The effects of consumption of organic and nonorganic red wine on low-density lipoprotein oxidation and antioxidant capacity in humans

The effects of consumption of organic and nonorganic red wine on low-density lipoprotein oxidation and antioxidant capacity in humans

Nutrition Research 24 (2004) 541–554 www.elsevier.com/locate/nutres The effects of consumption of organic and nonorganic red wine on low-density lipo...

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Nutrition Research 24 (2004) 541–554 www.elsevier.com/locate/nutres

The effects of consumption of organic and nonorganic red wine on low-density lipoprotein oxidation and antioxidant capacity in humans Yasemin Delen Akc¸aya,*, Hatice Kalkan Yıldırımb, Ulgar Gu¨venc¸b, Eser Yıldırım So¨zmena a

University of Ege, Faculty of Medicine, Department of Biochemistry, 35100 Bornova Izmir, Turkey b University of Ege, Faculty of Food Engineering, 35100 Bornova Izmir, Turkey Received 15 July 2003; accepted 6 December 2003

Abstract It is known that moderate red wine consumption can reduce the risk of cardiovascular disease. The protective effects of wine have been attributed to phenolic compounds that are efficient scavengers of free radicals and breakers of lipid peroxidative chain reactions. Besides antioxidant activity, phenols also have anti-inflammatory effects and may protect low-density lipoproteins (LDL) against oxidative modification. The aim of this study was to determine the effects of the so-called “organic” wines (i.e., those that are produced from genetically nonmodified grapes and without fertilization) and “nonorganic” red wines (i.e., those that are produced in a conventional manner) on LDL oxidation, antioxidant activity, and other antioxidant enzymes such as catalase and superoxide dismutase. Male subjects (n ⫽ 6) drank 200 mL and female subjects drank (n ⫽ 2) 100 mL of red wine (the so-called organic wine) wine, and after 6 weeks the experiment was repeated with the nonorganic red wine. Blood samples were obtained at baseline and after 60 and 360 minutes. Total phenol, erythrocyte superoxide dismutase (e-SOD), erythrocyte catalase (e-CAT), erythrocyte thiobarbituric acid reactive substances (eTBARS), serum total antioxidant activity (AOA), LDL-TBARS, and Cu-stimulated LDL-TBARS levels were determined. Although the Cabernet Sauvignon wine caused a significant increase in eSOD activity during hour 1 (P ⫽ 0.046) and hour 6 (P ⫽ 0.028) of the experiment compared to the baseline levels, it led to an insignificant increase in eCAT activity in hour 1 (P ⫽ 0.08) and hour 6 (P ⫽ 0.069). There was no significant difference between two types of wines with respect to LDL-TBARS blood levels, and only the nonorganic wine led to a decrease in Cu-stimulated

* Corresponding author. Tel.: (90) 232 388 1096; fax: (90) 232 373 9477. E-mail address: [email protected] u.tr (Y. Delen Akc¸ay). 0271-5317/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2004.04.004

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LDL-TBARS. There were noteworthy differences in the alcohol and phenol content of the organic and nonorganic wines. © 2004 Elsevier Inc. All rights reserved. Keywords: Red wine phenol; Organic wine; Nonorganic wine; Antioxidant enzymes; LDL oxidation

