Very high plasma homocysteine levels are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. We studied the association between circulating homocysteine levels and the risk of incident osteoporotic fracture in 2406 subjects, 55 years of age or older, who participated in two separate prospective, population-based studies. In the Rotterdam Study, there were two independent cohorts: 562 subjects in cohort 1, with a mean follow-up period of 8.1 years; and 553 subjects in cohort 2, with a mean follow-up period of 5.7 years. In the Longitudinal Aging Study Amsterdam, there was a single cohort of 1291 subjects, with a mean follow-up period of 2.7 years. Multivariate Cox proportional-hazards regression models were used for analysis of the risk of fracture, with adjustment for age, sex, body-mass index, and other characteristics that may be associated with the risk of fracture or with increased homocysteine levels. During 11,253 person-years of follow-up, osteoporotic fractures occurred in 191 subjects. The overall multivariable-adjusted relative risk of fracture was 1.4 (95 percent confidence interval, 1.2 to 1.6) for each increase of 1 SD in the natural-log–transformed homocysteine level. The risk was similar in all three cohorts studied, and it was also similar in men and women. A homocysteine level in the highest age-specific quartile was associated with an increase by a factor of 1.9 in the risk of fracture (95 percent confidence interval, 1.4 to 2.6). The associations between homocysteine levels and the risk of fracture appeared to be independent of bone mineral density and other potential risk factors for fracture. An increased homocysteine level appears to be a strong and independent risk factor for osteoporotic fractures in older men and women.

Hyperhomocysteinemia may contribute to the development of osteoporosis. The relationship of Hcy and vitamin B₁₂ with bone turnover markers, BUA, and fracture incidence was studied in 1267 subjects of the Longitudinal Aging Study Amsterdam. High Hcy and low vitamin B₁₂ concentrations were significantly associated with low BUA, high markers of bone turnover, and increased fracture risk. Introduction: Hyperhomocysteinemia may contribute to the development of osteoporosis. Vitamin B₁₂ is closely correlated to homocysteine (Hcy). The main objective of our study was to examine the association of Hcy and vitamin B₁₂ status and the combined effect of these two with broadband ultrasound attenuation (BUA), bone turnover markers, and fracture. Materials and Methods: Subjects were 615 men and 652 women with a mean age of 76 ± 6.6 (SD) years of the Longitudinal Aging Study Amsterdam (LASA). At baseline (1995/1996), blood samples were taken after an overnight fast for dairy products. Plasma Hcy was measured with IMx, serum vitamin B₁₂ with competitive immunoassay (IA) luminescence, serum osteocalcin (OC) with immunoradiometric assay (IRMA), and urinary excretion of deoxypyridinoline (DPD) with competitive IA and corrected for creatinine (Cr) concentration. CVs were 4%, 5%, 8%, and 5%, respectively. BUA was assessed in the heel bone twice in both the right and left calcaneus. Mean BUA value was calculated from these four measurements. CV was 3.4%. After baseline measurements in 1995, a 3‐year prospective follow‐up of fractures was carried out until 1998/1999. Subjects were grouped by using two different approaches on the basis of their vitamin B₁₂ concentration, normal versus low (15 μM) or highest quartile (Q4) versus normal quartiles (Q1‐Q3). Analysis of covariance was performed to calculate mean values of BUA, OC, and DPD/Cr_urine based on the specified categories of Hcy and vitamin B₁₂ and adjusted for several confounders (potential confounders were age, sex, body weight, body height, current smoking [yes/no], mobility, cognition). The relative risk (RR) of any fracture was assessed with Cox regression analysis. Quartiles were used when Hcy and vitamin B₁₂ were separately studied in their relationship with fracture incidence. Results: Fourteen percent of the men and 9% of the women had high Hcy (>15 μM) and low vitamin B₁₂ (<200 pM) concentrations. Women with vitamin B₁₂ levels 15 μM had lower BUA, higher DPD/Cr, and higher OC concentrations than their counterparts. In men, no differences were found between the different Hcy and vitamin B₁₂ categories in adjusted means of BUA, OC, or DPD/Cr_urine. Twenty‐eight men and 43 women sustained a fracture during the 3‐year follow‐up period. The adjusted RR for fractures (95% CI) for men with high Hcy and/or low vitamin B₁₂ concentrations was 3.8 (1.2‐11.6) compared with men with normal Hcy and vitamin B₁₂ concentrations. Women with high Hcy and/or low vitamin B₁₂ concentrations had an adjusted RR for fractures of 2.8 (1.3‐5.7). Conclusions: High Hcy and low vitamin B₁₂ concentrations were significantly associated with low BUA, high markers of bone turnover, and increased fracture risk.

