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The Journal of Clinical Endocrinology & Metabolism 89(3):1217–1221 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-030074

Low Serum Vitamin B-12 Levels Are Associated with Increased Hip Bone Loss in Older Women: A Prospective Study KATIE L. STONE, DOUGLAS C. BAUER, DEBORAH SELLMEYER,

AND

STEVEN R. CUMMINGS

Departments of Epidemiology and Biostatistics (D.C.B., S.R.C.) and Medicine (K.L.S., D.C.B.) and Division of Endocrinology and Metabolism (D.S.), University of California, San Francisco, California 94105 The purpose of this study was to test whether low serum vitamin B-12 levels are associated with more rapid bone loss in elderly women. We archived sera and measured calcaneal bone mineral density (BMD) in community-dwelling white women, aged 65 yr and over, who participated in the Study of Osteoporotic Fractures. BMD of the hip and subregions was measured 2 yr later. Repeat measurements of calcaneal and hip BMD were obtained after 5.9 and 3.5 yr of follow-up, respectively. Serum vitamin B-12 assays were performed in 83 randomly selected participants with initial and repeat measurements of BMD who were not taking estrogen replacement therapy at baseline. After adjusting for age, weight, and clinic

site, women with vitamin B-12 levels at or below 280 pg/ml (207.2 pmol/liter; lowest quintile) experienced an annual change of ⴚ1.6% (95% confidence interval, ⴚ2.4% to ⴚ0.8%) in total hip BMD, compared with ⴚ0.2% (ⴚ0.5% to 0.2%) in women with levels above 280 pg/ml (P ⴝ 0.003). Results were similar when subregions of the hip were analyzed separately. Serum vitamin B-12 levels were not significantly associated with calcaneal bone loss. We conclude that low serum vitamin B-12 levels are associated with increased rates of hip, but not calcaneal, bone loss in older women. (J Clin Endocrinol Metab 89: 1217–1221, 2004)

A

Subjects and Methods Experimental subjects

LTHOUGH VITAMIN B-12 is known to influence the hemopoeitic and nervous systems (1), little is known about the skeletal effects of vitamin B-12. Research concerning the association between serum vitamin B-12 levels and the risk of osteoporosis has been limited; a few crosssectional studies have shown low serum vitamin B-12 levels to be associated with decreased levels of markers of bone formation, such as serum alkaline phosphatase and osteocalcin (1, 2). Furthermore, Kim et al. (3) demonstrated an association between serum vitamin B-12 concentration and [3H]thymidine incorporation into human bone marrow stromal osteoprogenitor cells and UMR106 osteoblastic cells, suggesting a direct effect of vitamin B-12 on osteoblast function. A retrospective cohort study among postmenopausal women in Rochester, Minnesota, reported an almost 2-fold increase in the risk of hip and spine fractures and almost 3 times the risk of wrist fractures in women diagnosed with pernicious anemia compared with normal controls (4). There have been no prospective studies of the relationship between serum vitamin B-12 levels and rates of bone loss or fracture. To test the hypothesis that elderly women with lower vitamin B-12 levels experience more rapid bone loss, we measured serum vitamin B-12 levels in stored sera and determined rates of hip and calcaneal bone loss over 3.5 and 5.9 yr of follow-up, respectively, in a randomly selected cohort of older women.