1. Introduction Recent studies demonstrated an association between increased intake of wine and reduced risk for cardiovascular diseases [1–7]. The main polyphenolic groups that are found in wines are phenolic acids and flavonoides. Some of these, such as flavon and flavolols (quarcetin, myricetin), catechins [⫹] catechin and [⫺] epicatechin), proanthocyanidins, and benzoic acids may be medically important [8]. Polyphenols are efficient scavengers of free radicals and breakers of lipid peroxidative chain reactions [2,8 –10]. They exert their antioxidant activity by chelating transition metals such as iron and copper ions, which are involved in free radical generation. Besides this well known antioxidant activiy, polyphenols also have anti-inflammatory effects and may protect low-density lipoproteins (LDL) from oxidative modification [2,11,12]. Polyphenols are found in a number of vegatables, spices, and fruits (mainly grapes). The types and concentrations of the polyphenols present in grapes depend mainly upon ecological conditions, grape variety, the degree of grape ripening, and the techniques used in producing the must and in the winemaking itself [13–16]. To investigate the effect of wine processing on the polyphenol content and antioxidative effect, two different wine categories were considered: the so-called organic and nonorganic wines. The idea of organic wine evaluation derived from the increasing trend of consumption of “natural” foods. The main characteristics of the so-called organic wines compared to the nonorganic wines are as follows: grape origin (use of nonhybrid certified grapes), harvesting method (hand-harvesting method), crushing (nonmechanical equipment), yeast type (genetically nonmodified strains), and sulfur treatment (using of ⬍100 ppm for total SO2 and 30 ppm for SO2). Stabilizing and finening agents are prohibited in the production of wines labeled as organic. There are also some special requirements for storage tanks (stainless steel) and corks (high-quality natural corks). Several studies have reported the antioxidant activities of polyphenols of wine [3,4,17,18] but not compared the polyphenols of the so-called “organic” and “nonorganic” wines (referred to throughout article as simply organic and inorganic). Therefore, the present study was designed to determine the effects of polyphenols found in organic and nonorganic red wines (Cabernet Sauvignon) on LDL oxidation, antioxidant activity and other antioxidant enzymes (catalase and superoxide dismutase) in vivo. 2. Methods and materials 2.1. Materials The organic wine Cabernet Sauvignon (CS) was obtained by defined standards (certificated grapes of Vitis vinifera origin, nonmechanically crushed, with active yeasts in musts,

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5% 25 ppm SO2, without using of any stabilizing and fining agent). The nonorganically produced Cabernet Sauvignon wines were obtained by conventional manner (hand harvest, mechanical pressing “Com.Fad.Type G,” with commercial dry yeast 2% “Fermorouge,” 5% 50 ppm SO2, using of 250 g/h L gelatin as fining agent (Merk Darmstadt, Germany), and plate filter “Seitz,D.6800” for filtration). 2.2. Subjects Eight healthy volunteer subjects (six men and two women, aged 24 to 45 years), who were nonsmokers and were assessed as healthy based on their medical histories, participated in the study. Differences in body mass index (BMI) among the subjects were nonsignificant. None of the subjects were taking drugs or vitamin supplements. They were asked to keep their diet as constant as possible before and during the study period. One week before the experiment, the subjects reportedly did not consume any product rich in polyphenols (vegatables, tea, fruit, wine, etc). On the days of the experiment, the subjects consumed a standard breakfast. On the first test day of study, the men drank 200 mL (alcohol content 24 g) and the women drank 100 mL (alcohol content 12 g) of red the organic wine (CS) over the course of 15 minutes. Blood samples were taken in basal conditions as well as 60 and 360 minutes after the wine consumption. On the second test day of study (which was performed after a 6-week wash-out period), subjects were asked to drink from nonorganically produced red wine (CS). The dosage and time consumption for men was 200 mL in 15 minutes (alcohol content 22.4 g) and for women was 100 mL in 15 minutes (alcohol content 11.2 g). Blood samples were obtained again at the same intervals. 2.3. Methods Blood samples were collected in EDTA and heparin containing tubes (Vacuette, Greiner Labortechnic, Austria). Serum and plasma were immediately separated by centrifugation from cells. The serum total antioxidant activity (AOA) was determined immediately. Plasma and hemolyzates were stored at ⫺20°C. 2.4. Serum parameters Serum total cholesterol, triglycerides, and HDL-cholesterol were determined by enzymatic-colorimetric methods with commercialy available kits (Biocon Diagnostik, Germany). LDL-cholesterol levels were calculated according to the Friedman formula. Total antioxidant activity was measured spectrophotometrically (UV-160A, Shimadzu, Japan). The solution of 0.1 mmol/L DPPH (1,1 – diphenyl-2-pikrylhydrazin) was rapidly mixed well with serum. The decline in absorbance was recorded at 517 nm against ethanol blank over a period of 15 minutes. The decreases of absorbance corresponding to 100% radical scavenging was determined with a solution of pyrogallol in DMSO (⬃0.5 %), which caused complete scavenging within seconds. Percent radical scavenging was calculated as 100 ⫻ (Ao ⫺ At) / (Ao ⫺ Ap), where Ao is the initial absorbance and At and Ap are the

