consumption increases the total plasma antioxidant capacity in humans (26). Furthermore, in a previous study by our group (23), 5-caffeoylquinic acid, the most abundant phenolic in coffee, was undetectable in human plasma at any time after coffee consumption, whereas caffeic acid was present in plasma, mainly in bound forms as sulfates or glucuronides (19, 23); these findings indicated that an extensive metabolism of coffee phenolics occurs in vivo in humans. Therefore, the compounds responsible for the in vivo effects of coffee consumption likely are the metabolites of coffee phenolic acids, rather than coffee phenolic acids themselves. In the present study, we showed that coffee consumption extends the lag phase of metal-catalyzed LDL oxidation. A similar result was obtained by Yukawa et al (29) after 1 wk of coffee supplementation (3 cups coffee/d, each 200-mL cup made with 8 g powdered coffee). In contrast, McAnlis et al (28) did not find any modification in the LDL resistance to oxidation after 1 wk of coffee supplementation (5 cups coffee/d, each cup made with 2.1 g powdered coffee). The discrepancy between these 2 studies may depend on the difference in the quantity of coffee supplied per day (24 compared with 10.5 g powdered coffee/d). In the present study, we provided the subjects with 200 mL filtered coffee, corresponding to 12 g powdered coffee. The LDL resistance to oxidative modification was measured in particles separated from plasma before and 30 and 60 min after coffee consumption; the latter 2 times corresponded to the peak of absorption of phenolic acids (23). The increase in the resistance of LDL to oxidation did not correspond to a decrease in the proportion of LDL with a higher content of lipid hydroperoxides [ie, LDL()]. This result suggests that, in our experimental conditions, coffee consumption does not affect the concentrations of already oxidized lipids. Similarly, Mursu et al (27) reported no changes in serum LDLconjugated dienes after acute or chronic coffee consumption. The susceptibility of LDL to oxidative modification is determined by the concentrations of 1) baseline conjugated dienes and lipid hydroperoxides and 2) antioxidant species. In the present study, we showed for the first time that phenolic acids from coffee are incorporated into the LDL particle, which strongly suggests their role in the increased protection of LDL against metal-catalyzed oxidation. One hour after coffee consumption, the concentrations in LDL of caffeic, p-cumaric, and ferulic acids were 36.1, 9.1, and 12.9

