Millen AE, Voland R, Sondel SA, Parekh N, Horst RL, Wallace RB, Hageman GS, Chappell R, Blodi BA, Klein ML, Gehrs KM, Sarto GE, Mares JA, CAREDS Study Group. Vitamin D Status and Early Age-Related Macular Degeneration in Postmenopausal Women. Arch Ophthalmol. 2011;129(4):481-489. doi:10.1001/archophthalmol.2011.48
The relationship between serum 25-hydroxyvitamin D (25[OH]D) concentrations (nmol/L) and the prevalence of early age-related macular degeneration (AMD) was investigated in participants of the Carotenoids in Age-Related Eye Disease Study.
Stereoscopic fundus photographs, taken from 2001 to 2004, assessed AMD status. Baseline (1994-1998) serum samples were available for 25(OH)D assays in 1313 women with complete ocular and risk factor data. Odds ratios (ORs) and 95% confidence intervals (CIs) for early AMD (n = 241) of 1287 without advanced disease were estimated with logistic regression and adjusted for age, smoking, iris pigmentation, family history of AMD, cardiovascular disease, diabetes, and hormone therapy use.
In multivariate models, no significant relationship was observed between early AMD and 25(OH)D (OR for quintile 5 vs 1, 0.79; 95% CI, 0.50-1.24; P for trend = .47). A significant age interaction (P = .002) suggested selective mortality bias in women aged 75 years and older: serum 25(OH)D was associated with decreased odds of early AMD in women younger than 75 years (n = 968) and increased odds in women aged 75 years or older (n = 319) (OR for quintile 5 vs 1, 0.52; 95% CI, 0.29-0.91; P for trend = .02 and OR, 1.76; 95% CI, 0.77-4.13; P for trend = .05, respectively). Further adjustment for body mass index and recreational physical activity, predictors of 25(OH)D, attenuated the observed association in women younger than 75 years. Additionally, among women younger than 75 years, intake of vitamin D from foods and supplements was related to decreased odds of early AMD in multivariate models; no relationship was observed with self-reported time spent in direct sunlight.
High serum 25(OH)D concentrations may protect against early AMD in women younger than 75 years.
Age-related macular degeneration (AMD), a chronic, late-onset disease that results in degeneration of the macula, is the leading cause of adult irreversible vision loss in developed countries.1 Age-related macular degeneration affects approximately 9% (8.5 million) of Americans aged 40 years and older.2 Earlier stages of AMD, which increase the odds of developing advanced disease,3 are the most common, affecting 8% of persons aged 43 to 54 years and 30% of those older than 75 years.4 There is no cure for this condition.5 Limited treatment is available to slow its progression, and no established means of prevention exits.5 Therefore, it is important to identify modifiable risk factors that may reduce disease occurrence or prevent progression to advanced stages.
The pathogenesis of AMD is likely to involve a complex interaction of multiple factors including light damage,6 oxidative stress,7 inflammation,8 possible disturbance in the choroidal blood vessels,9 and genetic predisposition.10 Nonmodifiable genetic risk factors,11,12 especially those associated with inflammatory response, and the modifiable risk factor of smoking,12,13 appear to explain a large percentage of the variation in risk of AMD. Recently, a strong protective association between vitamin D status, as reflected by serum concentrations of 25-hydroxyvitamin D (25[OH]D), and the prevalence of early AMD was reported in a nationally representative, cross-sectional study.14 Research suggests that vitamin D affects immune modulation and perhaps the prevention of diseases with inflammatory etiologies.15 Currently there is evidence that vitamin D deficiency and insufficiency exist in individuals worldwide and that the risk of developing many chronic diseases of aging have been shown to be inversely associated with vitamin D status.16
The purpose of this study was to investigate whether the previously observed protective association of vitamin D status and AMD could be confirmed in a second study, the Carotenoids in Age-related Eye Disease Study (CAREDS), in which 25(OH)D concentrations were assessed 6 years prior to AMD status. The CAREDS study is an ancillary study within the Women's Health Initiative Observational Study (WHIOS), which was initiated to investigate relationships of carotenoids in the diet, serum, and retina to AMD17 and cataract.18 Using CAREDS data, the relationship between individually measured serum 25(OH)D concentrations at WHIOS baseline (1993-1998) and the prevalence of early AMD, assessed an average of 6 years later at the CAREDS baseline (2001-2004), was investigated. Additionally, analyses sought to determine whether associations between all sources of vitamin D (sunlight, food, and supplements) and AMD supported associations observed between serum 25 (OH)D and AMD.
