Vitamin C, Vitamin E, Selenium, and β-Carotene and … Other Carotenoids: Overview, Antioxidant...

Copyright © National Academy of Sciences. All rights reserved. 35

2Vitamin C, Vitamin E,

Selenium, and β-Caroteneand Other Carotenoids:Overview, Antioxidant

Definition, and Relationshipto Chronic Disease

OVERVIEW

This report focuses on vitamin C, vitamin E, selenium, and β-carotene and other carotenoids (α-carotene, β-cryptoxanthin, lu-tein, lycopene, and zeaxanthin). These compounds have frequentlybeen called dietary antioxidants since in some cases they counteractoxidative damage to biomolecules (Halliwell, 1996), and the possi-bility exists that increased intakes of these compounds may protectagainst chronic disease. Although the term dietary antioxidants is aconvenient description, these compounds are multifunctional, andsome of the actions observed in vivo may not represent an antioxi-dant function, even though the compounds have been classified asantioxidant nutrients (Sies and Stahl, 1995).

Therefore, in this report the above compounds were evaluatedwith respect to their role in human nutrition, without limiting theinvestigation to antioxidant properties. Information was reviewedregarding the minimum amount of these nutrients required to pre-vent deficiency diseases, as well as the amounts that might impacton chronic diseases, regardless of whether the effect was an antioxi-dant effect or not. Resolution of any impact of these compounds onchronic disease will require evaluation of the many human inter-vention trials that are still under way (Table 2-1).

Four main tasks were assigned to the Dietary Reference IntakesPanel on Dietary Antioxidants and Related Compounds. The firsttask was to develop a definition of a dietary antioxidant; the second

Copyright © National Academy of Sciences. All rights reserved.

36

TABLE 2-1 Intervention Trials: Antioxidants and ChronicDiseases

Study Country Study Typea Study Population

Skin Cancer U.S.c Secondary prevention; 1,805 men and womenPrevention randomized, with recent nonmelanomaStudy double-blind, skin cancer; aged 40–89 y(Greenberg placebo-controlledet al., 1990) intervention

Linxian Cancer China Primary prevention; 29,584 poorly nourishedPrevention randomized, men and women,Study double-blind, aged 40–69 y(Blot et al., placebo-controlled1993) intervention

α-Tocopherol, Finland Primary prevention; 29,133 male cigaretteβ-Carotene randomized, smokers, aged 50–69 yCancer double-blind,Prevention placebo-controlledStudy intervention(ATBC CancerPreventionGroup, 1994)

Polyp U.S. Secondary prevention; 864 men and women withPrevention randomized, recent nonmelanomaStudy double-blind, skin cancer(Greenberg placebo-controlledet al., 1994) intervention

The β-Carotene U.S. Primary prevention; 14,254 heavy smokers andand Retinol randomized, 4,060 asbestos workers,Efficacy Trial double-blind, aged 45–69 y(Omenn et al., placebo-controlled1996) intervention

Cambridge U.K.e Secondary prevention; 2,002 patients withHeart randomized, coronary atherosclerosis,Antioxidant double-blind, mean age 62 yStudy placebo-controlled(Stephens interventionet al., 1996)


37

Duration Primaryof Treatment Disease(y) Daily Dose Outcome Resultsb

5 50 mg β-carotene Skin No effect on occurrence ofcancer new nonmelanoma skin

cancers

5.25 15 mg β-carotene, Cancer 9% reduction in total30 mg α-tocopherol, mortality; 13% decrease in50 mg selenium cancer mortality; 21%

decrease in stomach cancerdeaths; 10% decrease incerebrovascular mortality(nonsignificant)

6 50 mg α-tocopherol Lung 50% increase in hemorrhagicand/or 20 mg cancer stroke deaths amongβ-carotene vitamin E group; 11%

increase in ischemic heartdisease deaths amongβ-carotene group; 18%increase in lung canceramong β-carotene group;no effect of vitamin E onlung cancer

