AJCN Tufts Nutrition Symposium, Boston & Online Sept 2009
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American Journal of Clinical Nutrition, Vol. 87, No. 5, 1290-1297, May 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Food selection based on total antioxidant capacity can modify antioxidant intake, systemic inflammation, and liver function without altering markers of oxidative stress1,2,3

Silvia Valtueña, Nicoletta Pellegrini, Laura Franzini, Marta A Bianchi, Diego Ardigò, Daniele Del Rio, PierMarco Piatti, Francesca Scazzina, Ivana Zavaroni and Furio Brighenti

1 From the Department of Internal Medicine and Biomedical Sciences (SV, LF, DA, and IZ) and the Department of Public Health (NP, MAB, DDR, FS, and FB), University of Parma, Parma, Italy, and the Medicine Division, Diabetology, Endocrinology and Metabolic Disease Unit, Scientific Institute San Raffaele, Milano, Italy (PP)

2 Supported by the COFIN 2004 project from the Italian Ministry of University and Research and by the EC project "PIPS—Personalised Information Platform for Life & Health Services" (IST-2004-507019).

3 Address reprint requests to F Brighenti, Human Nutrition Unit, Department of Public Health, University of Parma, Via Volturno 39, 43100 Parma, Italy. E-mail: furio.brighenti{at}unipr.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: It is unknown whether diets with a high dietary total antioxidant capacity (TAC) can modify oxidative stress, low-grade inflammation, or liver dysfunction, all of which are risk factors for type 2 diabetes and cardiovascular disease.

Objective: We studied the effect of high- and low-TAC (HT and LT, respectively) diets on markers of antioxidant status, systemic inflammation, and liver dysfunction.

Design: In a crossover intervention, 33 healthy adults (19 men, 14 women) received the HT and LT diets for 2 wk each. Dietary habits were checked with a 3-d food record during both diet periods and the washout period.

Results: Fruit and vegetable, macronutrient, dietary fiber, and alcohol intakes did not differ significantly between the 2 diets, whereas dietary TAC, {alpha}-tocopherol, and ascorbic acid were significantly (P < 0.001) higher during the HT diet. Plasma {alpha}-tocopherol rose during the HT and decreased during the LT diet (P < 0.02 for difference) without changes in markers of oxidative stress except plasma malondialdehyde, which decreased unexpectedly during the LT diet (P < 0.05). Plasma high-sensitivity C-reactive protein, alanine aminotransferase, gamma-glutamyltranspeptidase, and alkaline phosphatase concentrations decreased during the HT compared with the LT diet (mean ± SEM for pre-post changes: –0.72 ± 0.37 compared with 1.05 ± 0.60 mg/L, P < 0.01; –1.73 ± 1.02 compared with 2.33 ± 2.58 U/L, P < 0.01; –2.12 ± 1.45 compared with 5.15 ± 2.98 U/L, P < 0.05; and 1.36 ± 1.34 compared with 5.06 ± 2.00 U/L, P < 0.01, respectively).

Conclusion: Selecting foods according to their TAC markedly affects antioxidant intake and modulates hepatic contribution to systemic inflammation without affecting traditional markers of antioxidant status.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Systemic inflammation and oxidative stress appear to be involved in the pathogenesis of type 2 diabetes and cardiovascular disease (CVD) in the context of the metabolic syndrome. On one hand, obesity, hypertension, insulin resistance, and nonalcoholic fatty liver disease are all conditions associated with higher levels of inflammatory proteins, increased markers of oxidative stress, and lower plasma concentrations of antioxidants (1–4). On the other hand, prospective cohort studies have linked the consumption of fruit and vegetables to a decreased risk of cardiovascular events (5) and the intake of green leafy or dark-yellow vegetables and coffee to a reduced risk of type 2 diabetes (6–8), which suggests a protective effect of dietary antioxidants (9).

Indeed, public campaigns have been launched in several countries to increase the amount of fruit and vegetables consumed by the general population (10–12) as a tool for the primary prevention of chronic disease, partly because of the contribution of fruit and vegetables to overall antioxidant intake. Fruit and vegetables are the main dietary sources of ascorbic acid and carotenoids and contain a large number of phenolic antioxidants (which are more ubiquitous in foods such as coffee, chocolate, tea, red wine, whole-grain cereals, pulses, and nuts) (13, 14). However, even if antioxidant compounds, including the scarce bioavailable phenolics, are thought to play a role in disease prevention, the results of intervention studies with single antioxidants administered as supplements have been poor so far (15, 16).

