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 the mitochondria. Mitochondria are the "power plants" in our cells that provide the energy needed to maintain normal body 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 are very reactive species, because electrons like to pair up to form stable two-electron bonds (a landmark discovery made many decades ago by Linus Pauling, culminating in his Nobel Prize in Chemistry in 1954). Because of its radical character, superoxide is also called a "Reactive Oxygen Species" (ROS).

The production of superoxide by the mitochondrial respiratory chain occurs continuously during normal aerobic metabolism. It has been estimated that one to two percent of all the electrons traveling down the mitochondrial respiratory chain never make it to the end, but instead form superoxide. In addition to the mitochondrial respiratory 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 large amounts 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 cigarette smoke and possibly ozone) do not contribute significantly to the total load of free radicals and ROS to which we are exposed (or to cancer rates either).

Under normal metabolic conditions, each cell in our body is exposed to about 1010 molecules of superoxide each day. For a person weighing 150 pounds, this amounts to about 4 pounds of superoxide per year, a substantial amount! 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 cells and tissues. The recent, though as yet unconfirmed, evidence that excess iron intake may be linked to increased risk of heart disease and cancer may, in part, be explained by the role iron plays in the conversion of superoxide to hydroxyl radicals, resulting in oxidative damage to arteries and genes.

Oxidative Damage
The 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 and fat 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 damaged by ROS, leading to structural changes and loss of enzyme activity (an enzyme is a protein that catalyzes the rate of a chemical reaction without itself being changed). Oxidative damage to DNA also occurs at a high rate under normal metabolic conditions. It has been estimated that the DNA in each cell of our body suffers 10,000 "oxidative hits" per day, leading to the formation of more than twenty different oxidative DNA lesions. Many of these lesions are known to cause mutations. There are a number of DNA repair enzymes that can remove these lesions, but their repair success is not perfect. Therefore, oxidative DNA damage and mutations accumulate with age, which may contribute to cancer.

Sources of ROS:

mitochondrial respiratory chain, activated leukocytes, indoor and outdoor air (cigarette smoke, radon, ozone), ultraviolet light, etc.

----  X --->
----  D --->

Oxidative Damage to:




Causal Contribution to:

aging, heart disease, cancer, Alzheimer's disease, inflammatory-immune injuries/autoimmune diseases (rheumatoid arthritis, lupus, diabetes), AIDS, adult respiratory distress syndrome, etc.

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 of heart attacks and strokes are by far the No.1 killer in the United States. Interestingly, only polyunsaturated fatty acids can undergo lipid peroxidation, while saturated fats do not. This is quite ironic, because polyunsaturated fat is considered beneficial for keeping LDL levels low in the blood, while saturated fat increases these levels. The solution 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 abundant monounsaturated dietary fat is oleic acid, present in olive oil. The high consumption of olive oil may explain why people living in Mediterranean countries like Greece and Italy have such low rates of heart disease (in addition to some other factors, for example, the regular consumption of moderate amounts of alcohol).

Antioxidant Protection
Antioxidants protect us because they can scavenge ROS before they cause damage to the various biological molecules, or prevent oxidative damage from spreading, e.g. by interrupting the radical chain reaction of lipid peroxidation. 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 discovered that 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 other antioxidant enzymes (e.g., catalase, glutathione peroxidase) attests to the importance of oxidative damage as a real threat to cellular and organismal survival. Otherwise, organisms would not go through the considerable trouble and energy expenditure of evolving and synthesizing these enzymes. Before the discovery of the enzyme superoxide dismutase in 1969 by Irwin Fridovich, scientists did not believe that free radicals and ROS are even generated 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 in antioxidant defense systems. These small-molecule antioxidants are particularly important in blood and the fluids present 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 antioxidants are localized to cellular membranes and lipoproteins, whereas the water-soluble antioxidants are present in aqueous 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 day substantially lowers the risk of heart disease. A very plausible mechanism for this effect of vitamin E is the protection of LDL against oxidation, which, as mentioned above, is a critical step in the development of atherosclerosis. Vitamin E may also be useful in the treatment of heart disease, as indicated by a recent study from England in which a reduction by about 75% of a second, non-fatal heart attack was noted in a cohort of heart disease patients given 400-800 IU of vitamin E per day.

Other lipid-soluble antioxidants are beta-carotene (a vitamin A precursor) and related substances called carotenoids, 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-carotene supplementation 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 given beta-carotene supplements, a result that awaits a scientific explanation.

Vitamin C, the prominent water-soluble antioxidant, has a number of well-defined biological functions, including collagen, catecholamine, and carnitine biosynthesis. Vitamin C also very effectively scavenges a wide array of ROS and free radicals. Obviously, the role of vitamin C in human health and disease was one of Dr. Pauling's main interests in the last 25 years of his life and is also an area in which I have published extensively. For example, our studies have shown that vitamin C forms the first line of antioxidant defense in plasma against many different types 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 risk of cancer; heart disease, and cataract. One such study showed that men and women with the highest intake of vitamin C, defined as 50 mg or more from the diet plus regular supplements, had a risk of dying from heart disease or cancer that was 34% and 18%, respectively, lower than expected. Unfortunately, however, only limited information is available from large, well-controlled trials studying the potential benefits of vitamin C supplementation in disease prevention or treatment. For example, only a single trial testing vitamin C as an isolated 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. These ROS 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 degenerative diseases, such as heart disease and cancer. Antioxidants, both enzymatic and non-enzymatic, limit oxidative 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-related degenerative diseases.

Last updated November, 1997

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