Hormonal Predictors of Bone Loss in Elderly Women:A Prospective Study

KATIE STONE,1 DOUGLAS C. BAUER,1,2 DENNIS M. BLACK,1 PETER SKLARIN,3

KRISTINE E. ENSRUD,4 and STEVEN R. CUMMINGS1,2 FOR THE STUDY OF OSTEOPOROTICFRACTURES RESEARCH GROUP*

ABSTRACT

To test the hypotheses that baseline concentrations of sex steroids, sex hormone binding globulin (SHBG), andcalciotropic hormones predict rates of bone loss in elderly women, sera were stored at 2190°C, and calcaneal bonemineral density (BMD) was measured in 9704 community-dwelling white women aged 65 and over (1986–1988).Hip BMD was measured 2 years later (1990). Repeat measurements of calcaneal and hip BMD were obtained in1993–1994, after 5.7 and 3.5 years of follow-up, respectively. In 1994, sera were assayed for circulating hormonelevels in random subcohorts of 231 and 218 women who did not report current use of hormone replacementtherapy at baseline. Lower levels of endogenous estrogens and higher SHBG concentrations were associated withmore rapid subsequent bone loss from both the calcaneus and hip. After adjusting for age and weight, women withhigh SHBG levels (highest quartile > 2.3 mg/dl) experienced an average of 2.2% (95% confidence interval 5 1.6%,2.9%) calcaneal bone loss per year compared with 1.2% (0.7%, 1.2%) among women with low SHBG concentrations(lowest quartile < 1.1 mg/dl; p < 0.01). This association was independent of concentrations of other sex hormones.Women with estradiol levels > 10 pg/ml averaged only 0.1% (20.7%, 0.5%) annual hip bone loss while women withlevels below 5 pg/ml averaged 0.8% (0.3, 1.2) hip bone loss per year. Lower 25-hydroxyvitamin D levels wereassociated with increased hip but not calcaneal bone loss. Levels of parathyroid hormone, 1,25-dihydroxyvitaminD, and calcium were not significantly associated with bone loss from the calcaneus or hip. (J Bone Miner Res1998;13:1167–1174)

INTRODUCTION

HYPOTHESES REGARDING bone loss in elderly women havefocused primarily on age-related changes in parathy-

roid hormone (PTH) and other calciotropic hormones, re-sulting in part from decreased calcium absorption withage.(1,2) Although one small prospective study has shownlow levels of 25-hydroxyvitamin D (25(OH)D) to be asso-ciated with increased bone loss from the distal radius inelderly women,(3,4) in general results of studies concerningthe role of calciotropic hormones in osteoporosis amongelderly women have been inconclusive; research has been

limited primarily to cross-sectional studies of the associa-tion between levels of PTH, 25(OH)D, or 1,25-dihydroxyvi-tamin D (1,25(OH)2D), and bone density,(4–8) or to retro-spective studies comparing hormone levels among fracturecases and controls.(2,9,10)

Less emphasis has been placed on the roles of endoge-nous sex hormone concentrations and sex hormone bindingglobulin (SHBG) on bone loss in elderly women. While it isrecognized that the rapid loss of bone in women during andimmediately following menopause is related to declininglevels of endogenous estrogens,(11,12) little is known aboutthe influence of concentrations of endogenous sex hor-

*Universities of California (San Francisco), Pittsburgh, Minnesota (Minneapolis), and Kaiser Center for Health Research, Portland.1Department of Epidemiology and Biostatistics, University of California, San Francisco, California, U.S.A.2Division of General Internal Medicine, University of California, San Francisco, California, U.S.A.3Department of Endocrinology, University of California, San Francisco, California, U.S.A.4Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, Minnesota, U.S.A.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 13, Number 7, 1998Blackwell Science, Inc.© 1998 American Society for Bone and Mineral Research

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mones and SHBG on rates of bone loss in elderly women.The importance of estrogen in preserving bone in elderlywomen is suggested by several prospective studies that havedemonstrated that the use of exogenous estrogen preventsbone loss and fractures.(13–15) Recently, a small prospectivestudy found higher levels of both estrogens and androgens,but not lower SHBG concentrations, to be protectiveagainst femoral bone loss in elderly women.(16) However,results of cross-sectional studies correlating circulating lev-els of sex hormones or SHBG with bone density have beeninconclusive,(11,12,17–20) as have studies comparing hormoneconcentrations in fracture cases and controls.(21,22)

To test hypotheses regarding the influence of circulatinglevels of sex steroids, SHBG, and calciotropic hormones onbone loss in elderly women, we measured hormone levels instored sera and determined hip and calcaneal bone lossover 3.5 and 5.9 years of follow-up, respectively, in a ran-domly selected cohort of older women.

