Abstract
Context:
Hyperhomocysteinemia is an independent risk factor for premature atherosclerosis and thromboembolism. 25-Hydroxyvitamin D [25(OH)D] may modulate the expression of genes involved in homocysteine metabolism.
Objective:
Little is known about the relationship between homocysteine and 25(OH)D. We hypothesized an inverse and nonlinear association between 25(OH)D and homocysteine.
Design:
We analyzed data from the continuous National Health and Nutrition Examination Survey 2001–2006 for asymptomatic adults (≥18 y).
Setting:
Linear regression models with spline adjusted for cardiovascular disease risk factors were used to explore nonlinearity.
Main Outcomes Measure:
Mean change (β-coefficients with 95% confidence intervals) in homocysteine was reported per 10 ng/mL change in 25(OH)D.
Results:
Mean (SD) age and homocysteine levels of 14 630 participants were 47.2 (20) years and 8.8 (4.7) μmol/L, respectively, whereas the median (interquartile range) of 25(OH)D was 21 (15–27) ng/mL. Without using spline, we observed an inverse relation between homocysteine and 25(OH)D both in simple [−0.25 (−0.34 to −0.02) μmol/L] and multivariable [−0.13 (−0.18 to −0.01) μmol/L] regression. With spline, in a univariate model, an increase in 25(OH)D was associated with a significant decrease in homocysteine [−0.56 (−0.75 to −0.37) μmol/L] until 25(OH)D reaches but not if above its median (21 ng/mL). Similarly, in multivariable spline models, the inverse relationship between homocysteine and 25(OH)D remain significant [−0.49 (−0.67 to −0.31) μmol/L] only below the population median of 25(OH)D.
Conclusions:
From a large community-based cohort of asymptomatic adults, we found an inverse relation between 25(OH)D and homocysteine among those with 25(OH)D concentration of 21 ng/mL or less. We did not observe any statistical decrease in homocysteine once 25(OH)D concentration rose above 21 ng/mL.
Hyperhomocysteinemia is an independent risk factor for venous thromboembolism (1, 2) and premature cerebral (3, 4), peripheral, and coronary atherosclerotic vascular disease (5, 6). Homocysteine, a highly reactive amino acid, is a product of methionine metabolism, whereas its metabolic pathway involves several enzymes. Mutations in genes that code for enzymes involved in methionine metabolism (such as methylenetetrahydrofolate reductase and methionine synthase reductase) and homocysteine (eg, cystathionine β-synthase) result in hyperhomocysteinemia (6–9). In addition, deficiencies in serum folate and vitamin B12 levels are among the common nutritional causes of hyperhomocysteinemia (10–12). However, efficacy of treatments with these vitamins for reducing homocysteine levels in the primary prevention of cardiovascular disease remains unclear (13–16).
Activated vitamin D and its analogs manifest their diverse effects through binding with a high-affinity vitamin D receptor (VDR), which also acts as a ligand-activated transcription factor and modulates transcription of several genes (17–20). It has been shown that cystathionine β-synthase (CBS) is a target gene of VDR regulation, which makes activated vitamin D analogs a potential modifier of homocysteine metabolism (21). Although a nonlinear trend was observed in the relationship between 25-hydroxyvitamin D [25(OH)D] and all-cause and cardiovascular mortality, a body of literature suggest that low-serum vitamin D concentrations may be a risk factor for cardiovascular disease (22, 23). Given that both lower 25(OH)D and elevated homocysteine levels are potential risk factors for premature cardiovascular diseases and that activated vitamin D may regulate the gene expression of enzymes involved in homocysteine metabolism, we hypothesized that vitamin D status may also have an impact on serum homocysteine levels, independent of traditional cardiovascular disease risk factors as well as serum folate and vitamin B12 status. We further hypothesized that the relationship between vitamin D status and homocysteine is nonlinear, such that there may be no beneficial effect of the vitamin D status on serum homocysteine concentration once 25(OH)D levels increase beyond a certain threshold. We tested our hypothesis in a large sample of the adult US population without established cardiovascular disease.
Materials and Methods
We analyzed data from publically available continuous National Health and Nutrition Examination Survey (NHANES) from 2001 through 2006. Continuous NHANES is an ongoing, multistage probability sample survey designed to assess the health and nutritional status of the civilian, noninstitutionalized population of the United States. Detailed interviews, physical examinations, and blood samples were obtained from more than 31 000 individuals in the surveys conducted for 2001 to 2006 biannual cycles. Details of sampling procedures and data collection techniques have been described previously and are available online (http://www.cdc.gov/nchs/nhanes/about_nhanes.htm [accessed August 24, 2013]).
