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Department of Epidemiology and Public Health, Yale University School of Medicine and Yale Cancer Center, New Haven, CT 06520
2 To whom correspondence should be addressed. E-mail: Susan.Mayne{at}Yale.Edu.
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ABSTRACT |
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 TOP ABSTRACT Biomarkers of exposure for...Biomarkers of oxidative stress...Methodologic issuesImplications and future...LITERATURE CITED |
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KEY WORDS: • antioxidants • biomarkers • diet assessment • epidemiology • nutrition • oxidation • oxidative stress
All cells in the body are exposed chronically to oxidants from both endogenous and exogenous sources but come equipped with an antioxidant defense system. Nutrients, both water soluble and lipid soluble, comprise an important aspect of the antioxidant defense system with which humans have evolved. The antioxidant nutrients that are the focus of this review include ß-carotene and other carotenoids (
-carotene, lycopene, lutein and zeaxanthin, ß-cryptoxanthin), -tocopherol or vitamin E, ascorbic acid or vitamin C, and the trace mineral selenium. There are many other dietary constituents that may have either direct antioxidant activity such as flavonoids or indirect antioxidant activity such as zinc and manganese (constituents of antioxidant enzymes such as superoxide dismutase), that are not covered in this review.
Sies (1) has defined oxidative stress as "a disturbance in the prooxidant-antioxidant balance in favor of the former." Thus oxidative stress is essentially an imbalance between the production of various reactive species and the ability of the organism's natural protective mechanisms to cope with these reactive compounds and prevent adverse effects. Oxidative stress has been suggested to be involved in the etiology of a host of chronic diseases including cancer, cardiovascular disease, cataracts, age-related maculopathies and aging in general (2).
Reactive oxygen and nitrogen species can attack various substrates in the body including lipids, nucleic acids and proteins. Oxidation of any of these substrates, if unchecked, can theoretically contribute to chronic disease development. For example, oxidatively modified low density lipoprotein (LDL) has been hypothesized to be a causative agent in the development of cardiovascular disease, as is reviewed elsewhere (2).3 Oxidatively modified DNA may also play a role in human carcinogenesis, although a causal relationship has not yet been firmly established (3). Cataracts are thought to result from photooxidation of lens proteins that results in protein damage, accumulation, aggregation and precipitation in the lens (4).
Together these chronic diseases of aging account for an enormous amount of morbidity and mortality; therefore prevention of these diseases is a critical public health priority. A voluminous body of research on the potential role of antioxidant nutrients in the prevention of chronic diseases has accumulated over the past several decades. Despite this effort, there is much that remains unknown. Biomarker research in this field has the potential to help fill the gaps in current knowledge.
With this background in mind, this article reviews both biomarkers of exposure of the selected antioxidant nutrients for use in epidemiologic studies as well as biomarkers of oxidative stress. This review concludes with some methodologic issues and future directions for biomarker research concerning antioxidant nutrients and their relationship with chronic disease risk.
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Biomarkers of exposure for antioxidant nutrients |
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TOPABSTRACTBiomarkers of exposure for...Biomarkers of oxidative stress...Methodologic issuesImplications and future...LITERATURE CITED |
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ß-Carotene and other carotenoids.
Epidemiologic studies of carotenoids and disease can rely upon either dietary intake assessment or carotenoid levels in plasma or tissues to estimate exposure. Beginning with intake estimation, the U.S. Department of Agriculture has released (5) and subsequently updated (6) a food-composition database for the major dietary carotenoids: ß-carotene, -carotene, lycopene, lutein + zeaxanthin and ß-cryptoxanthin. These carotenoids are concentrated in fruits and vegetables (and in some cases in relatively few fruits and vegetables) which makes intake assessment via questionnaire feasible. The National Academy of Sciences/Institute of Medicine released intake estimates of these dietary carotenoids from the Third National Health and Nutrition Examination Survey (NHANES III) (7), therefore normative data for the intake of these nutrients in various age- and gender-specific strata of the U.S. population are now available.
