Vitamin B12 is important to DNA synthesis and may affect bone formation. We examined the association between this vitamin and BMD in 2576 adults. Men with plasma B12 < 148 pM had significantly lower BMD at the hip, and women at the spine, relative to those with higher B12, and trends were similar for both at all sites. Low vitamin B12 may be a risk factor for low BMD.
Introduction: Vitamin B12 is important to DNA synthesis and may affect bone formation. It has been linked to osteoblastic activity in clinical studies and cell culture.
Materials and Methods: We examined the relationship between plasma vitamin B12 status and BMD in 2576 adult participants in the Framingham Offspring Osteoporosis Study (1996–2001). BMD was measured by DXA at the hip and spine. Plasma vitamin B12 was measured by radioassay. Mean BMD measures were estimated for four categories of vitamin B12 concentration, based on commonly used cut-offs, using analysis of covariance, adjusted for age, BMI, physical activity score for the elderly (PASE), alcohol use, smoking status, total calcium and vitamin D intake, season of bone measurement, and for women, menopause status and current estrogen use. Further adjustment for protein intake and total homocysteine concentration was also performed.
Results: Both men and women with vitamin B12 concentrations <148 pM had lower average BMD than those with vitamin B12 above this cut-off. These differences were significant (p < 0.05) for men at most hip sites and for women at the spine. Significance remained after further adjustment for protein intake and plasma homocysteine.
Conclusions: Vitamin B12 deficiency may be an important modifiable risk factor for osteoporosis.
OSTEOPOROSIS AND related fractures represent major public health problems that are expected to increase dramatically as the population ages. It has been estimated that the lifetime risk of fracture exceeds 40% for women and 13% for men.(1) Among elders, hip fractures are associated with excess mortality of up to 20%, with costly long-term nursing home care for most survivors.(2) It is therefore of great importance to identify risk factors for osteoporosis to inform interventions that may reduce the likelihood of fracture. Dietary risk factors are particularly important, because they are modifiable. While calcium and vitamin D have received the most attention in relation to maintenance of BMD with aging, new research suggests that many nutrients may be important to bone.(3)
One nutrient thought to contribute to bone health, but which has been considered in only a few studies, is vitamin B12. This vitamin has been associated with osteoblast activity and bone formation,(4-6) and patients with pernicious anemia have been shown to have greater risk of fracture.(7,8) Recently, two population studies have reported associations between vitamin B12 status and BMD.(9,10) We therefore examined the association between plasma vitamin B12 and BMD at several sites, with data from >2500 men and women participating in the Framingham Osteoporosis Study.
MATERIALS AND METHODS
This study used data from participants in the Framingham Osteoporosis Study, drawn from the Framingham Offspring Cohort. The original population-based Framingham Heart Study began in 1948 to examine risk factors for heart disease. The original cohort included a two-thirds systematic sample of the households in Framingham, MA.(11) The offspring cohort, established in 1971, consists of the children (and their spouses) of the original cohort members. Offspring participants return every 4 years for a physical examination, where they also complete a series of questionnaires and tests. At the sixth examination cycle (1996-1999), there were 3532 participants (1657 men and 1875 women, 30-87 years of age). BMD measurement was conducted from 1996 to 2001 during the end of the sixth and beginning of the seventh examination cycles. A total of 1123 men and 1453 women who provided a blood sample for vitamin B12 analysis during cycle 6 also had BMD measurements. This study was approved by the Institutional Review Board for Human Research at Boston University and the Institutional Review Board at the Hebrew Rehabilitation Center for Aged (Boston, MA, USA). Written informed consent was obtained from all participants.