To create a large nationally representative sample, data from three 2-year cycles of the continuous NHANES were combined for years 2001–2002, 2003–2004, and 2005–2006. Analyses were performed with adjustments for the complex survey sampling methods of NHANES data. In continuous NHANES, primary sampling units represent variance (sampling units used to estimate sampling error) units. The sampling weights were assigned to each person, reflecting adjustment for the unequal probability of selection, nonresponse, and adjustments to independent population controls. Participants were oversampled for certain population subgroups such as African and Mexican Americans to ensure reliability and precision of health estimate indicators in these subgroups. Masked variance units were constructed to protect the confidentiality of data obtained from sample persons. Masked variance units, used to define strata, were created for each 2-year cycle of the continuous NHANES, allowing any combination of data cycles without recoding by the users. Sample weights were constructed with the rescaling of weights such that the sum of weights matched the survey population at the midpoint of each 2-year survey period. For this study, sample weights were constructed for 6 years of combined survey data.
Demographic information was ascertained from self-reported responses to the questionnaire administered by trained interviewers. Analysis was limited to participants 18 years of age or older who were free of clinical cardiovascular disease(s) and had homocysteine and 25(OH)D concentrations measured. Body mass index (BMI) was calculated by dividing body weight in kilograms with height in meters squared. Obesity was defined as a BMI of 30 kg/m2 or greater and overweight if BMI was 25 kg/m2 or greater but less than 30 kg/m2. Blood pressure (BP) was calculated as an average of up to four readings. Hypertension was defined as a mean systolic BP of 140 mm Hg or greater, a mean diastolic BP of 90 mm Hg or greater, a diagnosis of hypertension, or current use of antihypertensive medications. Glomerular filtration rate was calculated using Modification of Diet in Renal Disease equation (24). Participants were categorized as smokers if they were actively smoking or had smoked more than 100 cigarettes in their life time.
Detailed laboratory methods for individual specimen collection and analysis have been described previously and are available (http://www.cdc.gov/nchs/nhanes/search/datapage.aspx?Component=Laboratory&CycleBeginYear=2001). Briefly, serum glucose was measured using a Beckman Synchron LX20 test on refrigerated specimens, whereas total serum cholesterol was measured enzymatically in a series of coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of cholesterol. Diasorin RIA (Nutritional Biochemistry Branch, National Center for Environmental Health, Atlanta, Georgia), and a two-step procedure was used to assay 25(OH)D. The first procedure involved extraction of 25(OH)D and other hydroxylated metabolites from serum with acetonitrile. The treated sample was then assayed using an equilibrium RIA procedure, based on an antibody specific to 25(OH)D. Updated and adjusted data files were used for 25(OH)D to address assay drift. Details on quality control and other laboratory procedures for 25(OH)D RIA can be found at http://www.cdc.gov/nchs/data/nhanes/nhanes_05_06/vid_d_met_vitamin_d.pdf (accessed September 30, 2013).
C-reactive protein (CRP) was quantified by latex-enhanced nephelometry using a Behring Nephelometer II analyzer. Serum folate and vitamin B12 were measured by using the Bio-Rad Laboratories Quantaphase II folate/vitamin B12 RIA kit.
Serum homocysteine was measured by using an Abbott Homocysteine assay on the Abbott AxSym analyzer, a fully automated fluorescence polarization immunoassay method from Abbott Diagnostics. Briefly, dithiothreitol reduces homocysteine bound to albumin and to other small molecules, homocysteine, and mixed disulfides to free thiol. S-adenosyl-homocysteine (SAH) hydrolase catalyzes conversion of homocysteine to SAH in the presence of added adenosine. Subsequently the specific monoclonal antibody and the fluoresceinated SAH analog tracer constitute the fluorescence polarization immunoassay detection system. Serum homocysteine concentrations were calculated by the Abbott Axsym immunoassay analyzer using a machine-stored calibration curve.
Statistical analyses were performed on Stata/IC version 12.1 (Stata Corp LP), using survey-specific commands. We used a t test to estimate differences in means between groups and a χ2 test to estimate the difference in proportions between groups. Median values were reported with a corresponding interquartile range (IQR). Selected variables were log transformed to meet assumptions of residual normality. Adjusted β-coefficients with corresponding 95% confidence intervals (CIs) were reported using linear regression models. Models were adjusted for demographic variables, BMI, CRP, hypertension, serum glucose, total cholesterol, smoking status, renal function, and folate and vitamin B12 levels.
