Free Radical Biology & Medicine 46 (2009) 769774

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Free Radical Biology & Medicine

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Antioxidant activity of blueberry fruit is impaired by association with milk

Mauro Serani a,, Maria Francesca Testa a, Debora Villao a, Monia Pecorari a, Karin van Wieren b, Elena Azzini a, Ada Brambilla c, Giuseppe Maiani a
a b c

Antioxidant Research Laboratory, Unit of Human Nutrition, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, 00178 Rome, Italy Friesland College, Leeuwarden, The Netherlands Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Istituto Sperimentale per la Valorizzazione Tecnologica dei Prodotti Agricoli, Milan, Italy

a r t i c l e

i n f o

a b s t r a c t
The antioxidant properties of dietary phenolics are believed to be reduced in vivo because of their afnity for proteins. In this study we assessed the bioavailability of phenolics and the in vivo plasma antioxidant capacity after the consumption of blueberries (Vaccinium corymbosum L.) with and without milk. In a crossover design, 11 healthy human volunteers consumed either (a) 200 g of blueberries plus 200 ml of water or (b) 200 g of blueberries plus 200 ml of whole milk. Venous samples were collected at baseline and at 1, 2, and 5 h postconsumption. Ingestion of blueberries increased plasma levels of reducing and chain-breaking potential (+ 6.1%, p b 0.001; +11.1%, p b 0.05) and enhanced plasma concentrations of caffeic and ferulic acid. When blueberries and milk were ingested there was no increase in plasma antioxidant capacity. There was a reduction in the peak plasma concentrations of caffeic and ferulic acid (49.7%, p b 0.001, and 19.8%, p b 0.05, respectively) as well as the overall absorption (AUC) of caffeic acid (p b 0.001). The ingestion of blueberries in association with milk, thus, impairs the in vivo antioxidant properties of blueberries and reduces the absorption of caffeic acid. 2008 Elsevier Inc. All rights reserved.

Article history: Received 22 May 2008 Revised 25 November 2008 Accepted 27 November 2008 Available online 11 December 2008 Keywords: Blueberry Milk Antioxidant capacity Caffeic acid Bioavailability Proteins Free radicals

The human diet provides a wide array of plant-derived phenolics with antioxidant activity that helps the body to cope with oxidative stress [1]. The biological response to antioxidant-rich foods is critically determined by the bioavailability of active molecules. The most abundant phenolics in the diet are not necessarily those able to reach the highest levels in human circulation, owing to the considerable differences in bioavailability. Human intervention studies have shown that absorption of phenolics in the gastrointestinal tract is, typically, between 1 and 5% of the ingested dose [2]. Polyphenols can be absorbed in the stomach and at the small intestine level by passive diffusion or active transport [3,4]. Once absorbed, the metabolism of polyphenols in humans implies a profound biotransformation through enzymatic conjugation with sulfate, methyl, or glucuronide groups in both the small intestine epithelial cells and the liver [4]. These metabolites can reach a maximum concentration in plasma (Cmax) 1 2 h after ingestion of phenolic-rich foods [57] and appear principally in urine after 48 h [8,9]. Variable amounts of avonoids, not absorbed in the upper gastrointestinal tract, reach the colon, where they are subjected to the action of the colonic microora, resulting in cleavage of glycosidic

Abbreviations: AUC, area under the curve; FRAP, ferric reducing antioxidant potential; TAC, total antioxidant capacity; TRAP, total radical-trapping antioxidant parameter. Corresponding author. Fax: +390651494550. E-mail address: serani_mauro@yahoo.it (M. Serani). 0891-5849/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.11.023

