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Lecture 8

Biochemistry of Oxidant Stress in Health
and Disease Antioxidants: Endogenous and Exogenous

The human genome dates back over 40,000 years ago when man was a "hunter-gatherer" and consumed a diet consisting of over 90 percent plant-derived foods compared to about 30 percent in the modern post-industrial era (1). Our modern diet is largely derived from animal sources which are higher in fat, lower in fiber, and poorer in micronutrients especially antioxidants. While our modern diet has virtually eliminated malnutrition in the majority of the population, there is an epidemic of obesity (affecting 35% of Americans in the latest Department of Agriculture survey in 1989) and an increased incidence of heart disease, breast cancer, prostate cancer, and colon cancer by comparison with populations eating less meat and more fruits, vegetables, cereals and grains. The increasing incidence of chronic diseases and obesity in Japan which has adopted a more Western diet over the last 30 years is strong evidence of the importance of nutrition in disease prevention. There are three levels at which the diet of an individual can be assessed: 1) the overall caloric content, and macronutrient profile (i.e. protein, carbohydrate, and fat composition); 2) the vitamin and micronutrient adequacy for prevention of deficiency diseases; and 3) the adequacy of antioxidants including carotenoids, vitamin C, vitamin E, and selenium for prevention of heart diseases and common forms of cancer.

Oxidants and Antioxidants
The process of oxidation occurs in any oxygen-rich environment where substrates are exposed to ultraviolet light or heat. Examples of commonly occurring chemical oxidation processes include an apple turning brown following brief exposure to air, a car rusting in the open air, and the formation of rancid fats in poorly preserved foods. These processes are all mediated by highly reactive oxygen radicals (oxygen atoms with a single unpaired electron). In order to understand the mechanisms by which antioxidants can prevent chronic diseases, it is necessary to understand the actions of free radicals and how antioxidants limit free radical reactions (2).

Oxygen involved in the oxidation of substrates to produce energy in normal metabolic processes can produce oxygen radicals. They can have a beneficial roles as in phagocytes where they protect against bacteria and parasites. However, if natural antioxidant mechanisms are not adequate to quench excess oxygen radicals then they can react with cell structures. Metabolism is not the only source of free radicals. Environmental pollutants are sources for free radicals including nitrogen dioxide, ozone, cigarette smoke, radiation, halogenated hydrocarbons, heavy metals and certain pesticides. Alcohol consumption can induce oxidative reactions in the liver. Certain chemotherapeutic agents including doxorubicin, cyclophosphamide, 5-fluorouracil, methotrexate, and vincristine can produce oxygen radicals at doses used in cancer patients. Increased physical activity can generate free radicals as the result of increased oxygen consumption during exercise. Oxygen radicals in the human body react with proteins, lipids, carbohydrates and nucleotides.

When free radicals attack polyunsaturated fats in the presence of oxygen, lipid peroxides are formed in the a chain reaction that results in amplification of the original oxidative damage. For example, low density lipoprotein that is oxidized is more easily taken up by macrophages in the endothelial wall of blood vessels promoting atherogenesis (3). A DNA base oxidation product (8-hydroxydeoxyguanosine) can be detected in the urine of humans exposed to oxidant stresses raising the possibility that oxidation can alter genetic information. These oxidative processes have been associated with degenerative changes occurring with aging, and the development of cardiovascular disease and cancer. Free radical reactions occur continuously in living cells, but most of the changes resulting are repaired. Damage that escapes these repair systems may accumulate over long periods of time and play a role in degenerative diseases.

The body's susceptibility to oxidant damage is thought to depend on the balance between the extent of pro-oxidant stress and the antioxidant levels of body tissues. Most antioxidants have a large number of alternating double bonds which can act as electron traps. In some cases, this quenching reaction can lead to increased oxidation. This occurs when a polyunsaturated fat neutralizes an oxygen radical but becomes a fatty acid radical which then attacks another lipid leading to a chain reaction. Other antioxidants can also act as pro-oxidants after quenching an oxygen radical. On balance, a number of studies have shown that various antioxidants act as a cooperative system of antioxidant defense. Vitamin C quenches free radicals in aqueous systems, but also regenerates cellular vitamin E which helps to control lipid peroxidation (4). Beta-carotene also traps free radicals in concert with vitamin E. The selenium-containing enzyme glutathione peroxidase destroys peroxides before they can damage cell membranes and interacts synergistically with vitamin E (5). In a number of animal studies, the administration of antioxidants ameliorated damage from experimental oxidant stress. Furthermore, the antioxidant requirement in these studies was directly proportional to the increased tissue concentrations of free radicals. While not proven, promotion of antioxidation through consumption of antioxidant-rich fruits and vegetables as well as dietary supplements is a habit many Americans consider health-enhancing.