Downloaded from www.ajcn.org by on January 18, 2009

608

NATELLA ET AL
of the cholesterol controversy, part IV: the 1984 coronary primary prevention trial ends italmost. J Lipid Res 2006;47:114. Berliner JA, Navab M, Fogelman AM, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 1995; 91:2488 96. Hu FB. Plant-based foods and prevention of cardiovascular disease: an overview. Am J Clin Nutr 2003;78(suppl):544S51S. Ziouzenkova O, Asatryan L, Sahady D, et al. Dual roles for lipolysis and oxidation in peroxisome proliferation-activator receptor responses to electronegative low density lipoprotein. J Biol Chem 2003;278:39874 81. Parasassi T, Bittolo-Bon G, Brunelli R, et al. Loss of apoB-100 secondary structure and conformation in hydroperoxide rich, electronegative LDL(). Free Radic Biol Med 2001;31:829. Sevanian A, Hwang J, Hodis H, Cazzolato G, Avogaro P, Bittolo-Bon G. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations. Arterioscler Thromb Vasc Biol 1996;16:784 93. Moro E, Zambon C, Pianetti S, Cazzolato G, Pais M, Bittolo Bon G. Electronegative low-density lipoprotein subform (LDL) is increased in type 2 (non-insulin-dependent) microalbuminuric diabetic patients and is closely associated with LDL susceptibility to oxidation. Acta Diabetol 1998;35:161 4. Ziouzenkova O, Sevanian A. Oxidative modification of low-density lipoprotein (LDL) in HD patients: role in electronegative LDL formation. Blood Purif 2000;18:169 76. Andersen LF, Jacobs DR Jr, Carlsen MH, Blomhoff R. Consumption of coffee is associated with reduced risk of death attributed to inflammatory and cardiovascular diseases in the Iowa Womens Health Study. Am J Clin Nutr 2006;83:1039 46. Happonen P, Voutilainen S, Salonen JT. Coffee drinking is dosedependently related to the risk of acute coronary events in middle-aged men. J Nutr 2004;134:2381 6. Kleemola P, Jousilahti P, Pietinen P, Vartiainen E, Tuomilehto J. Coffee consumption and the risk of coronary heart disease and death. Arch Intern Med 2000;160:3393 400. Panagiotakos DB, Pitsavos C, Chrysohoou C, Kokkinos P, Toutouzas P, Stefanadis C. The J-shaped effect of coffee consumption on the risk of developing acute coronary syndromes: the CARDIO2000 case-control study. J Nutr 2003;133:3228 32. Viani R. The composition of coffee. In: Garattini S, ed. Caffeine, coffee, and health. New York: Raven Press, 1993:17 41. Azuma K, Ippoushi K, Nakayama M, Ito H, Higashio H, Terao J. Absorption of chlorogenic acid and caffeic acid in rats after oral administration. J Agric Food Chem 2000;48:5496 500. Wittemer SM, Ploch M, Windeck T, et al. Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans. Phytomedicine 2005;12:28 38. Lafay S, Gil-Izquierdo A, Manach C, Morand C, Besson C, Scalbert A. Chlorogenic acid is absorbed in its intact form in the stomach of rats. J Nutr 2006;136:11927. Lafay S, Morand C, Manach C, Besson C, Scalbert A. Absorption and metabolism of caffeic acid and chlorogenic acid in the small intestine of rats. Br J Nutr 2006;96:39 46. Mateos R, Goya L, Bravo L. Uptake and metabolism of hydroxycinnamic acids (chlorogenic, caffeic, and ferulic acids) by HepG2 cells as a model of the human liver. J Agric Food Chem 2006;54:8724 32. Nardini M, Cirillo E, Natella F, Scaccini C. Absorption of phenolic acids in humans after coffee consumption. J Agric Food Chem 2002; 50:5735 41. Nardini M, Natella F, Gentili V, Di Felice M, Scaccini C. Effect of caffeic acid dietary supplementation on the antioxidant defense system in rat: an in vivo study. Arch Biochem Biophys 1997;342:157 60. Nardini M, DAquino M, Tomassi G, Gentili V, Di Felice M, Scaccini C. Inhibition of human low-density lipoprotein oxidation by caffeic acid and other hydroxycinnamic acid derivatives. Free Radic Biol Med 1995; 19:54152. Natella F, Nardini M, Giannetti I, Dattilo C, Scaccini C. Coffee drinking influences plasma antioxidant capacity in humans. J Agric Food Chem 2002;50:6211 6. Mursu J, Voutilainen S, Nurmi T, et al. The effects of coffee consumption on lipid peroxidation and plasma total homocysteine concentrations: a clinical trial. Free Radic Biol Med 2005;38:52734. McAnlis GT, McEneny J, Pearce J, Young IS. Black tea consumption

pmol/mg protein, respectively. Incorporation into LDL was already shown for only a few other phenolic compoundsie, quercetin and catechin (40), daidzein and genistein (41), rutin and quercetin (42), and tyrosol (43). In 2 of these studies, the concentration of phenolic compounds in LDL was 1000 pmol/mg protein (40, 43). These high concentrations could depend on the methods used, because LDL samples were not subjected to any kind of purification (by filtering or dialyzing), and thus, the unbound phenolic compounds could remain in the aqueous phase, as shown by Tikkanen et al (41). To test whether caffeic acid, at the concentration observed in LDL after coffee consumption, is able to modulate LDL oxidation, we conducted an in vitro experiment using caffeic acid concentrations from 1 to 100 nmol/L. As shown in Figure 4, even at concentrations similar to those we observed in vivo (nanomolar concentrations), caffeic acid is capable of modulating the in vitro LDL oxidation. It is important to underline that caffeic acid is not the only phenolic acid incorporated into LDL after coffee consumption. We are aware that phenolic acids incorporated in vivo in LDL are not in free form but are in bound form. Because at the moment we do not know the nature of these bound forms, we cannot speculate on whether these forms are more or less active than their respective free forms, even if polyphenol metabolites have been reported to have lower in vitro antioxidant activities than the parent molecules (44). Further studies will be necessary to identify the bound forms and the nature of the bonds of phenolic acids to LDL particle. Data presented here indicate that the consumption of 200 mL (1 cup) coffee improves the resistance to oxidative modification of LDL in humans, and this effect can be explained by the quick incorporation of phenolic acids in LDL. The role of coffee in CVD risk is controversial. Some epidemiologic studies indicate a J-shaped relation between coffee consumption and CVD risk (13, 14, 16). This relation is probably the result of the opposite action of positive and negative molecules present in coffee. Through their antioxidant action, phenolic acids can represent one of the positive contributors to the beneficial effects of coffee.
The authors thank Kariklia Pascucci for her support in the daily laboratory work. The authors responsibilities were as followsFN, MN, and CS: the study design, endpoint assays, data analyses and interpretation, and writing of the manuscript; FB: assistance in the conduct of the experiments; and all authors: review of the manuscript and scientific and editorial input. None of the authors had a personal or financial conflict of interest.