linkages and the breakdown of the avonoid heterocycle into phenolic acids and aldehydes [1014]. These microbial catabolites are absorbed into the circulatory system from the large intestine, reaching a Cmax ca. 5 h postconsumption and, consequently, are able to exert biological actions [2]. Flavonoids are generally consumed in foods along with other macronutrients such as proteins, lipids, glucose, or ethanol and these constituents may have an impact on avonoid bioavailability. It has been proposed that the linking of phenolics with proteins during food ingestion can reduce their absorption [15]. A large number of in vitro studies have suggested that these interactions involve both the formation of hydrogen bonds and hydrophobic interactions between hydroxyl groups of the phenolic compounds and the carbonyl groups of the proteins [16]. Recently, it has been shown with human volunteers that an improvement in ow-mediated dilation, a marker of vascular function, brought about by the ingestion of 500 ml of black tea did not occur when the tea was consumed with milk [17]. Intervention studies with humans showed reduced bioavailability of phenolics and in vivo antioxidant activity after the ingestion of tea and chocolate with milk. However, other studies have failed to detect an impact of milk and as a consequence the topic is somewhat controversial [15,1821] and one that requires further investigation. In the current study, blueberries (Vaccinium corymbosum L.), which increase plasma antioxidant defenses and delay chemically induced LDL and liposome oxidation ex vivo, were chosen as a dietary source of phenolic antioxidants [2227]. The aim of the investigation was to

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assess whether the consumption of blueberries in association with milk, compared with the ingestion of blueberries alone, affects in vivo antioxidant levels and bioavailability of phenolic acids. In vitro experiments were also carried out to investigate the effects of various types of milk on the antioxidant capacity of a blueberry extract. Materials and methods Chemicals and reagents Hydrochloric acid, methyl alcohol, and acetonitrile solvents were purchased from Carlo Erba (Milan, Italy). Glacial acetic acid, 85% v/v orthophosphoric acid, metaphosphoric acid, ferric chloride hexahydrate (FeCl36H2O), sodium acetate trihydrate (C2H3NaO23H2O), and sodium phosphate monobase (NaH2PO4) were obtained from BDH (Poole, England). 2,2-Azobis (2-amidinopropane) dihydrochloride (ABAP) was purchased from Wako Chemical (Germany), 2,4,6-tri(2pyridyl)-s-triazine (TPTZ) was from Fluka (Switzerland), and ferrous sulfate hexahydrate (FeSO46H2O) and formic acid were from Merck (Darmstadt, Germany). R-phycoerythrin (R-PE), phosphate-buffered saline (PBS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, and -glucuronidase type HP2 from Helix pomatia were provided by SigmaAldrich Srl (Milan, Italy). Commercial standards of gallic acid, chlorogenic acid, cyanidin 3-glucoside, caffeic acid, ferulic acid, quercetin 3-galactoside, quercetin 3-glucoside, and o-coumaric acid were also from SigmaAldrich Srl. Doubledistilled water (Millipore, Milan, Italy) was used throughout the study. Food matrices A mixture (1/1) of two varieties of blueberry (V. corymbosum L.), cv. Berkeley and Coville, provided by the Istituto per la Valorizzazione Tecnologica dei Prodotti Agricoli from Milan (Italy) was used. Whole milk, semiskimmed milk, and skimmed milk were supplied by Centrale del Latte di Roma (Rome, Italy). The nutritional composition of the milks is displayed in Table 1. Sample phenolic extraction Extraction of phenolic compounds was performed, with slight modications, based on previous studies [25,28]. Blueberries (15 g) were added to a solution containing acetone, water, and formic acid (60:30:10 v/v) and homogenized using an Ultraturrax blender (IKA Labortechnik, Staufen, Germany) at 20,300 rpm. The homogenate was left at room temperature for 15 min and then centrifuged at 25C at 4025 g for 5 min. The supernatant was collected and the pellet was further washed with 25 ml of the extraction solvent; after further centrifugation, the two supernatants were combined and the organic solvent was removed by vacuum leaving an aqueous extract. Hydrophilic and amphiphilic extracts of blueberry fruits for the assessment of in vitro ferric reducing antioxidant potential (FRAP) and total radical-trapping antioxidant parameter (TRAP) were obtained according to the method of Pellegrini et al. [29]. Blueberry fruits were pooled, mixed, and homogenized under nitrogen ux. A weighed amount of sample (1 g) was extracted twice with 4 and 2 ml of water in a shaking bath for 15 min at room temperature. The homogenates
Table 1 Nutritional composition of the milks used in the studya Whole milk Energy content (kcal) Proteins (g) Carbohydrates (g) Lipids (g) Calcium (mg)
a