Abbreviations: BAP, Bone-specific alkaline phosphatase; BMD, bone mineral density; 25(OH)D, 25-hydroxyvitamin D. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Subjects were participants in the Study of Osteoporotic Fractures, the details of which have been previously described (5). During 1986 –1988 (baseline), a total of 9704 community-dwelling white women ages 65 yr and above were recruited from population-based listings at four clinical centers in the United States: Baltimore, MD; Minneapolis, MN; Portland, OR; and the Monongahela Valley near Pittsburgh, PA. As part of a larger case-cohort study, we randomly selected 119 subjects (i.e. the random cohort sample), without restrictions, from among those who attended the baseline visit (1986 –1988) and provided a serum sample at that time. Among the original random sample, there were 114 women who were nonestrogen users and had calcaneal bone mineral density (BMD) measured at the baseline examination. Our final sample for the calcaneal bone loss analysis was further restricted to 83 women (90% of the 92 women returning for follow-up BMD measures in 1993–1994) who had technically adequate calcaneal BMD measures at both time points. Similarly, of 98 nonestrogen users who had hip BMD measured at the 2 yr examination in 1990, 81 women returned for follow-up hip BMD measures in 1993–1994, and 77 women (95%) had technically adequate hip BMD measurements at both time points for inclusion in the analysis. The study protocols were approved by the institutional review boards of the participating centers, and all study subjects provided appropriate written informed consent to participate. At the baseline visit in 1986 –1988, a detailed questionnaire was administered in which subjects were asked about current or previous use of estrogen, use of multivitamin supplements containing vitamin D, and history of medical conditions and procedures, such as gastric surgery. Participants were also asked about personal habits, such as smoking and alcohol consumption, and current physical activity was assessed using the Paffenbarger questionnaire (6). A 23-item food frequency questionnaire (Block Dietary Data Systems, Berkeley, CA) was administered to ascertain dietary intakes of calcium and protein. Subjects were examined to obtain height and weight measurements. Calcaneal bone mass was measured using single photon absorptiometry (OsteoAnalyzer, Siemens-Osteon, Wahiawa, HI) at baseline and at a follow-up visit in 1993–1994, after an average of 5.9 yr. Bone density of the hip and its subregions was measured using dual x-ray absorptiometry (QDR 1000, Hologic, Inc., Waltham, MA) at a second visit in

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1988 –1990 among 82% of the original cohort who survived. Repeat hip bone density was measured at follow-up in 1993–1994, after an average of 3.5 yr. The mean coefficient of variation of 1.2% between centers for both the calcaneus and femoral neck was estimated using multiple measurements for two staff members who visited each site. Spine bone density measurements were not performed on a sufficient number of subjects (n ⫽ 4) to warrant inclusion in this study. Further details of these bone densitometry measurements were described previously (7).

Serum samples At the baseline examination, blood was drawn between 0800 –1400 h, and serum was stored at ⫺20 C. Within 2 wk, samples were shipped to Biomedical Research Institute (Rockville, MD) and stored in liquid nitrogen at ⫺190 C. In 1994, sera for selected participants were thawed and assayed for levels of vitamin B-12, bone-specific alkaline phosphatase (BAP), osteocalcin, albumin, serum creatinine, and calcium. Laboratories performing the assays were blinded to the participant’s fracture status.

Biochemical assays Serum vitamin B-12 levels were measured using a microparticle enzyme intrinsic factor assay (Endocrine Sciences, Calabasas, CA). The sensitivity of this assay is 60 pg/ml. Interassay coefficients of variation range from 3.9 – 4.5% and intraassay coefficients of variation range from 6.4 – 8.5%. Serum total osteocalcin was measured with a human-specific immunoradiometric assay (ELSA-OSTEO, Cis Biointernational, Baglos/Ceze, France), which recognizes a large N-terminal midfragment in addition to the intact molecule (8). Serum BAP was measured with an immunoradiometric assay using two monoclonal antibodies directed against the human bone isoenzyme and BAP purified from human SAOS-2 osteosarcoma cells as a standard (Ostase, Hybritech, Inc., San Diego, CA). This assay has a 16% crossreactivity with the circulating liver isoenzyme (9). Serum calcium, 25-hydroxyvitamin D [25(OH)D], PTH, and creatinine assays were performed by the Calciotropic Hormone Reference Laboratory (University of California, San Francisco, CA). Serum creatinine and calcium were measured using an automated chemistry analyzer. Serum intact PTH was assayed in duplicate using a two-site immunoradiometric assay, with intraassay variation of 5.2% and interassay variation of 8.5%. Serum 25(OH)D was assayed in duplicate using a competitive protein binding assay after purification by methanol extraction and chromatography. The intraassay variation was 10%, and the interassay variation was 15%. Serum albumin was measured by serum protein electrophoresis using a commercially available kit (Beckman Instruments, Brea, CA).