4 25 mg β-carotene, Colorectal No reduced incidence of1,000 mg vitamin C, cancer adenomas400 mgα-tocopherol

4 30 mg β-carotene, Lung 28% increase in lung25,000 IU retinol cancer cancer; 26% increase in(as retinyl palmitate) CVDd (nonsignificant);

17% increase in totalmortality among treatmentgroup

1.4 400 or 800 IU CVD 77% decrease in risk of(268 or 537 mg) death or subsequent nonfatal MI;α-tocopherol nonfatal no benefit on

MIf cardiovascular mortality

continued


38 DIETARY REFERENCE INTAKES

TABLE 2-1 Continued


Physicians’ U.S. Primary prevention; 22,071 male physicians,Health Study randomized, aged 40–84 y(Hennekens double-blind,et al., 1996) placebo-controlled

intervention

Nutritional U.S. Secondary prevention; 1,312 men and womenPrevention randomized, with history of basal orof Cancer double-blind, squamous cell carcinoma;Study placebo-controlled aged 18–80 y(Clark et al., intervention1996, 1998)

GISSI- Italy Secondary prevention; 11,324 patients with recentPrevention randomized, MITrial (GISSI- double-blind,Prevenzione placebo-controlledInvestigators; intervention1999)

Women’s U.S. Primary prevention; 39,876 healthy women,Health Study randomized, aged ≥45 y(Lee et al., double-blind,1999) placebo-controlled

intervention

Heart Canada Secondary prevention; 9,541 high-risk men andOutcomes randomized, women, aged ≥55 yPrevention double-blind,Evaluation placebo-controlledStudy intervention(HOPE StudyInvestigators,2000)

Studies In Progress

MRC/BHF U.K. Secondary prevention 20,536 high-risk men andHeart trial women, aged 40–80 yProtectionStudy(MRC/BHF,1999)


OVERVIEW, ANTIOXIDANT DEFINITION, CHRONIC DISEASE 39


12 50 mg β-carotene Cancer, No effects on CVD or cancer(alternate days) CVD including among smokers

4.5 200 µg selenium Skin No effect on incidence ofcancer, skin cancer; 63% reductionprostate in prostate cancercancer incidence; reduction in

total cancer mortality andtotal cancer incidence

3–5 300 mg α-tocopherol Total No benefit from vitamin E;and/or 1 g ω-3 mortality 15% decrease in risk ofPUFAg death, nonfatal MI, and

stroke from ω-3 PUFA

2.1 50 mg β-carotene MI, stroke, No effect on incidence of(alternate days) or CVD cancer, CVD, or total

death mortality

4–6 400 IU (268 mg) MI, stroke, No benefit from vitamin Eα-tocopherol, or CVDACEh inhibitor death

≥5 20 mg β-carotene, Total No results yet600 mg mortalityα-tocopherol,250 mg vitamin C

continued



TABLE 2-1 Continued


Women’s U.S. Secondary prevention; 8,000 women with priorAntioxidant randomized, CVD event or ≥3 coronaryCardiovascular double-blind, risk factors, aged ≥40 yStudy placebo-controlled(Manson et al., intervention1995)

Women’s U.S. Primary prevention; 39,876 healthy women,Health Study randomized, aged ≥45 y(Buring and double-blind,Hennekens, placebo-controlled1992) intervention

Physician’s U.S. Primary prevention; 15,000 healthy maleHealth Study randomized, physicians, aged ≥55 yII double-blind,(Hennekens, placebo-controlled1998) intervention

SUVIMAX France Primary prevention; 12,735 men and women,(Hercberg randomized, aged 35–60 yet al., 1998) double-blind,

placebo-controlledintervention

a A primary prevention trial is one in which the study participants have no history ofthe disease outcome being investigated. Participants in a secondary prevention trialhave had a prior occurrence of the outcome being investigated.

b Unless noted otherwise, results are statistically significant.c U.S. = United States.d CVD = cardiovascular disease.