were then centrifuged at 1000 g for 10 min and the supernatants collected. The residue from pulp was extracted twice following the same procedures using 4 and 2 ml volumes of acetone. Analysis of blueberry phenolics The phenolic prole of the blueberry extracts was assessed by an RP-HPLC system equipped with a diode array detector [30]. The column, an Inertsil ODS-3 column (4.6 250 mm i.d., particle size 5 m), was held at 40 C and eluted at a ow rate of 0.5 ml min 1. The mobile phases consisted of acetonitrile (solvent A) and water:formic acid (90:10 v/v) (solvent B); all solvents were HPLC grade. Elution was performed following linear gradient steps: start condition 8% A in B, then 11% A in 25 min, 23% A in 22 min, 45% A in 16 min, 75% A in 3 min, 75% A for 8 min, and 8% A in 5 min. Before injection (20 l), samples were diluted 1:10 (v/v) in mobile phase B and ltered through a 0.45 m HA membrane lter (Millipore Corp., Bedford, MA, USA). Anthocyanin glycosides were identied on the basis of their absorbance spectra compared to published data [30]. All of the anthocyanin monoglycosides were quantied as cyanidin 3-glucoside equivalents [31]. Phenolic acids (chlorogenic acid, o-coumaric acid, ferulic acid, gallic acid) and avonols (quercetin 3-galactoside, quercetin 3-glucoside) were identied by comparing absorbance spectra and retention times with those of authentic reference compounds. Total antioxidant capacity (TAC) assays Total antioxidant capacity in vitro (milk and blueberry extracts) and in vivo (plasma samples) was assessed using the FRAP and the TRAP assays, respectively, for the measurement of reducing and chainbreaking antioxidant potential [32,33]. The FRAP assay is based on the reduction of the Fe3+TPTZ complex to the ferrous form at low pH monitored at 595 nm by a Sunrise absorbance plate reader (Tecan Italia Srl, Segrate, Italy) [32]. Briey, 160 l of FRAP reagent, prepared daily, was mixed with 30 l of water and 10 l of diluted sample; the absorbance at 595 nm was recorded after a 30-min incubation at 37C. FRAP values were obtained by comparing the absorption changes in the test mixture with those obtained from increasing concentrations of Fe2+ and expressed as mol of Fe2+ per liter of plasma or mol g 1 extract. The TRAP method is based on the protection provided by antioxidants on the uorescence decay of R-phycoerythrin (lag phase) during a controlled peroxidation reaction [33]. In brief, 50 l of diluted sample was added to 75 l of PBS (pH 7.4), 15 l of R-PE (fc 4.3010 3 g l 1), and 60 l of ABAP (fc 7.5 mM); the reaction kinetic at 38 C was recorded for 60 min (ex =495 nm, em = 570 nm) by a Tecan GENios standard uorescence plate reader spectrometer (Tecan Italia Srl). The length of the lag phase, automatically calculated, was used to assess TRAP values, expressed as mol g 1 or mol L 1 for extracts and plasma, respectively. In vitro study on milk addition to blueberry extracts The hydro- and amphiphilic extracts of blueberries were used for the in vitro study of milk association. Stock solutions of 50-folddiluted milk were prepared in MilliQ water. To 300 l of blueberry extract, 1000 l of diluted milk and 700 l of MilliQ water were added [34]. After an incubation for 15 min at room temperature, the samples were centrifuged for 15 min at 2683 g at 4C and the supernatant was collected. The pellet was redissolved in 500 l of acetate buffer for analysis. Both supernatant and pellet obtained from each hydro- and amphiphilic extract were analyzed with the FRAP method as described above. In vivo study design The acute ingestion model is a reliable tool to test the contribution of antioxidant-rich food on the endogenous antioxidant defenses. The

Semi-skimmed milk 46 3.15 4.85 1.55 120

Skimmed milk 33 3.20 4.90 0.10 120

64 3.20 4.80 3.60 120

100 ml of product.

