Download - Wine, Diet, Antioxidant Defenses, and Oxidative Damage

Ann. N.Y. Acad. Sci.

957: 136–145 (2002). ©2002 New York Academy of Sciences.

Wine, Diet, Antioxidant Defenses, and Oxidative Damage

DRUSO D. PÉREZ,

a

PABLO STROBEL,

a

ROCÍO FONCEA,

a

M. SOLEDAD DÍEZ,

a

LUIS VÁSQUEZ,

a

INÉS URQUIAGA,

a

OSCAR CASTILLO,

b

ADA CUEVAS,

b

ALEJANDRA SAN MARTÍN,

a

AND

FEDERICO LEIGHTON

a

a

Department of Cellular and Molecular Biology and

b

Department of Nutrition, Catholic University of Chile, Santiago, Chile

A

BSTRACT

: Oxidative stress is a central mechanism for the pathogenesis ofischemic heart disease and atherogenesis, for cancer and other chronic diseas-es in general, and it also plays a major role in the aging process. Dietary anti-oxidants constitute a large group of compounds that differ in mechanism ofaction, bioavailability and side effects. A systematic analysis of the role of thevarious antioxidants in chronic diseases is hampered by the difficulty ofemploying death or clinical events as end points in intervention studies. There-fore, valid markers for oxidative stress, which show dose response and are sen-sitive to changes in dietary supply of antioxidants, are potentially of greatvalue when trying to establish healthy dietary patterns, or when one compo-nent, like red wine, is evaluated specifically. To evaluate potential oxidativestress markers we have studied the effect of different diets plus wine supple-mentation on antioxidant defenses and oxidative damage. In three experimen-tal series, four groups of young male university students, one of older menand other of older women, 20–24 volunteers each, received Mediterranean oroccidental (high-fat) diets alone or supplemented with red wine, white wine,or fruits and vegetables. Measurements included, leukocyte DNA 8-OH-deoxyguanosine (8OHdG), plasma 7

�

-hydroxycholesterol, TBARS and well-characterized antioxidants, and plasma and urine polyphenol antioxidants. Inall experimental groups that received red wine, consumption resulted inmarked decrease in 8OHdG. The changes observed in 8OHdG correlate posi-tively with the other markers of oxidative damage, and shows a clear inversecorrelation with the plasma level of well established antioxidants and withmeasurements of total antioxidant capacity. Urinary total polyphenol contentas well as the sum of some specific plasma species also correlate inversely with8OHdG. In conclusion, the results identify 8OHdG as a very promising generalmarker of oxidative stress in nutrition intervention studies in humans, and redwine shows a remarkable protective effect.

K

EYWORDS

: 8-OH-deoxyguanosine; oxidative stress; antioxidants

Address for correspondence: Federico Leighton, M.D., Faculty of Biological Sciences,Catholic University of Chile, Casilla 114-D, Santiago, Chile. Voice: 56-2-222 2577; fax: 56-2-222 2577.

[email protected]

137PEREZ

et al.

: WINE AND OXIDATIVE DAMAGE

INTRODUCTION

Human organisms are necessarily exposed to a certain degree of oxidative dam-age, a consequence of aerobic life and energy metabolism. The intensity of this oxi-dative stress will depend on the rate of generation of free radicals and other activeoxygen and nitrogen oxidative species, and on antioxidant defenses. Diet composi-tion influences both, oxidative damage and antioxidant mechanisms and thisexplains, at least in part, the relationship among diet and some chronic diseases likeatherosclerosis and cancer.

1–3

Diets rich in fruits and vegetables are associated withdecreased risk of cancer and cardiovascular disease. Biomarkers of oxidative DNAdamage and lipid peroxidation can be used to establish the role of antioxidant in thisprotection and the optimal intake of those antioxidants. Oxidative DNA damage is asignificant contributor to the age-related development of some cancers. Also lipidperoxidation play a key role in the development of cardiovascular disease. This is thebase of the oxidative hypothesis of atherogenesis that proposed oxidized LDL as theprincipal agent of damage, being the endothelial cell the main target for the oxidizedLDL-mediated damage.

4,5

Many studies have evaluated the effectiveness of antiox-idant vitamins supplementation.

1,2

Also the lower incidence of coronary artery dis-ease associated to diets rich in fruits, vegetables and wine, could be largely explainedby the antioxidant content of these diets.

