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Human Nutrition and Metabolism

Dietary Modification and Moderate Antioxidant Supplementation Differentially Affect Serum Carotenoids, Antioxidant Levels and Markers of Oxidative Stress in Older Humans^1,2

Jerusha L. Nelson³, Paul S. Bernstein^*,3, Matthew C. Schmidt, Mark S. Von Tress and E. Wayne Askew

Division of Foods and Nutrition, University of Utah, Salt Lake City, UT 84115; ^* The Moran Eye Center, University of Utah, Salt Lake City, UT 84112; and Alcon Research, Fort Worth, TX 76134

³To whom correspondence should be addressed. E-mail: jnelson{at}usoe.k12.ut.us.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Oxidative stress is thought to be a factor in the progression of chronic diseases, and even the aging process itself. We examined the effect of two moderate vitamin, mineral and phytochemical supplements and a dietary intervention on markers of oxidative stress and antioxidant status in older humans. Subjects (n = 60, 65–85 y old) were enrolled in a 5-wk, double-blind, placebo-controlled study. The subjects were randomly assigned to a group with one of four interventions: antioxidant supplement capsule, antioxidant supplement tablet, high carotenoid diet or placebo. Postintervention serum levels of some of the supplemented carotenoids and -tocopherol were higher in all treated groups compared with the placebo group and with preintervention levels within groups. Markers of oxidative stress or antioxidant capacity were not significantly different from preintervention levels, although the diet and supplement capsule groups had lower levels of some of those markers than the placebo group postintervention. The results suggest that both moderate antioxidant supplementation and a diet high in carotenoids elevate serum carotenoids and antioxidant levels in an older adult population, although with different specific effects.

KEY WORDS: • oxidative stress • carotenoids • antioxidant supplements • oxygen radical absorptive capacity (ORAC) • high carotenoid diet

Oxidative stress has been implicated in diseases ranging from cancer (1) to cardiovascular disease (2) to age-related macular degeneration (AMD)⁴ (3) and even to the aging process itself (4).

Because many diseases related to oxidative stress have potentially serious consequences and limited treatments, research has focused on arresting the oxidative damage that may be involved in disease initiation or progression. Recent research has measured both markers of oxidative stress and whole-body antioxidant capacity in response to diet and vitamin, mineral and phytochemical supplementation, particularly with respect to the bioefficacy (5) and bioavailability (6) of carotenoids. Some of the efforts have been based on the results of previous research documenting the anticarcinogenic role of carotenoids (7), the possible effect of carotenoids on oxidative damage following stroke (8) and the fact that persons who consume more fruits and vegetables (foods rich in antioxidants including carotenoids) have decreased rates of digestive, urinary and respiratory tract cancers (9–12), lower incidence of cardiovascular and cerebrovascular damage (13), exhibit lower blood pressure, have a lower frequency of stroke (14) and reduced rates of AMD (15).

Markers of oxidative damage measure damage to DNA, protein and lipids. Measures of antioxidant capacity determine serum response to oxidizing agents in vitro. Serum levels of antioxidant vitamins and phytochemicals are also used to predict defense against oxidative stress because they play a major role in the defense of cells and tissues. Although these various measures are helpful in estimating the potential for oxidative stress and antioxidant status, no one marker can provide an accurate picture of either antioxidant status or oxidative stress in an organism, and no marker or group of markers has been established as a standard (16).

Several studies have focused on raising antioxidant capacity and serum antioxidant levels and lowering markers of oxidative stress in younger populations. Supplementation with varying levels of the macular pigments (MP) (17), lutein (L) and zeaxanthin (Z) have led to serum and MP density increases in humans (18–20). Dietary carotenoid interventions have resulted in both decreases in some markers of oxidative stress and increases in serum carotenoids levels (21,22). Other researchers found an increase in serum antioxidants but no accompanying change in oxidative stress after a dietary intervention (23,24). Studies involving antioxidant supplementation resulted in increases in serum antioxidant levels, but not in measures of overall serum antioxidant capacity (25,26). Dietary intervention in older adults in one study led to an increase in a measure of antioxidant capacity, but the study did not focus on carotenoid or other antioxidant levels (except vitamin C) or markers of oxidative stress (27). The great majority of these studies have focused on younger, rather than older populations.

AMD, a disease of the macula of the eye, is of particular interest to researchers in nutrition and ophthalmology because it is the leading cause of irreversible blindness in people > 65 y old (28), has extremely limited options for clinical intervention (29), and epidemiologic studies indicate the possibility that persons with higher levels of dietary and serum L and Z and antioxidants may have higher MP densities, and/or be at decreased risk for AMD or related pigment abnormalities (30–32), although not for age-related maculopathy, a precursor of AMD (33,34). L and Z appear to protect the macula against damage from oxidative and photooxidative stresses (35), and higher MP densities are associated with a lower incidence of AMD (36).