The CAREDS population consists of women (ages, 50-79 years) who were enrolled in the observational study of the Women's Health Initiative at 3 of 40 sites: the University of Wisconsin, Madison, the University of Iowa, Iowa City, and the Kaiser Center for Health Research, Portland, Oregon. Participants with baseline WHIOS (1993-1998) intakes of lutein plus zeaxanthin above the 78th and below the 28th percentiles, as assessed at WHIOS baseline (1993-1998), were recruited. Of the 3143 women who fulfilled these criteria, 96 died or were lost to follow-up between the selection year (2000) and enrollment in CAREDS (2001-2004). Those who remained were mailed letters inviting them to participate.
A total of 1042 women declined participation, and 2005 were enrolled (64%). Of those enrolled, 1894 participated in study visits. Gradable fundus photographs were obtained for 1853 participants; an additional 4 participants were included who did not have AMD photographs but had a doctor's confirmation of AMD. One participant was excluded because her lutein data were determined to be unreliable. Sixty-nine participants were further excluded because of missing important AMD risk factor data. Of the remaining 1787 participants, 474 women had insufficient serum for assays, leaving a sample size for the analysis of 1313. All procedures conformed to the Declaration of Helsinki and were approved by the institutional review board at each university.
Serum 25(OH)D is the preferred biomarker for vitamin D status, as it reflects vitamin D exposure from both oral sources and sunlight.16 Serum samples were drawn at WHIOS baseline after a fast of 10 or more hours and stored at −80°C.19 From 2004 to 2005, serum lutein and zeaxanthin concentrations were determined at Tufts University, Boston, Massachusetts,17 where samples were stored at −70°C and thawed at room temperature. The remaining serum was refrozen at −70°C and remained frozen until the day of vitamin D assay (in fall 2008), at which time they were thawed at room temperature and assayed within 2 to 3 hours for serum 25(OH)D concentration (nmol/L) using the LIAISON (DiaSorin, Stillwater, Minnesota) chemiluminescence method. Previous research shows that blood 25(OH)D concentrations are minimally affected by multiple freeze-thaw cycles20 or extended years in storage.21,22 C-reactive protein (CRP) (mg/L) concentrations were assessed using the high-sensitivity CRP assay kit (DiaSorin) on separate days from the 25 (OH)D assessment. C-reactive protein has been shown to remain stable for up to 5 freeze/thaw cycles.23 Both 25(OH)D and CRP assays were conducted by Heartland Assays, Inc (Ames, Iowa). The coefficient of variation determined using blind duplicates was 8.9% for 25(OH)D and 18.8% for CRP.
As sun exposure, and thus 25(OH)D concentrations, vary by season at Northern climates, 25(OH)D concentrations were adjusted for month of blood acquisition. Residuals from local regression of 25(OH)D on month of blood draw, with application of the local regression (LOESS) procedure (PROC LOESS in SAS v.9.2, SAS Institute, Cary, North Carolina),24 were added to the overall population mean (57.31 nmol/L). The LOESS method applies a nonparametric curve to smooth the means between adjacent months using weighted polynomial regression. Means for each month determined from the smooth curve are used for the adjustment.