3,6

To evaluate the prooxidant and antioxi-dant effect of currently used diets and the eventual contribution of moderate wineconsumption, volunteers were studied, choosing oxidative leukocyte DNA damage(DNA 8-OH-deoxyguanosine content) as a marker of systemic oxidative stress,

7,8

together with TBARS and 7

β

-hydroxycholesterol as markers of lipid oxidativedamage,

9,10

plasma antioxidants levels

11

and TAR and TRAP

12

as markers of anti-oxidant status, and endothelial function

13,14

as a marker of oxidative stress andatherogenic risk. The results show a close correlation among plasma antioxidant andoxidative damage, especially with plasma levels of vitamin C and polyphenols. Thisconfirm the hypothesis that the antioxidants present in wine, fruits and vegetables;vitamin C, polyphenols and others, contribute to the healthy effect of some diets.

METHODS

Six groups of 20–24 volunteers each, participated in three diet and wine interven-tion studies, in the years 1998, 1999, and 2000. Subjects were healthy male univer-sity students aged 20–28 years, and older men and women aged 53–73 years from agerontology center, who signed an informed consent. The Faculty of Medicine ethicscommittee approved the study protocols. For the 1998 and 2000 experimental proto-cols, the volunteers received for a period of 90 or 60 days, respectively, either a high-fat (occidental diet) or a diet rich in fruits and vegetables (Mediterranean diet).

15

Inthe 1999 experimental protocol, we supplied for 90 days two high fat diets (40% ofcalories from fat), which differed in the vegetable oil employed: sunflower oil(PUFA diet, rich in polyunsaturated fatty acids) and olive oil (MUFA diet, richin monounsaturated fatty acids). They received 14.5 or 4.2 percent calories fromPUFA and 12.6 or 22.2 percent calories from MUFA. During the wine or fruit andvegetables supplementation periods, that lasted three or four weeks each, men

138 ANNALS NEW YORK ACADEMY OF SCIENCES

received 240 mL/day of white wine or red wine and women 120mL/day, or alterna-tively a supplement of fruits and vegetables, keeping constant the total caloric intake.No other alcoholic beverage was allowed during the entire study.

Blood samples were collected after overnight fasting. Plasma, serum and leuko-cytes samples were stored following common laboratory procedures until they wereanalyzed.

The endothelial function was measured noninvasively as flow-mediated vasodila-tion of the brachial artery, as previously described.

15

The 8-OHdG and 2-deoxyguanosine (dG) in DNA from peripheral blood leuko-cytes were measured by HPLC with electrochemical (EC) and diode array (DAD)detection.

16

Briefly, DNA (100–150

µ

g) was solubilized in 20mM acetate buffer(pH 5.3), digested with nuclease P1 (1mg/mL) and hydrolyzed with alkaline phos-phatase (1.5 units). The hydrolysate was filtered and DNA bases were separated byHPLC using a Supelco LC-18 column with 50mM KH

2

PO

4

(pH 5.5) containing10% methanol (1 mL/min) as eluent. The 8-OHdG levels were quantitated with anelectrochemical detector and compared to the amount of dG quantitated by DAD.The amount of 8-OHdG in the sample was expressed as 8-OHdG per 10

5

dG. Vitamin E (as

α

-tocopherol),

β

-carotene, lycopene, and ubiquinol were measuredin plasma using HPLC with electrochemical detection.

11

Serum vitamin C (ascor-bate) was detected by a spectrophotometric procedure based on the reduction offerric chloride.

17

Total plasma antioxidant capacity was determined as TAR (TotalAntioxidant Reactivity) and TRAP (Total Reactive Antioxidant Potential) fromluminol-enhanced chemiluminescence measurements.

12

Results were expressed as

µ

M Trolox equivalent.Plasma polyphenols were analyzed by HPLC using electrochemical and diode

array detection and results were expressed as

µ

M rutin equivalent.

18

The Folin-Ciocalteau method was used to determine total polyphenols in urine and resultsexpressed as gallic acid equivalents.

19

Lipid peroxidation was assessed by measuring the levels of thiobarbituric acidreactive substances (TBARS),

20

and plasma 7

β

-hydroxycholesterol by gas chroma-tography method.

21

Results are presented as mean

±

SEM. The measurements within the same group,at different times, were compared by paired

t

-test and analysis of variance (ANOVA)for repeated measurements with Bonferroni adjustment. Bivariant correlation anal-ysis was performed according to Pearson. Values

p

<

0.05 were considered statisti-cally significant.

RESULTS AND DISCUSSION

Oxidative Damage Markers

Although free radicals and reactive oxygen species (ROS) are normally generatedduring cell metabolism they are implicated in the pathogenesis of several humandiseases.

7,8

ROS can interact with biological molecules, such as DNA, lipids and pro-teins, with potentially deleterious consequences. One of the products of DNA oxida-tion, 8-hydroxy-2-deoxyguanosine (8-OHdG), has been extensively investigated

139PEREZ

et al.

: WINE AND OXIDATIVE DAMAGE

because it can be measured with high sensitivity, and its levels in target tissues arecorrelated with oxidative stress and with the incidence of cancer.