Researchers and consumers alike are interested in the possibility that AMD may be retarded and/or prevented by supplementation because supplementation with L and Z has been shown to increase both serum levels of these carotenoids and MP density (18–20), and dietary consumption of L and Z is associated with increased serum levels (30–32). In the Age-Related Eye Disease Study (37), antioxidant supplementation, containing neither L nor Z, which were not commercially available at the outset of the trial, was shown to reduce progression of the disease, thereby heightening interest in the effect that supplementation may have on AMD. Nevertheless, high level ß-carotene use may have associated risks (38), and effective lower level supplementation and/or moderate dietary modifications could be of benefit.

Because so few studies have focused on the elderly population that is most at risk for oxidative stress in general and AMD in particular, we considered the effect of a 5-wk antioxidant supplementation and dietary addition of foods high in carotenoids in older humans on serum antioxidant levels, markers of oxidative stress and a measure of antioxidant capacity to determine whether supplementation either by tablet or capsule or by dietary modification would increase serum carotenoid and antioxidant vitamin levels, decrease markers of oxidative stress and increase antioxidant status.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Subjects and study design.

The Institutional Review Boards of the University of Utah Health Sciences Center and St. Mark’s Hospital approved the study protocol in Salt Lake City, UT. Written informed consent was obtained from each subject before participation.

Volunteers were recruited from an older adult population (ages 65–85 y) in the Salt Lake City, UT metropolitan area. Persons using supplements who were unwilling to eliminate them from their diets for the duration of the study were excluded. Persons using cholesterol-lowering drugs that act as bile acid binders were excluded because these drugs might interfere with fat-soluble vitamin and carotenoid absorption. Persons with diagnosed renal disease or diabetes were excluded from the study because the diet intervention had the potential to compromise control of their disease.

A total of 91 subjects were prescreened for serum carotenoids and oxygen radical absorptive capacity (ORAC, a global measure of serum antioxidant capacity) to identify subjects who might be at higher risk for oxidative stress and possibly at higher risk for AMD. Serum L and Z levels were used as the final criteria for selecting the 60 subjects for the full intervention trial because the literature indicates that individuals with lower levels of serum L and Z may be at greater risk of AMD (31), whereas there does not appear to be any documented relationship between serum ORAC and disease. The 60 subjects with the lowest serum L and Z were selected for the full 5-wk study. Of these, 55 completed the study.

Procedures.

Appointments for an initial blood draw were made with prospective subjects responding to advertisements for study participation. The subjects were sent information packets, which included instructions to discontinue using any vitamin/mineral supplements (except calcium alone) for a 2-wk washout period before their initial appointments. In this packet, they received written instructions to eat what they considered their own typical diets and to avoid any unusual eating behaviors for 3 d before the appointment. Each subject recorded what he or she ate on a 3-d diet record that was included in the information packet. Subjects were also instructed to avoid eating or drinking anything other than water overnight before their appointments. Three subjects fasted for only 4 h; the early morning meal that they consumed appeared to have little or no influence on the subsequent level of carotenoids in the blood sampled. An examination of the values for individuals who had fasted only 4 h revealed values that were similar to those of test subjects who had fasted overnight.

A total of 91 subjects participated in the initial screening. At the screening appointment, each subject’s height, weight and body composition [using bioelectrical impedance analysis (Tanita; Body Fat Analyzers, Ft. Lauderdale, FL)] were measured. The 3-d diet records provided by all subjects were stored for later reference to provide an individually standardized 3-d diet for each subject before any later data collection.

A phlebotomist collected blood samples by venipuncture into 10-mL Tiger-top serum-separator tubes, which were refrigerated at 4°C, without exposure to light, for no longer than 2.5 h. The tubes were then centrifuged at 1200 x g to separate the serum, which was immediately pipetted into 2-mL cryotubes, frozen at -80°C and stored until all samples were collected. Samples were shipped to Genox Laboratories (Baltimore, MD) for serum carotenoid and ORAC analysis.

After the prescreening serum samples were analyzed, and 60 subjects were selected for the full 5-wk intervention; they were randomly assigned to one of three treatment groups [an antioxidant capsule, an antioxidant tablet, (Table 1) or dietary addition of carotenoids (Table 2)] or a placebo group. This was a double-blind, placebo-controlled clinical trial, with the exception of the diet group.