Biological markers of exposure to these compounds are also widely used in studies of carotenoids and disease. Carotenoids are known to increase in plasma or serum in response to dietary interventions [e.g., see McEligot et al. (8), Rock et al. (9), Micozzi et al. (10), Campbell et al. (11), Martini et al. (12)], and their levels can be readily measured in plasma or in serum using high performance liquid chromatographic (HPLC) approaches (13). The U.S. National Institute of Standards and Technology has coordinated a micronutrient measurement proficiency testing program for many years, whereby participating laboratories measure various carotenoids and other micronutrients in test samples of human plasma over time. This type of quality assurance program is important in assuring both intra- and interlaboratory reliability in assay results. Available HPLC assays can readily detect numerous carotenoids in humans (14). Because carotenoids can occur in both the all-trans and in cis configurations, an emerging laboratory issue surrounds the ability to adequately discriminate between the various carotenoid isomers (15). There is considerable interest, for example, in quantitating cis- versus trans-lycopene and its relationship to prostate cancer risk (16). In refining carotenoid assay methodology for epidemiologic studies, investigators must balance the chromatographic time needed to adequately resolve structurally similar compounds against the costs associated with longer assay times; different assays may be appropriate, depending on the needs of a specific study.
Plasma carotenoid concentrations generally correlate significantly with dietary intake estimates, but the magnitude of the correlation is generally modest (< 0.5) (17). This is because carotenoid concentrations in plasma are influenced by many factors as reviewed elsewhere (18). For example, bioavailability of ß-carotene from supplements is dramatically better than that from foods (10,18). Also, the food matrix can affect bioavailability, ranging from poor bioavailability of carotenoids in raw green leafy vegetables to intermediate bioavailability in cooked vegetables and fruits and high bioavailability in oil preparations such as red palm oil (18). Mild cooking and steaming of plant foods generally increases carotenoid bioavailability; for example, bioavailability of lycopene from tomato juice is improved by heat treatment (19). Fat also facilitates absorption of carotenoids. Lycopene has dramatically better bioavailability in the form of cooked tomato sauce as compared to tomato juice or raw tomatoes, which could result from mechanical or heat disruption of cells and extraction of the carotenoid into the lipophilic phase (20). Thus the food form of the carotenoid can greatly influence bioavailability. In addition, host factors such as adiposity, gender, smoking status, ethanol consumption and plasma cholesterol concentrations are also known to affect carotenoid concentrations in plasma (21). Thus both diet and biomarker approaches are valid ways of estimating exposure, but their interpretations are somewhat different. That is, the biomarker approach to exposure assessment is an indicator of dietary intake over a relatively short time period, but it is also a reflection of the amount of the carotenoid in blood after absorption, metabolism, tissue distribution and excretion. In contrast, using diet to estimate exposure to carotenoids has clear advantages in terms of translating associations with chronic disease risk to nutrient recommendations and prudent dietary patterns.
Carotenoids have also been measured in various tissues, especially adipose tissue (22), and studies have reported reasonable correlations between plasma and adipose tissue concentrations of carotenoids (23,24). In general, correlations between tissue and plasma exceed those between diet and plasma when measured in the same subjects (25). One large epidemiologic study evaluated both plasma and adipose tissue carotenoid concentrations as predictors of risk for myocardial infarction, and results indicated that lycopene in adipose tissue (26) but not in plasma (27) was inversely associated with risk. Adipose tissue is a logical tissue to sample to estimate systemic exposure to lipid-soluble nutrients such as carotenoids and vitamin E, because these substances are known to accumulate in adipose tissue. The advantage of using adipose tissue as compared to plasma is that adipose tissue reflects longer-term exposure to lipid-soluble nutrients. However, the use of adipose tissue (needle) biopsy for epidemiologic studies might adversely affect participation rates, and adipose tissue samples generally require saponification before HPLC analysis, which adds to analytic costs.
