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Effect of Vitamin D Replacement on Musculoskeletal
Parameters in School Children: A Randomized
Controlled Trial
Ghada El-Hajj Fuleihan, Mona Nabulsi, Hala Tamim, Joyce Maalouf, Mariana Salamoun,
Hassan Khalife, Mahmoud Choucair, Asma Arabi, and Reinhold Vieth
Calcium Metabolism and Osteoporosis Program (G.E.-H.F., J.M., M.S., M.C., A.A.), Department of Medicine, Department of
Pediatrics (M.N., H.K.), School of Health Sciences (H.T.), American University of Beirut, 113-6044 Beirut, Lebanon; and Mt.
Sinai Hospital, Toronto University (R.V.), Toronto, Ontario, Canada M5G 1X5
Background: Despite the high prevalence of hypovitaminosis D in
children and adolescents worldwide, the impact of vitamin D defi-
ciency on skeletal health is unclear.
Methods: One hundred seventy-nine girls, ages 10–17 yr, were ran-
domly assigned to receive weekly oral vitamin D doses of 1,400 IU
(equivalent to 200 IU/d) or 14,000 IU (equivalent to 2,000 IU/d) in a
double-blind, placebo-controlled, 1-yr protocol. Areal bone mineral
density (BMD) and bone mineral content (BMC) at the lumbar spine,
hip, forearm, total body, and body composition were measured at
baseline and 1 yr. Serum calcium, phosphorus, alkaline phosphatase,
and vitamin D metabolites were measured during the study.
Results: In the overall group of girls, lean mass increased signifi-
cantly in both treatment groups (P ⱕ 0.05); bone area and total hip
BMC increased in the high-dose group (P ⬍ 0.02). In premenarcheal
girls, lean mass increased significantly in both treatment groups, and
there were consistent trends for increments in BMD and/or BMC at
several skeletal sites, reaching significance at lumbar spine BMD in
the low-dose group and at the trochanter BMC in both treatment
groups. There was no significant change in lean mass, BMD, or BMC
in postmenarcheal girls.
Conclusions: Vitamin D replacement had a positive impact on mus-
culoskeletal parameters in girls, especially during the premenarcheal
period. (J Clin Endocrinol Metab 91: 405– 412, 2006)
V
ITAMIN D IS essential for bone growth and develop-
ment in children and for skeletal health in adults (1).
Although rickets is rare in developed countries, it is one of
the five most prevalent diseases in developing countries. We
and others have reported a high prevalence of more subtle
degrees of vitamin D insufficiency in normal children and
adolescents worldwide (2–10). We have demonstrated that
girls were at higher risk for low vitamin D levels than boys
due to lower sun exposure and decreased exercise (2). This
problem was most prevalent in boys and girls of low socio-
economic status and in veiled girls (2).
In adolescents, there is an inverse relationship between
serum 25-hydroxyvitamin D [25(OH)D] levels and PTH lev-
els (2, 5, 7, 10) and a positive association between serum
25(OH)D levels and bone mineral density (BMD) (7, 9), sim-
ilar to what has been reported in adults (11). In the elderly,
vitamin D supplements increase grip strength (12), and mus-
cle mass is an excellent predictor of BMD (13).
Nutrition guidelines targeted to children and adolescents
to optimize bone health have focused on calcium and exer-
cise, but have neglected vitamin D (14, 15). To date, there is
no recommended dietary allowance for vitamin D for chil-
dren and adolescents (16, 17) due to the lack of any evidence
for a beneficial effect of supplementation in this age group.
In this study, we hypothesized that treatment with high-
dose vitamin D would optimize gains in muscle mass, BMD,
and bone mineral content (BMC) in adolescent girls com-
pared with low-dose vitamin D and placebo. We anticipated
that the most substantial increments would be noted in pre-
menarcheal girls.
Subjects and Methods
Subjects
Three hundred sixty-three healthy children and adolescents were
recruited between December 2001 and June 2002. Recruitment took place
in four schools from the greater Beirut area to ensure balanced repre-
sentation geographically and socioeconomically (18). The age group
chosen was 10 –17 yr, a critical age for accretion of bone mass (19). In
boys, there was no consistent positive effect of vitamin D supplemen-
tation on lean mass, BMD, or BMC (20). Therefore, in this paper we
report the results of the trial in 179 girls.