Recently, the total antioxidant capacity (TAC) of foods, which describes the ability of food antioxidants to scavenge preformed free radicals, has been suggested as a tool for investigating the health effects of antioxidants present in mixed diets (17). We reported an inverse and independent cross-sectional relation between dietary TAC and markers of systemic inflammation [C-reactive protein (CRP) and leukocytes], particularly in subjects with hypertension (17). This suggests that dietary antioxidants could protect against CVD by decreasing CRP concentrations, primarily in subjects at risk. Because CRP is primarily synthesized in the liver, and increasingly so in subjects with intrahepatic fat accumulation, oxidative stress, and liver dysfunction, we hypothesize that dietary antioxidants could modulate low-grade systemic inflammation by counteracting hepatic inflammation and liver dysfunction (18, 19). An additional observation in the above-mentioned study was that almost one-half of the variability in habitual TAC intake was explained by differences in antioxidant content among single food items within any given food group, leading to the hypothesis that fruit, vegetables, grains, and drinks could have different effects on health depending on the antioxidant content of the single items actually consumed (17).

To test the above hypotheses, we conducted a crossover intervention study to investigate the effects of a diet naturally rich in antioxidants compared with a diet low in antioxidants, both containing the same amount of fruit, vegetables, alcoholic beverages, and fiber, on markers of antioxidant status, systemic inflammation, and liver dysfunction. Secondary endpoints were traditional risk factors for CVD (namely insulin resistance, blood pressure, and the lipid profile) and adipokines.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Nineteen men and 15 postmenopausal women from a cohort of apparently healthy workers and exworkers of a local food company who were enrolled in a follow-up survey on diabetes and CVD (20) volunteered to participate in the present intervention study. Exclusion criteria were diabetes mellitus, cardiovascular events, evidence of hepatitis B virus or hepatitis C virus infection, chronic liver diseases or nephropathies, cancer, organ failure, smoking, last menses within the past 12 mo, taking cholesterol-lowering or anti-inflammatory medications, and having taken hormone replacement therapy for the past 12 mo. The protocol was approved by the Ethics Committee for Human Research of the University of Parma.

Study design
Subjects consumed a diet with high TAC (HT) and a diet with low TAC (LT) for 2 wk each, with a 2-wk washout (WO) period in between. The order of diets was randomly assigned. The subjects were also instructed to maintain their usual level of physical activity and to not consume supplements of any type during the study. In the last week of the HT, LT, and WO periods, the subjects completed a 3-d food record to assess dietary habits and compliance. At the beginning and the end of each diet, a medical history to check health status and medication use, a brief physical examination including anthropometric variables and blood pressure, and a blood draw for biochemical analyses after the subjects had fasted overnight for 12 h were taken. Liver steatosis was assessed by echography, but the subjects were not recruited on the basis of that characteristic.

Description of diets
Two dietary interventions (HT and LT) were designed to be comparable for energy, macronutrient, fiber, and alcohol intakes but to differ substantially in TAC. To achieve this objective, the subjects were asked to consume a minimum of 5 medium-sized portions of fruit and vegetables daily in both diets, but choices of only high-TAC items were permitted during the HT diet and choices of only low-TAC foods were allowed during the LT diet (21, 22). Similar changes were introduced regarding beverages, sweets, and dressings (Table 1Go). To control for dietary sources of TAC and to enhance compliance, a wide choice of food items permitted during each dietary period was delivered biweekly to the volunteers at home free of charge and in sufficient amounts to cover the intended consumption of each volunteer and his or her household. Volunteers were also instructed to follow suggestions regarding the consumption of first courses, with particular attention to seasoning (ie, use of tomato sauce, olive oil, vinegar, and spices). Finally, the subjects were asked to consume their usual diet during the WO period and to maintain their usual dietary habits relative to the consumption of meat, fish, milk and dairy products, eggs, cereal products, sweets, cakes, and alcohol throughout the whole 6-wk study period.