BMD was measured using DXA (Lunar DPX-L; Lunar Radiation, Madison, WI, USA) at the right hip (total hip, trochanter, Ward's area, and femoral neck) and at the lumbar spine (L2-L4). The precision (CV) was 1.7% at the femoral neck, 2.5% at the trochanter, and 0.9% at the spine, similar to the range of 1.8-1.9 reported by others.(12,13)
Plasma vitamin B12
Blood had been drawn from subjects at the sixth examination cycle and stored at −80°C. Plasma vitamin B12 concentrations were measured using the BioRad Quantaphase II radioassay (Hercules, CA, USA). Pooled plasma was used for quality control. The CV for this assay in our laboratory is 7%. There is currently no clearly accepted cut-off for vitamin B12 deficiency. A commonly used clinical cut-off for vitamin B12 deficiency is 148 pM (250 pg/ml).(14) However, there is evidence that the sensitivity of this clinical cut-off is poor and that many individuals with concentrations previously labeled as “low normal” do have clinical symptoms.(15,16) We therefore also created a group between 148 and 185 pM (250 pg/ml) as a group at high risk of vitamin B12 deficiency, a group between 185 and 258 pM (350 pg/ml) as a group at moderate risk of deficiency, and >258 pM, a cut-off generally considered to be indicative of vitamin B12 adequacy.(15)
Measurement of confounders
Factors known to affect BMD were considered as covariates. Specifically, these included body mass index (BMI), height, physical activity, alcohol use,(17) smoking,(18) dietary intake of calcium and vitamin D and use of supplements containing calcium or vitamin D,(19) season of measurement,(19,20) osteoporosis medication use, and estrogen use by women.(21) Because protein intake and high homocysteine have recently been identified as possible risk factors for osteoporosis,(22,23) we included these measures in a second set of models.
Usual dietary intakes of calcium, vitamin D, protein, and total energy were assessed with a semiquantitative 126-item food frequency questionnaire.(24,25) Questionnaires were mailed to the subjects before the sixth examination, and they were asked to complete them and bring them to the exam. The questionnaire also included questions about use of vitamin and mineral supplements, allowing for the calculation of total nutrient intakes. We created variables for calcium and vitamin D supplement use based on average daily intakes from supplements. We created categorical variables for calcium and for vitamin D supplement use. These were coded as 1 for use up to the amount that may be found in a single multivitamin/mineral supplement (250 mg for calcium and 15 μg for vitamin D) and 2 for higher intakes; nonusers comprised the referent group. This food frequency questionnaire has been validated for many nutrients, and in several populations, against multiple diet records and/or blood measures.(24-27) Questionnaires resulting in energy intakes <600 and >4000 kcal (2.51-16.74 MJ)/day or with more than 12 food items left blank were considered invalid and excluded from further analysis.
The questionnaire also asked subjects to quantify their usual intake of liquor, wine, and beer. From this information, total grams of alcohol consumed per week were estimated. We created a variable where subjects were classified as nondrinkers, moderate drinkers (based on the current recommendations for moderate intake - up to one drink per day for women and two drinks per day for men), or heavy drinkers (if intakes were greater than these cut-offs). Smoking was defined as current smoker, past smoker, or nonsmoker, based on questionnaire responses. Physical activity was measured with the physical activity score for the elderly (PASE) physical activity scale for the elderly.(28,29) Use of medications for osteoporosis was treated as a dichotomous (yes/no) variable and include bisphosphonates, selective estrogen receptor modulators, and calcitonin. Because previous research has shown that there are seasonal changes in BMD in New England, we created a categorical variable for season of BMD measurement.(19,20) July, August, and September were coded as summer; October, November, and December as fall; January, February, and March as winter; and April, May, and June as spring. Total homocysteine was measured using high-performance liquid chromatography with fluorometric detection.(30)
For women, estrogen use was defined as continuous use for at least 2 years versus never or past users, based on evidence that past use does not sustain bone benefits.(21) Postmenopausal status was defined as women reporting no menstrual period during the preceding year (with no pregnancy) or those >55 years of age. We included two indicator variables - women who were menopausal but were not using estrogen and women who were menopausal and were using estrogen. Both of these were compared with premenopausal women.
All statistical analyses were performed using SAS for Windows (version 8.2; SAS Institute, Cary, NC, USA). Measures of BMD at the hip and spine were regressed on plasma vitamin B12 concentrations separately for men and women, adjusting for a full set of potential confounders using the regression procedure in SAS. These variables included age, BMI, PASE, alcohol use, smoking status, calcium intake from diet, vitamin D intake from diet, energy intake, use of calcium supplement (none, ≤250 mg, >250 mg), use of vitamin D supplement (none, ≤15 μg, >15 μg), use of osteoporosis medications, season of bone measurement, and for women, menopause status and current estrogen use. For visual presentation and to assess the form of the relationship, we also created vitamin B12 groups for plasma vitamin B12 concentrations. Bone measures were each regressed on these vitamin B12 categories along with the set of confounding variables using the general linear models covariance procedure in SAS to obtain least squares means by vitamin B12 status category.
Finally, because we have previously found that greater protein intake was associated with BMD,(22) elevated homocysteine was associated with fracture risk,(23) and both of these are associated with vitamin B12, we repeated all models with the inclusion of total protein intake and plasma homocysteine concentration.