6. 7. 8.

9. 10.

11.

12. 13.

Downloaded from www.ajcn.org by on January 18, 2009

14. 15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25.

REFERENCES
1. Steinberg D. Thematic review series: the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy: part I. J Lipid Res 2004;45:158393. 2. Steinberg D. Thematic review series: the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy: part II: the early evidence linking hypercholesterolemia to coronary disease in humans. J Lipid Res 2005;46:179 90. 3. Steinberg D. Thematic review series: the pathogenesis of atherosclerosis: an interpretive history of the cholesterol controversy, part III: mechanistically defining the role of hyperlipidemia. J Lipid Res 2005;46: 203751. 4. Steinberg D. Thematic review series: the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy, part V: the discovery of the statins and the end of the controversy. J Lipid Res 2006;47:1339 51. 5. Steinberg D. The pathogenesis of atherosclerosis. An interpretive history

26. 27. 28.

COFFEE INCREASES LDL RESISTANCE TO OXIDATION


does not protect low density lipoprotein from oxidative modification. Eur J Clin Nutr 1998;52:202 6. Yukawa GS, Mune M, Otani H, et al. Effects of coffee consumption on oxidative susceptibility of low-density lipoproteins and serum lipid levels in humans. Biochemistry (Mosc) 2004;69:70 4. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:134553. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:26575. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med 1992;13:34190. Hodis HN, Kramsch DM, Avogaro P, et al. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL). J Lipid Res 1994;35:669 77. Nardini M, Natella F, Scaccini C, Ghiselli A. Phenolic acids from beer are absorbed and extensively metabolized in humans. J Nutr Biochem 2006;17:14 22. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:93356. Krisko A, Kveder M, Pifat G. Effect of caffeine on oxidation susceptibility of human plasma low density lipoproteins. Clin Chim Acta 2005; 355:4753. Lee C. Antioxidant ability of caffeine and its metabolites based on the

609

29.

38. 39. 40.

30.

31. 32.

33.

41. 42. 43.

34.

35.

36.

44.

37.

study of oxygen radical absorbing capacity and inhibition of LDL peroxidation. Clin Chim Acta 2000;295:14154. Yokozawa T, Dong E. Influence of green tea and its three major components upon low-density lipoprotein oxidation. Exp Toxicol Pathol 1997;49:329 35. Hodgson JM, Puddey IB, Croft KD, et al. Acute effects of ingestion of black and green tea on lipoprotein oxidation. Am J Clin Nutr 2000;71: 11037. Hayek T, Fuhrman B, Vaya J, et al. Reduced progression of atherosclerosis in apolipoprotein E-deficient mice following consumption of red wine, or its polyphenols quercetin or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler Thromb Vasc Biol 1997;17:2744 52. Tikkanen MJ, Wahala K, Ojala S, Vihma V, Adlercreutz H. Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance. Proc Natl Acad Sci U S A 1998;95:3106 10. Lamuela-Raventos RM, Covas MI, Fito M, Marrugat J, de La TorreBoronat MC. Detection of dietary antioxidant phenolic compounds in human LDL. Clin Chem 1999;45:1870 2. de la Torre-Carbot K, Jauregui O, Castellote AI, et al. Rapid highperformance liquid chromatography-electrospray ionization tandem mass spectrometry method for qualitative and quantitative analysis of virgin olive oil phenolic metabolites in human low-density lipoproteins. J Chromatogr A 2006;1116:69 75. Natsume M, Osakabe N, Yasuda A, et al. In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic Res 2004;38:1341 8.

Downloaded from www.ajcn.org by on January 18, 2009

You might also like