MATERIALS AND METHODS

Subjects

Subjects were participants in the Study of OsteoporoticFractures (SOF), the details of which have been previouslydescribed.(23) At the baseline visit in 1986–1988, a total of9704 community-dwelling white women aged 65 years andover were recruited from population-based listings at fourclinical centers in the United States: Baltimore, Maryland;Minneapolis, Minnesota; Portland, Oregon; and theMonongahela Valley near Pittsburgh, Pennsylvania. Thisstudy is based upon a randomly sampled subcohort of 261SOF participants who had blood drawn at the baselineexamination and who had technically adequate calcanealbone mineral density (BMD) measured at both the baselineexamination and at follow-up in 1993–1994. Among these,241 women had technically adequate hip BMD measure-ments both at the year-2 examination in 1990 and at fol-low-up in 1993–1994. Final results are based upon subco-horts of 231 and 218 women with complete calcaneal andhip BMD scan pairs, respectively, who did not report cur-rent use of estrogen replacement therapy during the base-line interview. Sample sizes for individual assays vary due tomissing values. Also, sample sizes are smaller for estradiolthan for other assays because we added estradiol to the setof assays some time after the study was underway.

Measurements

At the baseline visit in 1986–1988, a detailed question-naire was administered in which subjects were asked aboutcurrent or previous use of medications such as estrogen,corticosteroids, and anticonvulsants and about use of di-etary supplements such as calcium and multivitamins con-taining vitamin D. Subjects were examined to obtain heightand weight measurements.

Calcaneal bone mass was measured using single-photonabsorptiometry (OsteoAnalyzer, Siemens-Osteon, Wa-hiawa, Hawaii, U.S.A.) at baseline and at a follow-up visit in

1993–1994, after an average of 5.9 years. Bone density ofthe hip and its subregions were measured using dual-energyX-ray absorptiometry (QDR 1000, Hologic, Inc., Waltham,MA, U.S.A.) at a second visit in 1988–1990, among 82% ofthe original cohort who survived. Repeat hip bone densitywas measured at follow-up in 1993–1994, after an averageof 3.5 years. Repeat measurements of both calcaneal andhip BMD were performed on the same instruments as wereused for the initial measurements. The mean coefficients ofvariation of 1.2% between centers for both the calcaneusand femoral neck were estimated using multiple measure-ments on staff members who visited each site. Spine bonedensity measurements were not performed on a sufficientnumber of subjects to warrant inclusion in this study. Fur-ther details of these bone densitometry measurements aredescribed elsewhere.(24)

Serum samples

At the baseline examination, blood was drawn between8:00 a.m. and 2:00 p.m. and sera stored at 220°C. Within 2weeks, samples were shipped to Biomedical Research In-stitute (Rockville, MD, U.S.A.) and stored in liquid nitro-gen at 2190°C. In 1994, sera for selected participants wasthawed and assayed for levels of circulating hormones andother biochemical measurements. Laboratories performingthe assays were blinded to the participant’s fracture status.

In an earlier substudy, the stability of selected hormonemeasurements over time were determined in a separategroup of 51 women by testing levels in fresh sera at baseline(1986–1988) and retesting again after 3.5 years of storage at2190°C. Comparisons between the measurement pairswere as follows: estradiol (r 5 0.94; means 5 11.8 and 10.9pg/ml for the initial and repeat measurements, respective-ly), estrone (r 5 0.98; means 5 2.4 and 3.2 ng/dl), totaltestosterone (r 5 0.99; means 5 29 and 28 ng/dl), midregionPTH (r 5 0.99; means 5 43 and 47 pg/ml), and 25(OH)D(r 5 0.88; means 5 23 and 22 ng/ml). There were nostatistically significant differences in mean hormone levels(initial compared with repeat values), and all correlationswere significant at p , 0.001. Although intact PTH wasmeasured in all 231 participants in the bone loss study,stability of PTH was determined using the midregion mea-surement because an immunoradiometric assay for PTHwas not available at the time the baseline measurementswere taken in 1986–1988. The correlation between PTH(immunoradiometric assay) and midregion in the samplesmeasured after 3.5 years of storage was 0.78.

