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Reactive Oxygen Species and Antioxidant Vitamins

Balz Frei, Ph.D.

Director, Linus Pauling Institute

Professor of Biochemistry & Biophysics

In aerobic organisms like humans, oxygen is converted to water at the end of the respiratory chain in themitochondria. Mitochondria are the "power plants" in our cells that provide the energy needed to maintain normalbody function and metabolism. However, in this same mitochondria respiratory chain, oxygen is "partially reduced"to form superoxide. Superoxide is a radical, i.e. a chemical species with an unpaired electron. Radicals usually arevery reactive species, because electrons like to pair up to form stable two-electron bonds (a landmark discoverymade many decades ago by Linus Pauling, culminating in his Nobel Prize in Chemistry in 1954). Because of itsradical character, superoxide is also called a "Reactive Oxygen Species" (ROS).

The production of superoxide by the mitochondrial respiratory chain occurs continuously during normal aerobicmetabolism. It has been estimated that one to two percent of all the electrons traveling down the mitochondrialrespiratory chain never make it to the end, but instead form superoxide. In addition to the mitochondrialrespiratory chain, there are other endogenous sources of superoxide production. In particular, when leukocytes(white blood cells) encounter microorganisms or other pathogens invading our bodies, they start to generate largeamounts of superoxide. Additionally, there are a few external sources of superoxide, especially cigarette smoke,which is "packed" with free radicals and ROS. However, most environmental pollutants (other than cigarettesmoke and possibly ozone) do not contribute significantly to the total load of free radicals and ROS to which weare exposed (or to cancer rates either).

Under normal metabolic conditions, each cell in our body is exposed to about 1010 molecules of superoxide eachday. For a person weighing 150 pounds, this amounts to about 4 pounds of superoxide per year, a substantialamount! Once formed, superoxide is converted to other ROS. In the presence of small amounts of iron or copper,hydroxyl radicals may be formed. Hydroxyl radicals are extremely reactive and can cause severe damage to cellsand tissues. The recent, though as yet unconfirmed, evidence that excess iron intake may be linked to increasedrisk of heart disease and cancer may, in part, be explained by the role iron plays in the conversion of superoxide tohydroxyl radicals, resulting in oxidative damage to arteries and genes.

Oxidative DamageThe ROS formed by the mechanisms explained above can cause oxidative damage to various biological molecules.For example, hydroxyl radicals can damage cell membranes and lipoproteins (the particles carrying cholesterol andfat in the blood stream) by a process called lipid peroxidation. Lipid peroxidation occurs by a radical chain reaction,i.e. once started, it spreads rapidly and affects a great number of lipid molecules. Proteins may also be damagedby ROS, leading to structural changes and loss of enzyme activity (an enzyme is a protein that catalyzes the rateof a chemical reaction without itself being changed). Oxidative damage to DNA also occurs at a high rate undernormal metabolic conditions. It has been estimated that the DNA in each cell of our body suffers 10,000 "oxidativehits" per day, leading to the formation of more than twenty different oxidative DNA lesions. Many of these lesionsare known to cause mutations. There are a number of DNA repair enzymes that can remove these lesions, buttheir repair success is not perfect. Therefore, oxidative DNA damage and mutations accumulate with age, whichmay contribute to cancer.

Sources of ROS:

mitochondrialrespiratory chain,activatedleukocytes, indoorand outdoor air(cigarette smoke,radon, ozone),

ultraviolet light,etc.

ANTIO

---- X --->I

---- D --->

ANTS

Oxidative Damageto:

DNAlipids

proteins---------->

---------->

Causal Contribution to:

aging, heart disease,cancer, Alzheimer'sdisease, inflammatory-immuneinjuries/autoimmunediseases (rheumatoidarthritis, lupus, diabetes),

AIDS, adult respiratorydistress syndrome, etc.

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Adapted with permission from Frei, Reactive Oxygen Species and Antioxidant Vitamins: Mechanisms of Action,The American Journal of Medicine, Vol. 97, pg. 3A-7S, 1994 by Excerpta Medica Inc.

While oxidative damage to DNA is implicated in cancer, oxidative damage to lipids in low-density lipoprotein (LDL,the "bad" cholesterol) plays an important role in atherosclerosis. Atherosclerosis and the resulting complications ofheart attacks and strokes are by far the No.1 killer in the United States. Interestingly, only polyunsaturated fattyacids can undergo lipid peroxidation, while saturated fats do not. This is quite ironic, because polyunsaturated fatis considered beneficial for keeping LDL levels low in the blood, while saturated fat increases these levels. Thesolution to this dilemma is to replace both saturated and polyunsaturated fat in our diet with monounsaturated fat,

which is neither easily oxidized nor exerts adverse effects on the blood level of LDL. The single most abundantmonounsaturated dietary fat is oleic acid, present in olive oil. The high consumption of olive oil may explain whypeople living in Mediterranean countries like Greece and Italy have such low rates of heart disease (in addition tosome other factors, for example, the regular consumption of moderate amounts of alcohol).