To address the hypothesis that the association between 25(OH)D and homocysteine is nonlinear, we introduced spline in the linear regression models, a single knot at the population median of 25(OH)D, hypothesizing that the association between 25(OH)D and homocysteine may exist up to the population median of 25(OH)D but not above it. Results were reported as change in serum homocysteine levels (micromoles per liter) for each 10 ng/mL change in serum 25(OH)D. A P value of < .05 was considered statistically significant.
Results
Of the 17 176 participants older than 18 years, we excluded those with missing values of 25(OH)D (n = 1993) and homocysteine (n = 553). The mean (SD) age and serum homocysteine levels of the study participants (n = 14 630) was 47.2 (20) years and 8.8 (4.7) μmol/L, respectively. Median (IQR) of serum 25(OH)D and CRP were 21 (15–27) ng/mL and 0.22 (0.08–0.51), respectively. Study sample included 7583 females (52%) and 7436 non-Hispanic whites (51%).
Table 1 shows the population characteristic by 25(OH)D level. There were 7473 participants (51%) with 25(OH)D concentrations of 21 ng/mL or less, and 7157 (49%) had 25(OH)D level above 21 ng/mL. Among the participants with 25(OH)D concentrations of 21 ng/mL or less, 2447 (55%) were non-Hispanic whites, whereas 4989 (85%) of those with 25(OH)D levels of 21 ng/mL or greater were non-Hispanic whites (P < .0001). There were only 509 non-Hispanic blacks (3%) among those with 25(OH)D concentrations greater than 21 ng/mL as compared with 2507 (21%) with 25(OH)D concentration of 21 ng/mL or less (P < .0001). Mean serum 25(OH)D levels were significantly higher in non-Hispanic whites as compared with other racial groups (mean difference 7.6 ng/mL, 95% CI 6.9–8.4).
Table 1.
Population Characteristics
| Covariates | 25(OH)D, ng/mL |
P Value | |
|---|---|---|---|
| ≤21 (7473) | >21 (7157) | ||
| Age, ya | 45 (44.7, 46) | 46 (45, 47) | .5 |
| Females, n, % | 3961 (54) | 3622 (49) | <.0001 |
| Homocysteine, μmol/litera | 9.01 (8.8, 9.2) | 8.6 (8.4, 8.7) | <.001 |
| Race, n, % | |||
| Non-Hispanic white | 2447 (55) | 4989 (85) | <.0001 |
| Mexican American | 1897 (11) | 1218 (5) | <.0001 |
| Non-Hispanic black | 2507 (21) | 509 (3) | <.0001 |
| Other Hispanics | 272 (5) | 233 (4) | .004 |
| Other race | 350 (8) | 208 (3) | <.0001 |
| Hypertension, n, %b | 2993 (38) | 2528 (33) | <.0001 |
| CRP, mg/dLa | 0.5 (0.46, 0.53) | 0.37 (0.35, 0.39) | <.001 |
| Serum glucose, mg/dL | 99 (98, 100) | 93.5 (93, 94) | <.001 |
| BMI, kg/m2a | 29.7 (29.4, 29.9) | 27.2 (26.9, 27.4) | <.001 |
| Smoker, n, % (current and ever) | 1352 (23) | 1142 (19) | .001 |
| GFR, ml/min·m2a | 100 (98, 101) | 93 (92, 94) | <.001 |
| Total cholesterol, mg/dLa | 199 (197, 201) | 201 (200, 202) | .02 |
| Vitamin B12, pg/mLa | 502 (482, 521) | 560 (535, 586) | <.001 |
| Serum folate, ng/mLa | 12 (11.7, 13) | 15.4 (15, 16) | <.001 |
Abbreviation: GFR, glomerular filtration rate, measured using Modification of Diet in Renal Disease equation. Because of rounding, not all percentages total 100.
Variable reported as mean values with 95% CIs.
Hypertension is defined as average systolic BP greater than 140 mm Hg or average diastolic BP greater than 90 mm Hg or individuals ever told they had hypertension or if participants were taking an antihypertensive.
Mean homocysteine levels were significantly lower (mean difference 0.46 μmol/L, P < .001) in participants with 25(OH)D greater than 21 ng/mL, as compared with 21 ng/mL or less. The mean serum (SD) homocysteine levels in females [8 (4.5) μmol/L] were significantly lower than their male counterparts [9.8 (4.6) μmol/L] (mean difference −1.4 μmol/L; 95% CI −1.6 to −1.2).
