Free

radical
LAWRENCE

tissue
J.
MACHLIN AND

damage:
1

protective
BENDICH

role of

antioxidant

nutrients
ADRIANNE

Clinical Nutrition, Hoffmann-La Roche Inc.,Nutley,

New Jersey

07110,

USA

ABSTRACT

Highly
tissue acids

reactive
damage in cellular

molecules called free radicals can cause by reacting with polyunsaturated fatty
membranes, nucleotides in DNA,

and

critical sulfhydryl bonds in proteins. Free radicals can originate endogenously from normal metabolic reactions
or exogenously as components of tobacco smoke and air

pollutants and indirectly through the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation. There is some evidence that free radical damage contributes to the etiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. Defenses against free radical damage include tocopherol (vitamin E), ascorbic acid (vitamin C), /3.carotene, glutathione, uric acid, bilirubin, and several metalloenzymes
including glutathione peroxidase (selenium), catalase

tions. There is, therefore, a critical balance between free radical generation and antioxidant defenses. Many of the protective antioxidants are essential nutrients or have essential nutrients as part of their molecule. In the field of nutrition, the term essential is given to those nutrients (like the vitamins) that must be consumed because the body cannot synthesize these compounds. The objective of this review is to identify the sources of free radicals and the consequences of their reactions with cellular components. In addition, the essential dietary nutrients that can protect against the damaging effects of these reactive species are discussed. Particular emphasis is placed on the health implications of free radical damage and the defensive role of dietary antioxidants.

SOURCES

OF

FREE

RADICALS

(iron), and superoxide dismutase (copper, zinc, manganese) and proteins such as ceruloplasmin (copper). The extent of tissue damage is the result of the balance between the free radicals generated and the antioxidant protective defense system. Several dietary micronutrients contribute greatly to the protective system. Based on the
growing usefulness interest of in free essential, radical safe biology nutrients and in the

lack of

effective therapies against further the adverse

for many of the chronic

diseases,
protecting

the

study.-MACHLIN,

effects of oxidative injury warrants L. J.; BENDICH, A. Free radprotective 1: 441-445; role 1987. of antioxidant

ical tissue nutrients.

damage:

FASEBJ

Key Words: freeradicals antioxidants micronutrients tissue damage

essential nutrients

THE

FORMATION

OF HIGHLY

REACTIVE,

oxygen-containing

molecular species is a normal consequence of a variety of essential biochemical reactions. If a reactive molecule contains one or more unpaired electrons, the molecule is termed a free radical. As a result of the relative instability of free radicals and their potential to damage cells and tissues, there are both enzymes and small-molecularweight molecules with antioxidant capabilities that can protect against the adverse effects of free radical reac0892-6638/87/0001-0441/$01 .50. FASEB

Endogenous sources of free radicals include those that are generated and act intracellularly as well as those that are formed within the cell and are released into the surrounding area. Intracellular free radicals are generated from the autoxidation and consequent inactivation of small molecules such as reduced flavins and thiols, and from the activity of certain oxidases, cyclooxygenases, lipoxygenases, dehydrogenases, and peroxidases. Oxidases and electron transport systems are prime, continuous sources of intracellular, reactive oxygenated free radicals. Electron transfer from transition metals such as iron to oxygencontaining molecules can initiate free radical reactions. The sites of free radical generation encompass all cellular constituents including mitochondria, lysosomes, peroxisomes, and nuclear, endoplasmic reticular, and plasma membranes as well as sites within the cytosol. Molecular species include, but by no means are limited to, hydroxyl, peroxy, hypochlorite, superoxide, and alkoxy radicals, and reactive molecules such as hydrogen peroxide and singlet oxygen, which are not free radicals but are certainly reactive and capable of causing damage (Fig. 1) (1). Exogenous sources of free radicals include tobacco smoke, certain pollutants and organic solvents, anesthet-

presented by the American Institute of Meeting of the Federation of American Societies for Experimental Biology, Washington, DC, April 2, 1987. Participants included B. A. Freeman, B. Goldstein, G. F. Combs, Jr., T. F. Slater, and L. J. MachIm.
Nutrition at the 71st Annual