Biochemical assays

Serum concentrations of estradiol, estrone, and total tes-tosterone were measured by radioimmunoassay (EndocrineSciences, Calabasas, CA, U.S.A.). The binding capacity ofSHBG was measured using a displacement technique (En-docrine Sciences). Free testosterone levels were calculatedas the product of total testosterone and the percentage ofnon–SHBG-bound steroid as determined from an ammo-nium sulfate precipitation procedure (Endocrine Sciences).Intact PTH, 25(OH)D, and 1,25(OH)2D were measured by

1168 STONE ET AL.

radioimmunoassay (Calciotropic Hormone Reference Lab-oratory, University of California, San Francisco, CA,U.S.A.). Precision for all assays is reported in Table 1, alongwith descriptive data on the means and standard deviationsas well as the quartile ranges for each hormone.

Statistical analyses

Baseline characteristics of the 231 participants with base-line and repeat calcaneal BMD measurements included inthis study were compared with those of 5140 women withbaseline and repeat calcaneal BMD measurements in thetotal cohort from which the participants were sampled,using t-tests for continuous variables and chi-square testsfor categorical variables. The baseline characteristics of the218 women with initial and repeat hip BMD measurementswere similar to those of the 231 women with calcaneal BMDpairs (of which they are a subset), and these results are notpresented separately.

All biochemical variables except estradiol were analyzedby quartiles and as continuous variables. Since 36% ofestradiol levels were less than the minimum level of detec-tion (5 pg/ml), estradiol values were divided into fourgroups by setting the undetectable levels as the lowestcategory and dividing the remaining detectable levels intotertiles. Estradiol categories are reported as quartiles in thetables for simplicity. For continuous models, undetectableestradiol levels were assigned zero values.

Linear regression analysis was used to analyze the asso-ciation between hormone levels and the annual percentagechange in BMD, adjusting for age and weight. Furtheradjustment for clinic site, current use of calcium supple-ments, physical activity, and smoking status yielded similarresults: all statistically significant results remained signifi-cant after adjustment for each covariate separately as wellas including all simultaneously. Therefore, the most parsi-monious models are presented, adjusting hormone levels

for age and weight. Results for annual absolute change inBMD were also similar and are not presented. Associationsbetween hormone levels and bone loss were weaker for thefemoral neck and similar for the trochanteric regions com-pared with the total hip, and therefore we present resultsonly for total hip bone loss. Age- and weight-adjusted av-erage percentage change in BMD and 95% confidenceintervals were calculated by quartiles of each hormoneusing the least squared means procedure, and a test fortrend across quartiles (adjusting for age and weight) wasused to assess statistical significance. The association be-tween continuous hormone levels and annual percentagechange in BMD are reported as standardized beta coeffi-cients, representing the difference in annual percentagechange in BMD per standard deviation increase in the levelof each hormone.

Multivariable models were constructed to determinewhich biochemical variables were independent predictors ofbone loss after controlling for levels of other hormones.Since estradiol and estrone levels are modestly correlated inthis cohort (r 5 0.64), these variables were only analyzed inseparate models.

RESULTS

Differences between the baseline characteristics of therandom subsample included in this study and those of thetotal cohort were not statistically significant (p . 0.10 for allcomparisons; Table 2). The mean age of women in thissubsample was 71.3 years. Average annual bone loss was;1.5% from the calcaneus and ;0.5% from both the totalhip and femoral neck.

The correlation between total and free testosterone was0.71, and the correlation between estrone and estradiol was0.64. The correlations among all other hormones ranged

TABLE 1. DESCRIPTIVE DATA ON BIOCHEMICAL ASSAYS

FactorIntra-assaycorrelation

Inter-assayvariability

(%) n* Mean (SD)Lowest

quartile† Q2† Q3†Highestquartile†

Sex steroids and SHBGestrone (ng/dl) 0.97 8–12.5 230 2.3 (1.2) , 1.6 1.6–, 2.3 2.3–, 3.0 $ 3.0estradiol (pg/ml) 0.96 6.2–7.0 168 6 (5) , 5 5–, 7 7–, 10 $ 10free testosterone (pg/ml) 0.79 10.7–15.5 230 2.2 (1.9) , 0.9 0.9–, 1.5 1.5–, 2.7 $ 2.7total testosterone (ng/dl) 0.97 6.1–13.4 230 20.2 (15.7) , 9.7 9.7–, 17.0 17.0–, 26.0 $ 26.0SHBG (mg/dl) 0.95 4.1–14.4 230 1.5 (0.8) , 1.1 1.1–, 1.5 1.5–, 2.4 $ 2.4

