Heart and Metabolism

Modifying oxidative stress by nutritional intervention

Inés Urquiaga, Federico Leighton
Facultad de Ciencias Biológicas, Universidad Católica de Chile, Santiago, Chile
Correspondence: Dr. Federico Leighton, Departamento Biología Celular y Molecular,
Facultad de Ciencias Biológicas, Universidad Católica de Chile, Casilla 114-D, Santiago, Chile Tel/Fax: +56 2 222 2577, e-mail: fleighto@bio.puc.cl

Abstract

Oxidative stress is the consequence of oxidative metabolism and of some spontaneous oxidative reactions in the organism. Its magnitude is reduced by endogenous antioxidant enzymatic processes and specific molecules, as well as by exogenous antioxidants, mostly of plant origin.
In chronic diseases inflammation causes oxidative stress, thus contributing to cell and tissue injury. In addition, specific oxidative processes appear as the main pathogenic mechanism in some
conditions like atherosclerosis, Parkinson’s disease, or ischemia-reperfusion tissue damage.
The magnitude of an oxidative stress condition can be estimated from measurement of ongoing oxidative processes or the magnitude and quality of antioxidant mechanisms. A more accurate estimation comes from the measurement of oxidative stress markers like 8-OH-deoxyguanosine in DNA, covalent protein modifications, lipid oxidation products, and oxidative stress sensitive processes like endothelial function. Food components other than antioxidant vitamins, mainly plant phenolic antioxidants with flavonoids among them, have been recognized in recent years as key components of a healthy diet. Moreover, the beginning of a systematic collection of data is allowing a better characterization of the dietary antioxidant supply, yet their bioavailability and overall effectiveness require more investigation. Part of the information will come from dietary intervention studies through antioxidant capacity, antioxidant levels and oxidative stress marker measurements, in normal and disease conditions. n Heart Metab. 2003;19:23–29.

Keywords: Antioxidants; oxidative stress; 8-OHdG; endothelial function; diet; wine;
mediterranean diet.

Oxidative stress
Oxidative stress, the consequence of a pro-oxidant imbalance between pro-oxidants and antioxidants in the human body, has been increasingly implicated during the last decade in the pathogenesis of chronic diseases and aging. Since antioxidants reduce oxidative stress, they could control the damage caused by reactive oxygen or nitrogen species and decrease the risk and consequences of chronic diseases. Diet composition influences both oxidative damage and antioxidant mechanisms, thus explaining, at least in part, the relationship between diet and chronic diseases such as atherosclerosis and cancer [1–3].

Mediterranean diet
The Mediterranean population has a low mortality rate, a fact attributed in part to the dietary habits of the region. Indeed, the Mediterranean diet has been proposed as a prototype for a healthy diet. The Mediterranean diet is high in monounsaturated fatty acids, fiber, and antioxidants, balanced in omega-6/omega-3 polyunsaturated fatty acids, and low in saturated fat. People from the Mediterranean regions of southern Europe eat more fish, white meat, olive oil, legumes, vegetables, and fruit; less red meat and animal fat; and consume a moderate quantity of red wine with meals [4–7]. Conversely, people from the USA and some other continental and northern European populations eat more red meat, animal fat, dairy products, and sugar; and fewer legumes, vegetables and fruit, and, in many populations, sea food. This diet, known as the occidental diet, is high in saturated fats and omega-6 polyunsaturated fatty acids, in refined or simple carbohydrates, and low in antioxidants and fiber [8–12].
Epidemiological studies have shown that the occidental diet is associated with a high incidence of cardiovascular disease and other chronic diseases, in contrast with diets rich in fruit and vegetables [13–15]. Wine, a rich source of antioxidants, has been associated with a low risk of cardiovascular disease [16, 17].

Biomarkers of oxidative stress
Lipid peroxidation and oxidative DNA damage constitute good biomarkers of oxidative damage [18]. Furthermore, oxidative DNA damage is mechanistically involved in cancer and aging, and lipid peroxidation plays a key role in cardiovascular disease. The oxidative hypothesis of atherogenesis postulates that oxidized LDL is the main agent of damage, and the endothelial cell the main target [19–21]. Recently, Natella et al [22] reported that supplementing a meal with grape seed proanthocyanidins, which are present in red wine, markedly reduces postprandial lipid peroxides. Oxidative DNA damage can be measured as 8-OH-deoxyguanosine (8-OHdG) in leukocytes and also shows good correlation with atherosclerosis status [23, 24]. In rabbits, dietary lipid lowering reduces oxidative DNA damage [25].