From

the Symposium

441

electron transport
cytochromes xanthine oxidase

system
b5

P450 and

hemoglobin
ENDOPLASMIC

oxidative burst myeloperoxidase enzyme system (phagocytes)

I

oxidases flavoproteins

PEROX/SOMES

reduced flavins transition

LIPID 8/LAYER OF ALL CELLULAR MEMBRANES

metals system

lectron transport lipid peroxidation

lipoxygenases prostoglandin synthetase

NADPH oxidase (phagocytes)

Figure 1. Cellular sources of free radicals. Adapted from ref 1.

hyperoxic environments, and pesticides. Some of these compounds as well as certain medications are metabolized to free radical intermediate products that have been shown to cause oxidative damage to the target tissues. Exposure to radiation results in the formation of free radicals within the exposed tissues (1-5). ics,

CELLULAR OF FREE
Prime

TARGETS AND CONSEQUENCES RADICAL DAMAGE

targets for free radical reactions are the unsaturated bonds in membrane lipids. Consequent peroxidation results in a loss in membrane fluidity and receptor alignment and potentially in cellular lysis. Free radical

damage

to sulfur-containing

enzymes

and

other

proteins

culminates in inactivation, cross-linking, and denaturation. Nucleic acids can be attacked. Subsequent damage to the DNA can cause mutations that may be carcinogenic. Oxidative damage to carbohydrates can alter any of the cellular receptor functions including those associated with hormonal and neurotransmitter responses (1-5). Free radicals such as peroxy radicals, the superoxide anion, and the hydroxyl radical are responsible for many of the damaging reactions. In addition, certain aldehydes such as malondialdehyde and hydroxynonenal, arising from the free radical degradation of polyunsaturated fatty acids, can cause cross-linkings in lipids, proteins, and
nucleic acids (1, 5-7).

can also react directly with singlet oxygen (13, 14). Vitamin C (ascorbic acid) is water soluble and, along with vitamin E, can quench free radicals as well as singlet oxygen. Ascorbic acid has been shown to react directly with superoxide (15, 16), hydroxyl radicals (17), and singlet oxygen (18). Ascorbic acid can also regenerate the reduced, antioxidant form of vitamin E. In the presence of transition metals, ascorbic acid can provoke the formation of free radicals. However, there is no evidence that the autoxidative effect of ascorbic acid leads to the promotion of lipid peroxidation (19). It is quite clear that vitamin E does function as an antioxidant in vivo as evidenced by the increased concentrations of aldehydes, peroxides, and lipofuscin in the tissues of vitamin E-deficient animals. Significantly increased levels of ethane and pentane in the exhaled air of vitamin E-deficient rats as well as from vitamin Cdeficient guinea pigs provide further evidence of increased lipid peroxidation when these nutrients are absent from the diet (20, 21). Recent work has shown that /3-carotene, a pigment found in all plants, is the most efficient quencher of singlet oxygen known in nature and can also function as A. Vitamin A, however, cannot quench singlet oxygen and has a very small capacity to scavenge free radicals (23, 24). /3-Carotene has been found in cellular membranes, including those of lysosomes (25). These three nutrients are also present in relatively high concentrations in the serum. The adrenal and pituitary glands, the brain, white blood cells, platelets, and the lens of the eye concentrate one or more of these nutrients at levels up to 20-fold of that found in the serum (21). There are, in addition, several nutritionally essential minerals incorporated into protective antioxidant enzymes. Zinc, copper, and manganese are required for the activity of the two types of superoxide dismutases. Selenium, an essential component of glutathione peroxidase, is important in the decomposition of hydrogen peroxide and lipid peroxides. Catalase, a hemeprotein,
MACHLIN AND BENDICH an antioxidant (22). precursor of vitamin /3-Carotene is the major carotenoid

ESSENTIAL ANTIOXIDANT

NUTRIENTS WITH FUNCTIONS nutrients can directly scavenge free E (a-tocopherol), the major lipidpresent in all cellular membranes,
peroxidation (8). Vitamin E can