Calciotropic hormones andcalcium

PTH (pg/ml) — 8.5 231 34.3 (23.7) , 23 23–, 31 31–, 41 $ 4125(OH)D (pg/ml) — 15.0 231 26.2 (9.8) , 21 21–, 25 25–, 32 $ 321,25(OH)2D (pg/ml) — 12.4 229 32.9 (9.7) , 26 26–, 32 32–, 40 $ 40calcium (mg/dl) — — 231 9.7 (0.5) , 9.5 9.5–, 9.8 9.8–, 10.0 $ 10.0

* n, mean (SD) and quartile cutpoints were calculated for the calcaneal bone loss cohort. n’s for the total hip bone loss cohort arereported in Table 4.

† Range of values for indicated quartile.

HORMONAL PREDICTORS OF BONE LOSS 1169

from 20.32 (between SHBG and free testosterone) to 0.42(between estradiol and free testosterone).

Sex hormones and SHBG

In age- and weight-adjusted linear regression models,only SHBG was a significant predictor of calcaneal boneloss (Table 3). Women with SHBG levels in the highestquartile ($ 2.4 mg/dl) experienced twice as much calcanealbone loss annually (mean change 5 22.2%; 95% CI 522.9%, 21.6%) compared with women with levels in thelowest quartile (, 1.1 mg/dl; mean change 5 21.2%;21.6%, 20.7%). This association remained strongly signif-icant even after adjustment for levels of serum estradiol,estrone, testosterone, and 25(OH)D (p , 0.002). Trendstoward increased calcaneal bone loss with decreasing hor-mone levels are apparent for all sex steroids (Table 3), butnone was statistically significant. There was a borderline(p , 0.10) inverse statistical association between serumestrone concentrations modeled as a continuous variableand calcaneal bone loss.

Lower levels of both estrone and estradiol were signifi-cantly associated with greater hip bone loss (Table 4). Forexample, after adjusting for age and weight, women withestradiol levels 10 pg/ml or higher experienced no signifi-cant change in bone mass during follow-up (mean annualchange 5 0.1%; 20.5, 0.7), while women with estradiollevels below 5 pg/ml experienced above average rates of hipbone loss (mean annual change 5 20.8%; 21.2, 20.3).

Examination of the mean annual percentage change inhip bone density by quartiles of testosterone levels sug-gested a threshold (Table 4): women with total testosterone

levels 26 ng/dl or higher (highest quartile) experienced anaverage annual percentage change of only 20.1% (20.5%,0.3%) compared with 20.6% (20.8%, 20.4%) amongwomen with below 26 ng/dl. This difference was statisticallysignificant (p , 0.05).

An increase in hip bone loss across quartiles of SHBGwas observed, although this trend was not statistically sig-nificant in age- and weight-adjusted regression models (p 50.27; Table 4). In a multivariable model including SHBG,estradiol, testosterone, and 25(OH)D levels in addition toage and weight, lower levels of total testosterone and higherSHBG concentrations were independently associated withmore rapid decline in hip BMD (Table 5). Because therewere a substantial number of women who did not haveestradiol measurements, it was unclear whether the signif-icant association between SHBG and hip bone loss in themultivariable model resulted from adjustment for otherhormone levels or whether a stronger association betweenSHBG and bone loss existed in this subcohort of women.Therefore, the age- and weight-adjusted association be-tween SHBG and hip bone loss was reexamined amongwomen who had estradiol measurements and was found tobe statistically significant (Table 5).

When expressed as a continuous variable, estradiol wasnot significantly associated with change in hip BMD eitherbefore or after adjustment for testosterone, SHBG, or25(OH)D. However, the test for trend across quartiles ofestradiol remained significant after controlling for levels ofSHBG, testosterone, and 25(OH)D, whereas the trendacross estrone quartiles was of borderline significance (p ,0.06).