Although adipose tissue and plasma can be used to estimate systemic exposure to the nutrients of interest, of great current interest in biomarker research is measurement of the nutrient of interest in the etiologically relevant target tissue for a given chronic disease. Lutein and zeaxanthin, for example, are two carotenoids that selectively accumulate in the human macula (retina). Some evidence suggests that higher dietary intake and plasma concentrations of these compounds are inversely associated with risk of age-related macular degeneration (28). Current research is focusing on measuring the concentrations of these compounds in the retina either invasively (in autopsy studies) (29) or noninvasively, (30,31), because retina is the target tissue of interest for this particular disease. The need to quantitate exposure in the target tissue of interest rather than in a surrogate (more easily obtained) tissue is suggested by a recent study that investigated the response to a 15-wk dietary intervention of lutein and zeaxanthin in seven subjects (32). The pattern of response in adipose tissue lutein concentrations was inverse to the pattern of macular pigment density. That is, adipose tissue lutein concentrations decreased at wk 4 relative to baseline and then significantly increased above baseline at wk 8. Macular pigment, in contrast, increased significantly at wk 4, which was followed by a decrease to a near-baseline value at wk 8. These results require confirmation in a larger group of subjects but emphasize the possibility of tissue-specific effects that should be considered in any human studies that use biomarkers of exposure.
Another tissue that has received increasing attention with regard to carotenoid concentrations is human skin. Carotenoids in skin have been measured using conventional HPLC methods (25), and newer methods such as reflection photometry (33) and resonance Raman spectroscopy (34) are also being proposed. These technologies have yet to be validated or used in epidemiologic research but do represent promising avenues of biomarker research.
-Tocopherol/vitamin E.
Vitamin E is a generic term that generally has referred to four different tocopherols and four different tocotrienols. -Tocopherol is the predominant form both with regard to biological activity and in that it is the only form that is specifically maintained in human plasma (2). Currently, most nutrient databases do not distinguish between the different tocopherols in foods but instead present the data as -tocopherol equivalents, thereby considering differences in bioavailability of the different forms. Supplements are generally either natural or synthetic forms of -tocopherol.
Several authors have attempted to quantitate intake of vitamin E from foods, but the intake estimates are generally considered to be somewhat unreliable as is detailed elsewhere (2). Vitamin E is concentrated in many vegetable oils and fats, and the amount of fats and oils added during food preparation is difficult to assess. Also, different oils have different concentrations of tocopherols, and food labels often do not provide the specific fat or oil in the product; also, manufacturers may substitute fat sources depending upon availability and cost. Food-composition tables also do not allow investigators to differentiate -tocopherol from 
-tocopherol, for example, which thus introduces additional errors in assessment.
Epidemiologic studies of vitamin E and health often rely upon biochemical markers of exposure. Plasma concentrations of -tocopherol in particular can be readily estimated using HPLC methods. With the use of multiple-wavelength (ultraviolet/visible) or photodiode-array detectors, it is common to measure tocopherols in the same chromatographic run used to measure carotenoids (35). As compared with intake estimates, plasma vitamin E concentrations may represent the more relevant biological exposure, but may correlate poorly with intake estimates. For example, Ford and Sowell (36) reported that plasma -tocopherol concentrations measured in NHANES III did not correlate with estimated vitamin E intake from 24-h dietary recalls. Others have also reported that plasma -tocopherol concentrations were not associated with intake estimates, or that the associations seen were largely due to supplemental vitamin E intake (2). If investigators choose to measure concentrations of -tocopherol in plasma as an index of exposure, then measurement of total plasma cholesterol should also be undertaken, because -tocopherol levels in plasma are highly correlated with plasma cholesterol, and adjustment for plasma cholesterol in multivariate analyses should be considered.
Adipose tissue obtained via needle biopsy can also be used to estimate exposure to vitamin E. Some investigators have estimated correlations between concentrations of vitamin E in plasma and in adipose or other tissues (24,25,27). Vitamin E levels in plasma are correlated with adipose tissue levels, and the magnitude of the correlation generally is higher than when one compares dietary vitamin E intake with adipose tissue concentrations (24).
Intake of vitamin E from supplements, unlike from foods, can be easily estimated by using a questionnaire. In response to supplementation, plasma -tocopherol concentrations generally increase, whereas plasma -tocopherol concentrations generally decrease (37). For every 10-fold increase in -tocopherol intake, plasma concentrations roughly double (38). Handelman et al. (37) reported that the adipose tissue ratio of -tocopherol/-tocopherol could be used to discriminate between vitamin E supplement users (
250 mg/d) and onusers, which suggests that this might be a suitable biomarker of exposure for supplemental vitamin E use.
Vitamin C.