Mean values (±SD) for all continuous independent variables and potential confounders used in these analyses are presented in Table 1. For categorical variables, percentages are presented by category. Vitamin B12 status was poor for many subjects in this Framingham Offspring sample (Table 2), and in general, those with higher plasma vitamin B12 had greater intakes of vitamin B12. It is noteworthy that mean intakes, even in the lowest plasma vitamin B12 group, were considerably greater than the current recommended daily allowance (RDA) of 2.4 μg. More than 4% had plasma concentrations below the widely used clinical cut-off of 148 pM. Importantly, >11% of women and 13% of men had plasma vitamin B12 <185 pM, suggesting marginal deficiency, and 41% of men and 37% of women had concentrations <259 pM, the lower cut-off generally considered to be adequate for most individuals.
Table Table 1.. Subject Characteristics (Mean ± SD or %)
Table Table 2.. Distribution of Plasma Vitamin B12 Concentrations and Associated Vitamin B12 Intake
After completing both linear and categorized regression models, it was evident that there was a nonlinear association between vitamin B12 status and BMD. Therefore, we present only the categorical results. There were significant differences in BMD for men at each of the hip sites and for total hip (Table 3). Men with vitamin B12 >259 pM had mean BMD at Ward's area that was >7% greater than those with vitamin B12 concentrations ≤148 pM. Differences between the highest and lowest vitamin B12 category at other hip sites ranged from 4.6% to 6.1%, and significance levels for these comparisons reached p < 0.01 at the femoral neck and for the total hip. Although similar in pattern, differences in spine BMD did not reach statistical significance in men.
Table Table 3.. Adjusted Mean BMD by Plasma Vitamin B12 Concentration in Men*
For women, similar patterns were seen, but differences were greater at the spine than at the hip (Table 4). Women with vitamin B12 concentrations >185 pM had spine BMD that was significantly (p < 0.01) greater than those in the lowest vitamin B12 category, reaching a 6.6% difference between the highest and lowest categories. Differences approached (p < 0.1), but did not achieve, significance at all of the hip sites except the femoral neck. Differences between the highest and lowest vitamin B12 categories ranged from 2.1% to 5.0%. The similarity in patterns for men and women, despite differences in significance, are shown in Figs. 1 and 2 for total hip and spine BMD, respectively.
Table Table 4.. Adjusted Mean BMD by Plasma Vitamin B12 Concentration in Women*
Further adjustment for dietary protein intake and plasma homocysteine slightly attenuated differences, but did not change any statistically significant (p < 0.05) results. Because of the clear nonlinear association, we also repeated analyses with a dichotomous vitamin B12 variable: <148 pM versus ≥148 pM. In these models, three of the four hip sites were significant in men: the femoral neck at p < 0.01 and Ward's area and total BMD at p < 0.05, whereas the trochanter approached significance (p < 0.1). In women, the spine was significant at p < 0.01 and Ward's area at p < 0.05, and the trochanter and total BMD measures approached significance at p < 0.1. Least squares mean BMDs were up to 6.2% (spine) higher in women and 5.6% (femoral neck) higher in men for those with vitamin B12 concentration ≥148 versus <148 pM.
To our knowledge, this is the first large population-based cohort to show an association between low vitamin B12 and low BMD in men and to confirm other reports of this association in women. The consistency of pattern across bone sites for both men and women, and after adjustment for multiple potential confounding variables, supports the likelihood that vitamin B12 influences BMD. In all cases, those with vitamin B12 <148 pM had lower average BMD that did those with higher vitamin B12 status. For men, this was significantly lower than those in the highest two groups (>185 pM) at most hip sites and for women at the spine. This significance remained after adjusting for a variety of confounders including age, height, BMI, smoking, alcohol use, physical activity, total energy intake, calcium intake, vitamin D intake, use of a calcium or vitamin D supplement, season of measurement, and for women, menopausal status and estrogen use. Furthermore, the results did not change with further adjustment for intake of protein and or plasma homocysteine concentration.
Although clinical associations between vitamin B12 deficiency and fracture have been noted in patients with pernicious anemia,(7,8) this association has not been thoroughly examined. With the exception of two recent small studies, one in a sample of 111 elderly women and 49 elderly men in the Netherlands(9) and one in 83 elderly women,(10) we know of no other population-based studies that have shown this association in community-dwelling adults. The Dutch study(9) found an association between vitamin B12 and BMD in women but not in men. Women with vitamin B12 concentrations <210 pM were 6.9 times more likely to have osteoporosis relative to those with vitamin B12 >320 pM after adjusting for age, weight, and calcium intake. Similarly, results from a subset of 83 women in the Study of Osteoporotic Fractures showed that women with vitamin B12 <207 pM had significantly greater loss in total hip BMD (but not of calcaneal BMD) relative to women with higher concentrations (annual change of −1.6% versus −0.2% over 3.5 years) after adjustment for age, weight, and clinic site. We identified associations between vitamin B12 concentration and BMD in a much larger sample of both men and women at several bone sites.