M. Serani et al. / Free Radical Biology & Medicine 46 (2009) 769774 Table 2 Phenolic composition of blueberry fruits Phenolic compound Cyanidin galactoside Cyanidin glucoside Cyanidin arabinoside Delphynidin galactoside Delphynidin glucoside Delphynidin arabinoside Malvidin galactoside Malvidin glucoside Malvidin arabinoside Petunidin galactoside Petunidin glucoside Petunidin arabinoside Peonidin galactoside Peonidin glucoside Anthocyanins total Chlorogenic acid o-Cumaric acid Ferulic acid Gallic acid Phenolic acids total Quercetin galactoside Quercetin glucoside Quercetin xb Flavonols total
a b

771

mg 100 g 1a 2.50 0.93 1.74 13.12 1.11 8.70 26.16 1.60 16.57 9.83 1.10 5.95 2.02 0.99 92.32 24.19 5.54 0.08 1.00 30.81 1.08 0.43 0.50 2.01

(3 N) 1:1 v/v. Polyphenols were extracted with 2 ml ethyl acetate, followed by stirring and sonication (23 min) before centrifugation at 1700 g for 5 min. The extraction procedure was repeated twice and the ethyl acetate extracts were combined, reduced to dryness in vacuo, and reconstituted in 200 l of HPLC mobile-phase methanol:H2O (1:1) for analyses. Quantitative analyses were performed with an ESA HPLC with a coulometric electrode array detector [37]. A Supelcosil LC-18 column (25 cm 4.6 mm, 5 m) was used, protected by a Perisorb Supelguard LC-18 guard cartridge. Chromatography was performed at 30 C with a ow rate of 0.8 ml/min. This method employs a binary gradient: solvent A, 0.02 M NaH2PO4H2O adjusted to pH 2.8 with 85% orthophosphoric acid; solvent B, methanol. It was programmed as follows: 10% B at the start, increasing to 30% B over 7 min and to 33% B over 28.5 min, increasing to 45% B over 19.5 min, held for 8.5 min, and reaching the nal condition of 100% 24 min later. The setting potentials were 60, 120, 200, 340, 480, 620, 760, and 900 mV. Sample peaks were identied by matching them with standard peaks (caffeic acid, ferulic acid; Sigma Chemical Co., St. Louis, MO, USA) on the basis of their retention time and the accuracy ratio between adjacent channels. Statistics The results obtained are expressed as means standard deviation of the mean (SDM) for the in vitro study, whereas values from the in vivo study are expressed as means standard error of the mean (SEM). The normal distribution of variables was conrmed by the KolmogorovSmirnov test. A two-tailed Student t test was performed in every sample, comparing the values of T0 with the various time points postingestion as in previous studies [21]. The area under the curve (AUC) of phenolic concentrations in plasma was calculated for the 5-h period after each food intervention and the comparison of AUCs between treatments was performed by t test. A p value of b0.05 was considered signicant. All statistical treatments were performed with the Kaleida Graph version 4.0 program. Results Phenolic composition and in vitro TAC of blueberry The phenolic composition of blueberry fruits is described in Table 2. Five anthocyanins were identied: galactosides, arabinosides, and

Extracted in acetone/acidied water. Glycosylated quercetin.