Vitamin C is a water-soluble nutrient found primarily in fruits and vegetables. Although adequate food-composition databases exist for quantifying the amount of vitamin C in various foods, it should be recognized that actual ingested levels vary according to food-handling and cooking practices. That is, vitamin C can be destroyed in foods as a result of exposure to high temperatures, oxidation or cooking in large amounts of water. Despite these limitations, vitamin C intake from foods, fortified foods and supplements is commonly estimated in numerous epidemiologic studies and is the usual approach for estimating exposure.
Plasma ascorbate can also be measured to estimate exposure to vitamin C; validated HPLC methods with documented performance over time are currently available (39). However, the use of plasma ascorbate to estimate exposure has several limitations in the context of epidemiologic research. First, plasma samples need to be specifically preserved at the time of sample collection to avoid degradation of the ascorbate. This renders many plasma archives unsuitable for epidemiologic studies. Metaphosphoric acid is often used; total ascorbic acid losses in samples preserved with metaphosphoric acid are >1%/y, and metaphosphoric acid-stabilized samples are compatible with both liquid chromatography (LC) and colorimetric methods (39). Second, ascorbate levels in plasma fluctuate in response to current intakes, which makes fasting blood samples essential (40). Plasma fluctuations also mean that concentrations may not indicate status over the long or even middle term. Third, plasma ascorbate is directly related to ascorbate intake between 50 and 90 mg/d; however at intakes greater than this, renal clearance increases sharply with plasma nearly fully saturated at intakes that are readily achievable by diet (e.g., by 200 mg/d) (41). Thus plasma ascorbate may reasonably predict intake at low levels but not at higher dietary levels or in supplement users.
Despite these limitations with plasma ascorbate, it has been measured in some epidemiologic studies. For example, Drewnowski et al. (42) evaluated serum ascorbate and ß-carotene as biomarkers of vegetable and fruit intake in a population of 361 males and 476 females from France. Although serum ß-carotene was more highly correlated with reported intake of vegetables (r = 0.30) than was serum ascorbate (r = 0.15), the latter was more highly correlated with reported fruit intake (r = 0.36 for serum ascorbate and total fruit intake versus 0.29 for serum ß-carotene and total fruit intake). Consistent with many other studies (2), smokers had lower levels of both ascorbate and ß-carotene in their sera compared with nonsmokers.
Selenium.
Selenium is widely present in the food supply with rich sources that include cereals and grains, meats and fish. The selenium content of food varies depending on the selenium content of the soil where the plant was grown or where the animal (which ingests local forage crops) was raised. The amount of selenium in soil can vary substantially, and the same food items may have a > 10-fold difference in selenium content (2). For these reasons, estimation of selenium exposure through dietary assessment is generally considered unreliable, and most epidemiologic studies rely on biomarkers for assessing selenium status.
Available biomarkers include either blood-based measures or toenail clippings. Plasma selenium, for example, can be measured by atomic absorption spectrophotometry (43). Plasma selenium is thought to reflect fairly recent exposure and rises in response to selenium supplementation (44). In concept, selenium-dependent glutathione peroxidase could also be measured in plasma, platelets and red blood cells. However, this enzyme saturates at levels of intake readily achieved by diet and is only appropriate for characterizing selenium status at suboptimal intakes. The measurement of selenium in toenail clippings is commonly used in epidemiologic studies, because toenail selenium content is unaffected by dietary selenium intake during the previous 3 mo but appears to provide a time-integrated measure of exposure over a period 26–52 wk (45). The length of the relevant exposure period with toenails as compared to plasma is an advantage for epidemiologic studies of chronic disease. Coupled with the ease of collecting and storing toenail clippings, it accounts for the widespread use of toenail selenium for assessing exposure in epidemiologic research.
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Biomarkers of oxidative stress status |
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TOPABSTRACTBiomarkers of exposure for...Biomarkers of oxidative stress...Methodologic issuesImplications and future...LITERATURE CITED |
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Biomarkers of lipid oxidation.