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  		TABLE 1. Dietary instructions for subjects during the high total antioxidant capacity (HT) and low total antioxidant capacity (LT) diets

 
Data collection
Anthropometric variables (height, weight, and waist circumference) were collected as previously described (17). Blood pressure was measured twice in a standard manner (17). Hypertension was defined as active treatment with blood pressure–lowering medications or systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥ 90 mm Hg on at least 2 occasions out of the 4 d in which blood pressure was measured. A liver ultrasonography was performed to assess the degree of liver steatosis (0, absent; 1, mild; 2, moderate; 3, severe) as defined by Saverymuttu et al (23). All ultrasonographies were performed by the same operator, who was blinded to dietary assignment and laboratory values, using a Hitachi AU 600 echographer equipped with a convex 3.5 MHz transducer (Hitachi Ltd, Tokyo, Japan).

Dietary data
At baseline, the subjects completed a food-frequency questionnaire specially designed to retrieve information on usual antioxidant intake (24). In addition, a certified dietitian trained the subjects to fill in a 3-d weighed-food record that included all foods, beverages, and supplements consumed during 2 nonconsecutive working days plus a weekend day the last week of the LT, HT, and WO periods. The food record relative to the WO period was considered to be representative of the subject's usual diet. The record was checked for completeness and portion sizes within 48 h of compilation by using a book of photographs and standard household measures. Nutrient intakes and TAC were calculated by using an in-house Microsoft ACCESS application (Microsoft Corp, Redmond, WA) linked to the food database of the European Institute of Oncology, which covered >700 Italian foods (25) integrated with the TAC values of >150 raw foods, measured as ferric-reducing antioxidant power (26). Moreover, compliance during the HT and LT periods was assessed by means of a food chart specifically designed to track the number of portions consumed daily of each food item permitted.

Biochemical analyses
High-sensitivity CRP (hs-CRP) was measured by using an ELISA kit (ICN Pharmaceuticals, New York, NY) with a minimum detectable concentration of 0.004 mg/L. Intra- and interassay CVs were 2.3% and 2.5%, respectively. Serum insulin concentrations were measured by microparticle enzyme immunoassay (IMX; Abbott Laboratories, Abbott Park, IL), with intra- and interassay CVs of 3.0% and 5.3%, respectively. For human leptin, adiponectin, and tumor necrosis factor-{alpha}, see the supplemental online material. Fasting plasma glucose, total cholesterol, HDL cholesterol, triacylglycerols, uric acid, aspartate aminotransferase, alanine aminotransferase (ALT), gamma-glutamyltranspeptidase (GGT), and alkaline phosphatase were assessed by a central laboratory using standard methods. LDL cholesterol was calculated by using the Friedewald formula. A complete blood cell count was performed at the Laboratory of Hematology by using a Beckman-Coulter Hmx analyzer (Beckman-Coulter, Miami, FL).

The TAC of plasma was estimated from its ability to reduce a Fe(III)-2,4,6-tri(2-pyridyl)-s-triazine complex to Fe(II)-2,4,6-tri(2-pyridyl)-s-triazine (26) by using a multiplate reader (Tecan, Maennedorf, Switzerland). Each determination was performed in triplicate on plasma obtained from EDTA-collected blood and was analyzed within 4 h of collection. Plasma {alpha}-tocopherol was quantified by reversed-phase HPLC with diode array detection according to the method of Gimeno et al (27), with slight modifications.

Plasma malondialdehyde was detected by the method of Del Rio et al (28). Oxidized LDLs were detected in plasma by a solid-phase two-site enzyme immunoassay (DRG International Inc, Mountainside, NJ), with 2 monoclonal antibodies directed against separate antigenic determinants on the oxidized apolipoprotein B molecule. Finally, protein carbonyls were quantified by the Protein Carbonyl Diagnostic kit (Biocell Corporation Limited, Papatoetoe, Auckland, New Zealand).