On the basis of population studies and animal studies, a number of governmental agencies including the National Research Council, the National Cancer Institute and the United States Department of Agriculture have recommended that Americans eat at least five servings a day of fruits and vegetables in part to increase the intake of beneficial antioxidants (see Table 1). Recent surveys indicate that only a small fraction of the general population follows this advice, leading some nutrition authorities to recommend dietary supplementation or food fortification with antioxidants to reduce chronic disease incidence. While there remains some uncertainty about the long term effects of antioxidant supplementation, there is accumulating evidence that the practice of dietary supplementation may be beneficial.

Carotenoids are found in green, yellow, and orange vegetables and some fruits. The commonly occurring carotenoids include beta and alpha carotene, lycopene, cryptoxanthin, lutein, and zeaxanthin (see Table 2). These all act as antioxidants, but only beta carotene can be converted to vitamin A, and only beta carotene is available as a dietary supplement. Beta carotene is classified as a generally safe natural food coloring. The only toxic effect in man is yellowing of the skin at doses of greater than 20 mg per day. It has been estimated that individuals eating five servings of fruits and vegetables per day would obtain about 6 mg of beta carotene from their diets. The average intake in Americans is about 3.5 mg per day currently. There is clearly a large gap between the recommended intakes and the level of toxicity making supplementation with up to 10 mg generally safe.

Vitamin E is a generic term that includes entities exhibiting the biological activities of d-alpha tocopherol. In nature, eight substances have vitamin E activity: d-alpha, d-beta, d-gamma, and d-delta tocopherols and d-alpha, d-beta, d-gamma, and d-delta tocotrienols. D-alpha tocopherol has the highest biologic activity in the vitamin E assay but all of these are antioxidants. There is also a synthetic form of vitamin E: d,l-alpha tocopherol. It bioactivity in the vitamin E assay is reduced (1.0 vs. 1.49). Vitamin E is present in small amounts in a large number of foods including vegetable oils (soybean, sunflower, and corn oil), wheat germ, whole grains, egg yolk, nuts, sunflower seeds, green vegetables, milk fat, and liver (see table 2). The Seventh RDA Committee in 1968 set the RDA for vitamin E at 20 mg (30 IU), but found that this was difficult to attain through normal diets without supplementation. Subsequent editions of the RDA halved the requirement to 10 mg (15 IU). It is estimated that 64 percent of the vitamin E intake of the American diet is supplied by salad oils, margarine, and shortening. Eleven percent is supplied by fruits and vegetables, and about 7 percent by grains and grain products. The average amount supplied by the American diet is estimated to be 7 to 9 mg (10.4 to 13.4 IU). Vitamin E in amounts considered to be protective are not easily consumed through the diet. For example, a quart of corn oil contains 200 IU of vitamin E but over 7,700 Calories.

Some commonly consumed dietary supplements with antioxidant activity are referred to as antioxidant vitamins although not all components of these combinations are vitamins. These preparations most commonly include a combination of vitamin E, vitamin C, and beta carotene. Beta carotene is an antioxidant that is converted to vitamin A only under conditions of vitamin A deficiency. Vitamin E and vitamin C only have antioxidant properties at doses well above the doses required to prevent vitamin deficiencies. Other carotenoids such as lycopene (the red pigment found in tomatoes) are not converted to vitamins but act as antioxidants. Certain antioxidants such as ubiquinone (also known as Coenzyme-Q10) and glutathione are synthesized in the body and are not essential dietary constituents. Most of the studies cited below have considered the three most commonly consumed antioxidants - Vitamin E, Vitamin C, and beta carotene. Other antioxidants such as the flavinoids may have other pharmacologic actions including anti-hormonal and anti-growth factor effects at a cellular level. For example genistein, an isoflavone found in soybean protein, binds to the type II estrogen receptor and interferes with EGF receptor tyrosine kinase phosphorylation in breast cancer cells. While there is much that is not known about antioxidants, there is evidence accumulating on the preventive role of antioxidants in the two most prevalent chronic diseases - heart disease and cancer.

Antioxidants in Heart Disease
Approximately 70 million Americans have one or more forms of cardiovascular disease including hypertension, coronary heart disease, stroke, and rheumatic heart disease. Every year it is estimated that 1.5 million Americans will have a heart attack, with approximately 500,000 to 600,000 deaths resulting. The estimated cost of coronary heart disease in 1993 was 51.6 billion dollars. Data from numerous studies indicate a beneficial effect of supplemental vitamins C, E, and beta carotene in significantly reducing coronary events, reperfusion injury, platelet aggregation, and low-density lipoprotein (LDL) oxidation. Two large-scale epidemiologic studies of men and women (6,7) show vitamin E consumption at levels greater than 100 IU per day to be associated with a reduced incidence of coronary artery disease. In the Nurses' Health Study, women who consumed more than 3 times the RDA of vitamin E had a 34% lower risk of heart attacks than women who consumed lesser amounts. Similar results were found in the Health Professionals Study which involved men.

