Intake of alcohol-free red wine modulates antioxidant enzyme activities in a human intervention study

Pharmacological Research 65 (2012) 609–614

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Intake of alcohol-free red wine modulates antioxidant enzyme activities in a human intervention study M.A. Noguer a , Ana B. Cerezo b , E. Donoso Navarro a , M.C. Garcia-Parrilla b,∗ a b

Servicio de Bioquimica Clinica H.U. Puerta de Hierro-Majadahonda, Madrid, Spain Área de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Sevilla, C/P. García González-2, 41012 Sevilla, Spain

a r t i c l e

i n f o

Article history: Received 22 January 2012 Received in revised form 7 March 2012 Accepted 7 March 2012 Keywords: Wine Alcohol-free wine Antioxidant enzymes SOD Catalase Glutathione reductase

a b s t r a c t Wine intake affects the antioxidant enzyme activities that contribute to the overall antioxidant properties of wine. The purpose of this study is to evaluate whether alcohol-free wine has any effect on antioxidant enzymes. The study was a randomized cross-over human intervention. A low phenolic diet (LPD) was designed to prevent interference from polyphenols in other food sources. In the first period, the volunteers ate only this low phenolic diet; in the second, they ate this diet and also drank 300 mL of alcohol-free wine. The enzymes under study were: superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The activities of glutathione reductase, superoxide dismutase and catalase decreased during the LPD period and increased in the LPD + dealcoholized wine period. On the third day of intervention, significant changes were observed in glutathione reductase and superoxide dismutase activity for both intervention periods under study. Catalase activity changed significantly on the seventh day of intervention. Antioxidant enzymes modulated their activity more easily than the endogenous antioxidants, which did not undergo any changes. Our results show for the first time that the increase in the activity of the antioxidant enzymes is not due to the alcohol content in wine but to the polyphenolic composition. Therefore, alcohol-free wine could be an excellent source of antioxidants to protect people suffering from oxidative stress (cancer, diabetes, alzheimer, etc.) who should not consume alcohol. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Polyphenols are well known dietetic antioxidant compounds that have in vitro radical scavenging properties against a wide range of radicals and tests [1,2]. However, their bioavailability is rather limited as they are poorly absorbed, and fully metabolized into methylated, sulphated or glucuronide metabolites in the enterocyte. The radical scavenging activity of polyphenol metabolites, if they have any, is much lower. On the other hand, dietary polyphenols greatly depend also on their transformation into aglycones, better absorbed in the intestine, by specific components of the gut microbiota via esterase, glucosidase, demethylation, dehydroxylation and decarboxylation activities [3–5]. Therefore, circulating polyphenols as such are very limited. Thus, the hypothesis that polyphenols exert a direct radical scavenging action on free radicals formed in the body is rather limited [4].

∗ Corresponding author. Tel.: +34 954556760; fax: +34 954233765. E-mail address: [email protected] (M.C. Garcia-Parrilla). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2012.03.003

However, various mechanisms have provided evidences supporting the efficacy of polyphenols regulating enzyme activities, such as resveratrol activating the catalytic activity of sirtuins in animal models [6–9] or anthocyanins reducing the activity of NADPH oxidase in dialysis patients [10] and glucose production via AMPK activation [11]. Antioxidant enzymes are the first line of defense against reactive oxygen species: that is to say, they prevent them from forming. In this regard, enzymatic systems in cells and body fluids control the level of reactive species which otherwise might generate a cascade of products and lead to attacking oxidants. The main classes of antioxidant enzymes in our antioxidant defense system are the superoxide dismutases (SOD), catalases (CAT) and glutathione peroxidases (GPx) [12]. SOD catalyzes the dismutation of superoxide to hydrogen peroxide, CAT catalyzes the conversion of H2 O2 to water (preventing hydroxyl radicals from being generated) and GPx reduces lipid hydroperoxides to their corresponding alcohols and free hydrogen peroxide to water [13]. Under oxidative stress conditions, antioxidant enzymes modulate their activities and play a role in vascular function [14]. Indeed, ecSOD expression is depressed by atherosclerosis [15]