Endothelial dysfunction
The loss of endothelial function can be considered as evidence of oxidative stress. Nitric oxide, essential for normal vasodilatation, has antiatherogenic properties and its concentration decreases in the presence of the radical superoxide. Endothelial dysfunction or reduced endothelial function measured as flow-dependent vascular reactivity is present in early atherosclerosis families, hypercholesterolemia, hypertension, diabetes, hyperhomocysteinemia, and in smokers. Antioxidant administration restores endothelial function, at least in part through prevention of nitric oxide interaction with superoxide [25–28].

Intervention studies
The use of 8-OHdG as a marker in dietary antioxidant intervention studies has recently been questioned [29]. However, we have accumulated experimental evidence that peripheral leukocyte 8-OHdG content is a reliable marker of oxidative damage [30, 31]. We have performed intervention studies using controlled diets, a Mediterranean and an occidental diet, with and without wine, to evaluate the changes on antioxidants and oxidative stress in volunteers. Among other parameters, we measured 8-OHdG in leukocyte DNA as a marker of systemic oxidative stress [32]; thiobarbituric acid-reacting substances (TBARS) and 7b-hydroxy-cholesterol as markers of lipid peroxidation [32]; plasma antioxidant levels [33], total antioxidant reactivity (TAR), and total radical antioxidant potential (TRAP) [34] as markers of antioxidant status; and endothelial function [35] as a marker of atherogenic risk. The results show close correlation between plasma antioxidants and oxidative damage and favor the hypothesis that diet effectively regulates oxidative stress in humans.

Total plasma antioxidant capacity
In an intervention study we measured the total plasma antioxidant capacity as TAR; the results are shown in Figure 1.

Figure 1. Effects of an occidental diet, Mediterranean diet, and wine consumption on plasma TAR. Values within the same diet, denoted by different capital or lower case letters, are significantly different (P < 0.003). *Values for the same time interval in different diets are significantly different (P < 0.05). Mean value ± SD. (Reproduced from Leighton et al [30].)

In this study we compared a Mediterranean diet with an occidental diet, and the effect of wine supplementation, administered as shown in Figure 2.

Figure 2. Intervention study design. Diets were supplied for 90 days; from days 30 to 60 red wine was supplied isocalorically. Clinical and biochemical evaluations were carried out at days 0, 30, 60, and 90.

TAR detects hydrosoluble antioxidants, with urate and ascorbate as the major known contributors. Volunteers were given either a Mediterranean diet or an occidental diet for 3 months. During the second month they additionally received 240 mL/day red wine of the Cabernet Sauvignon variety.
With the Mediterranean diet the TAR values increased above basal levels: 28% at day 30 and a further 56% increase after the addition of wine. The changes after commencing the Mediterranean diet and again after the addition of wine were statistically significant. By contrast, with the occidental diet the TAR values were unmodified, but a significant 23% increase above day 0 or basal level was observed with the addition of wine. Thus in the occidental diet only wine increased plasma TAR values above those corresponding to the volunteers’ usual diet.
When Mediterranean and occidental diet values were compared for the same time intervals, the former were all significantly higher than the latter: 29%, 37% and 31% at days 30, 60, and 90, respectively. Clearly the Mediterranean diet per se induces a higher antioxidant capacity, just as wine does when added to a Mediterranean or occidental diet.

Oxidative DNA damage
To evaluate the oxidative damage in DNA, the content of 8-OHdG was measured in DNA from peripheral blood leukocytes. The levels of 8-OHdG in both the Mediterranean and occidental groups, at different times of the study, are shown in Figure 3.

Figure 3. Effects of an occidental diet, Mediterranean diet, and wine consumption on 8-OHdG content in blood leukocytes. Results are presented as picomoles of 8-OHdG per 105 picomoles of deoxyguanosine (dG). Mean value ± SD. Different lower case letters within the same dietary group denote statistically different values (P < 0.05). (Reproduced from Leighton et al [30].)