Prevalent AMD was determined from stereoscopic retinal fundus photographs taken from 2001 through 2004. Of the 1857 participants with ocular data, 5% (n = 95) self-reported a diagnosis of AMD at the WHIOS year 3 follow-up (prior to fundus photography), 94% self-reported no AMD, and 1% (n = 24) had missing data. Photographs were graded by the University of Wisconsin Fundus Reading Center using the Age-Related Eye Disease Study protocol for grading maculopathy.25 Presence of AMD was classified as any, early, or advanced AMD (at least 1 eye). There were 241 cases of early AMD in 1287 women without advanced AMD. Early AMD was further classified as large drusen (≥1 large drusen [≥125 μm] or extensive intermediate drusen [area ≥360 μm when soft indistinct drusen are present or ≥650 μm when soft indistinct drusen are absent]) or pigmentary abnormalities (increased or decreased pigmentation accompanied by at least 1 drusen ≥63 μm). Of this sample, 26 women were classified as having advanced AMD (the presence of geographic atrophy in the center subfield or neovascular or exudative macular degeneration). Owing to the minimal number of advanced AMD outcomes, these analyses focus on early AMD.
At Women's Health Initiative baseline, vitamin D intake from foods was estimated from a self-administered food frequency questionnaire,26 to assess usual dietary intake during the previous 3 months. An interviewer-administered form was used to collect information on the dose, frequency, and duration of current supplement use at WHIOS baseline.27,28 Total vitamin D intake was calculated by summing vitamin D intake from foods and supplements. Using food frequency questionnaire data, dietary pattern scores were estimated for the 2005 Health Eating Index (HEI 2005) without inclusion of the oil subscore, as previously described.29
At the CAREDS baseline visit, participants were asked to report their sunlight exposure for each city/town in which they resided from 18 years of age to their age at CAREDS. Specifically, for each residence, they were asked to report the number of daytime hours (<1, 1-3, >3) spent in direct sunlight between 10 AM to 4 PM, in the months of April through September, during weekdays and leisure time. They also reported daytime activity on the water for 3 or more hours and whether they used protective gear (hats, sunglasses, and protective lenses). From these data, participants' estimation of reported time spent in direct sunlight at WHIOS baseline, corresponding in time to 25(OH)D assessment, was ascertained, and chronic ocular exposure to visible light in the last 20 years was estimated.30
Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for AMD by quintile of serum 25(OH)D, adjusting for age. Additional adjustment of the age-adjusted model for the following potential confounders of early AMD was investigated: study site, age, race/ethnicity, smoking pack years, recreational physical activity, body mass index (BMI; calculated as weight in kilograms divided by height in meters squared), and family history of AMD. Only BMI and physical activity changed the ORs by 10% or more. Although both measures of adiposity and physical activity have been reported as risk factors for AMD in the literature,31 they are also significant determinants of serum 25(OH)D status.32 Addition of BMI and physical activity to the model could potentially overadjust and explain the relationship of vitamin D status to early AMD. For this reason, the ORs were first investigated adjusted for early AMD risk factors identified a priori that were not strong determinants of serum vitamin D status : smoking pack years, iris pigmentation, self-reported family history of AMD, cardiovascular disease, diabetes, and hormone therapy use. In a second step, this multivariate model was further adjusted for BMI and physical activity. Next, we adjusted the multivariate model for CRP, a marker for systemic inflammation, to explore whether this association was potentially acting through an inflammatory pathway. As an exploratory analysis, we adjusted the multivariate model for other dietary factors highly correlated with 25(OH)D concentrations and associated with AMD in previous CAREDS analyses: dietary intake of lutein and zeaxanthin,17 dietary intake of polyunsaturated fat,33 and overall healthy diet, as indicated by the HEI 2005 score.34
Next, it was investigated whether consistent relationships were observed between early AMD and 2 sources of vitamin D: sunlight exposure and oral intake. The odds of early AMD were estimated among women self-reporting more than 3 and 1 to 3 compared with less than 1 h/d in direct sunlight at WHIOS baseline, and among women in high compared with low quintiles for baseline intake of vitamin D from foods, supplements, and foods and supplements combined.