working window of the study is free from interference by different variables (food intake, physical exercise, energy expenditure, etc.). The study was approved by the ethics committee of the Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione and all participants gave their written consent. Eleven healthy volunteers (six men, ve women), nonsmokers, normolipidemic, taking no supplements or medications, and free of any pathology, were recruited. Mean age and BMI of the subjects were 31 5 years and 22.1 3 kg m 2. For 2 days before the intervention, the subjects followed a lowantioxidant diet, avoiding phenolic-rich foods, namely, all fresh fruit and vegetables and derived products (tea, coffee, fruit juices, wine, chocolate). After an overnight fast, the volunteers were divided into two groups and, following a crossover design, they consumed either (a) 200 g of blueberry fruits plus 200 ml of water or (b) 200 g of blueberry fruits plus 200 ml of whole milk. The intervention was repeated after 1 week, this time swapping the treatments. Venous samples at baseline (T0) and at 1 h (T1), 2 h (T2), and 5 h (T5) after blueberry consumption were collected in EDTA- and heparinVacutainers and centrifuged at 2500 rpm for 10 min at 4C and the plasma obtained was stored at 80C for analysis. Biochemical analyses Plasma thiols were determined spectrophotometrically by monitoring the absorbance at 412 nm after addition of Ellman's reagent [35]. Uric acid, total cholesterol, and triacylglycerol concentrations in plasma were quantied using colorimetric kits (Giesse Diagnostics, Rome, Italy). Plasma vitamin C was immediately stabilized by adding metaphosphoric acid in the ratio of 1:1; ascorbic acid and dehydroascorbic acid were determined by HPLC with electrochemical detection [36]. Determination of phenolic acids in plasma samples Plasma phenolic acids were detected after enzyme and acid hydrolysis of the conjugated forms as described by Azzini et al. [37]. Hydrolysis of conjugates was performed by adding 100 l ascorbic acid (1%), 100 l acetic acid (0.2 M), and 50 l -glucuronidase type HP2 from Helix pomatia (134300 U/ml) to 500 l plasma. After incubation at 37 C for 2 h, proteins were precipitated with 700 l methanol:HCl

Fig. 1. In vitro total antioxidant capacity of blueberry extracts. Blueberry fruits were pooled, mixed, and homogenized under nitrogen ux. A weighed amount of sample (1 g) was extracted twice with 4 and 2 ml of water in a shaking bath for 15 min at room temperature. The homogenates were then centrifuged at 1000 g for 10 min, and the supernatants were collected (hydrophilic extract). The residue from pulp was extracted twice following the same procedures using 4 ml and 2 ml volumes of acetone (amphiphilic extract). Chain-breaking antioxidant potential (TRAP) and reducing power (FRAP) were determined as described under Materials and methods. Results are expressed as mol g 1 of extract (mean SDM; n = 3).

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Fig. 2. Effect of milk addition on the in vitro reducing power (FRAP) of blueberry extracts. Stock solutions of 50-fold-diluted milk were prepared in MilliQ water. To 300 l of blueberry extract, 1000 l of diluted milk and 700 l of MilliQ water (control) were added. After an incubation for 15 min at room temperature, samples were centrifuged for 15 min at 2683 g at 4C and the supernatant was collected. The pellet was redissolved in 500 l of acetate buffer for analysis. Both supernatant and pellet obtained from each hydro- and amphiphilic extract were analyzed with FRAP as described under Materials and methods. Results are expressed as means SDM of three independent experiments and represent the percentage of FRAP decrease in the supernatant of blueberry extracts after milk addition relative to water (0% decrease). SM, skimmed milk; SSM, semiskimmed milk; WM, whole milk.