Statistical analyses Baseline characteristics of the 83 women included in this substudy (Table 1) were compared with those of 5163 participants who had baseline and repeat measurements of calcaneal BMD and did not report current use of estrogen replacement therapy at baseline, using t tests for continuous variables and ␹2 tests for dichotomous variables. The least squared mean annual percent changes in BMD for the calcaneus and for the hip and subregions were calculated by quintiles of vitamin B-12 levels, adjusting for age, weight, and clinic site in linear regression models. Models using annual absolute change in BMD for all sites instead of annual percent change produced similar results and are not presented. Data were divided into two groups according to vitamin B-12 levels, low levels being defined as those in the lowest quintile (ⱕ280 pg/ml; 207.2 pmol/liter) and normal levels as those in the upper four quintiles (⬎280 pg/ml). The sample size was insufficient to analyze participants with vitamin B-12 levels more commonly recognized as clinically low (⬍200 pg/ml; n ⫽ 7) as a separate group. However, a study by Lindenbaum et al. (10) has shown that many elderly individuals with tissue deficiency of vitamin B-12 have serum B-12 levels in the low normal range (200 –300 pg/ml). Women with low vitamin B-12 levels were compared with those with normal vitamin B-12 levels with respect to a variety of potential con-

Stone et al. • Serum Vitamin B-12 and Bone Loss

TABLE 1. Characteristics of participants Characteristic

Bone loss cohort, biochemical samplea

No. 83 Age (yr) 71.1 (4.4)c Weight (kg) 68.0 (11.2) 26.5 (4.4) Body mass index (kg/m2) Gastric surgery [no. (%)] 2 (2.4) Current multivitamin use 36 (43.9) [no. (%)] Self-rated health excellent/ 75 (90.4) good vs. others [no. (%)] Physical activity (kcal/wk) 1621 (1457) Dietary protein intake (g/d) 51.9 (17.4) Total daily calcium intake 1075 (592) (mg/d) Current use of alcohol [no. (%)] 60 (72.3) Current cigarette smoker 5 (6.0) [no. (%)] Serum albumin (g/dl) 3.62 (0.41) Serum osteocalcin (ng/ml) 25.5 (10.9) Serum alkaline phosphatase 12.6 (5.4) (ng/ml) Serum calcium (mg/dl) 9.73 (0.45) Creatinine clearance (ml/min) 66.6 (15.9) Serum vitamin B-12 (pg/ml) 477 (236) Calcaneal BMD (g/cm2) 0.419 (0.085) 2 Total hip BMD (g/cm ) 0.774 (0.120) Femoral neck BMD (g/cm2) 0.669 (0.120) Calcaneal bone loss (%/yr) ⫺1.36 (1.76) Total hip bone loss (%/yr) ⫺0.41 (1.66) Femoral neck bone loss (%/yr) ⫺0.18 (1.79)

Total bone loss cohortb

5163 70.9 (4.8) 67.6 (12.3) 26.6 (4.6) 65 (1.3) 2250 (44.3) 4480 (86.8) 1705 (1662) 50.9 (18.9) 1083 (732) 3722 (72.1) 453 (8.8)

– – – 0.403 (0.089) 0.755 (0.125) 0.648 (0.105) d ⫺1.55 (1.57) ⫺0.60 (1.45) ⫺0.53 (1.79) d

a Data presented in this column are for the calcaneal bone loss cohort. Statistics for the hip bone loss cohort are similar and are not presented. b The total bone loss cohort that is used as the comparison group consists of all women in the cohort who were nonestrogen users and who provided both initial and repeat measures of calcaneal BMD that were technically adequate. c Mean (SD), unless otherwise indicated. d P ⬍ 0.10, comparison between B-12 bone loss group and total bone loss cohort.

founders or explanatory variables, such as self-rated health status; dietary protein intake; serum albumin, creatinine, osteocalcin, and BAP; and baseline BMD. Comparisons between the two vitamin B-12 groups were made using t tests for continuous variables and ␹2 tests for dichotomous variables. All comparisons (except age and weight) were repeated, controlling for age, weight, and clinic site, using multiple linear regression analysis for continuous variables and logistic regression for dichotomous variables. Variables that differed between low and normal vitamin B-12 groups with P ⬍ 0.2 in either unadjusted or multiple adjusted comparisons were included in multivariate regression analyses of the association between vitamin B-12 levels (independent variable) and change in bone density (dependent variable) along with age, weight, and clinic site.