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and increased by treatment with angiotensin-converting enzyme inhibitors [16]. It is also increased by estrogens which contribute to its vasoprotective effects [17]. Recently, the hypothesis that diet or dietetic components can modulate antioxidant enzyme activities has been explored. These components could be an underlying mechanism contributing to the vascular protective effect of polyphenolic compounds. In vitro and in vivo studies with animal models have shown that antioxidant enzymes can be modulated by polyphenolic compounds. Strawberry polyphenols activate the antioxidant enzymes SOD and CAT in the gastric mucosa of rats [18]. Surco-Laos et al. [19] have recently proved the influence of (+)-catechin and its methylated metabolites (200 ␮M) on the resistance to oxidative stress of C. elegans. Puiggròs et al. [20] proved that grape seed procyanidin extract (1 g/kg) increased the Cu/Zn-SOD activity in rats and Fao cell line hepatocytes (15 mg/L). Robb and Stuart [21] revealed that polyphenols, such as resveratrol, kaempferol, and genistein, upregulate the antioxidant MnSOD enzyme in myoblasts, fibroblasts and neuroblastoma cells, increasing their stress resistance. However, human intervention studies must be carried out to show whether a dietetic intake of polyphenolic compounds can modulate antioxidant enzymes or not. Our previous work revealed that SOD, CAT and glutathione reductase (GR) activities significantly increased after one week of wine consumption [22]. As alcohol is known to be an enzymatic inductor, the effect of wine consumption on antioxidant enzyme activities could also be due to ethanol. Thus, the aim of the present work is to evaluate whether the activity of antioxidant enzymes is modified by alcohol-free wine in a human intervention study due to its polyphenolic compounds.

Table 1 Diet list provided to the volunteers to follow during the intervention periods. Food

Devoided food

Vegetables

Garlic

Allowed food Spinach

Eggs and dairy products

Pumpkin Tomatoes Onion Broccoli Olive Cauliflower Aubergine Apple Melon Oranges Cherry Strawberry Peach Lemon Mango Kiwi Grapes Tea Wine Beer Chocolate Alcoholic drinks Juice Dairy products with fruits

Meat

Meat prepared with spices

Cereals and legumes

Lentil Chickpea Integral food Olive oil Ketchup Jam Spice Dried fruit

Fruit

Beverages

Miscellanea

Potatoes (not peel) Carrots Cucumber Mushroom Lettuce Pear Banana Pineapple

Water Coke Sprite Milk

Butter Cheese Yougurt Ice-cream All kind of meat, fish and seafood

Salt Vinegar Mayonnaise

2. Materials and methods 2.1. Alcohol-free wine The red-wine samples were purchased by E. Cantós (IFAPA, Jerez, Spain) and the antioxidant activity of the wine determined by FRAP method was 1982.70 ± 0.32 ␮mol Fe2+ /L wine. The alcohol was removed by rotary vacuum distillation (Laborota 4003-control (Heidolph) connected to a water jet vacuum system). A water bath maintained the temperature of the sample at 30 ◦ C during the extraction. 2.2. Study design and participants Eight volunteers aged 25–40 (28 ± 5.3) participated in the study. The subjects reported no previous cardiovascular, hepatic, gastrointestinal, or renal diseases. Four-week prior to the start of the intervention, they had taken no vitamin or mineral supplements and not any consumed drugs or antibiotics. Ethical approval for the study was obtained from the Ethical Research Committee of the University of Seville. Volunteers followed a low phenolic diet specially designed for the study; all volunteers drank the same alcohol-free wine. They were provided with a brochure listing all the foods they should consume or avoid during the intervention study. The foods withdrawn from the diet are listed in Table 1 as follows: almost all fruits, a large number of vegetables, virgin olive oil, tea and chocolate. They were asked to avoid all alcoholic drinks, during the intervention period. Volunteers filled in a 24-h dietetic questionnaire every day to make sure they had properly followed the recommendations. Fig. 1 displays the intervention design. The previous wash-out period lasted two days. Throughout this time volunteers did not drink wine or other alcoholic beverages and they followed the low phenolic diet (LPD) described above. The intervention was a