task was to select, in addition to vitamin C, vitamin E, andβ-carotene, other food components which might prove to be anti-oxidants and play a role in health; the third task was to assess therole of these compounds in health; and the fourth task was to devel-op Dietary Reference Intakes (DRIs) for the selected nutrients. Thepanel was asked to evaluate vitamin C, vitamin E, and β-caroteneand other antioxidants. Since other dietary carotenoids share many,




4 50 mg β-carotene, CVD No results yet600 IU α-tocopherol,500 mg vitamin C(alternate days)

NRi 600 IU α-tocopherol, MI, stroke, No results yet100 mg aspirin or CVD(alternate days) death

NR 50 mg β-carotene, CVD, No results yet400 IU α-tocopherol cancer,(alternate days), eye500 mg vitamin C, diseasesmultivitamin(centrum silver)

8 6 mg β-carotene, Cancer, No results yet30 mg α-tocopherol, ischemic120 mg vitamin C, heart100 µg selenium, disease20 mg zinc

e U.K. = United Kingdom.f MI = myocardial infarction.g PUFA = polyunsaturated fatty acid.h ACE = angiotension-converting enzyme.i NR = not reported.

although not all, of the properties of β-carotene, additionalcarotenoids (α-carotene, β-cryptoxanthin, lutein, lycopene, andzeaxanthin) were added as related nutrients that would be investi-gated. In addition, because of the important role that seleniumplays as a cofactor for oxidant defense enzymes, it was also includedas a related nutrient.



DEFINITION AND CRITERIA FOR A DIETARYANTIOXIDANT

Definition of a Dietary Antioxidant

The definition was based on several criteria: (1) the substance isfound in human diets; (2) the content of the substance has beenmeasured in foods commonly consumed; and (3) in humans, thesubstance decreases the adverse effects of reactive species, such asreactive oxygen and nitrogen species in vivo. Thus, the definition ofa dietary antioxidant is as follows:

A dietary antioxidant is a substance in foods that significantly decreasesthe adverse effects of reactive species, such as reactive oxygen and nitrogenspecies, on normal physiological function in humans.

This stringent definition, with supporting information, was slight-ly modified from the proposed definition published earlier (IOM,1998). The earlier report stated that the nutritional recommenda-tions that will be presented in the final report for some of thesedietary components may not be determined by or related to theirpossible action as antioxidants. The primary indicators for develop-ing the Dietary Reference Intakes (DRIs) are based on specific ac-tions and effects of the compounds that may or may not be attribut-ed to their antioxidant activity. In particular, criteria chosen uponwhich to base EARs and thus RDAs for any of the compounds inves-tigated are not related specifically to ameliorating chronic diseasesbecause a scientific justification for such a requirement was notfound.

The European Commission Concerted Action on Functional Foodalso adopted stringent criteria regarding the relationship betweenfree-radical events and human disease, and whether antioxidantsare capable of modulating these events and thus reducing the riskof disease (Diplock et al., 1998). The summary report concludedthat, “there is at present insufficient evidence available on which tobase a firm conclusion that antioxidants are capable of reducingrisk of disease” (Diplock, 1998). Recommendations for the nutri-ents in this report are similar. However, it should be pointed outthat a large number of human intervention studies are under wayusing the compounds discussed in this report, and the results ofthese studies may give rise to new conclusions.



Are Vitamin C, Vitamin E, Selenium, and β-Carotene and OtherCarotenoids Dietary Antioxidants?

Vitamin C functions physiologically as a water-soluble antioxidantby virtue of its very strong reducing power (high redox potential)and facile regeneration via ubiquitous reductants such as glutathione,nicotinamide adenine dinucleotide, and nicotinamide adeninedinucleotide phosphate. The primary method used to estimate therequirement relates to the vitamin C intake needed to maintainnear-maximal neutrophil ascorbate concentration with minimal uri-nary excretion. Because smokers suffer increased oxidative stressand metabolic turnover of vitamin C, their requirement is increased.Although there is ample evidence that vitamin C administrationcan result in decreases in markers of oxidative stress, a Recom-mended Dietary Allowance (RDA) derived from a direct antioxidantfunction of vitamin C could not be calculated because of the lack ofa quantitative relationship between this antioxidant function and ahealth-related endpoint. Since vitamin C reduces markers of oxida-tive stress, it meets the definition of a dietary antioxidant.