Results

The characteristics of the 83 participants in the vitamin B-12 bone loss subcohort were similar to those of the target population of 5163 participants in the overall cohort from which the sample was drawn (i.e. those who were nonestrogen users and had technically adequate measures of calcaneal BMD at both initial and follow-up visits). The exceptions were that women in the subsample had higher baseline femoral neck BMD and experienced significantly less femoral neck bone loss than those in the larger cohort from which the

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Stone et al. • Serum Vitamin B-12 and Bone Loss

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sample was drawn. In relation to the total study cohort recruited during the baseline examination, our sample is younger, is more physically active, and reported better health and greater use of multivitamin supplements. This was expected given that inclusion in our study required that participants survive and be sufficiently healthy to attend a follow-up visit several years after baseline. The average age of participants in our subsample was about 71 yr, and the mean weight was approximately 68 kg. Women with low vitamin B-12 levels (ⱕ280 pg/ml; n ⫽ 16) had lower creatinine clearance rates than women with higher levels (mean, 59.7 and 68.3 ml/min in low and normal B-12 groups, respectively; P ⫽ 0.05; Table 2), although this difference was no longer significant after controlling for age, weight, and clinic site (P ⫽ 0.12). Current use of multivitamins was more common among participants with normal vitamin B-12 levels (n ⫽ 33; 50.0%) compared with those with low B-12 levels (n ⫽ 3; 18.8%; P for comparison ⫽ 0.02), and this difference was unchanged by adjustment for age, weight, and clinic site. Among those reporting multivitamin use at baseline, the mean duration of use was approximately 1 yr among those with low B-12 levels compared with more than 12 yr in those with normal serum B-12 levels (data not TABLE 2. Descriptive data by levels of serum vitamin B-12 Characteristic

No. (%) Age (yr) Weight (kg) Body mass index (kg/m2) Gastric surgery [no. (%)] Current multivitamin use [no. (%)] Self-rated health excellent/ good vs. others [no. (%)] Physical activity (kcal/wk) Dietary protein intake (g/d) Total daily calcium intake (mg/d) Current use of alcohol [no. (%)] Current cigarette smoker [no. (%)] Serum albumin (g/dl) Serum osteocalcin (ng/ml) Serum BAP (ng/ml) Serum calcium (mg/dl) Creatinine clearance (ml/min) Serum PTH (pg/ml) Serum 25(OH)D (ng/dl) Calcaneal BMD (g/cm2) Total hip BMD (g/cm2) Femoral neck BMD (g/cm2) Calcaneal bone loss (%/yr) Total hip bone loss (%/yr) Femoral neck bone loss (%/yr)

Low B-12 group (ⱕ280 pg/ml)a

Normal B-12 group (⬎280 pg/ml)

16 (19.3)b 72.4 (4.3) 65.4 (10.9) 26.1 (4.3) 2 (12.5) 3 (18.8)

67 (80.7) 70.9 (4.4) 68.6 (11.2) 26.5 (4.4) 0 (0.0)d 33 (50.0)c

15 (93.8)

60 (89.6)

1507 (1382) 49.6 (15.9) 973 (626)

1648 (1483) 52.4 (17.8) 1101 (585)

9 (56.3)

51 (76.1)

1 (6.3)

4 (6.0)

3.62 (0.31) 27.0 (8.9) 11.9 (6.2) 9.64 (0.39) 59.6 (14.8)

3.62 (0.43) 25.1 (11.4) 12.7 (5.3) 9.75 (0.46) 68.3 (15.8)c

38.6 (17.6) 18.4 (6.4) 0.420 (0.089) 0.801 (0.128) 0.669 (0.107) ⫺1.49 (1.61) ⫺1.91 (2.32) ⫺0.95 (2.40)

36.3 (24.0) 28.3 (10.7)d 0.419 (0.085) 0.768 (0.118) 0.670 (0.123) ⫺1.32 (1.81) ⫺0.10 (1.31)c ⫺0.02 (1.62)

Data presented in this table are for the calcaneal bone loss cohort. Statistics for the hip bone loss cohort are similar and are not presented. a Corresponds to the lowest quintile of vitamin B-12 levels. b Mean (SD) unless otherwise indicated. c P ⬍ 0.05, comparison of low vs. normal vitamin B-12 groups. d P ⬍ 0.01, comparison of low vs. normal vitamin B-12 groups.

