Relation of blood homocysteine and its nutritional determinants to age-related maculopathy in the third National Health and Nutrition Examination Survey

¹ From the Departments of Ophthalmology and Visual Sciences (RAH, AIF, RK, BEKK, and JAM-P) and Preventive Medicine (MP), University of Wisconsin Medical School, Madison, and the Research Center on Aging, Tufts University, Boston (PFJ).

² Supported by National Institutes of Health grant EY11722 (to JAM-P) and in part by an unrestricted grant to the Department of Ophthalmology from the Research to Prevent Blindness.

³ Reprints not available. Address correspondence to JA Mares-Perlman, Department of Ophthalmology and Visual Sciences, University of Wisconsin, 610 North Walnut Street, 460 WARF, Madison, WI 53705-2397.

ABSTRACT  
Background: Blood homocysteine and its nutritional determinants folate and cyanocobalamin (vitamin B-12) have been shown to affect the risk of vascular disease. The pathogenesis of age-related maculopathy (ARM) is related to adverse vascular changes.

Objective: The objective was to evaluate the associations between homocysteine, its nutritional determinants, and ARM in persons aged 40 y participating in the third National Health and Nutrition Examination Survey.

Design: A nonmydriatic fundus photograph of one eye, taken in a mobile examination center, was used to ascertain ARM status. Phlebotomy was performed for measurement of homocysteine, cyanocobalamin, and erythrocyte folate in participants of phase 2 of the survey (n = 3828). Logistic regressions were used to compute odds ratios and 95% CIs by quintile of serum analyte by using sample weights and jackknife replication methods to adjust for the complex survey design. The final analyses were adjusted for potential risk factors that influenced odds ratios.

Results: Total serum homocysteine, red blood cell folate, and serum cyanocobalamin were unrelated to ARM in the overall sample. However, red blood cell folate was inversely related to one type of early ARM lesion (soft drusen) in non-Hispanic blacks.

Conclusions: ARM does not appear to be associated with homocysteine or its dietary determinants in this survey. There is a need for further investigation to rule out potential associations in subgroups with low folate status that may not have been detected because of the cross-sectional survey design.

Key Words: Homocysteine • age-related maculopathy • third National Health and Nutrition Examination Survey • NHANES III • elderly • vitamin B-12 • folate

INTRODUCTION  
Age-related maculopathy (ARM) is the most frequent cause of visual impairment in the elderly (1, 2). Atherosclerosis has been a suspected risk factor for the development of ARM (3, 4). However, atherosclerotic vascular disease has been associated with ARM (5, 6) in some but not in all (7–12) epidemiologic studies. Vingerling et al (13) reported that the prevalence of exudative ARM was 4.7-fold that in the presence of plaques in the carotid bifurcation. Risk factors for atherosclerotic disease, such as smoking (14, 15), hypercholesterolemia (16), decreased estrogen exposure (16), and high intakes of fat or cholesterol (17–19) have also been associated with ARM in some previous studies.

Elevated plasma concentrations of total homocysteine have been shown to increase the risk of vascular disease (20–26). Prospective studies have found an increased risk of myocardial infarction in hyperhomocysteinemia, and meta-analyses of this association confirmed the finding that only slight elevations in plasma homocysteine significantly affected the odds ratios across race and sex (27–29). Homocysteine metabolism is dependent on reactions involving vitamins B-6, B-12, and folate for transsulfuration and remethylation (30). Epidemiologic studies have shown inverse associations between hyperhomocysteinemia and blood concentrations and intakes of these vitamins (31–34). In addition, human experiments have shown that the addition of supplemental B vitamins significantly reduced plasma homocysteine concentrations (35).

Proposed mechanisms for the deleterious effects of hyperhomocysteinemia include promotion of vascular smooth muscle proliferation and effects on vascular coagulant mechanisms (36–38). In addition, aberrations in redox thiol status resulting from elevated homocysteine concentrations may increase prooxidant burdens and compromise systemic total antioxidant capacity (39). Similar underlying atherosclerotic and oxidative mechanisms have also been suggested in the development of ARM. It is possible that changes in vasculature and antioxidant status as a result of hyperhomocysteinemia may increase the risk of ARM. Therefore, in the present cross-sectional investigation, we examined the relations between ARM and homocysteine, vitamin B-12, and folate concentrations.

SUBJECTS AND METHODS  
Study population
The Centers for Disease Control and Prevention and the National Center for Health Statistics conducted the third National Health and Nutrition Examination Survey (NHANES III) between 1988 and 1994 among a nationally representative sample of the civilian noninstitutionalized US population. The survey method included 2 phases over a 6-y period and the use of a stratified probability sample, with oversampling of non-Hispanic blacks, Hispanics, and adults aged > 60 y, the details of which are published elsewhere (40). The survey included a household interview, medical examination that took place in a mobile examination center, and phlebotomy. During the household interview, demographic, socioeconomic, health-history, and dietary data were collected. Information regarding food intake was gathered with the use of 24-h dietary recall methods and a 60-item food-frequency questionnaire in a subsample of participants. The medical examination included a physical exam, fundus photography, urine collection, and blood draws for the measurement of many hematologic factors, including serum homocysteine.