Statistical analyses
Statistical analyses were performed by using SPSS (version 14.0; SPSS Inc, Chicago, IL). Preliminary data (17) were used for statistical power calculations (80% power and {alpha} of 5%), showing that 24 subjects had to complete both dietary treatments to detect a change of 2.5 mg/L in hs-CRP concentrations with an SD of 2.9 mg/L. A total of 34 subjects were recruited to allow for dropouts and for nonparametric statistical analysis (15% additional subjects required), because we anticipated that not all clinical and biochemical data would be normally distributed at all time points. This actually applied to certain variables (notably hs-CRP, tumor necrosis factor-{alpha}, and liver enzymes), and given that not all of them could be normalized, we preferred the more conservative approach of using nonparametric tests in all occasions. Clinical and biochemical variables at any given time point (pre- and postdietary variables) were expressed as means ± SDs when the data were normally distributed and as medians (interquartile range) when the data did not follow a normal distribution. Differences in clinical and biochemical variables between pre- and postintervention periods, being normally distributed, were all expressed as means ± SEMs. The homeostasis model assessment was used as surrogate of insulin resistance (29). For comparisons between sexes, the Mann-Whitney U test or chi-square statistics were used as appropriate. Comparisons between pre- and postdiet values and between the LT and HT periods were performed by using Wilcoxon's test for paired samples. All dietary variables were normally distributed. Comparisons among the HT, LT, and WO diets were performed by repeated-measures general linear models and Bonferroni post-hoc tests, with diet as a within-subject factor and the order of intervention as a between-subject factor.

Repeated-measures general linear models were also applied to investigate the interaction between treatment and presence of liver steatosis, after data transformation into rank proportion estimates according to Tukey's formula. All tests were two-sided, and significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immediately after the first visit, a woman randomly assigned to receive the HT diet as the first intervention dropped out for personal reasons. The characteristics at admission of the 33 subjects who completed the protocol are shown in Table 2Go. Five (4 male and 1 female) volunteers were taking antihypertensive medications, and 11 (9 male and 2 female) subjects fulfilled the criteria for having hypertension during the study. The prevalence of hypertension and plasma TAC were significantly higher, and plasma concentrations of HDL cholesterol, leptin, and adiponectin were significantly lower, in men than in women. No other significant difference between the sexes was observed at baseline for the variables studied. Liver steatosis was absent in 14 subjects (8 female), mild in 13 (1 female), moderate in 5 (3 female), and severe in 1 (female).


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  		TABLE 2. Demographic, clinical, and dietary characteristics of the volunteers at admission1

 
Dietary variables
Dietary data, as determined by the 3-d weighed-food record, are reported in Table 3Go. Dietary intakes of energy, macronutrients, and alcohol during the LT, HT, and WO periods were not significantly different. As aimed for in the study design, dietary fiber was not significantly different between the LT and HT periods but was significantly lower during the WO period than during the LT and HT periods (P < 0.01).


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  		TABLE 3. Average daily dietary intake and total antioxidant capacity (TAC) during the high-TAC diet (HT), the low-TAC diet (LT), and the washout period (WO)1

 
As expected, intakes of single antioxidants and dietary TAC was significantly higher during the HT than during the LT period (P < 0.001). The intake of some antioxidants and TAC were also higher during the WO period than during the LT period (P < 0.001 for vitamin E; P < 0.001 for vitamin C; P < 0.001 for TAC) but were lower than during the HT period (P < 0.001 for vitamin C; P < 0.001 for TAC).

The contribution of the antioxidant capacity of specific food categories to daily TAC in the LT, HT, and WO periods is reported in Figure 1Go. The major contributors to TAC during the WO period were alcoholic beverages (28.1%, red wine being the major contributor) and coffee and tea (34.2%, coffee being consumed overwhelmingly more than tea). During the LT diet, the reduction of daily TAC with respect to the WO period was mainly due to a reduced TAC intake from such beverages. Conversely, the increase in daily TAC during the HT period was primarily due to the highest TAC of fruit and vegetables, because the TAC from alcoholic beverages, tea, and coffee remained almost unchanged compared with the WO period. The number of actual portions of fruit and vegetable consumed, as calculated by the compliance charts, was 5.3 ± 0.6 and 5.0 ± 1.4 for the LT and HT periods, respectively (P = 0.604).


Figure 1
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  		FIGURE 1.. Contribution of food categories to daily dietary total antioxidant capacity (TAC) in the high-TAC (HT), low-TAC (LT), and washout (WO) periods. Vegetables represents the sum of vegetables, legumes, and spices; others includes the contribution of chocolate, cereals and cereal-derived products, nuts, sweets, soft drinks, and oils and dressings. FRAP, ferric reducing antioxidant power.