Thiobarbituric acid-reactive substances. Oxidation of lipids has been widely studied, and there are more biomarkers available for assessing oxidation of this substrate than for protein and DNA combined. Thiobarbituric acid-reactive substances (TBARS) comprise one of the earliest markers in use for human and animal studies (47). The TBARS assay is a simple spectrophotometric assay that measures a chromogen that is produced by the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA), which is an endproduct of lipid peroxidation. The spectrophotometric TBARS assay is extremely easy to use, but is nonspecific in that many other substrates (e.g., aldehydes) can react with thiobarbituric acid (38). The lack of specificity with the simple spectrophotometric method has rendered it rather obsolete. Instead, most researchers today use an HPLC modification of the TBARS method. This approach uses HPLC to separate the MDA-TBA adduct from interfering chromogens, thereby resulting in improved sensitivity, specificity and reproducibility (48). TBARS can be measured in tissues but is generally measured in plasma in the setting of epidemiologic research. As reviewed elsewhere (2), plasma MDA concentrations have been reported to respond to alterations in antioxidant nutrient status. For example, plasma MDA concentrations in patients with cystic fibrosis are affected by dietary carotenoid alterations or carotenoid supplementation (49–51). However, other studies of healthy individuals have reported no effects of supplemental ß-carotene on erythrocyte MDA (52).
Breath hydrocarbons. The measurement of breath hydrocarbons is another commonly used, noninvasive approach for estimating lipid peroxidation in vivo (53). Pentane, which is formed from peroxidation of (n-6) fatty acids, and ethane, formed from peroxidation of (n-3) fatty acids, are volatile compounds that are released into the breath. Potential sources of error in the measurement of breath hydrocarbons have been described. A main source of error involves contamination with ambient-air ethane and pentane (53). In a recent study (52), levels of breath pentane were higher in smokers than in nonsmokers and were reduced with supplemental ß-carotene in smokers but not in nonsmokers. Breath ethane output was not significantly affected by supplemental ß-carotene in this study. Another study (54) also reported that breath pentane output was significantly reduced by daily supplements of high-dose (120 mg/d) but not low-dose (12 mg/d) ß-carotene.
LDL resistance to oxidation.
The oxidative modification of LDL is thought to enhance atherogenicity. For this reason, the resistance of LDL to induced oxidative stress ex vivo has been used as a possible biomarker of oxidative defense, at least in the LDL particle itself. Lipid-soluble antioxidants such as -tocopherol and ß-carotene are known to be carried in LDL in vivo; therefore in concept LDL resistance to oxidation should reflect the antioxidant defense system particularly as it relates to lipid substrates and lipid-soluble antioxidant compounds. Unlike some assays used to quantitate oxidative stress, this assay involves challenge with exogenous oxidants. For example, copper is commonly used to induce oxidation and is followed by measurements of lag time before oxidation. As reviewed elsewhere (55), vitamin E has shown reasonably consistent effects with regard to increasing the resistance of LDL to oxidation. ß-Carotene appears to have only a mild effect or no effect (55). In interpreting results based on LDL oxidation, it should be appreciated that antioxidant efficacy can be affected by the choice of inducing agent used; both metal ion-dependent (e.g., cupric ions) oxidation and metal ion-independent [e.g., 2,2'-azobis(2-amidinopropane)dihydrochloride or AAPH] oxidation protocols are used. For example, Gaziano (56) found that supplemental ß-carotene shortened the lag phase of copper-induced lipid peroxidation (lowered resistance to oxidation) but not AAPH-induced lipid peroxidation in LDL.
F2 isoprostanes.
F2 isoprostanes are produced by free radical-induced peroxidation of arachidonic acid (57). These compounds are formed in phospholipids and then cleaved and released into the circulation before excretion in the urine as free isoprostanes (58). The most abundant F2 isoprostane is 8-isoprostaglandin F2 (8-iso-PGF2), which has been suggested to be a promising marker for oxidative injury (59). Urine is generally considered a better matrix than serum to quantify isoprostane status (60). Aspirin, for example, suppresses 8-iso-PGF2 in serum but not in urine (60), and serum samples but not urine samples stored for any length of time are generally considered inappropriate for use in isoprostane analyses. If serum or plasma samples are used, flash-freezing is usually suggested. Ideally, 24-h urine samples should be collected for isoprostane analyses, although spot urine samples have also been used. Standardization of urinary isoprostane concentrations by creatinine should be considered. One possible limitation to the use of isoprostanes in epidemiologic research is that typical laboratory analyses use gas chromatography/mass spectrometry (GC/MS) methods that are time consuming and therefore expensive for large numbers of samples; however the sensitivity and specificity are excellent (38). Immunoassays for quantifying 8-iso-PGF2 are also available, but these assays sacrifice specificity because of possible cross reactions with other prostanoids. Studies of the modulation of isoprostanes by antioxidant nutrients are becoming available. For example, Reilly et al. (60) reported that vitamin C administered for 5 d at 2 g/d to heavy smokers significantly reduced 8-iso-PGF2 excretion in urine. In this study, vitamin E had no effect at either 100 or 800 IU/d.
