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Lack of a relation between vitamin and mineral antioxidants and bone mineral density: results from the Women's Health Initiative^1,2,3

¹ From the Program in Nutrition, Department of Health and Behavior Studies, Teachers College, Columbia University, New York, NY (RLW); the Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA (JAC); the Fred Hutchinson Cancer Research Center, Seattle, WA (MP and AL); The Ohio State University; Columbus, OH (RJ); the Department of Internal Medicine, Brigham and Women's Hospital, Boston, MA (MSL); the Division of Preventive Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL (CEL); the Department of Epidemiology and Biostatistics, University of California at San Francisco, San Francisco, CA (MCN, JAS, and KLS); the General Internal Medicine Section, San Francisco VA Medical Center, San Francisco, CA (JAS); and the University of Buffalo, Buffalo, NY (JWW)

² Supported by training grant no. 5T32AG000181-15 from the National Institutes of Health.

³ Reprints not available. Address correspondence to RL Wolf, Department of Health and Behavior Studies, Teachers College, Columbia University, 525 West 120th Street, Box 137, New York, NY 10027. E-mail: wolf{at}exchange.tc.columbia.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Antioxidant defenses are one possible mechanism for decreasing oxidative damage and its potentially negative effects on age-related bone mass.

Objective: This study cross-sectionally examined whether higher dietary intakes, total intakes, and serum concentrations of antioxidants may be associated with higher bone mineral density (BMD).

Design: Total hip (and subregions), spine, and total-body BMDs were measured in 11 068 women aged 50–79 y enrolled in the Women's Health Initiative Observational Study and Clinical Trial at 3 clinics. Antioxidant intakes from diet (vitamin A, retinol, ß-carotene, vitamin C, vitamin E, and selenium) were estimated by using a self-reported food-frequency questionnaire. Antioxidants from supplements were estimated with an interviewer-administered questionnaire. A random subset (n = 379) had serum concentrations of retinol, carotenoids, and tocopherols measured.

Results: After adjustment for important BMD-related covariates, increasing intakes of antioxidants were not independently associated with BMD. A significant interaction effect was observed between intake of total vitamin C (lower three-fourths compared with highest one-fourth) and use of hormone therapy (HT) (P < 0.01). The beneficial effect of current HT use on femoral neck BMD appeared to be greater in women with higher concentrations of total vitamin C. This interaction was also significant for total-body (P < 0.045), spine (P = 0.03), and total-hip BMDs (P = 0.029).

Conclusions: Our results do not support independent associations between dietary intake, total intake, or serum concentrations of antioxidants and BMD in women participating in the Women's Health Initiative. The extent to which HT use may interact with vitamin C intake and BMD warrants further exploration.

Key Words: Antioxidants • diet • serum • bone mineral density • osteoporosis • Women's Health Initiative

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Osteoporosis, and its associated fractures, is a serious public health problem that affects the entire aging US population (1). More than 44 million women and men in the United States are now estimated to have osteoporosis or low bone mass and thus are at risk of clinical fractures (2). Several dietary factors have been identified as being associated with bone mineral density (BMD), but most studies have focused attention on calcium intake. Sufficient evidence exists that calcium is important for bone health (3-13). However, the cause of osteoporosis is multifactorial (14). Other dietary factors could be important in preventing osteoporosis.

Intake of antioxidants may influence BMD by acting as free radical scavengers, possibly by reducing the effects of oxidative stress that may be associated with bone loss. To date, studies of the relation between antioxidants and BMD have received little attention. If such relations exist, it may suggest alternative lines of investigation about the potential causes of, and ways to prevent, osteoporosis.