These epidemiologic studies, combined with evidence in laboratory studies that concentrations of vitamin E and beta carotene that can be achieved with vitamin supplementation resulted in decreased susceptibility to oxidation of LDL isolated from plasma following in vitro challenge with pro-oxidants (copper or iron), have led many cardiologists to recommend that their patients take antioxidant supplements. While additional studies are still required to reach a significant scientific agreement on the benefits of antioxidants according to the Food and Drug Administration, many physicians are advising their patients to take supplements. The determination of compliance with this advice as well as confirmation of the achievement of steady-state levels in the range observed to be beneficial in epidemiologic studies will require measurement of blood levels of antioxidants.

Antioxidants in Cancer
The common forms of cancer, including breast, colon, and prostate cancer, are the result of genetic-environmental interactions. While a small minority (5-10%) of patients have inherited forms of cancer due to alterations in the genes of the germ cell line, cancers are all assumed to have genetic changes at the somatic cell level. These genetic changes lead to unregulated growth through activation of growth-promoting genes (oncogenes) or inactivation of tumor suppressor genes. In some cases oncogenes code for growth factor receptor proteins and growth factors. Reactive oxygen radicals are thought to damage biologic structures and molecules including lipids, protein, and DNA, and there is evidence that antioxidants can prevent this damage.

For example, vitamin C has three proposed mechanisms of action in chemoprevention: 1) incorporation of ascorbic acid into the hyaluronidase inhibitor system; 2) prevention of the formation of nitrosamines in the gastrointestinal tract; and 3) inhibiting the potency of chemical carcinogens by enhancing the activity of the detoxifying cytochrome P-450 system. Beta carotene and retinol have been shown in vitro and in animal studies to suppress carcinogenesis. Vitamin E in cell culture has been shown to reduce expression of certain oncogenes (H-ras and c-myc) in tumor cells, and to inhibit cell proliferation in other cell lines . Selenium has been shown to prevent the metabolism of chemical carcinogens necessary for their activation. Rats fed a diet high in beta carotene, vitamin C, or selenium were demonstrated to have a reduced incidence of carcinogen-induced pancreatic cancer .

Although the antioxidant properties of vitamin C, vitamin E, and beta carotene have been considered the primary mode of action, other mechanisms may also be involved in their chemopreventive actions. These include effects on the immune system, on modulation of growth factors and growth factor receptors, and on intercellular interactions. Epidemiologic studies provide evidence on the relationship of the intake of antioxidants to the incidence of lung cancer. In Finnish studies carried out over periods of five to twenty years, there was an inverse relationship between serum levels of vitamin E and other antioxidants and the risk of cancer at several anatomic sites. In a study of 15,093 women ages 15-99 years and initially free of cancer, cancer was diagnosed in 313 women over an eight year follow-up. Low serum levels of vitamin E strongly predicted the risk of epithelial cancers, but demonstrated only slightly elevated risks of cancers in reproductive organs exposed to estrogens (9). Therefore, while the effects of antioxidants can be demonstrated in animal studies, human studies are more complex due to the known effects of obesity increasing estrogen levels in women, and other factors in the Western diet such as reduced fiber intake.

Smoking sometimes also appears to override the protective effects of antioxidant vitamins in some studies. A randomized double-blind prospective controlled trial carried out for five to eight years in 29,133 male smokers studied the effects of 50 mg synthetic vitamin E alone or in combination with 20 mg synthetic beta carotene (10). Among the 876 cases of lung cancer observed there was no decrease observed in the men supplemented with vitamin E. There were 52 fewer cases of prostate cancer. Unexpectedly, the incidence of lung cancer was 18% higher and the mortality due to lung cancer was higher in the beta carotene supplemented group. This result was unexpected and runs counter to the body of experimental evidence and a recent supplementation trial in China in which 29,584 adults ages 40 to 69 years were provided a variety of vitamin supplements including vitamin A, riboflavin, vitamin C, and a combination of beta carotene, selenium, and vitamin E. The combination of selenium (50 mcg), vitamin E (30 mg) and beta carotene (15 mg) was associated with a 13% decrease in cancer mortality . The major effect was on gastric cancer (21% reduction) which is the most common cancer in this population.