Fig. 3. In vivo effect of blueberry ingestion on markers of plasma antioxidant status and phenolic bioavailability. Plasma levels of caffeic acid () and ferulic acid () and concentration relative to baseline values of plasma FRAP () and TRAP () after ingestion of 200 g of blueberries plus 200 ml of water are shown. Experimental details can be found under Materials and methods. Results are expressed as means SEM (n = 11). Caffeic and ferulic acid plasma concentrations are signicantly different from baseline (paired t test; p b 0.001 for all levels except for ferulic acid at 5 h, p b 0.05). p b 0.001 for FRAP and p b 0.05 for TRAP for time comparison at 5 h vs baseline.

glucosides of malvidin, delphinidin, cyanidin, petunidin, and peonidin. Anthocyanin derivatives represent 73.4% of the quantied polyphenols, followed by phenolic acids with 24.5% (78.5% represented by chlorogenic acid) and avonols with 2.1%. The TAC values of blueberry fruits were 19.65 2.62 mol g 1 extract in the TRAP assay and 15.75 0.35 mol Fe+ 2 g 1 extract in the FRAP assay (Fig. 1). The water-soluble fraction contributed a higher percentage (69.7 and 60.3%, respectively, for TRAP and FRAP) than the amphiphilic fraction (30.3% for TRAP and 39.7% for FRAP). In vitro study on food association The addition of various types of milk to blueberry extracts caused a decrease in FRAP values of the supernatants, with whole milk reducing more effectively than skimmed and semiskimmed milk (Fig. 2). The extent of the antioxidant decrease was linearly associated with the amount of lipids from the different milks (r = 0.99901 for hydrophilic extract; r = 0.99659 for amphiphilic extract). Effects of blueberry ingestion on markers of plasma antioxidant status and lipid metabolism Plasma concentrations of ascorbic acid, uric acid, thiols, total cholesterol, and triacylglycerol did not change after blueberry supplementation (Table 3). Plasma markers of TAC rose signicantly after blueberry ingestion (Fig. 3), reaching a maximum peak at 5 h,

+6.1%, p b 0.001, and +11.1%, p b 0.05, respectively, for FRAP and TRAP assays. Plasma concentrations of caffeic acid and ferulic acid increased signicantly after blueberry ingestion at all time points. Different from TAC, caffeic and ferulic acid reached Cmax (96 17 and 49 10 nM, respectively) at 1 h postingestion and steadily decreased thereafter to a nal value of 42 7 nM for caffeic acid and 18 8 nM for ferulic acid 5 h postingestion (Fig. 3). In vivo effects of milk addition on markers of plasma antioxidant status and lipid metabolism After the ingestion of blueberries alone, plasma concentrations of vitamin C, uric acid, and thiol groups did not differ signicantly from the values obtained after consumption of milk and blueberries (Table 4). Total cholesterol concentrations increased signicantly from 170 10 mg dl1 up to a maximum value of 181 9 mg dl 1 at 5 h postingestion (t test; p b 0.001). Changes in plasma triacylglycerol concentrations were not detected. The overall extent of absorption of caffeic acid, but not of ferulic acid, as determined with estimates of AUC, was reduced signicantly by the addition of milk to the blueberries (t test; p = 0.039) (Fig. 4). Caffeic and ferulic acids appeared in plasma 1 h after ingestion of the berries (Fig. 5), although milk resulted in a marked reduction in their absorption. A signicant reduction of Cmax for caffeic acid (49.7%, p b 0.001) and ferulic acid (19.8%, p b 0.05) was observed with respect to the levels reached after ingestion of blueberries with water. The ingestion of blueberries

Table 3 Effects of supplementation with 200 g of blueberry plus 200 ml of water on plasma concentrations of single antioxidants and lipid markers in 11 healthy volunteers (means SEM) Baseline Ascorbic acid (mg dl 1) Uric acid (mg dl 1) Thiol groups (mg dl 1) Total cholesterol (mg dl 1) Triacylglycerols (mg dl 1) 1.01 0.12 4.30 0.40 612 25 184 7 61.3 6.9 1h 1.07 0.07 4.39 0.50 598 18 181 7 59.2 7.4 2h 1.05 0.12 4.43 0.50 605 22 191 9 57.7 7.0 5h 1.14 0.11 4.39 0.50 629 47 188 7 59.4 6.5