Subjects in the diet group were given special instructions. They received four lists of fruits and vegetables and were instructed to select and daily consume one item from each list (Table 2). The lists were prepared using the USDA carotenoid database (39) and another source (40) of quantified carotenoid content. Combined, a daily choice from each of the four groups provided an average of 6 mg L, 0.6 mg Z, 10 mg lycopene and 11 mg ß-carotene. Serving sizes were specified to ensure adequate carotenoid consumption. Carotenoid content of individual foods within each list varied somewhat to allow subjects a greater variety of choices within each list. Subjects were given notebooks and instructed to record daily which food they chose from each list, how much they ate and what form the food was in when it was measured.

After 5 wk, the subjects returned for a final blood draw. Each subject was sent a copy of the 3 d-diet record that he or she had submitted at the original blood draw, and was instructed to follow it for the 3 d immediately before the final blood draw, in addition to continuing whichever treatment he or she had been assigned. Subjects collected their urine for 24 h before their appointments, which they turned in with their notebooks. The blood and urine were collected, processed and stored at -80°C until assayed.

Sample analysis.

Serum was analyzed for L, Z, - and ß-carotene, ß-cryptoxanthin, lycopene, - and -tocopherol, vitamin C, ORAC and lipid peroxides (LPO, a marker of oxidative damage to lipids). Urine was analyzed for creatinine, 8-hydroxy-2'-deoxyguanosine (8-OHdG, a marker of oxidative damage to DNA), and total alkenals (malondialdehyde and 4-hydroxynonenal, markers of oxidative damage to lipids). All analyses were conducted by an independent reference laboratory (Genox, Baltimore, MD).⁵

The lipid-soluble carotenoids and tocopherols were measured together. Proteins in the serum were denatured with ethanol. The carotenoids and tocopherols were extracted with hexane and the analysis was performed with HPLC using a C₁₈ reversed-phase column with an isocratic mobile phase. Analysis of the carotenoids utilized a diode array detector, and analysis of tocopherols used fluorescence (41). Vitamin C was quantified using a spectrophotometric assay performed on a robotic chemical analyzer (42).

Urine creatinine was quantified using a spectrophotometric assay performed on a robotic chemical analyzer. The urine sample was reacted under alkaline conditions with picric acid to form a Janovski (a red picrate) complex (43).

For total alkenal quantification, 200 µL of urine, with 650 µL of working reagent were pipetted into a 1.8-mL Eppendorf tube. The blank was 200 µL of 1% sulfuric acid. The tubes with reagents and sample or the blank were incubated at 45°C for 40 min in a water bath. The samples were cooled on ice for 10 min, and then centrifuged for 5 min at 3000 x g. Absorbance was measured at 590 nm using a Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA) (44).

Urine samples were pretreated by centrifugation at 2000 x g for 10 min (for opaque samples only; clear samples did not receive pretreatment). Anti-8-OHdG monoclonal antibodies and samples, quality control and standards were added to the microtiter plate, which was precoated with 8-OHdG-protein conjugate and allowed to react for 1 h. Unbound anti-8-OHdG monoclonal antibodies were removed. An enzyme-labeled secondary antibody was then added to the microplate and bound to the monoclonal antibody that was bound to 8-OHdG coated on the microplate and allowed to react for 1 h. The unbound secondary antibodies were removed by a second wash step. A chromatic substrate was added to the plate and resulted in the development of yellow color. The color reaction was continuously recorded for 20 min with a total of 14 readings (the kinetic reading mode) and the optical density was measured at 490 nm (45).

To estimate serum antioxidant ORAC, an indicator protein sensitive to oxidative damage (ß-pycoerythrin) was added to serum and allowed to undergo oxidation after the addition of a water-soluble peroxyl radical generator, 2,2'-azo-bis (2 amidinopropane) dihydrochloride at 37°C. The oxidation of the fluorescent protein was monitored spectrofluorometrically at 560 nm emission (540 nm excitation) every 5 min until extinction. The presence of antioxidants in the serum decreases the rate of decline of the fluorescence of the protein. A water-soluble vitamin E analog, Trolox, was used to establish the standard curve. One ORAC unit is equivalent to the protection provided by 1 µmol of Trolox (46).

Statistical methods, data analysis, and interpretation.

The statistical analyses were computed using SPSS (Chicago, IL) for the analyses of the mean responses, and SAS Version 8.2 for Windows (SAS Institute, Cary, NC) for the factor analysis. The Wilcoxon rank-sum test was used to determine changes from baseline within treatment groups, and Mann-Whitney comparisons were conducted to determine differences among groups postintervention. Multivariate and covariate analyses were also employed to assess the relative change in oxidative stress by treatment from the beginning of the study to the end. Significant difference was defined as P 0.05. Values in the text are means ± SD.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Volunteers.