Subjects were included in the study if they were considered healthy
based on careful physical examination and absence of a history of any
disorders or medications known to affect bone metabolism (18). At entry,
all had normal serum calcium, phosphorus, and alkaline phosphatase
levels for age. The study was approved by the institutional review board,
and informed consent was obtained from all study subjects and their
parents.
Intervention
The subjects were randomly assigned in a double-blind manner to
receive weekly placebo oil or a vitamin D
3
preparation, given as low-
dose vitamin D (1,400 IU; 35
g/wk), i.e. the equivalent of 200 IU/d, or
First Published Online November 8, 2005
Abbreviations: BMC, Bone mineral content; BMD, bone mineral den-
sity; CV, coefficient of variation; 1,25(OH)
2
D, 1,25-dihydroxyvitamin D;
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 en-
docrine community.
0021-972X/06/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 91(2):405–412
Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/jc.2005-1436
405
on February 8, 2006 jcem.endojournals.orgDownloaded from
high-dose vitamin D (14,000 IU; 350
g/wk), i.e. the equivalent of 2,000
IU/d (Vigantol oil, Merck KGaA, Darmstadt, Germany) for 1 yr. The
randomization sequence, stratified by socioeconomic status, was gen-
erated by a computer at Merck headquarters, mailed to the study center,
and administered by a senior pharmacist. All students received identical
bottles of an oily solution containing diluent oil for the placebo group,
diluted vigantol oil for the low-dose group, or undiluted oil for the
high-dose group. The low dose, i.e. the equivalent of 200 IU/d, corre-
sponds to the current adequate intake for vitamin D in this age group
(16). The high dose, i.e. the equivalent of 2000 IU/d, was chosen as half
the dose demonstrated to be safe in adults, resulting in desirable serum
25(OH)D levels (21), and had been confirmed to be safe in a 12-wk pilot
study conducted in school children at our center. There were no dif-
ferences in months of recruitment among the three treatment arms. The
subjects were called by study personnel every 2 wk to prompt them to
take the study drug. Subjects returned the bottles and received new
bottles every 3 months. Compliance was checked by measuring the
volume and, therefore, the amount of vitamin D left in the returned
bottles. The percentage of the dose taken was calculated as (total vol-
ume ⫺ returned volume)/total volume ⫻ 100. Dietary calcium intake
was comparable across treatment groups and was not controlled for.
Quality assurance
The samples of oil solution prepared for the three treatment groups
were assayed at the clinical pathology laboratory of Mt Sinai Hospital
(Toronto, Canada; by R.V.). The vitamin D concentration in the three
solutions was within 10% of that anticipated based on the label on the
bottles and the dilution protocol.
Data collection
The subjects had a baseline physical examination, including height,
weight, and Tanner stages. Standing height was measured in triplicate
using a wall stadiometer; weight was recorded with the subjects wearing
light clothes without shoes using a standard clinical balance. Pubertal
status was determined by a physician (H.K., M.N., or M.C.), according
to the established criteria of Tanner (22). Calcium intake, exercise, sun
exposure, and history of fractures were assessed by questionnaire at
baseline and follow-up (18). Exercise frequency was assessed on the
basis of a questionnaire inquiring about the average number of hours
spent on sports per week. Calcium intake was assessed through a food
frequency questionnaire that stressed the consumption of dairy prod-
ucts by adolescents in our population. Sun exposure was assessed as the
average number of hours spent in the sun for weekdays and weekends,
and the prorated average was reported. Vitamin D dietary intake was
not evaluated. Grip strength was measured using a squeeze grip ball
with a pressure gauge to measure grip strength; the average of triplicates
measured at baseline and study end was used (pneumatic squeeze
dynamometer, catalogue no. FAB 12-0293, Ingrams, Kansas City, MO).
The mean (⫾sd) coefficient of variation (CV), based on 363 triplicate
measurements, was 3.3 ⫾ 3.2%. Information on sick days was obtained
from school records and by self report if school records were not avail-
able. These analyses were preplanned because of the reported effect of
vitamin D on the immune system (23).
The vitamin D dose was usually taken on the weekends. The timing
of blood drawing was not systematically standardized, but occurred on
any weekday except Sunday. Serum calcium, phosphorus, and alkaline
phosphatase were measured at baseline, 6 months, and 12 months. Blood
for hormonal studies was stored as serum at ⫺70 C. Serum 25(OH)D was
measured at baseline and 12 months by a competitive protein binding
assay using the Incstar Kit (Diasorin, Incstar, Saluggia, Italy), with intra-
and interassay CVs less than 13% at a serum concentration of 47 ng/ml.