Vitamin E functions as a chain-breaking antioxidant that preventsthe propagation of lipid peroxidation. To estimate the requirement,data were examined on the intake of vitamin E that would preventhydrogen peroxide-induced lysis of erythrocytes. Under these cir-cumstances, vitamin E is acting as an ex vivo antioxidant, maintain-ing a normal physiological function in humans. Although it is notyet possible to relate vitamin E intake to a lowering of chronic dis-ease risk, it still meets the definition of a dietary antioxidant.

Selenium functions through selenoproteins, several of which areoxidant defense enzymes. The criterion used to estimate the re-quirement for selenium relates to the intake needed to maximizethe activity of the plasma selenoprotein glutathione peroxidase, anoxidant defense enzyme. It is not clear if the diseases associatedwith selenium deficiencies, Keshan disease or Kashin-Beck disease,are due to oxidative stress. The selenium in several selenoproteinshas a biochemical role in oxidant defense, thus maintaining normalphysiological function, and as such plays a role as a dietary antioxi-dant.

β-Carotene and other carotenoids function as sources of vitamin Aand, due to this provitamin A activity, can prevent vitamin A defi-ciency. Because no other specific nutrient functions have been iden-tified at this time, no requirements have been established for any ofthe carotenoids. β-Carotene and the other carotenoids display invitro antioxidant activity, but the evidence that they act as in vivo



antioxidants in humans is still controversial. Therefore, β-carotenedoes not meet the definition of a dietary antioxidant. Similar con-clusions have been drawn for α-carotene, β-cryptoxanthin, lycopene,lutein, and zeaxanthin.

OXIDATIVE STRESS, ANTIOXIDANTS, ANDCHRONIC DISEASE

Oxidative Stress

There have been many proposals as to how oxidative stress can bedefined. Because all cells are exposed to oxidants, generated eitherendogenously from metabolism or exogenously from a variety ofenvironmental insults, the problem arises as to what constitutes anoxidative stress. Furthermore, different cells can be exposed to thesame level of oxidants, but depending on the level of antioxidantsor protective mechanisms available to the cell, they may or may notexperience an oxidative stress. Sies (1985) defined oxidative stressas “a disturbance in the prooxidant-antioxidant balance in favor ofthe former.” Others have amplified this definition to include “short-and/or long-term disturbance of the prooxidant-antioxidant bal-ance resulting in adverse effects that are due either to impairedantioxidation or to favored prooxidation” (Biesalski et al., 1997).Here, oxidative stress is defined as an imbalance between the pro-duction of various reactive species and the ability of the organism’snatural protective mechanisms to cope with these reactive com-pounds and prevent adverse effects.

The primary reactive species include reactive oxygen species(ROS) and reactive nitrogen species (RNS). These in turn react inthe body and generate radical intermediates of lipids, proteins, andnucleic acids that ultimately form the chemical end products ofoxidative stress. The physiological consequences of these end prod-ucts have been hypothesized to be the causes of many chronic dis-eases as well as the natural aging process (Ames, 1998; Halliwell,1997). The protective mechanisms include protective enzymes, an-tioxidant or quenching compounds produced by the organism, andsimilar compounds made available in the diet. The evidence thatchronic disease results from an imbalance between formation andremoval of reactive species is discussed below.