The purpose of this study was to investigate whether specific antioxidants were associated with BMD in women participating in the Women's Health Initiative (WHI). Shikany et al (15) reported that antioxidant supplements were widely consumed in WHI participants mostly through multivitamins. Use of single supplements of vitamin C and vitamin E were also popular in WHI participants, although the use of ß-carotene supplements was not. We were specifically interested in whether women with the highest dietary intake, total intake (diet plus supplements), or serum concentrations of antioxidants were associated with the highest BMD measurement. We considered hip, spine, and total-body bone mass. Because other investigations have described possible interactions among various antioxidants and calcium intake (16), smoking (17), and postmenopausal estrogen therapy (18) on BMD or fracture, we also were interested in exploring whether these factors modified the association between antioxidants and BMD.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study population
The WHI is a multisite observational study and prevention trial that focuses on reducing some of the most common causes of morbidity and mortality among postmenopausal women, including coronary artery disease, breast and colorectal cancers, and osteoporotic fractures. Participants were recruited at 40 US clinical centers into 3 randomized clinical trials and a longitudinal observational study. Specifics of the study design and methods have been described elsewhere (19). Here, we report baseline data on a subsample of women enrolled in both the observational and clinical trial studies at the 3 clinic sites (Pittsburgh, PA; Birmingham, AL; Tucson, AZ) that measured BMD. The BMD centers were chosen to provide maximum racial diversity. All procedures were approved by the Institutional Review Board at each center.

Sample selection
Our sample included 11 393 women aged 50–79 y recruited from 1993 to 1997. All women had data collected as part of the WHI baseline procedures (ie, interviewer and self-administered questionnaires, physical measurements, and a fasting blood draw). For this analysis, we excluded women taking oral glucocorticoids (n = 99), bisphosphonates (n = 155), calcitonin (n = 25), and tamoxifen (n = 53), leaving 11 068 participants.

Questionnaire and clinical measures
Baseline questionnaire data from the WHI included self-reported age, race, education, income, and health habits. Health habits included history of smoking (categorized as never smoked, past smoker, and current smoker). Past smokers were those who had smoked 100 cigarettes but were not currently smoking. History of alcohol intake was categorized as nondrinker, past drinker, and current drinker. Past drinkers were those who had consumed 12 alcoholic beverages in their life but did not currently drink. Current drinkers were further classified by current intake based on the sum of intake of beer, wine, and liquor, adjusted for portion size from the food-frequency questionnaire (FFQ). Coffee consumption was categorized by current number of cups consumed per day. Current use of thiazide diuretics and thyroid medications were categorized as yes or no. Use of postmenopausal hormone therapy (HT) (estrogen with or without progesterone) was categorized as current user and never or former user. Total expenditure from physical activity was assessed by questions on frequency and duration of 4 speeds of walking and of 3 other types of recreational activity classified by intensity (strenuous, moderate, or mild). Energy costs of the physical activities were assigned with the use of a standardized classification (20) and were expressed as total metabolic equivalents per week (in kcal/wk x kg).

Diet intake was based on self-report with the use of a semiquantitative FFQ that asked about frequency of consumption over the past 3 mo and portion size of 122 foods or food groups. The FFQ also covered food preparation practices and added fats. Nutrients were calculated with the use of a database derived from the University of Minnesota Nutrition Coding Center (MINNESOTA NUTRITION DATA SYSTEM, version 30; Minneapolis, MN). Nutrients of interest for this analysis included vitamin A, retinol, ß-carotene, vitamin C, vitamin E, and selenium. From previously published studies (16-18, 21-23), covariates of interest from the FFQ were total energy intake, dietary fat, saturated fat, protein, magnesium, calcium, and vitamin D. Dietary supplements were estimated by an interviewer-administered, computer-driven inventory of all nutritional supplements taken by the participant. Information was recorded on the nutrients from multivitamin supplements and from individual supplements. Total intake of antioxidants was obtained by summing the intakes from diet plus supplements.

Clinical measures included weight and height. Weight was measured in indoor clothes to the nearest 0.1 kg with the use of a balance-beam scale. Height was recorded to the nearest 0.1 cm with the use of a wall-mounted stadiometer and a standard expiration technique. Body mass index (BMI; in kg/m²) was calculated. Waist circumference was measured at the natural waist or narrowest part of the torso.

BMD of the total body, lumbar spine (L2–L4), and total hip with subregions of the femoral neck and trochanter was measured by dual X-ray absorptiometry (QDR 2000, 2000+, or 4500W; Hologic, Inc, Bedford, MA). Standard protocols for positioning and analysis were used by technologists who were trained and certified by the bone densitometry reading center of the University of California at San Francisco. The ongoing quality assurance program was similar to that used in other studies (24).