crossover design. Volunteers were randomly assigned. The first week, four volunteers followed the low phenolic diet and drank 300 mL dealcoholized wine (DW) every day during dinner for a period of 7 days and the other four just followed the low phenolic diet. During the second period they changed. In order to minimize dietetic interferences, we asked the subjects to consume the same foods as they did in the first period, and provided them with a copy of the questionnaires they had filled in during the first week of the intervention. 2.3. Blood sampling Four venous blood samples were taken from overnight-fasted (8 h) subjects: on day 0, before wine ingestion (baseline value), and on days 1, 3 and 7 after consuming alcohol-free wine every day. Blood samples were obtained by antecubital venipuncture into a sodium citrate vacutainer (BD Vacutainer® CPTTM , REF 362781). Two aliquots of blood were separated to determine the GPx activity and GSH/GSSG ratio. Blood samples were immediately centrifuged at 3500 rpm for 5 min, and unnecessary exposure to light was avoided. Plasma aliquots (for GR and CAT activity, antioxidant activity: ORAC and FRAP and biochemical laboratory determinations) and erythrocytes (for SOD activity determination) were obtained and stored at −80 ◦ C until analysis. 2.4. Biochemical laboratory determinations Hemoglobin, glucose, proteins, creatinine, ALT, AST, cholesterol, triglycerides, HDL, LDL, VLDL, urea, albumin, bilirubin, and uric acid were analyzed in plasma using automated photometric techniques (ADVIA 2400 Siemens). Reactive “C” protein (RCP) was measured by the BN II (Dade-Behring) Siemens automated nephelometric

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Fig. 1. Scheme of the intervention study.

method. The results for all volunteers were in normal ranges before the start of the study.

spectrophotometrically at 540 nm with 4-amino-3-hydrazino-5mercapto-1,2,4-triazole (Purpald) as the chromogen.

2.5. Antioxidant enzyme activity

2.6. Antioxidant activity

Enzymatic kits were purchased from Randox Laboratories Ltd, UK (Superoxide dismutase SD125, Glutathione reductase GR2368 and Glutathione peroxidase RS504); Bioxytech® OxisResearch (GSH/GSSG) and Cayman chemical (Catalase activity catalogue no. 707002). SOD, GR, GPx, CAT activity and GSH/GSSG were determined in a Genesys 10␮V Thermo electron corporation spectrophotometer. All determinations were performed in duplicate. SOD activity was spectrophotometrically determined in erythrocytes. The assay principle is based on the role that SOD plays in converting toxic superoxide radicals produced during oxidative energy processes to hydrogen peroxide and molecular oxygen. The method uses xanthine and xanthine oxidase (XOD) to generate superoxide radicals that react with 2-(4-iodophenyl)3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye, detectable at 560 nm. SOD activity is measured by the degree of inhibition of this reaction. Determinations were performed in duplicate. GR activity was determined in plasma. The method is based on the reduction of glutathione in the presence of NADPH which is oxidized to NADP+ . The absorbance at 340 nm was measured by a Genesys 10␮V Thermo electron corporation spectrophotometer. Determinations were performed in duplicate. GPx activity was measured in whole blood. The glutathione peroxidase enzyme catalyzes the oxidation of glutathione by cumen hydroperoxide. In the presence of glutathione reductase and NADPH the oxidized glutathione (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+ . The decrease in absorbance at 340 nm is measured. Drabkin’s reagent (Randox Cat n◦ MS181) was used to dilute blood. GSH/GSSG activity was measured in whole blood. Reduced glutathione (GSH) is an antioxidant that provides reducing equivalents for the glutathione peroxidase-catalyzed reduction of hydrogen peroxide and lipid hydroperoxides to water and the respective alcohol. During this process GSH becomes oxidized glutathione (GSSG). The GSSG is then recycled into GSH by glutathione reductase and ␤-nicotinamide adenine dinucleotide phosphate (NADPH). The method for determining CAT activity is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured

Antioxidant activity was measured in plasma by the Ferric Reducing Ability (FRAP) method. This method measures the reducing power of plasma [23]. Three milliliters of FRAP reactive (10:1:1) acetate buffer (300 nM, pH 3.6), TPTZ (10 mM in HCl 40 mM), and FeCl3 ·6H2 O 20 mM were used; 100 ␮L of problem solution and 300 ␮L of Milli Q water were also used. The absorbance was measured after 8 min at 593 nm. Results are expressed as ␮mol/L of ferric reducing antioxidant power (FRAP value) [23]. The equipment used was a UV–vis F-2500 Hitachi spectrophotometer connected to a bath to keep the temperature at 37 ◦ C. In the Oxygen Radical Absorbance Capacity (ORAC) method, fluorescein (FL) was used as a fluorescent probe [24]. AAPH was used as a peroxyl radical generator and TROLOX as an antioxidant standard. The fluorescence was recorded (excitation wavelength, 490 nm; emission wavelength, 515 nm) every 5 min for 60 min, until the final value was less than 5% of the initial one. The results are expressed as ORAC values or TROLOX equivalents (␮mol/L) and obtained by integrating the area under the curve (AUC) described by the fluorescence signal over time. The measures were taken in a Synergy HT multiplate reader (BioTek). 2.7. Statistical analysis An ANOVA paired-test was performed with the Statistica software package to assess if there were any differences within the measurements over time. A p-value ≤ 0.05 was considered significant. 3. Results and discussion Tables 2 and 3 show the mean values and standard deviations for endogenous antioxidants (uric acid, bilirubin, albumin) as well as the lipid profile (total cholesterol, HDL, LDL, triglycerides), hepatic damage (ALT, AST, GGT) and inflammatory marker (CRP) for the two dietetic intervention periods under study. CRP is an inflammatory marker related to cardiovascular disease. ANOVA did not show significant differences (p < 0.05) for any endogenous antioxidants, lipid profile, GGT or CRP in either period. These results agree with

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Table 2 Mean values and standard deviation of volunteers (n = 8) for lipid profile, hepatic profile and inflammatory marker. Period

Days

Lipid profile

Hepatic profile

Total cholesterol (mg/dL) LPD

LPD + DW

0 1 3 7 0 1 3 7

162 169 175 179 183 175 171 176

± ± ± ± ± ± ± ±

21 25 21 20 25 22 35 30

HDL (mg/dL) 43 42 43 44 53 51 50 51

± ± ± ± ± ± ± ±

LDL (mg/dL)

8 9 10 13 17 14 15 13

108 106 113 120 114 110 114 112

± ± ± ± ± ± ± ±

25 26 20 14 14 16 25 16

Triglycerides (mg/dL) 61 65 67 71 74 70 60 83

± ± ± ± ± ± ± ±

17 26 21 23 44 20 15 41

ALT (U/L) 18 17 17 16 19 19 17 22

± ± ± ± ± ± ± ±

4 3 3 4 4 4 3 8

Inflammatory marker

AST (U/L) 21 25 22 20 21 21 22 23

± ± ± ± ± ± ± ±

9 8 3 4 3 4 4 7

GGT (U/L) 15 14 14 15 14 13 14 16

± ± ± ± ± ± ± ±

8 7 6 9 7 8 9 9

CRP (mg/L) 1.3 0.9 0.8 0.5 0.6 0.5 0.3 0.3

± ± ± ± ± ± ± ±

0.3 0.1 0.1 0.1 0.1 0.3 0.2 0.1

Table 3 Endogenous antioxidants and antioxidant capacity results (mean values and standard deviation, n = 8). Days Endogenous antioxidants 0 LPD 7 0 LPD + DW 7 a