Fifty-five subjects completed the full study. Their age was 71.2 ± 5.5 y (range 65–85 y). The 34 women had a BMI of 27.8 ± 7.1 kg/m² (range 17.9–49.8 kg/m²), and had 39.6 ± 7.2% body fat (range 22.7–56.1%). The 21 men had a BMI of 26.9 ± 5.6 kg/m² (range 16.6–39.9 kg/m²) and had 25.4 ± 7.0% body fat (range 9.8–38.7%). Baseline serum and urine antioxidants, markers of oxidative stress and ORAC data were collected (Table 3). Compliance was self-reported and generally high; for the supplement tablet and capsule and placebo groups it was >96%, and it was 90% for all selections in the diet group.

Compared with the preintervention levels, the supplement tablet group had higher postintervention serum levels of L (P = 0.001), lycopene (P = 0.017), ß-carotene (P = 0.001), and -tocopherol (P = 0.001), and a lower postintervention serum concentration of vitamin C (P = 0.019). The supplement capsule group had higher postintervention serum levels of L (P = 0.001), Z (P = 0.004), lycopene (P = 0.016), -carotene (P = 0.004), ß-carotene (P = 0.001) and -tocopherol (P = 0.003), and a lower postintervention serum level of vitamin C (P = 0.031). The diet group had higher serum levels of L (P = 0.002), Z (P = 0.014), lycopene (P = 0.046), ß-carotene (P = 0.044), ß-cryptoxanthin (P = 0.005) and -tocopherol (P = 0.004). The placebo group had lower postintervention levels of -carotene (P = 0.001) and ß-carotene (P = 0.006) (Table 4).

Compared with the placebo postintervention, the postintervention supplement tablet group had higher serum levels of L (P = 0.001), ß-carotene (P = 0.001), ß-cryptoxanthin (P = 0.034) and -tocopherol (P = 0.004) and lower serum levels of -tocopherol (P = 0.001). Compared with the placebo group, the supplement capsule group had higher levels of L (P = 0.005) and ß-carotene (P = 0.006), and lower levels of 8-OHdG (P = 0.009) and total alkenals (P = 0.015). The diet group had higher levels than the placebo group of serum L (P = 0.010), lycopene (P = 0.038), ß-carotene (P = 0.002) and ß-cryptoxanthin (P = 0.001), and lower levels of 8-OHdG (P = 0.001) and total alkenals (P = 0.006) (Table 4).

The supplement tablet group had higher postintervention concentrations than the diet group for ß-carotene (P = 0.003) and -tocopherol (P = 0.022) and a lower serum level for ß-cryptoxanthin (P = 0.012). Compared with the supplement capsule group, the supplement tablet group had a higher serum level of ß-carotene (P = 0.013) and a lower serum level of (-tocopherol (P = 0.001).

Factor analysis.

The straightforward statistical analyses (Mann-Whitney) showed postintervention differences between the placebo group and the diet and supplement capsule groups. Because there appeared to be a treatment effect, a multivariate factor analysis of the data was performed. The factor analysis constructed composite variables of the measurements according to their correlation structure (determined using Spearman analysis), which were then analyzed using traditional analysis of covariance. Two measures of oxidative stress, urinary 8-OHdG and total alkenals, were highly correlated and this composite variable was referred to as "oxidative stress." A second composite variable referred to as the "vitamin factor" consisted of the serum carotenoids L, Z, ß-carotene and -tocopherol, another group of highly correlated variables. The oxidative stress factor did not differ among groups at entry, but all treated groups differed from the placebo group postintervention (P = 0.047) (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIGURE 1 Analysis of covariance between the Oxidative Stress Factor [composite of urinary 8-hydroxy-2'-deoxyguanosine (8-OhdG) and total alkenals] and the Vitamin Factor (composite of serum lutein, zeaxanthin, ß-carotene and -tocopherol) after diet modification (n = 13) or supplementation of older adults with antioxidant capsules (n = 15), antioxidant tablets (n = 13) or placebo (n = 14) for 5 wk. All treated groups had a lower mean Oxidative Stress Factor than the placebo group (P = 0.047) at exit (indicated with *).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Moderate carotenoid supplementation and a dietary intervention over a short period of time increased most serum carotenoids, especially the xanthophylls that may be related to AMD, L (increased in all treatment groups) and Z (increased in the diet- and supplement capsule-treated groups). The increases in Z in the diet and supplement capsule groups and lack of an increase in the supplement tablet group are probably attributable to the higher intake of Z in both the diet and capsule supplement arms, both providing broader ranges of nutrients that also may affect bioavailability of the total formulation. Additionally, compared with the placebo group, the treated groups had higher levels of most carotenoids postintervention.