Table 4 Effects of supplementation with 200 g of blueberries plus 200 ml of milk on plasma concentrations of single antioxidants and lipid markers in 11 healthy volunteers (means SEM) Baseline Ascorbic acid (mg dl 1) Uric acid (mg dl 1) Thiol groups (mg dl 1) Total cholesterol (mg dl 1) Triacylglycerols (mg dl 1) 1.08 0.10 5.1 0.50 586 13 170 10 56.6 7.0 1h 1.19 0.10 4.94 0.50 610 15 176 10 54.7 6.8 2h 1.10 0.12 4.95 0.50 607 12 178 11 55.9 5.6 5h 1.19 0.09 4.91 0.50 630 32 181 9 58.8 6.7

p b 0.001 compared with baseline (paired t test).

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other molecules such as phenolic metabolites might contribute to the antioxidant effects of blueberry fruits in vivo. In vivo and in vitro effects of milk addition The in vitro addition of milk to blueberry extracts resulted in a precipitation of blueberry antioxidants, leading to a decrease in TAC values of the supernatants, with full-fat milk showing the highest degree of inhibition. This nding is in agreement with the data from Langley-Evans [41] that showed the greatest reduction in in vitro FRAP values of black tea with the addition of whole milk (28% reduction) compared to semiskimmed (22% reduction) and skimmed milk (12% reduction). The data suggest a possible novel involvement of lipids in the interaction between milk, protein, and antioxidant ingredients of blueberry fruits. Polypeptide chains located around the fat globule membrane in the milk increase with the increasing of lipid content of the whole milk, supplying a more favorable environment for their linkage with phenolics compared to lower fat milks [42]. However, more in vitro experiments are needed to understand the role of lipids in the inhibitory effect of milk on antioxidant activity. In humans, in agreement with previous evidence obtained with tea and chocolate [15,19,21], we have shown that the milk reduces plasma TAC and the Cmax of caffeic acid and ferulic acid, occurring after the ingestion of blueberries. The plasma Cmax of caffeic acid 1 h after blueberry consumption was 8.7 ng/ml. This is equivalent to 0.08% of the 5-caffeoylquinic acid intake, and when the blueberry supplement was ingested with milk this fell to 0.04% of intake. The reduction of phenolics absorption in the presence of milk is in agreement with Reddy et al. [43] and Serani et al. [21], who reported that the peak concentrations of plasma avan-3-ols were signicantly lower after consumption of black tea with milk, milk chocolate, and chocolate with milk. However, other studies have failed to show signicant differences in plasma concentrations of epicatechin [44], total catechins [45], avonols [18], and TAC [20,46] after ingestion of chocolate and tea with milk. Our results on TAC are in agreement with other studies in which increases in plasma TAC after the ingestion of green tea [15], black tea [15,19], and chocolate [21] are reduced by coconsumption of milk. The increase in plasma TAC observed by Leenen et al. [20] with black tea was +2%, which is very close to the 3% coefcient of variation in the FRAP assay [32] and negligible compared to increases of +52 and +65%

Fig. 4. In vivo effect of milk addition on overall absorption of phenolics. The AUC of plasma concentrations of caffeic acid and ferulic acid for the 0- to 5-h period after consumption of 200 g of blueberries plus 200 ml of water or whole milk is shown. Experimental details can be found under Materials and methods. Results are expressed as means SEM (n = 11). p b 0.05 for between-treatment comparison.