Uric acid (mg/dL) 4.8 5.0 4.7 4.3

± ± ± ±

0.8 0.6 0.6 0.7

Albumin (g/dL) 4.8 4.3 4.3 4.6

± ± ± ±

1.2 0.4 0.4 0.1

Bilirubin (mg/dL) 0.4 0.4 0.5 0.3

± ± ± ±

0.1 0.1 0.2 0.2

FRAP (␮M Fe2+ /L) 476 550 524 593

± ± ± ±

95 32 12a 15a

ORAC (␮M trolox/L) 2657 2357 2586 3109

± ± ± ±

406 446 473 496

GSH/GSSG ratio 39 43 41 36

± ± ± ±

11 20 26 13

Significant differences (p < 0.05) compared with baseline activity at 0 day in each intervention phase.

many studies reporting no changes in the lipid profile of volunteers after a month of drinking wine or spirit [25]. Conversely, Giovanelli et al. [26] reported a significant increase in triglycerides and cholesterol after one month of a low proanthocyanidin diet and 500 mL of dealcoholized wine intake. GGT is the most sensitive marker of alcohol intake because its synthesis is induced by alcohol consumption. Chronic alcohol consumption increases aminotransferases with a characteristic AST/ALT ratio >2 [27]. Additionally, some authors consider that GGT is also a marker of oxidative stress [28] because it catalyzes the first step in the degradation of extracellular glutathione. Our data (Table 2) show that there are no significant changes in GGT, ALT or AST levels in either of the two periods under study for either the low phenolic diet or the antioxidant status. Antioxidant activity determined with the FRAP method had previously been found to be related to uric acid increase [22] after 300 mL of acute wine consumption. In the present intervention, FRAP increased during the week of LPD + DW consumption but uric acid did not. On the other hand, no changes on FRAP values were observed during the week of LPD. Red wine is known to increase uric acid levels by two mechanisms: it decreases the renal excretion of uric acid because the blood lactate level increases, and it increases the production of urate, which is secondary to the enhanced turnover of adenine nucleotides [29]. Otherwise, previous studies suggest that red wine increases FRAP values by two separate mechanisms: polyphenols and urate [30]. In the present study, uric acid presents no significant changes (Table 3) but FRAP does in the LPD + DW consumption period. The GSH/GSSG ratio does not change in any of our data sets (Table 3). Previous studies suggest that their levels depend on the non-alcoholic components of red wine. Indeed, there is an enhancement of the antioxidant defense potential in rat kidney and plasma after chronic red wine consumption [31]. The present study shows that the levels of lipid profile, hepatic damage and inflammatory markers, uric acid and GSH/GSSG ratio remain unchanged after the consumption of alcohol-free wine. Table 4 presents the mean values and standard deviation (n = 8) of the antioxidant enzymes under study (glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase).

Figs. 2–4 display the average values and significant changes in the activities of the enzymes – glutathione reductase, catalase, and superoxide dismutase – at the end of the week for both periods under study. The two interventions show opposite trends. Glutathione reductase, superoxide dismutase and catalase decrease their activities during the LPD period and increase during the LPD + dealcoholized wine period. On the third day of intervention, significant changes are observed in glutathione reductase and superoxide dismutase activity during both intervention periods (Table 4). Catalase activity changes significantly on the seventh day of intervention. Additionally, on the seventh day of intervention GR, SOD and CAT activities were statistically higher (p < 0.05) in the period that volunteers drank the alcohol-free wine (Figs. 2–4). A statistically significant effect of alcohol consumption has been observed [32] on plasma GPX activity (p < 0.05) after the analysis of different populations and dietetic intake questionnaires. GPX seems to be increased in drinkers. However, Estruch et al. [25] reported no significant changes in glutathione peroxidase activity after intervention trials with gin or red wine. Glutathione peroxidase does not change at all in our set of data, which agrees with the

Fig. 2. Average values and standard deviation in the activity of the glutathione reductase enzyme on days 0 and 7 of intervention for each period: low phenolic diet (LPD) and low phenolic diet plus dealcoholized wine (LPD + DW). *, Mean value at day 7 is significantly different from that at day 0 (p < 0.05) during the same period. 䊉, Mean value at day 7 in LPD + DW period is significantly different from that at day 7 in LPD period (p < 0.05).