The mean (SD) and median (IQR) serum 25(OH)D were 22 (8.4) and 22 (16–27) ng/mL in men and 22 (10) and 21 (14–28) ng/mL in females, respectively. There was no statistical difference in the vitamin B12 levels between females and males (mean difference 23.2 pg/mL; 95% CI −5.4 to 52); however, females had higher serum folate levels than males (mean difference 1.9 ng/mL; 95% CI 1.5–2.4).
We observed a statistically significant decrease in serum homocysteine levels with an increase in 25(OH)D levels in both univariate (mean change −0.25 μmol/L; 95% CI −0.34 to −0.02) and multivariable (mean change −0.13 μmol/L; 95% CI −0.18 to −0.01) linear regression models.
In the univariate linear regression using spline [single knot at 21 ng/mL of 25(OH)D], we found that an increase in serum 25(OH)D was associated with a robust and significant decrease in homocysteine levels (mean change −0.56 μmol/L; 95% CI −0.75 to −0.37) until 25(OH)D reached its population median levels of 21 ng/mL. An increase in serum 25(OH)D to a level above 21 ng/mL was associated with only a modest and nonsignificant decrease in homocysteine (−0.09 μmol/L; 95% CI −0.21 to 0.02) levels (Table 2).
Table 2.
Mean Change (β-Coefficient and 95% CI) in Serum Homocysteine (Micromoles per Liter) per 10 ng/mL Rise in 25(OH)D Concentration
| Linear Regression | Linear Regression With Spline [25(OH)D ≤ 21 ng/mL] | Linear Regression With Spline [25(OH)D > 21 ng/mL] | |
|---|---|---|---|
| Univariable | −0.25 (−0.34, −0.02) | −0.56 (−0.75, −0.37) | −0.09 (−0.21, 0.02) |
| Multivariable | −0.13 (−0.18, −0.01) | −0.49 (−0.67, −0.31) | 0.02 (−0.08, 0.11) |
Multivariable models were adjusted for race, gender, hypertension, smoking status, CRP, BMI, total cholesterol, renal function, glucose, serum folate, and vitamin B12.
Similarly, in multivariable linear regression models using spline, the inverse association between homocysteine and 25(OH)D below its population median remained significant (mean change −0.49 μmol/L; 95% CI −0.67 to −0.31). Additionally, we also observed a modest but statistically nonsignificant increase in homocysteine concentration once 25(OH)D crossed its population median level of 21 ng/mL (mean change 0.02 μmol/L; 95% CI −0.08 to 0.11) (Table 2).
Discussion
From this large community-based nationally representative sample of healthy adults, we report an inverse relation between homocysteine and 25(OH)D among individuals with serum 25(OH)D concentrations up to 21 ng/mL independent of traditional cardiovascular disease risk factors as well as serum folate and vitamin B12 status. There were no significant associations between the homocysteine and 25(OH)D concentrations, once the latter has reached the 21 ng/mL threshold (ie, population median). These findings support our hypothesis that achieving serum 25(OH)D of greater than 21 ng/mL or greater may not result in an additional decrease in homocysteine concentration and is probably not needed for the primary prevention of atherosclerotic vascular disease, especially among asymptomatic adults.
The cellular mechanism responsible for the relationship between homocysteine levels and 25(OH)D status is unclear and may include indirect or direct effects of vitamin D on homocysteine metabolism. Biological activities of activated vitamin D are mediated through their binding to a high-affinity VDR. VDR acts as a ligand-activated transcription factor that modulates transcription of several genes responsible for the diverse cellular effects of vitamin D analogs (17–20). Activated vitamin D analogs inhibit the proliferation of lymphocytes and decrease the production of several proinflammatory cytokines such as IL-2 and interferon-α and stimulate the effects of the T-helper type 2 lymphocytes, which leads to a reduction in matrix metalloproteinase, thus restricting atherosclerotic plaque progression (25, 26). Active forms of vitamin D resemble retinoids in their control of the transcription of thrombomodulin and tissue factor genes expression in cultured human monocytic cells, which demonstrates that activated vitamin D through its receptors may play a physiological role in the maintenance of antithrombotic homeostasis (19, 27).