TABLE 2. CHARACTERISTICS OF PARTICIPANTS IN CALCANEAL BONE LOSS COHORT

Characteristic

Calcaneal boneloss cohort,

biochemical sample

Calcaneal boneloss cohort,total sample

Number 231 5140Age, years, mean (SD) 71.3 (4.8) 70.9 (4.8)Weight, kg, mean (SD) 68.1 (11.7) 67.6 (12.3)Height, cm, mean (SD) 159.4 (6.3) 159.2 (5.9)Current calcium supplements, n (%) 102 (44.2) 2109 (41.1)Current vitamin D supplements, n (%) 100 (44.1) 2241 (44.3)Dietary calcium, mg/day, mean (SD) 687 (390) 710 (421)Current corticosteroid use, n (%) 3 (1.3) 71 (1.4)Current anticonvulsant use, n (%) 4 (1.7) 53 (1.0)Current smoking, n (%) 19 (8.3) 450 (8.8)Physical activity, kcal/week, mean (SD) 1698 (1791) 1703 (1661)Calcaneal BMD, g/cm2, mean (SD) 0.409 (0.088) 0.403 (0.089)Total hip BMD, g/cm2, mean (SD) 0.765 (0.124) 0.755 (0.125)Femoral neck BMD, g/cm2, mean (SD) 0.658 (0.108) 0.648 (0.105)Spine BMD, g/cm2, mean (SD) 0.850 (0.154) 0.845 (0.161)Calcaneal bone loss, %/year, mean (SD) 1.48 (1.80) 1.55 (1.57)Total hip bone loss, %/year, mean (SD) 0.49 (1.49) 0.59 (1.45)Femoral neck bone loss, %/year, mean (SD) 0.50 (1.70) 0.53 (1.78)

p . 0.10 for all comparisons of differences in baseline characteristics between the biochemical bone losscohort and the total bone loss cohort from which they were sampled.

1170 STONE ET AL.

TABLE 3. ASSOCIATIONS OF HORMONES WITH CALCANEAL BMD CHANGE

FactorDifference perSD increase* Lowest quartile Q2 Q3 Highest quartile

Sex steroids and SHBGestrone (ng/dl) 0.2 (0.0, 0.5)† 21.7 (22.1, 21.2) 21.5 (21.9, 21.0) 21.6 (22.0, 21.1) 21.0 (21.5, 20.6)estradiol (ng/dl) 0.2 (20.1, 0.5) 21.6 (22.0, 21.1) 21.3 (21.9, 20.6) 21.3 (21.9, 20.8) 21.0 (21.7, 20.3)free testosterone (pg/ml) 0.1 (20.1, 0.4) 21.9 (22.4, 21.3) 21.4 (21.9, 21.0) 21.6 (22.1, 21.2) 21.2 (21.6, 20.7)total testosterone (ng/dl) 0.0 (20.2, 0.2) 21.5 (21.9, 21.0) 21.8 (22.3, 21.4) 21.2 (21.7, 20.8) 21.3 (21.8, 20.9)SHBG (mg/dl) 20.6 (20.8, 20.3)‡ 21.2 (21.6, 20.7) 21.2 (21.6, 20.7) 21.7 (22.2, 21.3) 22.2 (22.9, 21.6)‡

Calciotropic hormones and calciumPTH (pg/ml) 0.0 (20.2, 0.2) 21.3 (21.7, 20.8) 21.5 (21.9, 21.0) 21.6 (22.0, 21.1) 21.6 (22.1, 21.2)25(OH)D (pg/ml) 20.1 (20.3, 0.1) 21.2 (21.7, 20.8) 21.8 (22.3, 21.4) 21.3 (21.8, 20.8) 21.5 (22.0, 21.0)1,25(OH)2D (pg/ml) 0.0 (20.2, 0.2) 21.3 (21.8, 20.8) 21.6 (22.0, 21.2) 21.7 (22.1, 21.2) 21.3 (21.8, 20.8)calcium (mg/dl) 0.1 (20.2, 0.3) 21.4 (21.8, 20.9) 21.6 (22.1, 21.2) 21.4 (22.0, 20.9) 21.5 (21.9, 21.1)

Adjusted annual average percent change in BMD (95% CI), adjusted for age and weight, excluding estrogen replacement therapy users.* Difference in annual percent change in BMD per SD increase in level of hormone/factor.† p , 0.10; ‡p , 0.01.Significance tests for differences per SD are based upon the test H0: b 5 0 on the linear parameter estimate for each hormone.Significance tests across quartiles are based upon tests for trend.