Blood measures
Serum antioxidant concentrations of retinol, - and ß-carotene, - and -tocopherol, ß-cryptoxanthin, lycopene, lutein, and zeaxanthin were collected on a random subset of women at baseline. Covariates of interest were total serum cholesterol and triacylglycerols because adjustment for these factors are suggested as the appropriate measure of biologic status of vitamins E and A (25). Six percent of women who enrolled in the clinical trial components of the WHI, and 1% of the observational study participants (n = 379) were randomly selected to have their blood analyzed for micronutrients. The random sampling procedure was stratified by clinical center, age, hysterectomy status, and ethnicity to oversample minority women. The methods and quality-control procedures for each blood measure are described elsewhere (26).

Statistical methods
Descriptive statistics were conducted on all variables to evaluate range, variance, frequencies, and normality of data. Examination of skewness of the data, as well as the histograms, determined which variables were to be mathematically transformed. Means and SDs are shown for continuous data. Frequencies and proportions are shown for categorical data. Correlation coefficients (parametric or nonparametric) were calculated to determine the direction and magnitude of the relations among dietary intake, total intake, and serum concentrations of antioxidants. Scatterplots were evaluated for threshold effects between BMD and antioxidant concentrations.

Multiple linear regression analysis was used to examine the relation between dietary intake, total intake, and serum concentrations of antioxidants as a continuous variable and BMD at the various sites. For analyses of dietary antioxidants from food alone, we excluded participants who reported using supplemental antioxidants, either from single supplements or multivitamins (50% of subjects, n = 5607). Dietary intake and total intake were entered into multivariate models as continuous data and then adjusted for age, BMI, waist circumference, race, education, income, alcohol consumption, coffee consumption, smoking status, clinic site, hormone use, thiazide use, thyroid medication use, physical activity, total caloric energy, dietary fat, dietary saturated fat, dietary protein, dietary magnesium, total calcium, total vitamin D, and other antioxidants. Serum antioxidant concentrations were entered into multivariate models as continuous data and then adjusted for age, BMI, waist circumference, race, education, income, alcohol consumption, coffee consumption, smoking status, clinic, hormone use, thiazide use, thyroid medication use, physical activity, total caloric energy, and total serum cholesterol and triacylglycerol concentrations. In addition, we created 2 composite variables to study the relation of total serum carotenoids (- and ß-carotene, ß-cryptoxanthin, lycopene, lutein, and zeaxanthin) and total serum tocopherols (-tocopherol plus -tocopherol) to BMD.

Examination of select interactions between dietary intake, total intake, and serum concentrations of antioxidants (mean for lower 3 quartiles compared with mean for upper quartile) and BMD were performed in 1) women reporting high (>500 mg/d) compared with low (<500 mg/d) intakes of calcium, 2) current smokers compared with never and past smokers, and 3) current HT users compared with never and former HT users. We tested for interactions between calcium intake, smoking status, and hormone therapy use by including cross-product terms in regression models. Adjusted means are presented for BMD within each category along with nominal P values for the interaction term.

Dietary and serum variables outside of plausible range were deemed unreliable and treated as missing data. FFQ data were excluded from the analysis for participants with FFQ energy estimates of <600 kcal (n = 338) or >3500 kcal (n = 271) and for participants with missing data (n = 27). All dietary nutrients (alone or with supplements) have been log-transformed. Tables present back-transformed values by using a standard approximation formula for the SD.

All analyses were completed for each skeletal site for each antioxidant for dietary intake, total intake, and serum concentrations. Results were similar, so we present only the results from the femoral neck BMD. Nominal two-sided P values are reported for all statistical tests. Because 22 statistical tests were performed for each skeletal site, one test would be expected to be significant at the 0.05 level by chance. Analyses were performed by using SAS (version 8.2; SAS Institute, Cary, NC).