shown). Among those with normal vitamin B-12 levels, there were no participants who reported a history of gastric surgery, whereas two women in the low vitamin B-12 group reported having had gastric surgery. However, exclusion from the analysis of women reporting gastric surgery did not alter the results. We found no significant difference in levels of osteocalcin or BAP between women with low and normal serum vitamin B-12 levels. Although PTH levels were similar among those with low and normal vitamin B-12 levels, 25(OH)D levels were lower among those with low vitamin B-12 levels compared with those with normal levels of B-12. There was a significant trend in rate of change in total hip BMD across quintiles of serum vitamin B-12 levels after adjustment for age, weight, and clinic site (P ⫽ 0.04; Table 3), although this trend was no longer statistically significant after adjustment for current use of multivitamin supplements and creatinine clearance. After adjusting for age, weight, and clinic site, women serum B-12 levels of 280 pg/ml or less experienced more rapid declines in total hip BMD (mean change, ⫺1.6%; ⫺2.4% to ⫺0.8%) compared with those with levels above 280 pg/ml (⫺0.2%; ⫺0.5% to 0.2%; P for comparison ⬍ 0.003). The difference in rates of total hip bone loss between the low and normal vitamin B-12 groups was attenuated by adjustment for current use of multivitamin supplements; the estimated difference was reduced from 1.4% to 1.2% (0.3% to 2.2%; P ⫽ 0.01). The magnitude of the difference was unchanged after further adjustment for creatinine clearance, serum PTH, and serum 25(OH)D (Table 4). Although there was a similar significant trend in rates of change in femoral neck BMD across quintiles of serum vitamin B-12 levels after adjustment for age, weight, and clinical center (women with low vitamin B-12 levels experienced more rapid femoral neck bone loss; P for trend ⫽ 0.03; Table 3), this trend was no longer significant after further adjustment for current use of multivitamin supplements. The rate of change in femoral neck BMD between those with low serum vitamin B-12 (⫺0.8%/year; 95% confidence interval, ⫺1.8% to 0.2%) was not statistically different from women with higher serum vitamin B-12 levels (⫺0.1%/yr; ⫺0.5% to 0.4%; P for difference ⫽ 0.20). Results were similar after further adjustment for 25(OH)D, creatinine clearance, and PTH. Serum levels of vitamin B-12 were not associated with rates of change in calcaneal BMD in this cohort (P for trend ⫽ 0.82; Table 3). We found no association between B-12 levels and crosssectional BMD at either the hip or the calcaneus. This was true whether BMD was analyzed as a test for trend across quintiles of B-12 levels or comparing those with low B-12 to others. Discussion

We found that women with lower serum vitamin B-12 levels experience more rapid bone loss from the hip than women with higher levels of vitamin B-12. After adjusting for multiple factors, including age, weight, and clinic site, participants with serum vitamin B-12 levels of 280 pg/ml (207.2 pmol/liter) or less experienced an average rate of total hip

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Stone et al. • Serum Vitamin B-12 and Bone Loss

TABLE 3. Annual average percent change in BMD by quintiles of serum vitamin B-12 Serum vitamin B-12 (pg/ml) quintiles

a

BMD

1st quintile (114 –280a; n ⫽ 16)

2nd quintile (281–380; n ⫽ 17)

3rd quintile (381–511; n ⫽ 16)

4th quintile (512– 615; n ⫽ 17)

5th quintile (616 –1415; n ⫽ 17)

P for trend

Total hip Femoral neck Calcaneal

⫺1.59 ⫺0.76 ⫺1.43

⫺0.08 ⫺0.40 ⫺1.37

⫺0.25 ⫺0.70 ⫺1.24

⫺0.27 0.25 ⫺1.61

⫺0.07 0.54 ⫺1.13

0.04 0.03 0.82

Range.