 
Biochemical variables
The study variables at the beginning and end of each dietary intervention, as well as the changes observed during each dietary period, are shown in Table 4Go. Pre-post changes in concentrations of hs-CRP, ALT, GGT, and alkaline phosphatase were significantly different between the HT period and the LT period. Because pre-LT and pre-HT values for hs-CRP differed (P < 0.05), we recalculated the changes in hs-CRP as a percentage of initial values and obtained similar results (data not shown). No significant differences between dietary treatments were observed for changes in body weight, plasma glucose, insulin, homeostasis model assessment of insulin resistance index, lipid profile, uric acid, aspartate aminotransferase, adipokines (leptin, adiponectin, tumor necrosis factor-{alpha}; see supplemental online material), or leukocytes.


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  		TABLE 4. Values for the study variables before and after the low and the high total antioxidant capacity (TAC) diets (LT and HT, respectively) and changes ({Delta}) observed during each dietary period1

 
Plasma {alpha}-tocopherol rose after the HT diet and decreased after the LT diet, and the changes from before to after were significantly different between treatments. Unexpectedly, plasma malondialdehyde decreased during the LT period and the changes from before to after were significantly different between treatments. Other markers of antioxidant status (TAC, antibodies against ox-LDL, and protein carbonyls) remained unaffected.

To explore whether the presence of liver steatosis played a role in the differential effect that both diets had on plasma concentrations of hs-CRP and liver enzymes, we performed a general linear models analysis considering liver steatosis as a between-subject factor. Neither the sex distribution nor plasma concentrations of hs-CRP or liver enzymes were significantly different between the groups with or without liver steatosis at baseline (data not shown). Moreover, the liver steatosis x treatment interaction was not significant (P = 0.125).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous dietary intervention studies performed to evaluate the biological effects of antioxidants in foods were aimed at increasing the intake in the intervention arm of single antioxidant-rich foods or food groups, while scarcely controlling for other dietary factors (30, 31). To our knowledge, this is the first study designed to extensively modify antioxidant intake from food sources by selecting, within a given food group, items with either the highest or the lowest TAC while maintaining underlying dietary habits. Indeed, the HT and LT diets did not differ regarding the intake of macronutrients, dietary fiber, alcohol, or daily servings of fruit and vegetables, but ensured significantly different intakes not only of dietary TAC but also of single antioxidant molecules.

Compliance with the intervention is supported by the reciprocal changes in plasma {alpha}-tocopherol during the LT and HT periods, which mirrored dietary intake and differed significantly between the diets. Nevertheless, plasma TAC was practically unmodified by dietary TAC, as already reported in the literature (31, 32). Steady plasma TAC concentrations corresponded with a relative stability of oxidative stress biomarkers, which either did not change during the dietary interventions or even showed slight but significant modifications in unexpected directions.

The lack of effect of an increased antioxidant intake on the balance between antioxidant status and oxidative stress could indicate a homeostatic mechanism in which endogenous antioxidants fluctuate to compensate for a reduced or increased influx of dietary antioxidants (33). The duration of treatment could also have played a role: 2 wk may be long enough to appreciate modifications in the plasma concentration of single antioxidant molecules, but may not be sufficient to allow substantial modifications in total antioxidant balance.

Conversely, a mild but significant beneficial effect of dietary antioxidants on systemic inflammation and liver dysfunction was observed. Specifically, we noted a decrease in ALT, alkaline phosphatase, and GGT activities as well as in plasma concentrations of hs-CRP with a diet naturally rich in antioxidants compared with a diet low in antioxidants. The biological plausibility of these results is sustained by previous evidence in different ways.

First, large cross-sectional studies have shown that ALT and GGT activities are inversely related to plasma antioxidants, and ALT and GGT have therefore been proposed as markers of oxidative stress (34–36). Actually, reactive oxygen species are increasingly produced in the liver after fat accumulation and are known to impair mitochondrial oxidation and function (19). Because antioxidant compounds can neutralize reactive oxygen species, an increased influx of antioxidants to the liver could be likely to induce a decrease in markers of liver damage, as in our study (37).