Other markers related to lipid peroxidation. The markers described above are some of those most commonly used for lipid peroxidation, but it should be recognized that other potential markers exist as reviewed elsewhere (38). These include the ferrous xylenol orange (FOX) assay and assays of plasma lipid hydroperoxides by chemiluminescence. Also, some assays are designed to measure the overall capacity of a plasma sample to scavenge or trap oxygen radicals [oxygen radical absorbance capacity (ORAC) (61) or total peroxyl radical-trapping antioxidant potential (TRAP)] (62). Assays such as ORAC allow investigators to use different reactive oxygen species generators to measure total antioxidant capacity of a sample under different conditions. For example, a radiation-related hydroxyl radical generator can be used to evaluate the capacity of serum to absorb hydroxyl radicals specifically (61).
Biomarkers of DNA oxidation.
8-OHdG. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most commonly used markers for assessing oxidative DNA damage. This compound is also sometimes referred to as 8-oxy-7-hydrodeoxyguanosine (8-oxodG). DNA can be oxidized to produce many oxidative products; however oxidation of the C-8 of guanine is one of the more common oxidative events, and results in a mutagenic lesion that produces predominantly G-to-T transversion mutations. 8-OHdG can be measured in human DNA samples (e.g., lymphocyte DNA, placental DNA, other) and in urine and has been suggested as a possible approach to the assessment of an individual's cancer risk due to oxidative stress (63). Several methods for quantitating this biomarker are available. HPLC with electrochemical detection (HPLC/ECD) and GC/MS methods are most widely used, although enzyme-linked immunosorbent assay (ELISA) techniques are also being employed (64). HPLC and ELISA methods were compared using placental tissue DNA. Values by both methods correlated well, but the ELISA values were higher than those determined by HPLC (64). This is likely due to the antibody cross reacting with compounds other than 8-OHdG.
Urine is readily obtained, and thus many epidemiologic studies use urinary 8-OHdG as a biomarker of interest. Although convenient, this biomarker also has limitations: 8-OHdG in urine may be influenced by metabolic rate (oxygen consumption) (65); also, 8-OHdG in urine is a function not only of oxidation of DNA but also of excision repair. For this reason, the use of urinary 8-OHdG as a sole biomarker should be undertaken with caution, particularly in intervention studies, because agents that increase repair would increase excretion of 8-OHdG in urine, which could be misinterpreted as an increase in DNA damage (66). Some epidemiologic studies have measured 8-OHdG in both cellular DNA and in urine. For example, Haegele et al. (67) measured lymphocyte 8-OHdG by HPLC/ECD and urinary 8-OHdG via ELISA in the setting of a 14-d fruit and vegetable intervention trial. Results indicated that plasma lutein and ß-cryptoxanthin, both of which are xanthophyll carotenoids, were significantly inversely correlated with urinary isoprostane excretion (8-iso-PGF2) and with 8-OHdG in lymphocyte DNA but not with urinary 8-OHdG. The authors noted shortcomings with the ELISA assay as well as extreme variability in the preintervention urinary 8-OHdG values. Several studies have also examined the effect of supplemental antioxidant nutrients on oxidative DNA damage as reviewed elsewhere (68). One of the largest studies to date is a 2 x 2-factorial trial (500 mg of vitamin C/d; 400 IU of vitamin E/d) in 184 nonsmoking adults where urinary 8-OHdG was used as the outcome of interest (ELISA assay). Supplementation with either nutrient had no main effect or interactive effect in this study (68).
Autoantibodies to oxidized DNA.