The primary defensive compounds are antioxidants that can in-teract with and quench reactive radical species, and enzymes thatcan inactivate these species or their products. All of the compoundsin this report—vitamin C, vitamin E, selenium in the form of se-lenoproteins, and β-carotene and other carotenoids—are in vitro



antioxidants, and some evidence exists that they have in vivo antiox-idant actions. The protective enzymes include, among others, super-oxide dismutase, catalase, and selenium-containing glutathione per-oxidases and thioredoxin reductase. Other antioxidant mechanismsinclude stimulation of the expression of antioxidant or repair en-zymes, as well as chelation of transition metals. A recent review ofthe relationship between antioxidant supplementation and oxida-tive damage concludes that, with the exception of vitamin E andpossibly vitamin C reducing markers of lipid peroxidation, the evi-dence is insufficient that antioxidant supplementation leads to amaterial reduction in oxidative damage in humans (McCall andFrei, 1999).

Biomarkers of Oxidative Stress

There have been numerous tests described to evaluate the level ofmarkers of oxidative stress in living systems. Many of these markersrepresent oxidative breakdown products of normal tissue compo-nents and metabolites. Products derived from oxidized lipids in-clude age pigments (lipofuscin), aldehydes, alkanes, and pros-tanoids such as the F2-isoprostanes derived from unsaturated fattyacids. Oxidation of proteins produces protein carbonyls and aminoacid derivatives such as methionine sulfoxide and nitrotyrosine, de-rived from the reaction of peroxynitrite (ONOO–) with tyrosine.Purine and pyrimidine metabolites that are derived from oxidizednucleic acids can be detected in tissues and in urine. Since virtuallyall tissues are exposed to ROS and RNS, there will always be a base-line production of these biomarker molecules. What is important inevaluating the extent of oxidative stress is the change in the level ofthe biomarker compared to a baseline or steady-state level.

It is quite clear that dietary change can alter the levels of some ofthe biomarkers described above, and this phenomenon has beenreported when the dietary intake of fruits and vegetables has beenincreased. Examples of such studies include reductions in biomark-ers of deoxyribonucleic acid (DNA) damage (Pool-Zobel et al.,1997) or of lipid peroxidation (Miller et al., 1998), following con-sumption of diets rich in fruits and vegetables. Many of these stud-ies have been reviewed recently (Halliwell, 1999).

A major problem with the use of biomarkers such as those de-scribed above is not knowing whether they reflect processes in theinitiation of a disease state or whether they are products of a diseasestate. The validity of biomarkers, either intermediate or final, willnot be settled until they are evaluated as part of intervention trials.



A causal relationship between the formation of a biomarker of oxi-dant stress and a chronic disease has not yet been validated.

Some attempts have been made to determine a more global mea-sure of oxidative stress, such as TRAP (total radical-trapping antiox-idant capability) (Wayner et al., 1985), ORAC (oxygen radical ab-sorbance capacity) (Cao et al., 1993), TEAC (trolox equivalentantioxidant capacity) (Miller et al., 1993), and FOX (ferrous oxida-tion/xylenol orange) (Jiang et al., 1992) assays. However, becausethese markers do not measure the same oxidants or antioxidantdefenses (Cao and Prior, 1998), it is difficult to validate them asuseful markers of oxidant stress.

Evidence of Oxidative Stress and Chronic Disease

Aerobic metabolism produces energy, waste products such as car-bon dioxide, and a small, but steady stream of radical by-productsthat are capable of reacting with all of the body’s constituents toform oxidative damage products (Ames et al., 1993; Chance et al.,1979). Calculations have been made of the number of radical spe-cies formed per cell per day, but it is not at all clear how thesenumbers relate to diseases. Since the entire population is exposedto oxidative stresses and only a small fraction develops a chronicdisease, it is clear that at this time, it is not understood how toevaluate the role of oxidative stress in the development of chronicdisease. The potential role of oxidative stress in six chronic diseaseareas and aging is described briefly below.