Only three essential radicals. Vitamin soluble antioxidant

protects against

lipid

act directly with a variety of oxy radicals, including the peroxy radical (ROO.), Cd3., and HO. (9, 10), as well as with the superoxide radical (Or) (11, 12). Tocopherol

442

Vitamins

C and E

/3-carotene

Glutathione Peroxidose Cu/Zn

SOD

Vitamin
OF ALL

LIPID

B/LAYER

Vitamin E
Vitamin

CELL ULAR

MEMBRANES

E+ i3-carotene

Peroxidase

Figure

2. Antioxidant

protection

within

the cell.

catalyzes the decomposition of hydrogen peroxide (26). Glutathione, in contrast to the other antioxidant defenses discussed, is a tripeptide composed of nonessential amino acids (Fig. 2). It is important to note that the antioxidant enzymes are primarily intracellular and thus extracellular free radicals, either endogenously produced or from the environment, must be inactivated by the circulating antioxidants such as the direct acting vitamins discussed above as well as by ceruloplasmin, a copper-containing protein (Table 1). The level of dietary intake of all the antioxidant micronutrients directly affects the circulating level of these nutrients and the activity of the antioxidant metalloenzymes. Thus, low intakes of one or more of these antioxidant nutrients could reduce the bodys defenses against free radical damage and increase susceptibility to health problems associated with free radical damage. ANTIOXIDANT In addition free radicals, teract with the reduced antioxidant experiments, and recently, INTERACTIONS

A study of animals demonstrated that dietary supplementation with vitamin E or selenium alone was ineffective in preventing a chemically induced mammary cancer. However, supplementation with both micronutrients prevented tumor development (29). These micronutrient interactions strongly indicate a requirement for optimal intake of all the antioxidant nutrients, because even a marginal deficiency in one can result in a subsequent decrease in the bioactivity of other essential micronutrients even though recommended levels

are consumed.
TABLE
1. Antioxidant Nutrient Vitamin C (ascorbic acid)
micronutrients

Activity Important water-soluble cytosolic chain-breaking antioxidant; reacts directly with superoxide, singlet oxygen; regenerates tocopherol from tocopheroxy

radical Vitamin E (a-tocopherol) Major membrane-bound, lipidsoluble chain-breaking antioxidant; reacts directly with superoxide, singlet oxygen
Most potent singlet oxygen quencher, antioxidant properties particularly at low oxygen pressure, lipid soluble Constituent superoxide Constituent peroxidase of cytosolic dismutase of glutathione

to direct quenching of reactive, damaging vitamin C has been clearly shown to inthe tocopheroxyl radical and to regenerate tocopherol. The evidence for this important function for ascorbic acid includes cell-free investigations with liposomal membranes, in vivo evidence of higher concentrations of vitamin E in tissues of guinea pigs fed high dietary levels of vitamin C (19, 27). Vitamin E can protect the conjugated double bonds of /3-carotene from oxidation. The sparing action of tocopherol on /3-carotene was first described in vivo in humans by Urbach et al. (23). Vitamin E can protect against many of the symptoms of selenium deficiency and vice versa (26, 28). These sparing as well as synergistic actions are thought to result from the ability of both tocopherol and selenium-dependent glutathione peroxidase to decrease the production of lipid peroxidation products (Fig. 3).

3-Carotene

Zinc

Selenium

Copper

Constituent of cytosolic superoxide dismutase and ceruloplasmin Constituent Constituent superoxide of catalase of mitochondrial dismutase

Iron Manganese

FREERADICALS AND ANTIOXIDANT NUTRIENTS

443

glutathione peroxidase (selenium) alcohols

PUFA peroxides Activated Oxygen

and

radicals

C
N.

GSSG

NADPH

GSH Vitamin C. Vitamin c)

Vitamin E

_(

PUFA
N.

alcoholsX

Vitamin

N. N.

Vitamin E (o-tocopheroI)

10
Activated______ /3-caroten N. diet
/

Vitamin C (ascorbate)

/3-caro!ene\
energy Figure

Vitamin n Is peroxidotion
defense system.