Nonmydriatic fundus photographs were taken at the mobile examination center for participants aged 40 y. One eye was randomly selected, and a single color photograph was taken. Photographic fields were subsequently graded with the use of a modified Wisconsin Age-Related Macular Degeneration grading system criteria. Phlebotomy was performed in both fasting and nonfasting participants, and serum homocysteine was assessed in phase 2 participants only (1992–1994). Blood samples were processed in accordance with standard protocols, and sera were frozen and stored at -70 °C for 8 mo to 3 y before analysis (41). The reversed-phase HPLC with fluorescence detection method of Araki and Sako was used to measure homocysteine concentrations at the US Department of Agriculture’s Human Nutrition Research Center on Aging (42). Red blood cell (RBC) folate and vitamin B-12 were measured with the use of the Bio-Rad Laboratory’s Quantaphase Folate radioassay kit (43). Urinary albumin concentrations were measured via solid-phase fluorescent immunoassay, and serum creatinine was assessed as part of a biochemistry profile with the use of a Hitachi model 737 multichannel analyzer from Boehringer Mannheim Diagnostics, Indianapolis (41, 44).

Serum homocysteine concentrations were available for 4145 phase 2 participants aged 40 y. Persons with hyperhomocysteinemia resulting from renal dysfunction were excluded from the analysis. The criteria used to determine probable renal insufficiency were a urinary albumin concentration > 3 mg/mmol albumin, an elevated serum creatinine (> 14 mg/L for men and > 12 mg/L for women) concentration, or both. This resulted in the exclusion of 286 persons with probable renal insufficiency and the removal of 31 additional persons for whom insufficient data regarding renal status were available. The final analyses were performed with the use of data for 3527 of the phase 2 participants for whom eye photographs and serum homocysteine, cyanocobalamin, and folate values were available. The sample included 1647 non-Hispanic whites, 820 non-Hispanic blacks, 880 Hispanics, and 180 respondents of other racial or ethnic origin.

Statistical methods
Participants were ranked into quintiles by concentration of serum homocysteine, serum vitamin B-12, and RBC folate. Relations between potential risk factors for ARM and quintile rank for homocysteine, vitamin B-12, and folate were assessed by using analysis of variance for continuous variables and the Cochran-Mantel-Haenszel test for trend for categorical variables. Variables were adjusted for age and sex in these comparisons.

Logistic regression was used to compute odds ratios for the highest compared with the lowest quintiles for serum values. Test for trend was performed by using the continuous range of nutritional variables. Age-adjusted and multivariate analyses were performed by race and with all race groups combined. Race interactions were found to be significant at P < 0.03 (maximum likelihood); thus, subsequent analyses were done for each race group separately. In the analyses using all race groups combined, the jackknife replication method of variance estimation was used to account for complex sampling methods used in NHANES III (PA Berglund, unpublished observations, 1997). Sample weights were applied to all analyses.

Several variables were considered as potential confounders: age, sex, smoking status, systolic and diastolic blood pressure, HDL cholesterol, eye color, and estrogen use. These variables were added to the model when a change of 10% was observed in the odds ratios for serum quintiles. Additional analyses were performed in which users of supplements containing vitamin B-6, vitamin B-12, or folate were excluded in an attempt to remove potential biases from recent supplement use. Further analyses were done in which the data were stratified by the age group and sex of the survey participants to account for possible selective mortality biases and to investigate potential effect modification, respectively. The SAS software package (version 6.12; SAS Institute Inc, Cary, NC) was used for all statistical analyses.

RESULTS  
Median serum homocysteine concentrations increased with age in whites (8.6 ± 4, 9.6 ± 6, and 10.7 ± 6 µmol/L), blacks (8.5 ± 6, 9.7 ± 5, and 11.3 ± 5 µmol/L), and Hispanics (8.3 ± 7, 10.1 ± 4, and 10.6 ± 10 µmol/L) aged 40–59, 60–79, and 80 y, respectively. The age-specific serum homocysteine concentrations were lower in women than in men in each age category, except for non-Hispanic blacks aged 80 y (data not shown).

The distribution of remaining potential risk factors for ARM after age, race, and sex standardization in the highest compared with the lowest quintiles of serum homocysteine, cyanocobalamin, and folate are shown in Table 1. Men and women in the highest compared with the lowest quintiles of homocysteine were more likely to have higher diastolic blood pressure, to have higher plasma fibrinogen concentrations, to have received a diagnosis of cardiovascular disease or hypertension, to have been former smokers, or to be current smokers. Women in the highest compared with the lowest quintiles of serum homocysteine were more likely to have been users of oral contraceptives and estrogen or have had a hysterectomy.