Cancer

DNA is subject to damage, and either an exogenous or an endog-enous mutagen can produce damage at a faster rate than the nor-mal protective process of enzymatic repair (Ames et al., 1995). Un-der these circumstances, elevated levels of excision products willappear in the urine. The observation that 8-oxy-7,8-dihydro-2′-deox-yguanosine (8-oxodG) is a major urinary product of both oxidativedamage to DNA and damage by ionizing radiation that producesROS, has served as the basis for the hypothesis that oxidative DNAdamage is carcinogenic (Ames et al., 1995). The presence of 8-oxodG in DNA would lead to a guanine-to-thymine transversionthat would result in a DNA mutation and, depending on its loca-tion, an altered gene product. 8-OxodG is also produced by ioniz-ing radiation, and since this process can be carcinogenic, it hasbeen assumed that any condition that produces 8-oxodG will also



be carcinogenic. However, a causal relationship between oxidativestress to DNA and cancer in humans has not yet been established(Poulsen et al., 1998).

A great deal of evidence based on epidemiological studies indi-cates that consuming diets rich in fruits and vegetables is associatedwith both a decrease in oxidative damage to DNA (Halliwell, 1998)and a lower risk of a number of common cancers. There are severalmechanisms that could account for these observations, in additionto antioxidant components that scavenge radical intermediates.Other mechanisms include the modification of carcinogen acti-vation by the inhibition of phase 1 enzymes, modification of car-cinogen detoxification by phase 2 enzymes, and suppression of theabnormal proliferation associated with preneoplastic lesions (War-govich, 1997).

Cardiovascular Disease

Of all the chronic diseases in which excess oxidative stress hasbeen implicated, cardiovascular disease has the strongest support-ing evidence. A coherent pathogenetic mechanism has been devel-oped to account for the earliest stage in atherogenesis, namely, thedevelopment of the fatty streak lesion. Hypercholesterolemia and,particularly, increased concentrations of low-density lipoproteins(LDLs) cause the accumulation of cholesterol-loaded “foam cells”beneath the endothelial lining of major arteries, which in turn de-velop into a fatty streak. This lesion is clinically benign but is theprecursor of later lesions (the fibrous plaque and the complex le-sion) that ultimately give rise to clinical manifestations (angina pec-toris and myocardial infarction) (Steinberg and Witztum, 1990).

An elevated LDL cholesterol level sufficiently increases the risk ofcardiovascular disease. Brown and Goldstein (1986) demonstratedthat the molecular defect in individuals with familial hypercholes-terolemia was the absence of functional LDL receptors. The histo-logical lesions in these patients cannot be differentiated from thatof lesions in individuals who have normal LDL receptors. The im-plication therefore is that normal LDL taken up by way of the nor-mal LDL receptor cannot be the basis for the formation of foamcells. In other words, the LDL must first be modified somehow andthe modified form must be taken up into the monocytes or mac-rophages via one or more alternative receptors ultimately and de-velop into foam cells. One such modification, and the most exten-sively studied, is LDL oxidation, and several macrophage receptorshave been shown to take up oxidatively modified LDL (oxLDL) and



thus develop foam cells (Navab et al., 1995; Steinberg, 1997; Stein-berg and Witztum, 1990; Steinberg et al., 1989; Steinbrecher, 1997).

In addition to its ability to cause foam cell formation, oxLDL cancontribute to atherogenesis by virtue of a number of properties thatdiffer from those of normal LDL (reviewed in Steinberg, 1997).The key question is whether these potentially proatherogenic prop-erties of oxLDL are of sufficient importance that inhibition of thegeneration of oxLDL by antioxidants will have a significant impacton the rate of progression of atherosclerotic lesions.

The finding that a relationship existed between LDL levels andthe incidence of cardiovascular disease was followed by the observa-tion that oxLDL was associated with the development of atheroscle-rotic lesions in experimental animals. This association led to thehypothesis that oxLDL is the causative agent in the development ofcardiovascular disease (Steinberg et al., 1989). This hypothesis hasserved as the basis for a number of human intervention trials, test-ing whether antioxidant agents capable of decreasing the extent ofoxidation of LDL and thus decreasing oxLDL concentration mightprove useful in decreasing the incidence of cardiovascular disease.