The effect modification of the associations between serum 25(OH)D status and early AMD by age, BMI, physical activity, lutein plus zeaxanthin intake, a healthy dietary pattern (HEI 2005 score), hormone therapy use, and self-reported family history of AMD was investigated. Effect modification of the association between total vitamin D intake and AMD by sun exposure was also investigated. P < .10 was considered statistically significant. Analyses were conducted stratified by identified effect modifiers.
All analyses were conducted using SAS version 9.2.
Participants with high compared with low vitamin D status after adjustment for month of blood draw were more likely to be white, have a higher income, consume more alcohol, engage in a higher level of recreational physical activity, report greater ocular visible sun exposure, have a family history of AMD, have a lower BMI, be less hypertensive, and have lower concentrations of CRP (Table 1). Participants with high vitamin D status were also more likely to have higher calorie consumption, lower intake of fat, greater fiber intake, and greater intake of antioxidant nutrients (P ≤ .20). They consumed a greater amount of fruit, milk, and fortified cereal servings, had higher scores on the HEI 2005, and were more likely to use supplements compared with individuals with low vitamin D status (Table 2).
Table 3 shows the odds of having AMD among participants in quintiles 2 through 5 compared with 1. In models adjusted for age and further adjusted for a priori early- AMD risk factors (multivariate model), there was no significant relationship between vitamin D status and early or advanced AMD. The same was observed for drusen and pigmentary abnormalities (data not shown). However, the association between early AMD and 25(OH)D concentration was modified by age (P for interaction = .002). The ORs for early AMD among participants younger than 75 years were in the opposite direction of ORs for early AMD among women aged 75 or older, suggesting selective mortality bias in the older age group. Subsequently, further analyses were conducted using the sample of individuals younger than 75 years without advanced disease.
In the multivariate model, participants younger than 75 years had 48% decreased odds of early AMD (OR for quintile 5 vs 1, 0.52; 95% CI, 0.29-0.91; P for trend = .02) (Table 3). In women younger than 75 years, there were 57% decreased odds of pigmentary abnormalities (OR for quintile 5 vs 1, 0.43; 95% CI, 0.18-0.96; P for trend = .02) and the OR for quintile 5 compared with 1 for large drusen was also less than 1.0 but not statistically significant. Further adjustment of these relationships for BMI and physical activity, determinants of 25(OH)D status as well as potential confounders, attenuated these relationships. Further adjustment of the multivariate model for CRP strengthened relationships.
The inverse association between early AMD and 25(OH)D in women younger than 75 years was not explained by dietary intake of lutein plus zeaxanthin or polyunsaturated fat (Table 4). After adjustment for HEI 2005 score, the statistically significant relationship between 25(OH)D and AMD was attenuated, although the OR was still less than 1.0. There was no statistically significant (P < .10) effect modification of the relationship between 25(OH)D status and early AMD in women younger than 75 years by BMI, physical activity, HEI 2005 score, hormone therapy, or self-reported family history of AMD. However, the relationship between early AMD and 25(OH)D was stronger among women with higher than lower intakes of lutein plus zeaxanthin (adjusted OR for early AMD among women in tertile 3 vs 1 for serum 25(OH)D: low intake, 0.94; 95% CI, 0.50, 1.76; high intake, 0.46; 95% CI, 0.22, 0.93; P for interaction = .04).
There was no observed protective effect of reported hours spent in direct sunlight at WHIOS baseline on early AMD, as hypothesized (Table 5). Although oral sources of vitamin D accounted for only a small variation in serum 25(OH)D concentrations (<10%), a 59% reduced odds of early AMD in quintile 5 compared with 1 for vitamin D from food and supplements combined (associated P for trend = .15) was observed. Decreased, but not statistically significant, odds of early AMD in high compared with low intake of vitamin D from foods was observed, with a significant P for trend of .04. The top food sources of vitamin D in this sample included milk, fish, fortified margarine, and fortified cereal. Exploratory analyses revealed no statistically significant effect modification of the relationship between early AMD and total vitamin D intake by sunlight exposure (data not shown).