TABLE 4. Multivariate linear regression modela comparing rates of change in total hip BMD among women with low and normal vitamin B-12 levels Variable

Difference in % change BMD/yr

P value

Vitamin B-12 level (ⱕ280 pg/ml vs. ⬎280 pg/ml) Age (per 5-yr increase) Weight (per SD, 11.2 kg increase) Creatinine clearance (per SD, 15.9 ml/min increase) PTH (per SD, 22.9 pg/ml increase) 25(OH)D (per SD, 10.7 ng/dl increase) Current multivitamin supplement use (yes vs. no)

⫺1.25 (⫺2.25; ⫺0.25) ⫺0.61 (⫺1.03; ⫺0.19) 0.37 (⫺0.18; 0.91) ⫺0.28 (⫺0.86; 0.30) 0.01 (⫺0.34; 0.35) ⫺0.01 (⫺0.41; 0.39) 0.68 (⫺0.08, 1.44)

0.02 0.006 0.20 0.34 0.97 0.97 0.08

a Clinical center is also included as a covariate in the model but coefficients (modeled using three dummy variables) are not presented. Differences in rates of total hip bone loss between clinical centers were not statistically significant in this model.

bone loss of 1.6% (0.2–2.8%) annually compared with a loss rate of 0.2% (⫺0.2% to 0.5%) among women with levels above 280 pg/ml. This difference remained significant after further adjustment for multivitamin supplements, creatinine clearance, and serum levels of PTH and 25(OH)D. Although vitamin B-12 deficiency is rare in young healthy people, the prevalence of vitamin B-12 deficiency is more common in the elderly (10 –14). Possible causes of vitamin B-12 deficiency are several, including absorptive disorders such as inability to manufacture intrinsic factor or absorptive problems resulting from gastric surgery (14). Several studies have also shown that dietary deficiency may also be a significant contributor to low serum levels (15, 16). The results of our study suggest the possibility that, at least for some older women, simple dietary assessment and modification or supplementation may prove effective in slowing rates of bone loss and potentially reducing rates of hip and other osteoporotic fractures. A randomized trial would be needed to determine whether treatment with supplemental vitamin B-12, either transdermally or orally, may reduce rates of bone loss in elderly women. Our bone loss results of this study are consistent with recently presented cross-sectional findings from the Framingham Study indicating that low vitamin B-12 is associated with bone density (17), although we were unable to detect an association between B-12 levels and BMD crosssectionally. Our findings also support the results of previous studies comparing postmenopausal women with pernicious anemia to normal controls; in a cross-sectional study based in Rochester, MN, women with pernicious anemia were found to have lower spine BMD but similar fractional calcium absorption levels compared with normal controls (4). A retrospective study in the same population of women found higher rates of fracture among those with pernicious anemia compared with normal controls (4). We found no relationship between serum vitamin B-12 levels and levels of osteocalcin or BAP, in contrast to early

work by Dutch researchers who found serum levels of total alkaline phosphatase to be lower among subjects with low serum vitamin B-12 levels compared with normal controls (2). More recently, Carmel et al. (1) demonstrated a similar association between serum vitamin B-12 levels and osteoblast-related proteins: levels of skeletal alkaline phosphatase and osteocalcin were lower in participants with low serum B-12 levels and rose with vitamin B-12 therapy in the majority of the participants. However, as our subjects were selected randomly from a study population consisting of healthy, community-dwelling, elderly women, there were few women with very low serum vitamin B-12 levels in comparison with these other studies in which subjects with pernicious anemia were identified for recruitment. This study has several strengths. It is a prospective study, measuring rates of change in bone mass rather than crosssectional bone mass measurements. Serum samples were obtained before measurement of change in bone mass. The sample size, although still relatively small, was considerably larger than in most other related studies, and the subjects were sampled from a cohort of elderly women living in four separate communities in the United States. This study also has limitations. As the study population was restricted to ambulatory, community-dwelling, white women aged 65 yr and above, the results may not be generalizable to other groups, such as men, younger women, other racial groups, or the institutionalized elderly. Furthermore, in comparison with the entire baseline cohort, our study sample is relatively healthy, given that they were required to have returned for repeat BMD assessment several years after the initial visit. Therefore, we expect that our sample has fewer subjects with B-12 deficiency than would be the case in a more representative population. However, we expect that this would have biased our estimates toward the null. The biochemical measurements were based on a single assay, and the change in bone mass is based on one pair of baseline and follow-up measurements. Variability in both the predictor and outcome