We expected the increases in the serum carotenoid levels with intervention because supplementation and dietary intervention trials with carotenoids produced serum increases in several previous studies (18–20,22). The results of this study, however, suggest that consuming a daily supplement containing only 4–6 mg L + Z or incorporating reasonable amounts of dietary carotenoids through commonly available foods may provide enough of these carotenoids to elevate serum concentrations to levels comparable to those achieved by much higher supplementation, and that these increased serum levels may be attained in an older population. These modest measures may be protective against AMD and other chronic diseases in which oxidative stress plays a role in a susceptible population.

The increases in -tocopherol over time in the supplement groups were expected because both supplements contained substantial amounts of -tocopherol. The increase in -tocopherol in the diet group was somewhat surprising because none of the fruits or vegetables consumed were particularly high in vitamin E. Supplementation with carotenoids may have increased nutrient absorption or bioavailability of the tocopherols. Alternatively, other antioxidants in the fruits and vegetables may have protected or regenerated vitamin E.

The decision to use subjects with the lowest preintervention L + Z scores led to a specifically selected target population that was likely to benefit from the L and Z in the interventions. This selection may have influenced the end measure of antioxidant capacity; the ORAC score was not increased by the treatments. This may have also been due to the chemical nature of the ORAC assay, in which carotenoids and vitamins E and C are not the largest contributors (46).

Although there was no significant change from baseline to the end of the study in any marker of oxidative stress for any treated group, there were some postintervention differences between the supplement capsule and dietary intervention groups and the placebo group for measures of oxidative stress. Both the dietary intervention group and the supplement capsule group had lower values for 8-OHdG and total alkenals than the placebo group postintervention, indicating that the subjects in these groups may have had less DNA damage and lipid peroxidation than the controls. When the relative magnitude of the change from baseline was examined by a multivariate factor analysis that took into consideration the relationship of all the indices employed in this study, this difference between the treated and placebo groups was more evident.

This study evaluated the effects of two vitamin and mineral supplements and a high carotenoid dietary intervention on measures of oxidative damage (serum LPO, urine 8-OHdG, urine total alkenals), antioxidant capacity of the serum (serum ORAC), and serum antioxidant concentrations (vitamins E and C and carotenoids) in an older adult population. Overall, the results of this study provide a rationale for moderate rather than high level L and Z supplementation for older adults who use supplements to combat diseases associated with oxidative stress, particularly AMD. This is especially important because supplement use among older adults is increasing (48), and lower level supplementation may be safer. The results also show that dietary addition of moderate amounts of foods that are high in carotenoids is as effective at raising serum carotenoids as supplementation. Benefits to the eye or any other tissue can only be inferred from the results of this study.

	ACKNOWLEDGMENTS

 
The authors thank Amy Reeder for her assistance in reviewing the diet component of the study.

	FOOTNOTES

 
¹ Portions of these data were previously presented in abstract form at Experimental Biology 02, April 2002, New Orleans, LA [Nelson, J. L., Bernstein, P. S., Schmidt, M. C. & Askew, E. W. (2002) Comparison of ocular antioxidant supplements and diet as a means to increase serum carotenoid concentrations. FASEB J. 16: A639 (abs.)] and at the 2002 Annual Meeting of the Association for Research in Vision and Ophthalmology, May 2002, Fort Lauderdale, FL [Nelson, J. L., Bernstein, P. S., Schmidt, M. C., Yeum, K. J. & Askew, E. W. Ocular antioxidants from supplementation and diet: comparison and analysis [abs.]. Abstract and Program Planner accessed at www.arvo.org. Abstract 2541.

² Funding provided by Alcon Laboratories, Ft. Worth, TX.

⁴ P.S.B. is a Sybil B. Harrington Research to Prevent Blindness (New York, NY) scholar in macular degeneration research.

⁵ Abbreviations used: AMD, age-related macular degeneration; L, lutein; LPO, lipid peroxides; MP, macular pigment; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; ORAC, oxygen radical absorptive capacity; Z, zeaxanthin.

⁶ Assays performed: Serum lutein, zeaxanthin, lycopene, - and ß-carotene, ß-cryptoxanthin, - and -tocopherol, vitamin C, ORAC, and lipid peroxides; Urine 8-OHdG, creatinine, and total alkenals.

Manuscript received 28 April 2003. Initial review completed 4 June 2003. Revision accepted 16 July 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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