The level of 8-OHdG detected in the Mediterranean group was lower than that in the occidental group at day 30 (P < 0.05), suggesting that the Mediterranean diet is able to decrease the level of DNA oxidative damage. Conversely, an occidental diet low in fruit and vegetables and rich in fat induces oxidative DNA damage.
During the period when wine was added, a substantially reduced level of oxidative DNA damage was observed equally in both diets. In fact, in the occidental group, which showed a diet-induced increase in the level of 8-OHdG at day 30, wine consumption led to a sharp decrease in the level of 8-OHdG to a value even lower than the basal level. These results show that the high oxidative DNA damage induced by an occidental diet can apparently be prevented with moderate wine consumption.
In the Mediterranean group, no statistically significant differences were seen between basal values and those after diet at day 90. Nevertheless, values after 30 days of diet were lower. When wine was added to the Mediterranean diet, the level of 8-OHdG was significantly lowered (day 60). These results suggest that, in contrast with an occidental diet, a Mediterranean diet leads to a decrease in oxidative DNA damage and that moderate wine supplementation confers additional protection.

Endothelial function
Endothelial function was assessed noninvasively as flow-mediated vasodilatation of the brachial artery. The measurements, expressed as percentages of arterial diameter change compared with the basal recording, were made 1 minute after releasing the blood flow arrest imposed by forearm arterial occlusion. Individual measurements are shown in Figure 4, and mean values and statistical analysis in Table I. [26].

Table I. Effects of an occidental diet, Mediterranean diet, and wine supplementation on endothelial function. (Reproduced from Cuevas et al [26].)

Figure 4: Effects of an occidental diet, Mediteranean diet, and wine supplementation on endothelial function. (Reproduced from Cuevas et al [26].)

Endothelial function was significantly reduced in the occidental diet compared with the Mediterranean diet (P = 0.014). After the period with wine this difference disappeared. In fact, in the occidental group a complete loss of vascular reactivity was observed, which reappeared with the addition of wine to the diet. Remarkably, after the addition of wine, both groups showed the same, normal endothelial function values.
The limited number of analyses and the variability of the detection procedure do not allow a definitive conclusion on the effect of wine on endothelial function in volunteers following a Mediterranean diet. Nevertheless, the results suggest that both a Mediterranean diet and wine preserve or increase endothelial function, while they clearly show that an occidental diet suppresses flow-mediated vascular reactivity, in a process fully reversed by wine supplementation.

Correlations between oxidative damage and plasma antioxidants
As stated above, oxidative damage is the consequence of an imbalance between pro-oxidant reactions, mediated by reactive oxygen and nitrogen species, and antioxidants or antioxidant activity present in the body. In several intervention studies, we evaluated oxidative damage using biomarkers such as 8-OHdG in human DNA leukocytes, plasma TBARS, and 7b-hydroxycholesterol and correlated the results with various plasma antioxidants [31].

Table II. Correlation of 8-OHdG with oxidative damage, plasma antioxidants, and antioxidant capacity. Pooled data from several intervention studies. (Reproduced from Perez et al [31].)

The results shown in Table II illustrate that the three biomarkers for oxidative damage behave in a similar fashion: 8-OHdG content in leukocyte DNA correlates significantly with plasma TBARS and plasma 7b-hydroxycholesterol (0.215, P < 0.002, and 0.152, P < 0.012, respectively). Also shown in Table II is the negative correlation between 8-OHdG in leukocytes and each of the plasma antioxidants measured. The Pearson correlation coefficients between the level of 8-OHdG in leukocytes and plasma polyphenol antioxidants was -0.448 (P < 0.001), the highest value; followed by b-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 < 0.003).
The plasma polyphenol antioxidants correspond to the sum of catechin, protocatechuic acid, and gallic acid, and appear to be at least as effective as the other antioxidants. Interestingly, urine total polyphenol content is also a good indicator of antioxidant capacity. Vegetables, fruit, and red wine supplied most of the polyphenols measured in plasma. In urine, polyphenols originate both from endogenous and exogenous sources. The total plasma antioxidant capacity values measured as TAR and TRAP also have a significant negative correlation with leukocyte 8-OHdG, a result that validates their use as indicators of antioxidant capacity. These results also support the validity of the biomarkers employed to assess oxidative damage in humans; yet in our hands, 8-OHdG was the most reliable.

REFERENCES

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