Analyses in the present sample of postmenopausal women confirm a protective association of vitamin D status to the prevalence of AMD, similar to that previously observed in the American population.14 In women younger than 75 years, having 25(OH)D concentrations higher than 38 nmol/L was significantly associated with a 48% decreased odds of early AMD. This association was consistent across subtypes of early AMD. Attenuation of the multivariate model after adjustment for BMI and physical activity is most likely explained by the strong correlation between these factors (predictors of vitamin D status) and 25(OH)D concentrations. Adjustment of the multivariate model for intake of lutein plus zeaxanthin, polyunsaturated fat, or CRP, a marker of systemic inflammation, did not explain the observed association, but the relationship was attenuated after adjustment for dietary pattern score. Some of the association between vitamin D status and early AMD may be explained by dietary patterns but may also have resulted in overadjustment of the multivariate model owing to multicolinearity between serum 25(OH)D and HEI 2005 score levels. A marginally statistically significant (P for trend = .05) direct association between 25(OH)D and early AMD was observed in women aged 75 years or older.
The observed significant age interaction is consistent with previous observations in CAREDS. Exposures (macular pigment density,35 lutein and zeaxanthin intake,17 and fat intake33) associated with decreased odds of early AMD in younger women were associated with increased odds in older women, suggesting selective mortality bias.36 We propose that, as people age, a greater proportion of those susceptible to early AMD than unsusceptible individuals with low 25(OH)D concentrations die of other chronic diseases prior to developing early AMD.36 Subsequently, a direct association between 25(OH)D and early AMD was observed in the oldest women. One way to avoid the influence of this bias is to examine associations in the youngest age group, as we have done in this investigation.
We observed a possible threshold effect with 50% decreased odds of early AMD in quintile 2 compared with 1 for 25(OH)D. The odds of early AMD did not further decrease after 25(OH)D concentrations rose higher than 38 nmol/L. This is higher than what is considered severely deficient, less than 25 nmol/L,37 but not high enough to be considered sufficient by some investigators who suggest that concentrations lower than 50 nmol/L16 or even 75 to 80 nmol/L are deficient.38 A previous cross-sectional analysis14 observed at least a 25% significant decreased odds of early AMD among persons with 25(OH)D concentrations of 54 nmol/L or higher. It is possible that measured serum 25(OH)D concentrations in this article slightly underestimated the true 25(OH)D concentrations due to degradation in storage, although this has been shown to minimally occur.22 If degradation occurred, it seems likely that it would have been fairly uniform across samples and not greatly affected the risk estimate.
We did not observe an association between early AMD and reported time spent outside in direct sunlight, although most circulating vitamin D in most individuals is derived from UVB-induced dermal production of vitamin D.39 Previous relationships between sunlight exposure and AMD have been investigated because chronic sunlight exposure is hypothesized to increase risk.40 Of the observational studies41- 53 investigating this relationship, only a few found direct associations between sunlight and AMD.43,45,50,53 Detrimental effects may be limited to blue light exposure,6,43 which is not measured in all studies. Two studies found that sunlight exposure was related to lower odds of developing AMD.46,48 In another cohort, AMD was directly associated with leisure time outdoors in summer,45,50,53 not associated with ambient UVB exposure, and (in some instances) inversely associated with UVB exposure after accounting for use of sunglasses and hats with brims.45,50,53 Perhaps some amount of UVB exposure, necessary for dermal vitamin D synthesis, may be protective for AMD when the eyes are also protected from blue light exposure. In the CAREDS, measurement error in assessment of sun exposure may have biased the results toward the null, or sunlight's protective effect via vitamin D synthesis and its detrimental effect from blue light ocular damage negate any findings between AMD and ambient sun exposure.