3.

Micronutrient

interactions

in the antioxidant

HEALTH RADICAL

IMPLICATIONS DAMAGE

OF

FREE

Cardiovascular diseases including atherosclerosis tissue injury after myocardial infarction been shown to result in part from free radicals

and
have

the cardiac

The range of antioxidant cell and extracellularly

defenses available witihin the should be adequate to protect against oxidative damage. However, the balance can be lost because of overproduction of free radicals, by exposure to sources that overwhelm the antioxidant defenses, or by inadequate intake of nutrients that contribute to the defense system.
The
lutants,

lungs

are continuously

exposed

to oxygen,

air pol-

of nonsmokers. Exposure to hyperoxic conditions after premature birth or degenerative lung disease increases the potential for oxidative damage. Continuous exposure to free radicalcontaining environmental pollutants has been associated with lung damage, emphysema, and cancer (30, 31). The circulating levels of selenium, vitamin C, and /3carotene are lower in smokers than in nonsmokers (32, 33). Lower consumption of foods containing vitamins C, E, and A, /3-carotene, and selenium has been associated in numerous epidemiological studies with increased risk for lung and other cancers, and a recent study correlated lower circulating levels of vitamin E and /3-carotene with significantly greater risk of lung cancer (34). In addition to lung cancer, lower risk of colon cancer has been associated with higher dietary intakes of vitamin E and C, /3-carotene, and selenium (35, 36). Lower circulating levels of /3-carotene, vitamin C, and vitamin E have been found in precancerous dysplasias of the cervix and intestine. In most of these studies, a complete
antioxidant however, is recommended nutritional analysis was not performed;

and tobacco

smoke,

even lungs

generated at the site of damage. Both vitamin E and superoxide dismutase have been used therapeutically with success in lowering the oxidative insult after ischemia (6). A large, multinational epidemiological study suggests that higher dietary intake and blood levels of the antioxidant vitamins and selenium are associated with reduced risk of mortality from cardiovascular disease (36, 37). In addition to the diseases mentioned above, the formation of cataracts, the photodermatoses, inflammatory diseases including arthritis, and the aging process itself have all been associated with free radical damage. Supplementation of laboratory animal diets with antioxidant nutrients has in many cases prevented the development of these degenerative conditions (6, 19, 24, 31, 38-40). REFERENCES 1. 2. 3. 4. 5. 6. B. A.; Ca.uo, J. D. Biology of disease: free radicals and tissue injury. Lab. Invest. 47: 412-426; 1982. SLATER, T. F. Free radical mechanisms in tissue injury. London: Pion; 1972. TAYLOR, A. E.; MATALON, S.; WARD, P. A., EDS. Physiology ofoxygen radicals. Bethesda: Am. Physiol. Soc; 1986. SIES, H. Oxidative stress: introductory remarks. Sies, H., ed. Oxidative stress. New York: Academic; 1985: 1-8. HALLIWELL, B.; GUTTERIDGE, J. M. C. Freeradicals inbiology and medicine. Oxford, England: Clarendon; 1985. FLOHE, L.; BECKMANN, R.; GIERTZ, H.; LOSCHEN, G. Oxygen-centered free radicals as mediators of inflammation. Sies, H., ed. Oxidative stress. New York: Academic; 1985: 405-437. SLATER, T. F. Free radical-mediated tissue damage. Nutrition 1987. Bethesda: Am. Inst. Nutr. In press. MACHLIN, L. J. Vitamin E: a comprehensive treatise. New York: Dekker; 1980.
FREEMAN,

based
for similar

on the interactions of these nutrients, it that future investigators evaluate their

correlations.

7. 8.

data

444

MACHLIN

AND

BENDICH

9. 10.

MCCAY,
cals and

P. B. Vitamin
ascorbate.

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21:

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FREE RADICALS

AND

ANTIOXIDANT

NUTRIENTS

445