Antioxidant ProtectionAntioxidants protect us because they can scavenge ROS before they cause damage to the various biologicalmolecules, or prevent oxidative damage from spreading, e.g. by interrupting the radical chain reaction of lipidperoxidation. The antioxidant defense systems in the human body are extensive and consist of multiple layers,which protect at different sites and against different types of ROS.

An important part of the antioxidant defense system inside cells are the antioxidant enzymes. For example,superoxide dismutase scavenges superoxide and converts it to less reactive species. It was recently discoveredthat the gene for superoxide dismutase is defective in patients with amyotrophic lateral sclerosis (ALS), better

known as Lou Gehrig's disease. The very existence of superoxide dismutase in biological systems, as well as otherantioxidant enzymes (e.g., catalase, glutathione peroxidase) attests to the importance of oxidative damage as areal threat to cellular and organismal survival. Otherwise, organisms would not go through the considerabletrouble and energy expenditure of evolving and synthesizing these enzymes. Before the discovery of the enzymesuperoxide dismutase in 1969 by Irwin Fridovich, scientists did not believe that free radicals and ROS are evengenerated in our bodies, let alone that they play a role in human degenerative disease.

In addition to the antioxidant enzymes, there are several small-molecule antioxidants that play important roles inantioxidant defense systems. These small-molecule antioxidants are particularly important in blood and the fluidspresent in the extracellular space, where antioxidant enzymes are absent or present only in small quantities.

The small-molecule antioxidants include lipid-soluble and water-soluble antioxidants. The lipid-soluble antioxidantsare localized to cellular membranes and lipoproteins, whereas the water-soluble antioxidants are present inaqueous fluids, such as blood and the fluids within cells and surrounding them.

Alpha-tocopherol, the biologically and chemically most active form of vitamin E, is by far the most abundant lipid-soluble antioxidant in humans. Recent evidence indicates that an intake of about 100-200 IU of vitamin E per daysubstantially lowers the risk of heart disease. A very plausible mechanism for this effect of vitamin E is theprotection of LDL against oxidation, which, as mentioned above, is a critical step in the development ofatherosclerosis. Vitamin E may also be useful in the treatment of heart disease, as indicated by a recent studyfrom England in which a reduction by about 75% of a second, non-fatal heart attack was noted in a cohort of heartdisease patients given 400-800 IU of vitamin E per day.

Other lipid-soluble antioxidants are beta-carotene (a vitamin A precursor) and related substances calledcarotenoids, such as alpha-carotene, lycopene (the red color in tomatoes), lutein, and zeaxanthine. However,carotenoids are much weaker antioxidants than vitamin E. It was not surprising, therefore, that beta-carotenesupplementation failed to affect cancer and heart disease rates in three recent, very large (and very expensive!)studies in Finland, Boston, and Seattle. If anything, lung cancer rates were increased in smokers givenbeta-carotene supplements, a result that awaits a scientific explanation.

Vitamin C, the prominent water-soluble antioxidant, has a number of well-defined biological functions, includingcollagen, catecholamine, and carnitine biosynthesis. Vitamin C also very effectively scavenges a wide array of ROSand free radicals. Obviously, the role of vitamin C in human health and disease was one of Dr. Pauling's maininterests in the last 25 years of his life and is also an area in which I have published extensively. For example, ourstudies have shown that vitamin C forms the first line of antioxidant defense in plasma against many differenttypes of ROS and radicals and, surprisingly, also protects LDL more effectively against oxidation than vitamin E.There is also strong evidence from epidemiological studies that an adequate intake of vitamin C can lower the riskof cancer; heart disease, and cataract. One such study showed that men and women with the highest intake ofvitamin C, defined as 50 mg or more from the diet plus regular supplements, had a risk of dying from heartdisease or cancer that was 34% and 18%, respectively, lower than expected. Unfortunately, however, only limitedinformation is available from large, well-controlled trials studying the potential benefits of vitamin Csupplementation in disease prevention or treatment. For example, only a single trial testing vitamin C as anisolated intervention in the treatment of heart disease is underway, the "Women's Antioxidant Cardiovascular

Disease Study" at Harvard University.

In summary, we are constantly exposed to ROS generated from endogenous and some exogenous sources. TheseROS react with biological molecules, such as DNA, proteins, and lipids, causing structural and functional damage.Oxidative damage accumulates in human tissues with age and can causally contribute to a number of degenerativediseases, such as heart disease and cancer. Antioxidants, both enzymatic and non-enzymatic, limit oxidative

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damage to biological molecules by various mechanisms. Dietary antioxidants, such as vitamins C and E,significantly contribute to antioxidant defense systems in humans and may help protect us from certain age-relateddegenerative diseases.

Last updated November, 1997

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