and milk was not associated with any increase in TRAP and FRAP plasma concentrations (Fig. 5). As after the ingestion of blueberries with water, there was no time association between markers of phenolic absorption and markers of antioxidant capacity (Fig. 5). Discussion Effects of blueberry ingestion on markers of plasma antioxidant status In agreement with previous studies [26,27], the results show that blueberry ingestion results in a signicant increase in (i) endogenous plasma antioxidant defenses and (ii) caffeic and ferulic acid levels. The presence of caffeic acid in plasma but not in the food matrix suggests that postingestion metabolism of the blueberry phenolics results in their appearance in the circulatory system. Caffeic acid might be derived from the hydrolysis of chlorogenic acid (5-caffeoylquinic acid). Previous intervention studies have reported that such a transformation occurs after ingestion of artichoke [37] and coffee [38] by human volunteers. As no 5-caffeoylquinic acid was detected in the plasma, we hypothesize that 5-caffeoylquinic acid is hydrolyzed and caffeic acid partly absorbed in the small intestine. The tested meal contained 48.38 mg of 5-caffeoylquinic acid, with an average intake of 0.74 mg/kg body wt. The plasma Cmax of caffeic acid 1 h postconsumption was 0.599 g/kg body wt. If we assume that one molecule of 5caffeoylquinic acid produces one molecule of caffeic acid, the Cmax of caffeic acid reached in plasma 1 h after ingestion of blueberry is equivalent to 0.08% of 5-caffeoylquinic acid intake. Despite the low bioavailability of phenolic acids and the lack of changes in the levels of ascorbic acid, thiol groups, and uric acid, we observed an increase in plasma antioxidant defenses. In both treatments, there was no association between plasma concentrations of caffeic and ferulic acids and markers of plasma TAC. After blueberry ingestion, phenolics peaked 4 h before the increase in TAC, and when milk was utilized, the increase in phenolics did not translate into any positive changes in plasma TAC. Maximum increase of phenolic acids after blueberry ingestion was in the midnanomolar range for caffeic and ferulic acid (96 and 49 nM), whereas the increases in plasma TRAP and FRAP were 101 and 49 M, respectively. This 100-fold difference makes it highly questionable whether these hydroxycinnamates contribute to the endogenous redox network. In agreement with previous evidence [39,40], our results highlight the lack of correlation between plasma endogenous antioxidant defenses and circulating levels of dietary phenolics. It is possible that synergistic interactions between the redox component of the network and/or the presence of

Fig. 5. In vivo effect of milk addition on markers of plasma antioxidant status and phenolic bioavailability. Plasma levels of caffeic acid () and ferulic acid () and concentration relative to baseline values of plasma FRAP () and TRAP () after ingestion of 200 g of blueberries plus 200 ml of whole milk are shown. Experimental details can be found under Materials and methods. Results are expressed as means SEM (n = 11). p b 0.05 for FRAP for time comparison at 2 h vs baseline.

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observed in previous studies [15,19], narrowing, in this way, the observational window. In other studies in which chocolate was utilized as the antioxidant source, Schroeter et al. [44] and Serani et al. [21] reached opposite conclusions, utilizing different proportions of milk lipids: 3% in the former, with no effect of milk, and 30% in the latter, with a clear inhibitory effect [47]. It is a matter of fact that the discrepancy of the results in humans is remarkable, with half the reports suggesting a lack of effect [18,20,4346] and the other half suggesting an inhibitory effect of milk [15,17,19,21]. The present study suggests, for the rst time, that consumption of a fruit rich in antioxidants, such as blueberry, in association with whole milk decreases its ability to increase plasma endogenous antioxidant defenses and to deliver into the circulation bioactive molecules such as caffeic acid. More extensive research is needed to clarify the role played by incorrect food association on the bioactivity of dietary antioxidants in vivo. Acknowledgments The authors thank Irene Fabbri for proofreading the manuscript, Professor Alan Crozier for critical comments, and all the volunteers for the in vivo study. This study was supported by the Italian Ministry of Agricultural and Forestry Policies, within the frame of the project qFood Quality.q D. Villano enjoys a postdoctoral research fellowship from the Ministerio de Educacin y Ciencia, Spain. References
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