Several biomarkers of oxidative stress have been used to evaluatethe extent of oxLDL formation and/or the extent of oxidative dam-age to lipids in general. OxLDL formation has been assessed aftertreating individuals with dietary antioxidants and evaluating thepresence of oxLDL. In addition there have been reports of a potenteffect of supplementary antioxidants in lowering the extent of exvivo oxLDL formation. The results are not consistent, and as such,it is still not possible to conclude that dietary antioxidants preventthe formation of oxLDL in vivo. In addition, this modulation ofLDL oxidation still must be validated as a marker of risk for cardio-vascular disease (Zock and Katan, 1998).

The appearance of F2-isoprostanes in urine has been suggested bya number of investigators as a reliable index of in vivo free radicalgeneration and oxidative lipid formation. There is very strong evi-dence from animal studies that F2-isoprostanes increase in plasmaand urine as a result of oxidative stress, and in humans, these prod-ucts are elevated in smokers (Morrow et al., 1995). Evidence is grad-ually accumulating that supplementary antioxidants, and vitamin Ein particular, can both affect the level of F2-isoprostanes in animalmodels of atherosclerosis and decrease the extent of arterial walllesions (Pratico et al., 1998). Similar results have been obtained inhumans (Hodis et al., 1995). Although some reviews point out thatthe protective role of antioxidants in animal models of atheroscle-rosis is only partially confirmed in human studies (Faggiotto et al.,



1998), others are much more supportive of a protective role forvitamin E in coronary heart disease (Pryor, 2000).

Cataracts

There have been a number of observational epidemiological stud-ies of the risk of developing cataracts in humans. Many of thesestudies indicate that the risk of cataracts may be inversely propor-tional to the serum level of antioxidants (Knekt et al., 1992; Tayloret al., 1995) or may be reduced by supplement use (Jacques et al.,1997). These studies, however, have been considered to be incon-clusive (Christen et al., 1996a; Leske et al., 1998). In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, 5 to 8 years ofdaily supplementation with either 50 mg of vitamin E or 20 mg of β-carotene or both resulted in no difference in the prevalence ofcataracts in the men in this study (Teikari et al., 1997).

Age-Related Macular Degeneration

This irreversible disease, which is the major form of blindness inthe elderly in the United States, Canada, and Europe, has beenrelated to antioxidants found in the diet. This is because the pig-ment in the macular region of the normal retina consists of the twoxanthophyll carotenoids, lutein and zeaxanthin (Bone et al., 1988;Handelman et al., 1988). This observation, coupled with the epide-miological observations of an inverse relationship between the riskof age-related macular degeneration (AMD) and the ingestion offruits and vegetables (Goldberg et al., 1988), led a number of groupsto propose that the basis of this chronic disease was a nutritionaldeficiency of green, leafy vegetables and yellow and orange fruitsand vegetables that were rich in lutein and zeaxanthin (EDCCSG,1992; Seddon et al., 1994; Snodderly, 1995). Another associationwas the observation that smokers, who have lower plasma levels ofcarotenoids, also have a lower macular pigment (lutein and zeaxan-thin) density (Hammond et al., 1996) and an increased risk of de-veloping AMD (Christen et al., 1996b). However, all of these re-ports are associative in nature and do not demonstrate a causalrelationship between deficiencies of lutein and zeaxanthin and de-velopment of AMD.



Central Neurodegenerative Diseases

There is increasing evidence that a number of common neuro-degenerative diseases, such as Alzheimer’s disease, Parkinson’s dis-ease, multiple sclerosis, and amyotrophic lateral sclerosis may in-clude adverse responses to oxidative stress. Small intervention trialsin patients with these diagnoses have reported some improvementwith either vitamin E (Muller, 1994; Sano et al., 1997) or vitamin C(Morris et al., 1998; Riviere et al., 1998), but it is still too early todraw any conclusions as to the usefulness of these compounds inthese diseases, or to their ability to delay onset of the disease.

Diabetes Mellitus

Cardiovascular complications are the major causes of death indiabetes. The incidence of coronary heart disease in type II diabe-tes is significantly higher than that in the general population (Kan-nel and McGee, 1979). In addition, individuals with diabetes expe-rience microvascular complications (retinopathy, neuropathy, andnephropathy) secondary to their hyperglycemia. Thus, oxidativeprocesses also may play an important role in the development orprogression of diabetes mellitus.