Homocysteine is metabolized by transsulfuration and remethylation (folate dependent) pathways. The transsulfuration pathway is important for homocysteine disposal in which it is converted to cystathionine in the presence of the enzyme CBS and cofactor vitamin B6. Deficiency of the CBS enzyme has been linked with hyperhomocysteinemia (6, 28). A significant increase in the lower basal levels of CBS mRNA and protein was seen after incubation with activated vitamin D in murine preosteoblasts, which suggests that CBS is a target gene of VDR, and vitamin D may modulate homocysteine metabolism and can affect its serum concentration (21).
Our finding of nonlinear relationship between 25(OH)D and homocysteine in healthy adults are in agreement with the recent observations reported by Kriebitzsch et al (21). Using data from the Longitudinal Aging Study Amsterdam, the authors reported a significant correlation (P < .05) between 25(OH)D and homocysteine. They further observed a U-shaped relationship with the lowest homocysteine levels when 25(OH)D status was between 20 and 24 ng/mL, whereas the transition point was estimated at 20.7 ng/mL (21). Additionally, the authors further reported a correlation between vitamins D and homocysteine of −0.1 (P < .05) below and 0.08 (P = .05) above the cut point of 20.7 ng/mL, respectively (21).
Our results of an inverse relationship between 25(OH)D and homocysteine in healthy adults with serum 25(OH)D levels less than or equal to but not greater than 21 ng/mL provides further support to recent recommendations by the Institute of Medicine that serum 25(OH)D of about 20 ng/mL meets the requirement of 98% of the population (29). Evidence from observational studies has shown a nonlinear relation between serum 25(OH)D and certain outcomes (such as cardiovascular disease, vascular calcification, and all cause mortality) with optimal values of 25(OH)D between 16.1 and 24 ng/mL (29, 30). Melamed et al (31) reported an 80% increase in the adjusted prevalence of peripheral artery disease among individuals with 25(OH)D less than 17.8 ng/mL (lowest quartile) when compared with individuals with levels of 29.2 ng/mL or greater (highest quartile). We have recently reported a nonlinear relation between 25(OH)D and serum CRP among asymptomatic individuals, concluding that the role of vitamin D supplementation in reducing systemic inflammation (as measured by elevated serum CRP levels) is probably beneficial among individuals with serum 25(OH)D of less than or equal to but not greater than 21 ng/mL (32).
The major strengths of our study include its generalizability due to large population size and wide age range (18–85 y) of participants from various ethnic groups. The sampling scheme of the continuous NHANES data allows obtaining better estimates and adjustment for various ethnic backgrounds. The effect of assay drifts over time on 25(OH)D, which could affect association for risk assessment (33), was also accounted for in our analysis. The data on geographic locations, weather, and latitude was limited, and we were unable to analyze the effects of these variables on the association between 25(OH)D and homocysteine. Additionally, vitamin B6 levels were not measured for the 2001–2002 NHANES survey, and the assay methods for 2003–204 and 2005–2006 cycles were different, which posed a technical challenge in combining the data; hence, we did not adjust for vitamin B6 in our analysis.
In conclusion, we report an inverse relationship between levels of homocysteine and 25(OH)D independent of traditional cardiovascular disease risk factors, serum folate, and vitamin B12 status among asymptomatic adults. We further show that this association is present only once serum 25(OH)D levels reach 21 ng/mL or less. Our study does not support that an increase in serum 25(OH)D levels above 21 ng/mL is associated with a reduction in the homocysteine level. Supplementation with 25(OH)D to reduce homocysteine for the prevention of atherosclerotic vascular disease and venous thrombosis may only benefit individuals who have serum 25(OH)D levels of 21 ng/mL or less at baseline. This epidemiological observation deserves further validation and exploration of underlying cellular mechanism(s). If studies in the future support a causal role of vitamin D status in determining serum homocysteine levels, long-term interventional trials may be considered for the primary prevention of cardiovascular diseases among asymptomatic adults with a serum 25(OH)D below 21 ng/mL.
Acknowledgments
Both authors had complete access to data and had an equal role in writing the original manuscript.
Present address for M.A.: Howard University Hospital, Washington, DC 20060. E-mail: mamer@huhosp.org.
R.Q. was supported by National Institutes of Health Grant 5K23HL105897-01.
Disclosure Summary: The authors have nothing to declare.
Footnotes
- BMI
- body mass index
- BP
- blood pressure
- CBS
- cystathionine β-synthase
- CI
- confidence interval
- CRP
- C-reactive protein
- IQR
- interquartile range
- NHANES
- National Health and Nutrition Examination Survey
- 25(OH)D
- 25-hydroxyvitamin D
- SAH
- S-adenosyl-homocysteine
- VDR
- vitamin D receptor.
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