TABLE 4. ASSOCIATIONS OF HORMONES WITH TOTAL HIP BMD CHANGE

Factor nDifference perSD increase* Lowest quartile Q2 Q3 Highest quartile

Sex steroid and SHBGestrone (pg/ml) 217 0.2 (0.0, 0.4)† 20.8 (21.2, 20.4) 20.7 (21.1, 20.3) 20.3 (20.7, 0.2) 20.1 (20.5, 0.3)‡

estradiol (pg/ml) 160 0.2 (0.0, 0.5) 20.8 (21.2, 20.3) 20.8 (21.2, 20.2) 20.3 (20.7, 0.2) 0.1 (20.5, 0.7)§

free testosterone (pg/ml) 217 0.3 (0.0, 0.5)§ 20.6 (21.0, 20.1) 20.5 (20.9, 20.1) 20.7 (21.1, 20.3) 20.3 (20.6, 0.1)total testosterone (ng/dl) 217 0.1 (20.1, 0.3) 20.6 (20.9, 20.2) 20.6 (21.0, 20.3) 20.6 (21.0, 20.2) 20.1 (20.5, 0.3)SHBG (mg/dl) 217 0.1 (20.4, 0.1) 20.4 (20.7, 20.1) 20.4 (20.7, 0.0) 20.7 (21.7, 20.3) 20.7 (21.2, 20.1)

Calciotropic hormones and calciumPTH (pg/ml) 218 0.2 (0.0, 0.4)§ 20.4 (20.8, 0.0) 20.6 (21.0, 20.2) 20.6 (21.0, 20.2) 20.3 (20.7, 0.1)25(OH)D (pg/ml) 218 0.2 (0.0, 0.4)§ 20.7 (21.1, 20.4) 20.6 (20.9, 20.2) 20.5 (20.9, 20.1) 20.1 (20.5, 0.3)§

1,25(OH)2D (pg/ml) 216 0.0 (20.2, 0.2) 20.5 (21.0, 20.1) 20.6 (20.9, 20.2) 20.5 (20.9, 20.1) 20.4 (20.7, 0.0)calcium (mg/dl) 218 0.2 (0.0, 0.3) 20.4 (20.7, 0.0) 20.7 (21.1, 20.4) 20.5 (21.0, 20.1) 20.3 (20.7, 0.0)

Adjusted annual average percent change in BMD (95% CI), adjusted for age and weight, excluding estrogen replacement therapy users.* Difference in annual percent change in BMD per SD increase in level of hormone/factor.†p , 0.10; ‡p , 0.01; §p , 0.05.Significance tests for differences per SD are based upon the test H0: b 5 0 on the linear parameter estimate for each hormone.Significance tests across quartiles are based upon tests for trend.

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Calciotropic hormones

Only 25(OH)D levels were significantly different duringwintertime (January–June) compared with the remainderof the year (July–December), with mean values of 24.0ng/ml during the winter and 26.9 ng/ml otherwise (p 50.01). There were no significant differences in calciotropichormone levels comparing measurements taken Maythrough October compared with November through April.

None of the calciotropic hormones or calcium levels wassignificantly associated with change in calcaneal BMD aftercontrolling for age and weight (Table 3). This remainedtrue even after adjustment for season (July–December vs.January–June) and clinic, and after either adjustment for orexclusion of current users of calcium or multivitamins con-taining vitamin D.

Lower levels of 25(OH)D were significantly associatedwith increased hip bone loss (Table 4). Women with25(OH)D levels in the highest quartile ($ 32 ng/ml) expe-rienced an annual average change in BMD of 20.1% (20.5,0.3) compared with 20.7 (21.1, 20.4) among women with25(OH)D levels in the lowest quartile (, 21 ng/ml). Thistrend remained significant after adjustment for clinic, sea-son, and use of calcium and multivitamins containing vita-min D. The association between 25(OH)D and hip boneloss also remained significant after adjustment for levels ofestradiol, total testosterone, and SHBG (Table 5).

There was no consistent association between PTH levelsand bone loss from either site (Tables 3 and 4). Examina-tion of the trend across quartiles of PTH reveals an unusualpattern with greater hip bone loss for PTH levels in thecentral range (second and third quartiles) and less bone lossamong women with more extreme values (lowest and high-

est quartiles). Although PTH when modeled as a continu-ous variable was significantly associated with hip bone loss,this test may be inappropriate: given the pattern acrossquartiles we tested the linearity assumption by adding aquadratic term to the continuous model. The quadraticterm was significant, implying a possible nonlinear associa-tion between PTH and hip bone loss. None of the othercalciotropic hormones was associated with hip bone loss.