	RESULTS

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Characteristics of the 11 068 women with data available for these analyses are presented in Table 1. Women ranged in age from 50 to 79 y (: 63.2 y) with an average BMI of 28.3. Among the women, 4.8% were osteoporotic at the total hip because of a BMD t score > 2.5 SD below young normal (27). Participants were from the WHI clinic sites in Pittsburgh, PA (31.7%), Birmingham, AL (33.2%), and Tucson, AZ (35.1%). The sample was ethnically diverse with approximately one-fourth from minority subgroups. Of the sample, 68.3% had education beyond high school. About one-fourth (25.9%) had family incomes >$50 000/y. The majority of women consumed <1 alcoholic drink/wk (71.5%) and >1 cup coffee/d (57.1%). A low percentage was current smokers or users of thiazide diuretics and thyroid medications, whereas approximately one-third were currently using postmenopausal HT. Average total intake of calcium (diet plus supplements) was 900.1 mg/d. On average, supplemental antioxidant users had mean total intakes that ranged 9.6% (selenium) to 272% (vitamin E) higher than mean dietary intakes of nonsupplement users.

Table 2

Table 2

Table 3

Table 4

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Vitamin C is a known potent antioxidant that could reduce effects of free radicals (28) and antioxidants, having been shown in laboratory studies to limit bone resorption (29). Other mechanisms through which vitamin C may contribute to BMD remain unclear, but they are speculated to be related to the role of vitamin C in collagen formation of bone matrix development (30, 31) and the effect of vitamin C on osteoblast growth or in promoting calcium absorption (32). However, despite reasonable biologic plausibility, we found no evidence of a relation between vitamin C and BMD by itself, but we did find an interaction with HT use. Other epidemiologic studies have found an association among dietary intake, supplemental intake, total intake (diet plus supplemental), or serum concentrations of vitamin C and BMD or fracture in postmenopausal women (16-18, 21, 33). Similar to our study, most relations have been in subgroups of women only. Other studies have found associations between vitamin C and BMD in women with either calcium intake < 500 mg/d (16), women who had never used estrogen (18), or current smokers (17). Those studies varied as to whether the association between vitamin C and BMD was found for diet only (16, 33) or for supplements only (18).

Simon and Hudes (21) reported that neither dietary intake of vitamin C nor serum concentrations were associated with BMD or self-reported fracture among postmenopausal women, despite a relation being found in premenopausal women. There were interesting interactions such that, among postmenopausal women with a history of smoking and estrogen use, serum concentrations of vitamin C were associated with decreased prevalence of fractures but, unexpectedly, were also associated with lower BMD measurements among postmenopausal women without a history of either smoking or estrogen use. In another study, Maggio et al (34) found vitamin C concentrations to be consistently lower in a group of osteoporotic women than in control subjects.

Although the explanation for the differences between our study and prior studies is unclear, and may be due to chance, the interaction was not entirely unexpected. Because estrogen-containing medication increases the turnover of ascorbic acid (35) and is associated with lowered concentrations of ascorbic acid in the leukocytes, platelets, and plasma of guinea pigs, primates, and humans (36-40), it is possible that the interaction that we detected reflects a biologic effect rather than a chance finding.

Vitamin E derivatives may inhibit a transcription factor that regulates osteoclastogenic cytokines, such as interleukin 6 (41), and vitamin E has been shown to protect against cellular lipid peroxidation in cartilage and bone cells (42). Results have been mixed among the few epidemiologic studies that have examined the relation between vitamin E and BMD. Melhus et al (17) found an association between low intakes of dietary vitamin E and increased risk of hip fracture, but only among women who were current smokers. Maggio et al (34) found plasma concentrations of vitamin E to be lower in osteoporotic women than in control subjects. In contrast, we found no association between vitamin E and BMD.

Conflicting research suggests a potentially complex relation with vitamin A and bone. Vitamin A in high doses seems to stimulate bone resorption and inhibits bone formation (43). Reports have indicated an increased risk of hip fracture (23, 44, 45) and low BMD (23, 46) in women with high dietary intake of vitamin A. One study contradicts those findings and found lower concentrations of serum vitamin A to be associated with osteoporotic women compared with control subjects (34). More recently, Opotowsky et al (47) described a U-shaped relation between serum concentrations of vitamin A and hip fracture such that both low and high serum concentrations of vitamin A may be associated with increased risk. We found no association between vitamin A and BMD. The extent to which our finding that total intake of ß-carotene and femoral neck BMD (P = 0.03) is real, or due to chance, is unclear. This finding was not consistent in that it was not significant for dietary intake alone, in serum concentrations, or for other BMD sites (spine, total hip, trochanter).