Although vitamin B12 deficiency may have direct effects on bone metabolism, few studies have been done to clarify the underlying mechanisms. As a required co-factor in methionine synthase reactions, inadequate vitamin B12 concentrations may affect DNA synthesis through the “methyl-folate trap.” Tetrahydrofolate is necessary for the synthesis of thymidine and the purines, whereas methionine synthase is critical for the regeneration of tetrahydrofolate from methyltetrahydrofolate. When vitamin B12 levels are inadequate, folate becomes trapped as methyltetrahydrofolate, limiting the availability of folate for DNA synthesis. As such, it is likely to be an important factor in bone remodeling, but because few studies have examined this directly, more work is needed to confirm this potential mechanism.(31)
Vitamin B12 deficiency has been associated with low levels of markers of bone formation, including serum skeletal alkaline phosphatase and osteocalcin.(4,5) A cell culture study tested reactions of human bone marrow stromal osteoprogenitor cells (jBMSC) and UMR106 osteoblastic cells with differing vitamin B12 concentrations. In the setting of low vitamin B12 concentrations, [3H]thymidine incorporation into cells was significantly reduced, and alkaline phosphatase activity was significantly lower in both cell types, suggesting that low vitamin B12 may suppress osteoblastic activity.(6) Interestingly, this study showed clear threshold effects of vitamin B12 concentration on this activity, as we did on BMD.
Clinical studies have shown that vitamin B12-deficient patients have a higher risk of fracture.(7,8) Goerss et al.(8) found that 131 patients diagnosed with pernicious anemia from 1950 to 1979 were 1.9 times more likely to have a fracture in the proximal femur, 1.8 times more likely to have a vertebral fracture, and 2.9 times more likely to have a distal forearm fracture, relative to the general community. Finally, vitamin B12 treatment improved BMD in a case report of a patient with pernicious anemia.(32) Together, these pieces of evidence converge to suggest the likely importance of vitamin B12 to bone formation and therefore maintenance of BMD with aging.
Most recently, two studies confirmed associations between elevated homocysteine concentration and fracture risk.(23,33) The most common cause of homocysteine elevation is poor B vitamin status, most notably of folate and vitamin B12. It is therefore possible that the observed homocysteine effects are in fact caused by vitamin deficiency rather than to the homocysteine per se. Because these are in a causal pathway, it is difficult to separate the effects of B vitamins and homocysteine with epidemiologic data. However, when we included homocysteine in the model with vitamin B12, the latter remained significant, although slightly attenuated, whereas the former did not, suggesting an independent contribution of vitamin B12 deficiency.
Vitamin B12 deficiency or marginal status is prevalent in older adults,(34-38) with estimates of deficiency ranging from 5.3%(34) to 24%.(36) Of subjects with BMD measures in this study, 4.5% had plasma vitamin B12 <148 pM and 12% had <185 pM. In the larger cohort, we previously reported that 17% had vitamin B12 below the latter cut-off,(37) suggesting that those who did not get their BMD measurements had poorer nutritional status than those included here.
Loss of stomach acidity with age, resulting over time in type B atrophic gastritis, has been implicated in impaired B12 status.(39) This condition, which has been estimated to affect up to 40% of older adults, is associated with impaired absorption of protein-bound vitamin B12, but unbound vitamin B12, as found in supplements, is better absorbed.(40) It is also possible that the increased use of acid blockers may contribute to the development of vitamin B12 deficiency.(41) Therefore, it is important for older adults with these conditions to obtain adequate quantities of vitamin B12 from supplements or fortified foods.(42)
This report, along with several previous clinical observations and two recent population-based studies,(9,10) suggest that vitamin B12 status is important for maintenance of BMD. Because low vitamin B12 status is prevalent and easily preventable, more attention should be given to this vitamin in the treatment and prevention of osteoporosis.
This study was supported by USDA Contract 53-3K06-5-10, NIH R01 AR/AG 41398, and NIH/NHLBI Contract N01-HC-25195.