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absorbances after 15 minutes with test solutions and with pyrogallol solutions, respectively [19]. Total phenolic content was analyzed according to the Folin-Ciocalteu method [20], calibrating against gallic acid standards, and the results were expressed as gallic acid equivalents (GAE). 2.5. Erythrocyte analyses 2.5.1. Preparation of hemolyzates After separation of plasma from blood samples with heparin, the packed erythrocytes were washed two times with 9 g/L NaCl solution and hemolyzed with ice-cold water (1/5, v/v). eSOD, eCAT activities, eTBARS, levels were determined in haemolyzates [21]. We measured SOD activities, in which inhibition of autooxidation of epinephrine by SOD at 480 nm, measured with an UV-160A spectrophotometer (UV-160A, Shimadzu, Japan) [21]. The assay was calibrated by using purified SOD. We determined eCAT activities according to the method of So¨ zmen et al., in which the degradation of peroxide is recorded spectrophotometrically at 240 nm. One unit of catalase was defined as the amount of enzyme that decomposes 1 mmol H2O2per minute under specific conditions. We measured eTBARS levels according to the method previously reported [21]. After dilution of hemolysates with physiologic saline, they were incubated with TBA–working solution (0.12 mol/L TBA in 15% TCA and 1% HCl) for 30 minutes at 95°C. TBARS concentrations was calculated using a calibration curve constructed from 1,1,3,3 tetra ethoxy propan. 2.6. LDL isolation and in vitro oxidation Plasma samples were incubated at room temperature for 30 minutes with a commercial precipitant reagent (Merck/Darmstadt, Darmstadt, Germany) [21]. After centrifugation at 1600 ⫻ g for 10 minutes, LDL samples were solubilized with 0.15 N NaOH. The LDL oxidation was determined by using TBARS levels directly, in LDL samples containing 200 mg protein. Protein measurements were made according to the method of Lowry et al. [22]. TBARS measurements were performed by incubation the isolated LDL with TBARS solution (0.12 mol/L TBA in 15% TCA and 1% HCl) for 30 minutes at 95° C [21]. In vitro oxidation of LDL was induced by incubating the isolated lipoproteins with 5 mmol/L CuSO4 and TBARS levels at 2 hours were also determined [21]. 2.7. Statistical analyses Statistical analyses of relationships among eCAT, eSOD enzyme activities, eTBARS, AOA, and LDL oxidation were performed by the statistical package SPSS for Windows, version 10.0 (SPSS, Chicago, IL). Using multivariate exploratory techniques, Principal Component Analysis (PCA) (with cases) analysis was performed.

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Fig. 1. Phenol levels of subjects at baseline and at hours 1 and 6 after wine consumption.

3. Results In our study, there was no statistically significant difference in data between male and female subjects therefore we evaluated and presented data of all subjects together. The cholesterol, tryglyceride, HDL-c, and LDL-c levels of subjects were found as 183 ⫾ 27 mg/dL, 78 ⫾ 44 mg/dL, 59 ⫾ 8 mg/dL, and 113 ⫾ 25 mg/dL, respectively. The total phenolic compounds (GAE) of wines were found to be 18.3 mg/mL (organic) and 40.2 mg/mL (nonorganic). Serum phenol levels of subjects are presented in Fig. 1. The data showed that phenol levels reached their peak during hour 1 in subjects who drank the organic wine and during hour 6 in those who drank the nonorganic wine. Although the the organic CS wine caused a significant increase in eSOD activity in hour 1 (P ⫽ 0.046) and hour 6 (P ⫽ 0.028) of the experiment compared to the baseline (Fig. 2), it led to an insignificant increase in eCAT activity during hour 1 (P ⫽ 0.08) and a significant increase in hour 6 (P ⫽ 0.015) (Fig. 3). The nonorganic CS wine led to an increase in eSOD activity in hour 6 of the experiment compared to baseline, and it had no effect on eCAT activity. Evaluation of e-TBARS results in Fig. 4, demonstrates the slow decrease in the organic wine drinkers during hour 1 and the subsequent increase in hour 6, with no observable change in e-TBARS of the nonorganic wine drinkers. Our data demonstrated significant differences for AOA values between the organic/basal, with organic/1 hour (P ⫽ 0.030) and with organic/6 hour (P ⫽ 0.048). The AOA values of the nonorganic wine drinkers decreased to 70% and then increased up to 82.5% during hour 6. The AOA values of the organic wine profiles did not change significantly after hour 1 (Fig. 5). In accordance with this finding, we found negative correlations between the AOA and