In vitro oxidation of LDL from patients with diabetes mellitusproceeds at an accelerated rate, which suggests that they are moresusceptible to the atherogenic process (Chisolm et al., 1992; Nishiga-ki et al., 1981; Reaven et al., 1995; Tsai et al., 1994). The several waysin which oxLDL is potentially more atherogenic than native LDLhave been discussed above (see section “Cardiovascular Disease”).

Diabetics tend to have smaller, denser LDL (associated with hy-pertriglyceridemia) and these LDLs are more susceptible to oxida-tive modification ex vivo (Feingold et al., 1992). The TRAP of plas-ma from patients with insulin-dependent diabetes is decreased (Tsaiet al. 1994), which may account in part for the greater susceptibilityof their LDL to oxidation.

Microvascular complications are believed to be the ultimate con-sequences of nonenzymatic glycosylation and the progressive accu-mulation of advanced glycosylation end products (AGEs). There isevidence that the formation of these complex carbohydrate-proteinand carbohydrate-lipid complexes is accompanied and acceleratedby oxidative processes, which may lead to diabetic complications(Brownlee et al., 1988; Dyer et al., 1993; Hunt et al., 1988; McCanceet al., 1993; Mullarkey et al., 1990). The glycosylation process isassociated with increased formation of free radicals, and the possi-



bility that treatment with antioxidants might slow the developmentof AGEs is under investigation.

The issue of whether modifications observed in plasma and tissueproteins in patients with diabetes are due to an oxidative stress or astress from reactive carbonyls has been discussed by Baynes andThorpe (1999). A number of studies have tried altering the patho-biology of diabetes by treating patients with either single antioxi-dant-type compounds or combinations of compounds with antioxi-dant properties. The results have been inconclusive, which mayreflect the fact that the underlying pathology is not caused exclu-sively by an oxidative stress, but by an inability to metabolize andinactivate reactive carbonyls appropriately. Under these circum-stances, the oxidative damage may be exacerbated, resulting in anincrease in many of the markers associated with oxidative stress butnot caused directly by oxidative stress (Baynes and Thorpe, 1999).

Aging

Aging is not in itself a chronic disease, but rather is characterizedby the active or passive presence of a chronic disease (cardiovascu-lar disease, cancer, cataracts, Alzheimer’s disease, etc.). It is notclear if an accumulation of chronic insults and weakened defensesrenders the aging individual more susceptible to various diseases.Do antioxidants play a role in preventing aging or prolonging life?There is no direct evidence in humans for such an effect, althoughvitamin E supplementation appears to improve some immune re-sponses in the elderly (Meydani et al., 1997). There have been sug-gestions that supplementing older animals with antioxidants mayimprove various physiological functions (Hagen et al., 1999), butthe only experimental intervention that has resulted in prolonga-tion of the life span of the animals has been the drastic reduction offood consumption (Pariza and Boutwell, 1987). Whether such aprotocol would delay aging in humans has not yet been studied.Whether dietary antioxidants can lead to healthier aging remains tobe proven.

CONCLUSIONS

There is little doubt that an imbalance in the production of freeradicals and other reactive species and the natural protective sys-tems available to organisms can lead to the production of oxidizedproducts of lipids, nucleic acids, and proteins. These oxidationproducts, or biomarkers of this imbalance, may be related to early



events in certain chronic diseases. However, they have not yet beenadequately validated as markers of the onset, progression, or regres-sion of any chronic diseases. Although vitamin C, vitamin E, andselenium have been shown to decrease the concentrations of someof the biomarkers associated with oxidative stress, the relationshipbetween such observations and chronic disease remain to be eluci-dated. As a consequence, it has not been possible to establish thatdietary antioxidants or other nutrients that can alter the levels ofthese biomarkers are themselves causally related to the develop-ment or prevention of chronic diseases.

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