Folate and its synthetic form folic acid function as donor of one-carbon units and have been, together with other B-vitamins, implicated in programming of epigenetic processes such as DNA methylation during early development. To what extent regulation of DNA methylation can be altered via B-vitamins later in life, and how this relates to health and disease, is not exactly known. The aim of this study was to identify effects of long-term supplementation with folic acid and vitamin B12 on genome-wide DNA methylation in elderly subjects. This project was part of a randomized, placebo-controlled trial on effects of supplemental intake of folic acid and vitamin B12 on bone fracture incidence (B-vitamins for the PRevention Of Osteoporotic Fractures (B-PROOF) study). Participants with mildly elevated homocysteine levels, aged 65–75 years, were randomly assigned to take 400 μg folic acid and 500 μg vitamin B12 per day or a placebo during an intervention period of 2 years. DNA was isolated from buffy coats, collected before and after intervention, and genome-wide DNA methylation was determined in 87 participants (n = 44 folic acid/vitamin B12, n = 43 placebo) using the Infinium HumanMethylation450 BeadChip. After intervention with folic acid and vitamin B12, 162 (versus 14 in the placebo group) of the 431,312 positions were differentially methylated as compared to baseline. Comparisons of the DNA methylation changes in the participants receiving folic acid and vitamin B12 versus placebo revealed one single differentially methylated position (cg19380919) with a borderline statistical significance. However, based on the analyses of differentially methylated regions (DMRs) consisting of multiple positions, we identified 6 regions that differed statistically significantly between the intervention and placebo group. Pronounced changes were found for regions in the DIRAS3, ARMC8, and NODAL genes, implicated in carcinogenesis and early embryonic development. Furthermore, serum levels of folate and vitamin B12 or plasma homocysteine were related to DNA methylation of 173, 425, and 11 regions, respectively. Interestingly, for several members of the developmental HOX genes, DNA methylation was related to serum levels of folate. Long-term supplementation with folic acid and vitamin B12 in elderly subjects resulted in effects on DNA methylation of several genes, among which genes implicated in developmental processes.

Subclinical vitamin B-12 deficiency is common in the elderly. Encouraged by early indications, we investigated the plasma vitamin B-12 status in association with bone mineral content (BMC) and bone mineral density (BMD) in frail elderly people. Data of 194 free-living Dutch frail elderly (143 women and 51 men) were available. BMC and BMD were measured by dual energy X-ray analysis. Biochemical analyses were performed on plasma or serum including vitamin B-12, methylmalonic acid, homocysteine, 25-hydroxy vitamin D and parathyroid hormone. Women had higher plasma vitamin B-12 (288 and 238 pmol/L, respectively) and lower plasma homocysteine levels (15.8 and 21.3 μmol/L, respectively) than men. Of the total explained variance of BMC and BMD in women (46 and 22%, respectively), 1.3–3.1% was explained by plasma vitamin B-12, in addition to weight and height or energy intake. In men, the variance of BMC and BMD was explained by weight, smoking and/or height (total R² was 53 and 25%, respectively), but not by plasma vitamin B-12. Osteoporosis occurred more often among women whose vitamin B-12 status was considered marginal or deficient than in women with a normal status, i.e., the prevalence odds ratios (after adjustment for weight, age and calcium intake) (95% confidence intervals) were 4.5 (0.8;24.8) and 6.9 (1.2;39.4), respectively. These results suggest that vitamin B-12 status is associated with bone health in elderly women. Future studies on bone health should take into account a possible role of vitamin B-12 status in different populations.