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Table 4 Antioxidant enzymes (GR, CAT, SOD, GP) activities (mean values and standard deviation of volunteers, n = 8).

LPD

LPD + DW

a b

Days

GR (U/L)

0 1 3 7 0 1 3 7

40 36 30 24 28 36 41 50

± ± ± ± ± ± ± ±

5a 12 7a 13a , b 4a 12 11a 14a , b

CAT (nM/min/mL) 9 7 5 3 5 6 7 16

± ± ± ± ± ± ± ±

4a 3 1 1a , b 2a 1 2 5a , b

SOD (U/mL) 58 89 16 8 40 60 150 182

± ± ± ± ± ± ± ±

16a 48 3a 14a , b 13a 20 101a 136a , b

GP (U/L) 8863 11 898 7970 7522 11 346 9992 7158 9721

± ± ± ± ± ± ± ±

413 553 449 499 401 465 330 668

Significant differences (p < 0.05) compared with baseline activity at 0 day in each intervention phase. Significant differences (p < 0.05) between days 7 of both periods.

Fig. 3. Average values and standard deviation in the activity of the catalase enzyme on days 0 and 7 of intervention for each period: low phenolic diet (LPD) and low phenolic diet plus dealcoholized wine (LPD + DW). *, Mean value at day 7 is significantly different from that at day 0 (p < 0.05) during the same period. 䊉, Mean value at day 7 in LPD + DW period is significantly different from that at day 7 in LPD period (p < 0.05).

results obtained by other authors [31] in rat kidney with alcoholfree red wine. We obtained similar results in a previous intervention study testing the effect of wine on antioxidant activities for one week [22]. The experimental design also included dietetic control with LPD. Identical changes were found for catalase and superoxide dismutase, glutathione reductase increased during the wine period and no changes were found during the LPD period. These data allow us to conclude that the LPD does affect our antioxidant enzymes. The intake of wine phenolics increases their activities. As alcohol is known to be an enzyme inductor, the effect of wine consumption on antioxidant enzyme activities could be due to ethanol. In the present study no alcohol was taken. Thus, our results show that

the increase in the activity of the antioxidant enzymes is not due to the alcohol content in wine but to the polyphenol composition. It is shown that antioxidant enzymes modulate their activity more easily than the endogenous antioxidants, which present no change. In our opinion, other oxidative markers require a more intense damaging effect than a mere week-long diet to change. On the other hand, the results from this intervention show that phenolic compounds in diet can exert their antioxidant effects by modulating antioxidant enzymes in humans. The present study demonstrates for the first time that the polyphenol content is responsible for the preventive outcomes of the wine, as well as that alcohol-free wine possesses the same effect on the antioxidant enzymes than wine [22]. Additionally, alcohol-free wine allows people that cannot drink alcohol to enjoy the benefits of the phenolic compounds in wines [33]. Alcoholfree wine could therefore be an excellent source of antioxidants to protect people suffering from oxidative stress (cancer, diabetes, alzheimer, etc.) who should not consume alcohol. 4. Conclusions This paper proves that human antioxidant enzymes (superoxide dismutase, catalase and glutathione reductase) modulate their activities as a result of the diet that the volunteers ate. Alcohol-free wine increases their activities after a week of consumption. This effect can contribute to the overall antioxidant properties of wine. Acknowledgments Authors thank the Ministerio de Ciencia e Innovación (Project AGL 2007-64622), the Junta de Andalucía (Project AGR-07-02480), and the Fondo Europeo de Desarrollo Regional for financial support. Authors are deeply grateful to all volunteers participating in this study as well as to Dr. E. Cantos (IFAPA) for providing the wine. References

Fig. 4. Average values and standard deviation in the activity of the superoxide dismutase enzyme on days 0 and 7 of intervention for each period: low phenolic diet (LPD) and low phenolic diet plus dealcoholized wine (LPD + DW). *, Mean value at day 7 is significantly different from that at day 0 (p < 0.05) during the same period. 䊉, Mean value at day 7 in LPD + DW period is significantly different from that at day 7 in LPD period (p < 0.05).

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