The inverse association between early AMD and 25(OH)D was supported by analyses of vitamin D intake. Significantly decreased odds of developing early AMD of similar magnitude to that observed with serum 25(OH)D was observed among persons in quintile 5 (estimated intake of 18 μg/d [720 IU/d]) compared with 1 for total vitamin D intake. This is greater than the Dietary Reference Intakes, which recommend 600 IU/d.54
Inflammation is thought to be involved in the pathogenesis of AMD. Vitamin D, because of its anti-inflammatory, immune-modulating properties,55 may suppress the cascade of destructive inflammation that occurs at the level of the retinal pigment epithelium–choroid interface in early stages of AMD.8 The vitamin D receptor is expressed on cells of the human immune system and 1,25-dihydroxyvitamin D has been shown to suppress proinflammatory cytokines in vitro, perhaps in part by altering T-cell function toward a T-helper 2 (anti-inflammatory) rather than a T-helper 1 (proinflammatory) response.15,55 A possible role of vitamin D in ocular functioning is supported by evidence that the vitamin D receptor is located in vertebrate retinal tissue56,57 and is expressed in human cultured retinal endothelial cells.58 Additionally, vitamin D may play a role in preventing AMD progression from early to neovascular AMD; however, we could not assess this in CAREDS. Vitamin D has been shown to inhibit angiogenesis in cultured endothelial cells59 and within the retinas of animal models of retinoblastoma60 and oxygen-induced ischemic retinopathy.61
Although we saw an inverse association between prevalent early AMD and 25(OH)D assessed 6 years earlier, we cannot firmly establish causality with this study design. Conclusions from this analysis can only be extrapolated to US postmenopausal women and white women, as CAREDS had limited minority representation and was not a nationally representative sample. Additionally, CAREDS is limited by a lack of measures for genetic risk factors which strongly predict risk of AMD.11
As previously described,17 selection bias is a potential concern in this study. As eligibility of participation in CAREDS was based on lutein plus zeaxanthin intake, to maximize dietary diversity, we investigated the associations between AMD and serum 25(OH)D stratified by lutein plus zeaxanthin intake (low and high). Regardless of intake, the relationship between AMD and vitamin D status was inverse, although stronger in those with higher lutein and zeaxanthin intake, suggesting that serum vitamin D status and lutein plus zeaxanthin intake may synergistically be important to eye health.
Thirty-six percent of participants who were eligible (n = 3143) for CAREDS declined to participate. Those persons younger than 75 years who participated were healthier with respect to dietary and lifestyle factors and had slightly greater self-reported AMD (3.4% vs 2.0%) at the Women's Health Initiative year 3 follow-up. For this reason, we suspect that nonparticipation would have biased our results toward the null. We cannot completely dismiss the possibility of selection bias in this study. This association needs to be observed in other longitudinal studies.
This is the second study to present an association between AMD status and 25(OH)D, and our data support the previous observation that vitamin D status may potentially protect against development of AMD. In CAREDS we were able to adjust for the major nongenetic risk factors for AMD as well as explore relationships between other surrogate measures for vitamin D status such as oral sources of vitamin D and sun exposure. In conclusion, vitamin D status may significantly affect a woman's odds of developing early AMD. More studies are needed to verify this association prospectively as well as to better understand the potential interaction between vitamin D status and genetic and lifestyle factors with respect to risk of early AMD.
Correspondence: Amy E. Millen, PhD, Department of Social and Preventive Medicine, University at Buffalo, Ferber Hall 240, Buffalo, NY 14214 (firstname.lastname@example.org).
Submitted for Publication: March 5, 2010; final revision received July 13, 2010; accepted July 23, 2010.
Author Contributions: Amy E. Millen had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Group Information: A list of the Women’s Health Initiative Investigators was published online December 13, 2010, in Arch Ophthalmol.
Financial Disclosure: Dr Gehrs reports currently serving as a consultant for Sequenome.
Funding/Support: This study was supported by grants EY13018, EY016886, and DK07665 from the National Institutes of Health and by Research to Prevent Blindness. It was part of the Carotenoids and Age-Related Eye Disease Study (CAREDS), an ancillary study of the Women's Health Initiative (WHI). The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, and the US Department of Health and Human Services through contracts N01WH22110, 24152, 32100-2, 32105-6, 32108-9, 32111-13, 32115, 32118-32119, 32122, 42107-26, 42129-32, and 44221.
Additional Contributions: We thank the women who generously contributed their time to participate in the CAREDS.