22,23

Thus 8-OHdGis a useful and biological relevant marker for the study of oxidative stress. Often stud-ies are performed on DNA isolated from blood leukocytes, and it is assumed thatchanges reflect oxidative stress in organisms.

1

Lipid peroxidation

in vivo

is likely to contribute the development and progressionof atherosclerosis and other chronic diseases.

4,6,9

Lipid oxidation in human can beassessed by measurement of lipid oxidation products, such as 7

β

-hydroxychol-esterol, a major oxidation product of cholesterol, and lipid hydroperoxides asthiobarbituric acid-reactive substances (TBARS). Both apparently are markers forcardiovascular risk.

9,10

Oxidative stress markers, or biomarkers, are needed to investigate which diet orparticular dietary constituents are capable of reducing the oxidative damage to DNA,lipids and proteins

in vivo

.

1

The present study summarizes the effects of different diets and of wine supple-mentation on oxidative damage and plasma antioxidants in healthy volunteers. Theoxidative damage associated to different diets and the eventual protective effect ofred wine supplementation were evaluated with biomarkers, such as 8-OHdG contentin human DNA leukocytes, plasma TBARS and 7

β

-hydroxycholesterol. The valuesfor 8-OHdG in leukocytes correlate significantly with TBARS and with 7

β

-hydroxy-cholesterol, 0.215 (

p

=

0.002) and 0.152 (

p

=

0.012), respectively (see F

IGURE

1 andT

ABLE

1). These results support the validity of these markers to assess oxidativedamage in humans.

T

ABLE

1. Correlation of 8-OHdG with oxidative damage, plasma antioxidants andantioxidant capacity; all studies combined

R

p n

TBARS 0.215 0.002 211

a

7

β

-OH cholesterol 0.152 0.012 269

b

Vitamin C

−

0.236

<

0.001 363

Vitamin E

−

0.158 0.003 363

β

-carotene

−

0.425

<

0.001 363

Lycopene

−

0.362

<

0.001 363

Ubiquinol

−

0.336

<

0.001 363

Cat + prot + gallic.

−

0.448

<

0.001 266

b

Polyphenol ur.

−

0.183 0.001 348

TAR

−

0.113 0.032 363

TRAP

−

0.240

<

0.001 363

a

Data from 1998 and 2000 studies.

b

Data from 1998 and 1999 studies.


Plasma Antioxidants

Several dietary antioxidants have been characterized in terms of their effective-ness, bioavailability and recommended dietary allowances. The main ones are vita-min C, vitamin E,

β

-carotene, lycopene, and ubiquinol. The changes in the levels ofthese plasma antioxidants will be reported elsewhere, together with other biochem-ical parameters. As shown in T

ABLE

1 and F

IGURES

2 and 3, the content of 8-OHdGin leukocytes, in the three experimental groups combined, correlates negatively witheach of the plasma antioxidants in a statistically significant fashion. The Pearsoncorrelation coefficients between the level of 8-OHdG in leukocytes and the sum ofsome plasma polyphenols was

−

0.448 (

p

<

0.001) the highest value followed by

β

-carotene,

−

0.425 (

p

< 0.001); lycopene,

−

0.362 (

p

<

0.001); ubiquinol,

−

0.336(

p

< 0.001); vitamin C,

−

0.236 (

p

< 0.001) and vitamin E

−

0.158 (

p

< 0.001). So thecorrelation among the concentration of catechin, protocatechuic acid, and gallic acidappears at least as effective as the other antioxidants.24 These antioxidants were sup-plied by vegetable foods, fruits and also red wine.25

FIGURE 1. Oxidative damage markers. Correlation between 8-OHdG content in DNA leu-kocytes (8-OHdG per 105dG) and plasma concentration of TBARS and 7β-hydroxycholesterol.

141PEREZ et al.: WINE AND OXIDATIVE DAMAGE

The results indicate that 8-OHdG levels are closely correlated with plasma anti-oxidants. Vitamin C and vitamin E, the main exogenous plasma antioxidants, arecapable of reducing the risk of atherogenesis at various levels. Ascorbate is essentialand acts directly as an antioxidant while vitamin E, which is also essential, actsvia ascorbate, which are both capable of reducing oxidized vitamin E and also ofcontrolling the potential prooxidant capacity of vitamin E.26 It has been reportedthat long-term effects of vitamin E, and the combination of vitamins E and Creduced serum 7β-hydroxycholesterol by 50.4% (p = 0.013) and 44.0% (p = 0.041),respectively; and enhanced the oxidation resistance of isolated lipoproteins and totalserum lipids.27

There are some epidemiological studies that have evaluated the associationbetween β-carotene and the risk of cardiovascular disease and cancer. Apparently,intervention studies do not show benefits in the prevention of cardiovascular