Another approach that has been proposed as a possible biomarker of oxidized DNA is the measurement of serum autoantibodies that recognize 5-hydroxymethyl-2'-deoxyuridine (HMdU), which is a product of thymine oxidation. Frenkel et al. (69) found that humans produce autoantibodies to this compound, and titers of anti-HMdU autoantibodies can be measured in a simple ELISA assay. This marker was evaluated in blood samples from 169 women, with the samples obtained 0.5–6 y before diagnosis of breast, colon or rectal cancer. Anti-HMdU autoantibody levels were significantly higher in the cases as compared to the age-matched controls. Limited evidence suggests that dietary supplementation with -tocopherol (60 or 200 mg/d) can reduce levels of this biomarker (70).
Comet assay. The Comet assay, also referred to as the single-cell microgel electrophoresis or SCGE assay, was developed to detect DNA strand breaks. Breaks in DNA allow supercoiled loops of DNA to relax and move out to form what looks like a comet with a tail under the conditions of the assay. The proportion of DNA in the tail is indicative of the frequency of breaks. More recently, modifications to the Comet assay have been developed that allow for the detection of oxidized DNA bases. More specifically, an intermediate step is introduced into the assay, using endonuclease III, which introduces breaks in the DNA at sites of oxidized pyrimidines (71). This modified Comet assay is currently being used as a biomarker of DNA oxidative damage. For example, Pool-Zobel (72) demonstrated that supplementation of the diet with tomato, carrot or spinach products significantly reduced endogenous levels of strand breaks in lymphocyte DNA. Duthie et al. (73) investigated supplements rather than diet, and reported that supplementation with vitamin C (100 mg/d), vitamin E (280 mg/d) and ß-carotene (25 mg/d) resulted in a significant decrease in endogenous oxidative base damage in lymphocyte DNA of both smokers and nonsmokers at 20 wk but not at 10 wk.
Biomarkers of protein oxidation.
Protein carbonyls. Biomarkers of protein oxidation are often applied when a battery of markers of oxidative stress status is being studied. Elevated markers of protein oxidation have also been associated with diseases such as Alzheimer's disease, Parkinson's disease, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, rheumatoid arthritis and progeria (74). Protein oxidation is of etiologic interest for cataractogenesis (75) and also for the investigation of exercise-induced oxidative stress in muscle (76).
The biomarker that is generally used to estimate protein oxidation is protein carbonyls. The conventional assay is a colorimetric procedure that involves dinitrophenylhydrazine (77). More recently, an ELISA method has been developed (78) that correlates well with the colorimetric assay for quantifying protein carbonyls in plasma samples (r2 = 0.70) (78). However, absolute values of protein carbonyls differ in the two assays. Thus these assays are best used for comparing samples that were all analyzed under the same set of conditions.
An example of the application of this biomarker in the context of antioxidant intervention research is work by Marangon et al. (79), which investigated the effects of oral supplementation with -tocopherol and lipoic acid on various measures of oxidative stress in 31 healthy subjects. Lipoic acid reportedly significantly decreased plasma carbonyl levels after AAPH (induced) oxidation, whereas -tocopherol had no effect on plasma carbonyls. Both lipoic acid and -tocopherol prolonged LDL lag time and decreased urinary F2 isoprostanes in this study. As another example, exposure of plasma ex vivo to whole cigarette smoke or gas-phase cigarette smoke produced a steady increase in protein carbonyls, and addition of vitamin C to the plasma had no effect on protein carbonyl formation (80).
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Methodologic issues |
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TOPABSTRACTBiomarkers of exposure for...Biomarkers of oxidative stress...Methodologic issuesImplications and future...LITERATURE CITED |
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The first such issue concerns the potential for artifacts in estimates of baseline levels of oxidation markers. That is, oxidation of samples can occur during normal sample handling, processing and analysis such that measured levels are not at all reflective of the levels encountered in vivo. This issue has plagued investigations of many different biomarkers of oxidation. For example, there is a growing literature on artifacts in assays of DNA oxidation markers (83); extreme variability in reported levels of markers across studies is likely a consequence of artifacts. The magnitude of this problem varies with the biomarker and with the assay. For example, 8-OHdG has been seriously overestimated when GC/MS and 32P-postlabeling and immunoassay measurements were used (83). Laboratory-based approaches to minimize this problem are now being emphasized (63,83,84).