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Fig. 2. eSOD activities of subjects at baseline and at hours 1 and 6 after wine consumption.

e-TBARS levels of the organic wine drinkers (r ⫽ ⫺0.713, P ⬍ 0.05) and the nonorganic wine drinkers (r ⫽ ⫺0.897, P ⬍ 0.05) at the first hour. Interestingly, although e-TBARS levels decreased in the organic wine drinkers, it showed a small increase in the nonorganic wine drinkers. Although there was no significant effect of the organic and the nonorganic wines on LDL-TBARS levels during the study period (Fig. 6), the nonorganic wine led to a decrease in the Cu-stimulated LDL oxidation during hours 1 and 6 of treatment, whereas the organic wine had no effect. The comparison of the organic and the nonorganic wine effects was not able to show any statistically significant difference of either wine on any parameter. The effect of the organic wine on copper-mediated LDL oxidation levels (P ⫽ 0.063) during hour 6 was higher than with nonorganic wines. The projection of analysed parameters (e-SOD, e-CAT, e-TBARS, AOA, LDL-TBARS, Cu-LDL-TBARS, total phenols) on the factor plane of the first two factors (36.78% ⫻ 19.09%) are given in Fig. 7. As evident in the figure, three different groups are formed. AOA could be accepted alone and separated from the other groups. Very near to it are found

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Fig. 3. eCAT activities of subjects at baseline and at hours 1 and 6 after wine consumption.

members of the second group (total phenols, e-SOD, e-CAT, and LDL-TBARS) and next the third group (e-TBARS and Cu-LDL-TBARS). 4. Discussion It is known that a moderate red wine consumption is associated with a decrease in risk of coronary heart disease and consequent mortality [1–7]. The protective effects of wine depends on its phenolic compounds (phenolic acids and flavonoids), which have an important role not only as a quality criterion concerning some organoleptical attributes (color, astringency) of wine [23], but also as compounds with medical properties such as antioxidant activity, anti-inflammatory action, inhibition of platelet aggregation, and antimicrobial activities [24]. In this study, we investigated the antioxidant capacity and the effects on antioxidant– oxidant system of wines produced by conventional methods (i.e., the so-called nonorganic wines) with those produced by organic methods. Each subject acted as their own control and three measurements were made at baseline and at hours 1 and 6 of consumption of the organic and nonorganic wines after a 6-week wash-out period.

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Fig. 4. eTBARS levels of subjects at baseline and at hours 1 and 6 after wine consumption.

We determined the serum phenol levels of subjects to exclude individual variations. The total phenolic compounds (GAE) of wines were determined to be as 18.3 mg/mL for organic and 40.2 mg/mL for nonorganic wines. However, there was no statistically significant difference in serum phenol levels between the subjects who drank the organic or the nonorganic wine and during consumption. Although phenol levels showed a peak at hour 1 in subjects who drank the organic wine (351 mg/L GAL), phenols of the nonorganic wine drinkers (349 mg/L GAL) were found to be higher at hour 6. Berteli et al. also demonstrated that resveratrol (a polyphenol) reaches to its peak concentration at 60 minutes after wine ingestion. Experimental studies showed that flavonoids are generally poorly absorbed, with less than 1% of the total administered dose reaching the systemic circulation [25–27]. A possible explanation for this difference in phenol levels may be attributed to the alcohol content of red wine, which was found to be 12.0% and 11.2%, respectively. Chopra et al. suggested that the absorption of some nutrients in wines could have been influenced by the alcohol level [28]. Variations in the intestinal absorption rate of different wine types might be influenced by differences in methods of production. During production, minimal processing is used in the organic wines and no chemical additives are added. The organic winemakers pay particular attention to three factors: the use of yeasts, the filtration/fining method,

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Fig. 5. AOA activity of subjects at baseline and at hours 1 and 6 after wine consumption.