FIGURE 2. Oxidative DNA damage and antioxidants. Correlation between 8-OHdGcontent in DNA leukocytes (8-OHdG per 105dG) and plasma concentration of vitamin C,vitamin E, β-carotene, lycopene and ubiquinol.


disease.28 Some studies show that high blood levels of carotenoids are associatedwith a decreased incidence of certain forms of cancer, yet recent results have beencontradictory.29 The role of lycopene, an acyclic form of β-carotene, has been lessevaluated with regard to the risk of cardiovascular disease. In a recent study, low lev-els of plasma lycopene were associated with a 17.8% increment in intima-mediathickness of the common carotid artery wall (p = 0.003), in men. The authors con-clude that low plasma lycopene concentrations are associated with early atheroscle-rosis, evaluated as increased intima-media thickness of the common carotid arterywall.30 Also epidemiological studies suggest that high lycopene levels are associatedwith a decreased risk of prostate cancer.31 Absorption of dietary carotenoids isincomplete and depends on food mixture and food processing. Lycopene is poorlyabsorbed from raw tomatoes, but more is taken up from cooked tomatoes or tomatopaste.32

FIGURE 3. Oxidative DNA damage and polyphenols in plasma and urine. Correlationbetween 8-OHdG content in DNA leukocytes (8-OHdG per 105dG) and plasma concentrationof some free polyphenols, represented by the sum of catechin, protocatechuic acid, and gallicacid (µM rutin equivalents), and urine total polyphenols (mM gallic acid equivalents).


Extensive use of 8-OHdG as marker of oxidative damage has validated its usewith this purpose. Characteristically, different laboratories and different experimen-tal series show differences in basal values or in the magnitude of the changes detect-ed; however there is growing consensus in the explanation for these changes which,with adequate controls, do not invalidate 8-OHdG as a biomarker for oxidativestress.33

The DNA protection observed with diets rich in fruits and vegetables as well aswith red wine, and to some extent even white wine, is presumably due to the supplyof bioavailable antioxidants. Obviously ascorbate is present in fruits and vegetables,but in addition, polyphenol antioxidants should play a major role; they are charac-teristically abundant in red wine, 1 to 4 g/L, and white wine has 1/10 to 1/5 of thisamount.34 Phytochemicals are considered as valid tools in the prevention of coro-nary heart disease.35

There is considerable epidemiological evidence that diets rich in fruits, grains andvegetables are protective against several human diseases, especially cardiovasculardisease and some types of cancer. The vitamins C and E contribute to this protectiveeffect, but there are other constituents that exert additional antioxidants effects, orprotect like carotenoids and plant polyphenols.1

Plasma Antioxidant Capacity

The total plasma antioxidant capacity values were measured as total antioxidantreactivity (TAR) and total radical antioxidant potential (TRAP). TAR measures theconcentration of free radicals that can be initially trapped in the sample. It dependson the quantity and quality of the antioxidants present in the sample. TAR detectshydrosoluble antioxidants, particularly urate and ascorbate. Other components con-tribute to TAR but have not yet been identified. TRAP measures the total quantity offree radicals that can be trapped in the sample. It depends on the quantity of the anti-oxidants present in the sample and also detects hydrosoluble antioxidants.36 Ourresults (TABLE 1) show that both TAR and TRAP have a significant negative corre-lation with leukocyte 8-OHdG. This suggests that the plasma antioxidant capacityeffectively evaluates protection for oxidative damage.

Polyphenols in Plasma and Urine

Plasma polyphenol levels and urine total polyphenols as presented in FIGURE 3show a significant inverse correlation with leukocyte 8-OHdG content. The plasmapolyphenols illustrated represents the sum of catechin, protocatechuic acid and gal-lic acid expressed as µM rutin equivalents. These three compounds were selectedafter a preliminary evaluation of the changes in concentration observed. Other com-pounds are currently being studied.

The total polyphenols content in urine was measured with a simple method andit could be consider as a procedure for measuring polyphenol metabolism and anindex of antioxidant protection status.

Among the large number of polyphenols present in food and wine, some havebeen detected in plasma and urine;37 and there is more work to be done in relationto polyphenol metabolism and bioavailability.


Increased levels of plasma polyphenols would protect from oxidative damage, assuggested by our results. Other evidence also supports the hypothesis that wine, aswell as fruits and vegetables, decreases oxidative cellular damage to DNA and lip-ids.38 In conclusion, the high content of polyphenol antioxidants in red wine contrib-utes to reduce cancer and cardiovascular risk39 thanks to their antioxidant properties.

ACKNOWLEDGMENT

The work described was supported by Project PBMEC-PUC 1997–2000.

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