DISCUSSION

We found lower levels of serum estrogens to be signifi-cantly associated with increased hip bone loss in elderlywomen, even after controlling for age, weight, and levels ofSHBG and testosterone. In fact, women with estradiol lev-els 10 pg/ml or higher experienced no significant change inbone mass during follow-up (mean annual change 5 0.1%;95% CI 5 20.5, 0.7), while women with undetectable es-tradiol levels (, 5 pg/ml) lost bone from the hip at the rateof almost 1% per year (20.8%; 21.2, 20.3). Since bone lossmay be an important risk factor for hip fracture in elderlywomen,(25) these findings suggest that even moderate in-creases in levels of circulating hormones may be effective inreducing rates of bone loss and fractures. Our results sup-port the recent work of Slemenda et al. who found estradiolto be a strong predictor of midradius bone loss in post-menopausal women.(16) These findings are also consistentwith the results of several cross-sectional studies that havereported positive associations between endogenous estro-gen levels and BMD in elderly women.(11,12,17) Although asimilar trend was observed between serum estradiol levelsand bone loss from the calcaneus, this association was notstatistically significant. However, the power to detect astatistically significant relationship between estradiol andbone loss was limited in this study because estradiol wasmeasured in only a portion of the cohort.

SHBG concentrations were strongly associated with cal-caneal and hip bone loss, independent of age, weight, andsex hormone levels. These results are contrary to those ofSlemenda et al. who found no relationship between SHBGand bone loss among postmenopausal women after adjust-ing for age, weight, and levels of other hormones.(16) How-ever, our study is larger and has more power to detect suchassociations, particularly in older women. Our results areconsistent with those of van Hemert et al. who reported thatSHBG was a strong and independent predictor of change inmetacarpal cortical area among women ages 65–75years.(22) SHBG binds serum estradiol and testosteronewith great affinity, and this association may reflect an in-creased risk of bone loss with decreasing levels of bioavail-able sex hormones. However, the strength of this associa-tion even after adjusting for levels of serum estradiol andtestosterone suggest that there may be other influences ofSHBG on bone.

We found lower 25(OH)D levels to be related to morerapid bone loss from the hip, but not from the calcaneus. Toour knowledge, the only other prospective study by Lukertet al. found that decreased 25(OH)D concentrations werepredictive of increased rates of bone loss from the distal

TABLE 5. MULTIVARIABLE MODEL: SEX HORMONES, SHBGAND TOTAL HIP BONE LOSS

Factor

Adjusting forage and weight

only*Multivariable

model†

SHBG(per SD increase)

20.3 (20.6, 0.0)§ 20.3 (20.6, 0.0)‡

Estradiol(per SD increase)

0.2 (0.0, 0.5) 0.1 (20.1, 0.4)

Total testeosterone(. 25 ng/dlvs. others)

0.6 (0.0, 1.2)‡ 0.6 (0.0, 1.3)‡

25(OH)D(per SD increase)

0.3 (0.0, 0.5)§ 0.3 (0.0, 0.5)§

Difference in percent change in hip BMD.All analyses include only women who had estradiol measure-

ments for comparability.* The first column presents results of separate regression models

for each hormone, adjusted for age and weight.† The second column presents results of a single regression

model containing SHBG, estradiol, total testosterone, age, andweight.

‡ p , 0.05.§ p 5 0.05.

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radius.(3,4) Results of cross-sectional studies have beenmixed; two studies found postmenopausal women withlower 25(OH)D levels to have lower BMD,(6,26) but at leastone other cross-sectional study found no significant associ-ation between 25(OH)D and BMD at the distal radius, themidradius, or the lumbar spine.(8) However, this negativestudy by Tsai et al.(8) had a broad age range (33–94 years)and did not report associations separately for elderlywomen.

PTH levels were not associated with bone loss from thecalcaneus or the hip in our study. This was true even afteradjustment for season and exclusion of women taking cal-cium or vitamin D supplements. Although there was someevidence of a possible nonlinear association between PTHand bone loss from the hip, we are unaware of any potentialbiological explanations, and this pattern may have occurredby chance.