A second methodologic issue that is unique to biomarkers of oxidation concerns the role of substrate, reactive oxygen species and purported antioxidant activity. Different reactive oxygen species have different affinities for substrates and similarly, the effectiveness of a given biological antioxidant may depend on the reactive oxygen species involved. Carotenoids, for example, are highly efficient antioxidants when the oxidizing species is singlet oxygen, but their effectiveness for other reactive oxygen species is more questionable. As discussed by Halliwell and Gutteridge (85), "the relative importance of these [antioxidants] as protective agents depends on which reactive oxygen species is generated, how it is generated, where it is generated, and what target of damage is measured."
This is relevant in the setting of chronic disease research in that the key oxidizing species are rarely known, although there are a few limited situations wherein the etiology is clearly known (e.g., erythropoietic protoporphyria is known to be a singlet oxgyen-mediated condition). On a related note, this review has focused on biomarkers of oxidation that are related to reactive oxygen species, but current research now recognizes a potential role for reactive nitrogen oxide species in human disease. Biomarkers of nitration are also available, such as 3-nitrotyrosine (86). This compound has been identified as a stable end-product that is formed when reactive nitrogen oxide species such as peroxynitrite react with the amino acid tyrosine (free or protein bound). Antibodies raised against peroxynitrite-treated proteins are available, which allows for immunohistochemical analyses of tissue samples. However, the use of 3-nitrotyrosine in inflammatory conditions may underestimate reactive nitrogen species activity (87). Nonetheless greater usage of markers of reactive nitrogen oxide species in epidemiologic research can be anticipated.
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Implications and future directions |
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TOPABSTRACTBiomarkers of exposure for...Biomarkers of oxidative stress...Methodologic issuesImplications and future...LITERATURE CITED |
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In the absence of a positive control, at best one can speculate about the likely utility of a given biomarker. Halliwell (66) speculated that "mass spectrometric measurements of various families of isoprostanes and of multiple DNA base oxidation products are probably the most promising biomarkers for use in human nutritional intervention studies." Although this may be true, data in support of this conclusion remain unavailable. Also, technological advances in methodology (e.g., mass spectrometric measurement) can have important cost implications such that the most sensitive and specific assays may be essentially unaffordable in the setting of large-population studies.
In summary, the field of antioxidant nutrients, biomarkers of oxidation and chronic diseases is immature and in need of much further study. The status of this field is succinctly summarized by a recent report of the Institute of Medicine [(2), pp. 51–52], which concluded:
There is little doubt that an imbalance in the production of free radicals and other reactive species and the natural protective systems available to organisms can lead to the production of oxidized products of lipids, nucleic acids, and proteins. These oxidation products, or biomarkers of this imbalance, may be related to early events in chronic diseases. However, they have not yet been adequately validated as markers of the onset, progression, or regression of any chronic diseases. Although vitamin C, vitamin E, and selenium have been shown to decrease the concentrations of some of the biomarkers associated with oxidative stress, the relationship between such observations and chronic disease remain to be elucidated. As a consequence, it has not been possible to establish that dietary antioxidants or other nutrients that can alter the levels of these biomarkers are themselves causally related to the development or prevention of chronic diseases.
The challenge that lies ahead is formidable and requires a multidisciplinary approach to further our understanding of the complex relationships among oxidation, antioxidants and human disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AAPH, 2,2'-azobis(2-amidinopropane)dihydrochloride; ECD, electrochemical detection; ELISA, enzyme-linked immunosorbent assay; FOX, ferrous xylenol orange (assay); GC/MS, gas chromatography/mass spectrometry; HMdU, 5-hydroxymethyl-2'-deoxyuridine; HPLC, high-performance liquid chromatography; 8-iso-PGF2, 8-isoprostaglandin F2; LC, liquid chromatography; LDL, low-density lipoprotein; MDA, malondialdehyde; NHANES III, Third National Health and Nutrition Examination Survey; 8-OHdG, 8-hydroxy-2'-deoxyguanosine (also referred to as 8-oxodG); ORAC, oxygen radical absorbance capacity; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances; TRAP, total peroxyl radical-trapping antioxidant potential.
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