Clearly, much further research needs to be done on the effects of antioxidants, but the above studies indicate a potential protective effect on cancers of the aerodigestive system. In the presence of other major etiologic factors (e.g. estrogen for breast cancer, or smoking for lung cancer), the beneficial effects of antioxidants are not demonstrable. In patients at increased risk of cancer, including the population that has undergone primary treatment of cancer, antioxidant supplementation is being recommended by many physicians.

Future Directions of Development
Antioxidants work by supplementing significant host defenses against oxidant stress. The measurement of the host response to oxidant stress remains problematic. There are a number of proposed markers of oxidant stress including measurement of lipid oxidation products such as conjugated dienes, malondialdehyde or thiobarbituric acid reactive substances (TBARS) in blood or urine; modified DNA bases (8-hydroxydeoxyguanosine) and/or DNA adducts in peripheral blood cells or urine; vitamin E or vitamin C levels in blood fractions; catalase or dismutase levels in blood fractions; volatile gases such as pentane or ethane in expired breath; total peroxyl radical trapping antioxidant power of serum (TRAP assay); auto-oxidative (non-cyclooxygenase-derived) eicosanoids in plasma; and the in vitro oxidation of blood fractions such as LDL. Future refinement of these methods with clinical correlation will provide important information for designing supplement strategies for disease prevention.

While a number of government agencies recommend that Americans consume more than 5 servings per day of fruits and vegetables it is clear that only a minority are able to follow this advice. Supplementation of usually consumed foods with antioxidants is another potential strategy. It is already true that breakfast cereal is a major dietary source of folic acid for Americans who consume too little of green leafy vegetables to obtain the Recommended Dietary Allowances. It may be that foods can be developed which provide protective amounts of antioxidants in lieu of the capsules currently available. Additional research on the differences among the various antioxidants and how they work must also be done and reduced to dietary changes for the population.

Finally, normal ranges for different age groups referenced to dietary intake need to be developed. It is clear that antioxidant levels in the blood are influenced by dietary intake including seasonal variation in the intake of fruits and vegetables. A workshop sponsored by the Food and Drug Administration in 1993 outlined a pro-active strategy to be utilized in evaluating data on the positive impact of nutrient and non-nutrient antioxidants on human health. The World Cancer Research Fund and the American Institute of Cancer Research issued a 740 page analysis of international data supporting new dietary guidelines which indicate that between 400 and 800 gm per day of vegetables may be preventative. The overall strategy of these groups includes engaging the scientific community through advisory councils workshops and other mechanisms. It is likely that much new information on the benefit of antioxidants will be developed in the next few years providing clinicians with improved guidelines for following the antioxidant status of their patients at risk of cardiovascular diseases and cancer.



1. Eaton SB, and Konner M. Paleolithic nutrition. New Engl J Med 312:283-289, 1985.
2. Harman D. Free radical theory of aging.: the free radical diseases. Age 7: 111-131, 1984.
3. Rifici VA, Khachadurian AK. Dietary supplementation with vitamins C and E inhibits in vitro oxidation of lipoproteins. J. Am. Coll. Nutr. 12: 631-637,1993.
4. Watson RR and Leonard TK. Selenium and vitamins A, E, and C: nutrients with cancer prevention properties. J Am Diet Assoc 86:505-510,1986.
5. Wefers H and Sies H. The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E. Eur J Biochem 174:353-357, 1988.
6. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. New Engl J Med 328:1450-1456, 1993.
7. Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary artery disease in women. New Engl J Med 328: 1444-1449,1993.
8. Prasad K, Edwards-Prasad J, Kumar S, Meyers A. Vitamins regulate gene expression and induce differentiation and growth inhibition in cancer cells. Arch Otolaryngol Head Neck Surgery 119: 1133-1140,1993.
9. Knekt P. Serum vitamin E level and risk of female cancers. Int J Epidemiol 17:281-286, 1988.
10. Alpha Tocopherol Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer in chronic smokers. New Engl J Med 330: 1029-1035,1994.
11. Blot WJ, li J-Y, Taylor PR, Guo W, Dawsey S, Wnag G-Q, Yang CS, Zheng S-F, Gail M, Li G-Y, Yu Y, Liu B-Q, Tangrea J, Sun Y-H, Liu F, Fraumeni JF, Zhang Y-H, Li B. Nutrition intervention trials in Linxiang, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population. J Nat Cancer Inst 85:1483-1492,1993.

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Lecture 1
:Introduction to Nutrition in Western Civilization
Lecture 2:
Dietary Macronutrients, Body Fat, and Blood Lipids
Lecture 3:
Digestion and Absorption of Macronutrients
Lecture 4:
Basic Principles of Nutrient Metabolism
Lecture 5:
Lecture 6:
Fuel Utilization During Exercise
  Lecture 7:Biochemistry of Oxidant Stress in Health and Disease Antioxidants
Lecture 8:Nutrition for the 21st Century






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