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Stone et al. • Serum Vitamin B-12 and Bone Loss

variables could lead this study to underestimate the strength of the associations we observed, particularly given the relatively small sample size and the few women with low B-12 levels. However, the relatively long follow-up for this study (5.9 and 3.5 yr for calcaneus and hip, respectively) would be expected to minimize the influence of precision error in rates of bone loss. Nonetheless, a larger study would be useful to confirm our findings and to increase the precision of the estimates. We did not have measurements of methylmalonic acid or homocysteine to confirm tissue deficiency of vitamin B-12; we would expect inclusion of these measurements to strengthen the observed association between B-12 levels and rates of bone loss. The first hip BMD measurement was not taken until about 2 yr after sera were collected. Again, this would tend to weaken the observed association. Because this is an observational study, participants were not selected on the basis of evidence of vitamin B-12 deficiency. Therefore, we had very few subjects with serum B-12 levels considered to be clinically low (e.g. ⬍200 pg/ml). However, it has been shown that many individuals with tissue deficiency of B-12 have serum B-12 levels in the low normal range (200 –300 pg/ml) (10). Finally, it is possible that other unmeasured dietary correlates of vitamin B-12 intake, such as zinc, calcium, vitamin D, animal protein, or other micronutrients in breakfast cereals, may account for the observed association between serum vitamin B-12 levels and rates of bone loss. In conclusion, we have found that lower levels of serum vitamin B-12 are associated with more rapid bone loss from the hip in elderly women. Our data also indicate that longterm use of multivitamins may be an effective means of reducing B-12 deficiency in older women. Further research is needed to determine the relationship between serum vitamin B-12 levels and fractures and to confirm whether this association is due to direct effects of vitamin B-12 or whether serum vitamin B-12 may be a biomarker for dietary intake or absorption of other important nutrients. Additional studies are also needed to validate these results, in particular given the discrepancy between the results for hip and calcaneus, for which we have no explanation at present. However, the results of this study raise the possibility that supplementation with vitamin B-12 or, for some elderly women, dietary assessment and modification may slow rates of bone loss. Acknowledgments Investigators in the Study of Osteoporotic Fractures Research Group, University of California, San Francisco (Coordinating Center): S. R. Cummings (principal investigator), M. C. Nevitt (coinvestigator), D. C. Bauer (coinvestigator), K. L. Stone (coinvestigator), D. M. Black (study statistician), H. K. Genant (director, central radiology laboratory), T. Blackwell, B. Blunt, M. Dockrell, S. Ewing, C. Fox, M. Jaime-Chavez, S. Litwack, L. Y. Lui, P. Mannen, L. Nusgarten, L. Palermo, M. Rahorst, C. Schambach, J. Schneider, and R. Scott; University of Maryland: M. Hochberg (principal investigator), L. Makell (project director), C. Boehm, L. Finazzo, R. Nichols, T. Page, S. Trusty, and B. Whitkop; University of Minnesota: K. Ensrud (principal investigator), M. Homan (coinvestigator), P. Bowman (project coordinator), S. Love (clinical research director), E. Mitson (clinic coordinator), C. Bird, D. Blanks, C. Burchkhardt, M.

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Cardenas, J. Holmes, F. Imker-Witte, K. Jacobson, K. Moen, N. Nelson, H. Peterson, and M. Slindee; University of Pittsburgh: J. A. Cauley (principal investigator), L. H. Kuller (coprincipal investigator), M. Vogt (coinvestigator), L. Harper (project director), L. Buck (clinic coordinator), C. Bashada, N. Chiarvalle, A. Githens, M. Gorecki, D. Lee, D. Medve, C. Newman, D. Stewart, and N. Watson; The Kaiser Permanente Center for Health Research (Portland, OR): T. Hillier (principal investigator), E. Harris (coprincipal investigator), E. Orwoll (coinvestigator), H. Nelson (coinvestigator), M. Aicken (coinvestigator), J. Van Marter (project administrator), M. Rix (clinic coordinator), K. Canova, T. Constantin-Suvalcu, R. Garza, P. Legarda, K. Pedula, K. Redden, J. Rehinhardt, J. Rizzo, K. Snider, and J. Wallace. Received January 22, 2003. Accepted November 24, 2003. Address all correspondence and requests for reprints to: Dr. Katie L. Stone, University of California, 74 New Montgomery Street, Suite 600, San Francisco, California 94105. E-mail: [email protected]. This work was supported by USPHS Grants AG-05407, AR-35582, AG-05394, AR-35584, and AR-35583.

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