View this table:
TABLE 1 . Distribution of potential risk factors among participants of the third National Health and Nutrition Examination Survey in the high (Q5) compared with low (Q1) quintiles of serum homocysteine, vitamin B-12, and red blood cell (RBC) folate¹  
Serum homocysteine concentrations were not significantly related to early or late ARM in any age or race group tested (Table 2). Although the odds ratios were > 1 for non-Hispanic blacks and Hispanics, as hypothesized, they were not significant. There were no significant relations for individual ARM lesions, and odds ratios were similar across age groups (data not shown). Exclusion of supplement users who may have had recent reductions in homocysteine concentrations did not influence associations.

View this table:
TABLE 2 . Adjusted odds ratios (ORs) for age-related maculopathy (ARM) among participants of the third National Health and Nutrition Examination Survey for high (Q5) compared with low (Q1) quintiles of serum homocysteine, red blood cell (RBC) folate, and vitamin B-12 in persons aged >40 y¹  
RBC folate increased in all race and sex groups with advancing age. Median RBC folate concentrations were 12.7 ± 15, 18.1 ± 18, and 19.9 ± 26 nmol/L in whites; 10.2 ± 9, 12.0 ± 10, and 16.0 ± 13 nmol/L in blacks; and 10.4 ± 7, 11.3 ± 10, and 12.3 ± 14 nmol/L in Hispanics aged 40–59, 60–79, and 80 y, respectively. After standardization for age, sex, and race, higher RBC folate was related to higher serum HDL, lower LDL, lower fibrinogen, lower rates of hypertension, and higher rates of estrogen use among women. It was also inversely related to current smoking status (Table 1).

RBC folate concentrations were not related to early ARM, and there were too few cases to evaluate the effect in late ARM. Associations with early ARM were marginally significant after participants who reported supplement use that could have influenced recent folate concentrations were excluded. Adjusted odds ratios were 0.6 (95% CI: 0.4, 1.0; P for trend = 0.06) in high compared with low quintiles. RBC folate concentrations were inversely related to drusen in non-Hispanic blacks, for whom distributions of the vitamin were lowest, such that odds were 60% lower in the high than in the low quintiles. In Hispanics, for whom distributions of the vitamin were also lower, adjusted odds ratios for soft drusen were less than unity (0.7; 95% CI: 0.2, 1.2) but were not significant (P for trend = 0.40) (Table 3).

View this table:
TABLE 3 . Adjusted odds ratios (ORs) for specific age-related maculopathy (ARM) lesions among participants of the third National Health and Nutrition Examination Survey for high (Q5) compared with low (Q1) quintiles of red blood cell (RBC) folate¹  
The distribution of potential risk factors for ARM in the high and low quintiles of vitamin B-12 are shown in Table 1. Lower vitamin B-12 concentrations were associated with lower diastolic blood pressure. Serum cyanocobalamin concentrations were not significantly related to early or late ARM (Table 2). Adjustment for diastolic blood pressure or other potential confounders did not alter the directions of the associations.

DISCUSSION  
ARM was not significantly related to serum homocysteine, folate, or vitamin B-12 in the overall NHANES III population. This may reflect a true lack of association or an inability to detect an association in this population. Hypothesized direct associations with homocysteine may have been difficult to observe for a variety of reasons. One such reason could be the cross-sectional nature of the study design because there was limited information on persons who changed their diet and lifestyle on diagnosis with other comorbid conditions before being surveyed. Second, differences in the participants in the fasted state at the time of the blood draw in the mobile examination unit would have increased variability and may have altered our ability to detect relations. The exclusion of persons with missing homocysteine, folate, and cyanocobalamin data (who were more likely to be in the older age groups, in whom ARM is most often seen) could have also biased the associations observed. Third, selective mortality could have influenced our results. Respondent biases may also have been introduced, because persons who are visually impaired as a result of ARM would be less likely to have participated in the full examination for NHANES III.

It was of interest to note an inverse relation between RBC folate concentrations and soft drusen, one type of early ARM, in non-Hispanic blacks. In Hispanic subjects, the trend was in a similar direction but was not significant. The folate distributions were lower and wider in these 2 racial groups than in non-Hispanic whites. Also, homocysteine concentrations and early ARM showed direct, albeit nonsignificant, relations in these race groups, in keeping with biologically plausible mechanisms. However, non-Hispanic blacks and Hispanic Americans have also been shown to have lower rates of more advanced stages of ARM than their white counterparts (45). Although the associations between folate status and drusen deserve further attention, the importance of this association, given the lower prevalence of ARM in black and Hispanic Americans, particularly in the later stages, is uncertain.

Despite suggestions from previous epidemiologic studies that hyperhomocysteinemia plays a role in the development of vascular disease and that atherosclerotic vascular disease plays a role in the pathogenesis of ARM, concentrations of homocysteine, its nutritional determinants, or both appear not to be associated with ARM in the NHANES III population. However, the results, which are inconsistent across race and age groups, indicate the need for further investigation. The relations between ARM and homocysteine, as well as its dietary determinants folate and cyanocobalamin, need to be investigated in an effort to clarify the issues outlined herein.

ACKNOWLEDGMENTS  
We acknowledge Jacqueline Wright (Division of Health Examination Statistics National Center for Health Statistics) for her contributions to this project.

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