and the use of sulfur dioxide. However, temperature control during the winemaking process is well in use. Minimizing the use of sulfur dioxide as an antioxidant is stringently observed. Each of these parameters could have affected the total phenol content and respectively its absorption. The organic CS wine caused an increase in eSOD activity at hours 1 and 6 of the experiment compared to basal levels, and an insignificant increase in eCAT activity at hour 1 and a significant increase at hour 6. Martinez et al. also showed that resveratrol found in red wine inhibits superoxide radicals and hydrogen peroxide production [29]. Jovanovic et al. demonstrated that flavonoids scavenge the superoxide, hydroxyl, and peroxyl radicals at a high rate of constant reactivity with hydroxyl radical [30]. Therefore, flavonoids have potential SOD-like activity [31], as supported by our findings. However, the nonorganic CS wine led to an increase in eSOD activity at hour 6 of the experiment compared to basal levels, whereas it had no effect on eCAT activity. These differences in peak time of antioxidant enzymes may be due to some factors that affect the intestinal absorption of antioxidant molecules. Besides these factors, possible effects of grape origin and wine-making procedures could be considered. The differences in fermentation medium (homogeneous for

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Fig. 6. LDL-TBARS and Cu-stimulated LDL-TBARS levels of subjects at baseline and at 1 and 6 hours after wine consumption.

nonorganic and heterogeneous for the organic wines); the amounts of added SO2 (⬍100 ppm total and ⬍30 ppm free SO2for the organic wine); the absence of fining agents for the organic wine and the differences in alcohol content of both wine types could affect, either directly or indirectly, intestinal absorption of antioxidant. It is also proposed that storage tanks and corks could have some effects; however, to exclude the possibility of such effects, we used newly produced wine in our experiments. AOA levels of the nonorganic and the organic wine drinkers were higher at hours 1 and 6, respectively, in relation to the elevation in serum phenol levels. The strong negative correlations between the AOA and e-TBARS levels in the organic wine drinkers (r ⫽ ⫺0.713, P ⬍ 0.05) and the nonorganic wine drinkers (r ⫽ ⫺0.897, P ⬍ 0.05) during hour 1 are in agreement with our current knowledge of antioxidant– oxidant status. Durak et al. showed that plasma AOA values were elevated to a significant extent at hour 4 [32]. Various factors such as using of different enzymes, fining with gelatin or betonite, and aging may affect the phenol levels as well as AOA [23,33,34]. Considering the fact that total phenolic content vary by weight and composition, depending on different treatments in production (duration of must fermentation; use of different yeast strains, enzymes, and fining agents), the differences in antioxidant capacity of wines could be expected. Although our data provide evidence for the acute antioxidant effect (SOD activity) of red wine (especially the the organic ones), the two wine types showed no statistically significant differences in LDL oxidation. Cacetta et al. found a significant increase in plasma polyphenol concentration, as presented in this study, but they revealed that this concentration is sufficient to influence LDL oxidation ex vivo [35]. Although several studies suggest that red wine has an antioxidant effect on LDL oxidation, the data obtained from the in vivo studies are conflicting [7,36,37]. In accordance with the study by De Rijke et al. [37], we demonstrated

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Fig. 7. Principal component analysis of study data.

that both the organic and the nonorganic wines had no effect on basal and Cu-stimulated LDL oxidation. In vitro studies have indicated that the required amount to produce an effect on the ex vivo oxidation of LDL varies widely depending on phenolic types. Although we determined the serum total phenol levels, it is not clear at what amount or which phenolic compounds are absorbed from red wine. Further research needs to be done to understand the pharmacokinetics, absorption, and metabolism of wine flavanoids and to explain the potential antioxidant effect of wines in humans. A wide variation in bioavailability has been reported both for catechins and isoflavones

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and interindividual differences in plasma epigallocatechin gallate after consumption of red wine [38]; however, the metabolism and effective dose, as well as their biokinetics, are still incompletely understood [5]. The in vivo antioxidant potential of individual components is not clearly understood; and because the concentrations of the main phenolic components of red wine can vary widely, this leads to discrepancies in the results. From our data, it could be concluded that the long-term consumption of red wine may has more potent antioxidant activity rather than does a single dose, possibly due to the storage ability of flavonoids in the body. In agreement with this conclusion, Ferro-Luzzi and Maiani [38] suggested that excess flavonoids can be stored in body tissues and mobilized in response to physiological requirements. Current data also emphasize regular red wine consumption, rather than an acute dose, as beneficial in regard to the antioxidant potential on LDL oxidation.

Acknowledgments We are very grateful to Ebru Sezer, Erkin Bozdemir, Mehmet Mutlu, Yigˇ it Uyanıkgil, ¨ zgo¨ nu¨ l, Su¨ leyman Kus¸, and Bu¨ lent So¨ zmen for their participation in this study. Mert O

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