For selenium, a potent antioxidant, we hypothesized that higher concentrations of selenium would be associated with higher BMD. Melhus et al (17) did not find an association between low compared with high intakes of dietary selenium and risk of hip fracture. Those findings are not surprising, given that there is probably too little selenium in our bodies to act as a direct antioxidant without the use of supplements. Regardless, our study did not find an association between diet or total intake of selenium and BMD.

Collectively, these studies present mixed results. Taken together, they are difficult to interpret because studies varied as to the exposure measured (ie, diet alone, supplement alone, diet plus supplements, serum), the outcome measured (ie, BMD compared with fracture), the site measured (eg, hip compared with spine), and the various confounding factors included in the analysis (eg, total energy intake). The current study is unique it that it allowed us to look at dietary intake, total intake, and serum concentrations of a variety of antioxidants, at various BMD sites, after adjustment for a host of potentially important confounding factors.

The null associations observed in our study may be due to several factors. One possibility is that the variation in antioxidant concentrations was insufficient to see an association. WHI participants were healthy subjects who were clearly well nourished. Future studies should consider investigating the relation between antioxidants and BMD in populations with lower ranges of antioxidant concentrations. Second, a relation could be undetected because of the temporal relation between the measured exposure and the outcome did not encompass the true latent period. Of interest, Shikany et al (15) recently reported that most antioxidant use in WHI participants has been recent, within the past 1–4 y before enrollment into the study. It will be interesting to see whether changes in dietary and supplemental antioxidant use over time reflect changes in BMD and fracture risk. Third, there may be unmeasured variables that we did not control for influencing the association between antioxidants and BMD. Fourth, only several selected antioxidants were evaluated in these analyses, and not all dietary estimates had serum equivalents. In the case of vitamin C, for which serum concentrations were not available, we were trying to identify associations with the use of a FFQ at baseline that we assumed provided an estimation of usual vitamin C intake over previous years during which bone density developed. The correlation between FFQ estimates of vitamin C intake and serum concentrations are 0.55–0.58 (48), so there will be some misclassification, which, if nondifferential, will result in decreased statistical power to detect an association. Fifth, our participants were mostly women with normal range BMD. If associations are present primarily in osteoporotic women or women with low BMD, we might have missed these associations. Finally, our hypotheses were primarily based on the assumption that women with lower BMD may be in a state of oxidative stress, which may be ameliorated by increased antioxidants. A recent study by Maggio et al (34) found antioxidant concentrations to be lower in osteoporotic women than in control subjects; however, markers of oxidative stress (ie, malondialdehyde) were not different in osteoporotic women than in control subjects. These findings imply that there may be other potential mechanisms by which antioxidant nutrients may have an effect on bone.

We believe our study had several important strengths. First, this study investigated a broad spectrum of antioxidants; measured several BMD sites; included dietary intake, total intake, and serum concentrations of antioxidants; and controlled for a wide range of potential confounders, including energy intake. Measurements of dual-energy X-ray absorptiometry were state of the art with good quality control. Our sample size was large and allowed us to examine several interactions. Although our sample of women in the WHI study were volunteers and, thus, may not be representative of the general population of postmenopausal women, our sample was ethnically diverse, including US women from 3 geographic regions. Most other studies were conducted in primarily white samples.

Our results do not support an association between dietary intake, total intake, or serum concentrations of select antioxidants and BMD at any of the skeletal sites measured. Whether the beneficial effect of HT use on BMD among women with higher than with lower total concentrations of vitamin C is real, or due to chance, needs further exploration. The WHI participants continue to be followed prospectively, making it possible to explore dietary intake, total intake, and serum concentrations of antioxidants on changes in BMD and, ultimately, fracture risk. Researchers should consider exploring the relation of specific whole foods and food groups (eg, fruit, vegetables, or both) and their relation with BMD, instead of continuing to focus on individual antioxidants.

	ACKNOWLEDGMENTS

 
RLW was the lead author for this manuscript and contributed to the conception of the study question, the design, the data interpretation, and the writing of the manuscript. MP conducted all of the statistical analyses. JAC, MP, AL, RJ, MSL, CEL, MCN, JAS, KLS, and JWW contributed significantly to the interpretation of the study findings and to the writing and editing of the manuscript. None of the authors had a conflict of interest.

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TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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