This study has several strengths. It is a prospective study,measuring rates of change in bone mass rather than cross-sectional bone mass measurements. Serum samples wereobtained prior to measurement of change in bone mass.The sample size was considerably larger than in most otherrelated studies, and the subjects were sampled from a co-hort of elderly women living in four separate communitiesin the United States. This study also has limitations. Sincethe study population was restricted to ambulatory commu-nity-dwelling white women aged 65 years and older, theresults may not be generalizable to other groups, such asmen, younger women, other racial groups, or the institu-tionalized elderly. We did not have spine bone loss mea-surements in enough women to warrant inclusion in thisanalysis. The biochemical measurements were based on asingle assay, and change in bone mass is based on one pairof baseline and follow-up measurements. Variability in boththe predictor and outcome variables could lead this study tounderestimate the strength of the associations we observed.However, the relatively long follow-up for this study (5.9and 3.5 years for calcaneus and hip, respectively) would beexpected to minimize the influence of precision error inrates of bone loss.(27) The first hip BMD measurement wasnot taken until about 2 years after sera were collected.Although we have found fairly high concordance betweenhormone levels over a 2-year period (correlations rangingfrom 0.6 for estradiol to 0.9 for SHBG and testosterone),this could weaken the observed association between hor-mone levels and hip bone loss and potentially explain someof the negative findings observed in this study. While thesample size is relatively large for detecting associationsbetween a single hormone level and bone loss, the power todetect the influence of combinations of hormone levels onsubsequent bone loss is limited. Finally, it is possible thatthere are additional confounders we did not measure thatmay account for the observed associations between hor-mone levels and bone loss.

In conclusion, we have found that SHBG and endoge-nous estrogens are important determinants of bone loss inelderly women. Lower 25(OH)D levels are associated withmore rapid bone loss from the hip, but not from the calca-neus. PTH and other calciotropic hormones do not signif-icantly influence bone loss in elderly women.

ACKNOWLEDGMENTS

Investigators in the Study of Osteoporotic Fractures Re-search Group:

University of California, San Francisco (CoordinatingCenter:) S.R. Cummings (principal investigator), M.C. Nev-itt (coinvestigator), D.M. Black (coinvestigator), K.L. Stone(project director), H.K. Genant (director, central radiologylaboratory), C. Arnaud, D. Bauer, W. Browner, L. Chris-tianson, M. Dockrell, C. Fox, C. Gluer, S. Harvey, M.Jergas, Mario Jaime-Chavez, R. Lipschutz, G. Milani, L.Palermo, A. Pressman, R. San Valentin, H. Tabor, D.Tanaka, C. Yeung.

University of Maryland: J.C. Scott (principal investiga-tor), R. Sherwin (coprincipal investigator), M.C. Hochberg(coinvestigator), J. Lewis (project director), Cheryl Bailey,(clinic coordinator), A. Bauer, L. Finazzo, G. Greenberg,D. Harris, B. Hohman, S. Kallenberger, E. Oliner, T. Page,A. Pettit, S. Snyder, L. Stranovsky, S. Trusty.

University of Minnesota: K. Ensrud (principal investiga-tor), R. Grimm Jr. (coinvestigator), C. Bell (project direc-tor), E. Mitson (clinic coordinator), M. Baumhover, C.Berger, S. Estill, S. Fillhouer, J. Hansen, K. Jacobson, K.Kiel, C. Linville, N. Nelson, E. Penland-Miller, JayneGriffith.

University of Pittsburgh: J.A. Cauley (principal investi-gator), L.H. Kuller (coprincipal investigator), M. Vogt (co-investigator), L. Harper (project director), L. Buck (cliniccoordinator), C. Bashada, A. Githens, A. McCune, D.Medve, M. Nasim, C. Newman, S. Rudovsky, N. Watson,J. Carothers.

The Kaiser Permanente Center for Health Research,Portland, Oregon: E. Harris (principal investigator), W.M.Vollmer, E. Orwoll, H. Nelson, (coinvestigators), J. Blank(project director), S. Craddick (clinic coordinator), R.Bright, J. Wallace, Heinith, K. Moore, K. Redden, C. Ro-mero, C. Souvanlasy.

This work was supported by Public Service Grant K08AG00629 (NIA).

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Address reprint requests to:Katie L. Stone

University of California at San Francisco74 New Montgomery Street, Suite 600

San Francisco, CA 94105 U.S.A.

Received in original form September 2, 1997; in revised formOctober 30, 1997; accepted March 10, 1998.

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