Second, ALT, GGT, and alkaline phosphatase have shown a strong and direct relation with hs-CRP in population studies (18, 38), which in turn appears to be inversely related to dietary TAC independently of other factors known to decrease hs-CRP, including single antioxidant vitamins and carotenoids (17). Thus, our observation that a high-TAC diet has the simultaneous effect of decreasing liver enzymes and hs-CRP compared with a low-TAC diet further supports the contribution of hepatic inflammation to low-grade systemic inflammation associated with the metabolic syndrome (18, 38) and confirms the possibility of modifying such independent risk factors for type 2 diabetes and CVD with a diet naturally rich in antioxidants (39–43).

The present study, a controlled intervention aimed at drastically changing antioxidant intake from foods and beverages while maintaining the same dietary pattern, may help to elucidate the dissociation between the theoretical benefit from antioxidants in the prevention of nonalcoholic fatty liver disease, type 2 diabetes, and CVD risk, and the practical lack of evidence from intervention studies, with the possible exception of effects of high doses of vitamin E given as a supplement on hs-CRP (44) and markers of nonalcoholic steatohepatitis (45, 46). Actually, most interventions have provided supplements of antioxidant vitamins or carotenoids, which may not be as efficient in restoring the redox network as are antioxidants present in mixed diets (47, 48).

Esmaillzadeh et al (49) recently reported an inverse relation between plasma CRP, the risk of metabolic syndrome, and the quintile of fruit and vegetable intake of a group of 486 Iranian female teachers. Those authors concluded that dietary recommendations to increase fruit and vegetable intake are a primary preventive measure against CVD. However, an indication given by the present work is that the effect of increasing the amount of fruit and vegetables on human health may greatly depend on the TAC of such food items, and this should be taken into consideration when exploring the effects of fruit and vegetable consumption on disease prevention. An indication in this direction was already given by 2 prospective cohort studies in which a lower incidence of type 2 diabetes and lower concentrations of glycosylated hemoglobin were not linked to the consumption of fruit and vegetables per se but specifically to the consumption of those with the highest antioxidant capacity (6, 7). Similarly, Agudo et al (50) recently reported that, beside the amount, the TAC from fruit and vegetables was associated with reduced mortality in the EPIC Spanish cohort.

Obvious limitations of the present study are the small sample size, the short duration of the intervention, and, consequently, the use of surrogate markers of disease as endpoints. For example, we cannot exclude an effect of dietary antioxidants on blood pressure, lipid profile or adipokines, because sample sizes were not calculated for these secondary outcomes, nor on insulin resistance, because the clamp technique was not available. Similarly, some evidence supports an association between improved aminotransferase concentrations and liver biopsy findings in nonalcoholic fatty liver disease patients under treatment (51), as well as a benefit of reduced hs-CRP and liver enzymes on the risk of type 2 diabetes and CVD (39–43). However, whether risk of the above chronic diseases can effectively be reduced through dietary antioxidants needs to be confirmed in larger, long-term intervention trials.

In conclusion, qualitatively selecting food items on the basis of their TAC was a useful and effective approach to demonstrate that, in addition to the quantity, the quality of certain food groups may be crucial to decreasing hepatic and systemic inflammation, both of which are independent risk factors for chronic disease. That such a result was achieved without modifying biomarkers of antioxidant status opens new perspectives to investigate the mechanisms of action of antioxidant-rich foods. In the meantime, giving preference to foods naturally high in TAC could be a simple approach to further improving dietary habits.


   ACKNOWLEDGMENTS
 
We thank Elisa Campanini and Dirce Gennari from the Department of Public Health and the Department of Internal Medicine and Biomedical Sciences, respectively, for their skilful assistance in laboratory analyses; Filippo Numeroso from the Department of Internal Medicine and Biomedical Sciences for performing the liver ultrasound examinations; and Serena Marks for kindly revising our English writing.

The contributions of the authors were as follows—SV, LF, DA, and MAB: participated in requesting approval from the ethics committee, subject recruitment, collection and interpretation of the clinical data, and subjects' management; IZ: responsible for requesting approval from the ethics committee, subject recruitment, collection and interpretation of the clinical data, and subjects' management; DDR, MAB, NP, and FS: responsible for the collection and interpretation of dietary and laboratory data of antioxidant status biomarkers; PP: supervised the analysis and interpretation of biochemical data; SV, NP, DDR, and FB: were the primary authors of the manuscript, and all other authors provided input; FB and NP: were responsible for study concept and design; FB and IZ: secured the funding for the study. None of the authors had any personal or financial conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication May 23, 2007. Accepted for publication December 20, 2007.





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