Serum 1,25-dihydroxyvitamin D (1,25(OH)
2
D) was measured by RIA
using the IDS kit with intra- and interassay CVs less than 10% at serum
concentrations between 10 and 100 pg/ml (IDS Immuno-Diagnostic
Systems, Boldon, UK). Hypovitaminosis D was defined as 25(OH)D
below 20 ng/ml (24). All samples from an individual subject were
assayed together in the same run at the end of the study. BMD and BMC
of the lumbar spine, hip, and forearm and subtotal BMD, BMC, and
composition were measured at baseline and 1 yr using a Hologic 4500A
densitometer (Hologic, Bedford, MA; software version 11.2:3). The soft-
ware determines BMC, fat mass, and nonfat soft tissue mass, identified
in the software as lean mass. Because inclusion of the head BMD in the
calculation of total body BMD may lower the predictive value of some
parameters for this variable, subtotal body measurements, excluding the
head, were used in the analyses (25). In our center, the mean ⫾ sd
precision of the BMD measurements, expressed as the CV, for 280
same-day duplicate scans performed during the study duration was less
than 1.2 ⫾ 0.9% for the spine, total hip, femoral neck, trochanter, and one
third radius. Similar values were obtained for total body BMD and BMC,
lean mass, and fat mass. The mean ⫾ sd precisions of the 280 duplicate
BMC measurements, expressed as the CV, were 1.2 ⫾ 1.1% for the spine,
1.5 ⫾ 1.8% for the total hip, 2.1 ⫾ 1.7% for the femoral neck, 2.8 ⫾ 2.1%
for the trochanter, and 1.1 ⫾ 1.1% for the forearm.
Statistical analyses
Primary efficacy outcomes were percent change in lean mass and
percent change in BMD and BMC at the lumbar spine and total body;
these were the most established skeletal sites in children at the time the
study was started.
Secondary efficacy outcomes were percent changes in bone mass at
other skeletal sites. Exploratory preplanned analyses in subgroups de-
termined by menarcheal status at study entry were performed. Because
of the significant impact of growth in children on bone size and areal
BMD and the potential impact of vitamin D supplementation on bone
growth and lean mass, both strong correlates of areal BMD and BMC,
we also evaluated the impact of the intervention on bone size, as assessed
by bone area and height. To dissect the physiological pathway mediating
the impact of vitamin D on skeletal parameters, the impact of treatment
on BMD and BMC was assessed after adjusting for changes in lean mass
and bone area. This was done when there was a significant effect of
treatment on areal BMD or BMC, lean mass, or bone area in the unad-
justed analyses, using linear regression analysis. Because the effect of
treatment, low-dose vs. high-dose vitamin D, on percent change in bone
mass could not be assumed to be linear, treatment was entered as a
dummy variable.
The results reported are those based on an intent to treat analysis,
which were identical with the results of per protocol analyses in view
of the very high compliance of subjects and the fact that those who
retuned for follow-up visits and BMD measurements were all taking the
study medications (Fig. 1).
To demonstrate a difference of 3% (sd, 5%) in the percent change in
lumbar spine BMD between the placebo and any of the two treatment
groups, 44 subjects/treatment group would be needed (
␣
⫽ 0.05; power,
80%; Instat PRISM, Applied Biosystems, San Diego, CA). This would
translate into 132 subjects. We aimed to recruit 180 subjects to allow for
potential dropouts and for exploratory analyses by pubertal stages.
ANOVA was used for evaluating the difference among the three
treatment groups. The least significant difference test was implemented
to explore the differences among subgroups (placebo vs. low-dose vi-
tamin D, placebo vs. high-dose vitamin D, and low-dose vitamin D vs.
high-dose vitamin D). The nonparametric Kruskal-Wallis and Mann-
Whitney U tests were used to detect differences between treatment
groups for premenarcheal girls due to the small sample size.
Analyses were carried out using SPSS software, version 11.0 (SPSS,
Inc., Chicago, IL). There were a total of 64 siblings among the subjects;
however, only 16 siblings fell within comparable treatment arms by
pubertal status (as specified in Table 1). Thus, cluster analyses were
performed using STATA version 7 (STATA, College Station, TX) to
adjust for lack of independence among siblings due to heredity and
possible familial resemblance. The STATA program does that by in-
creasing the estimated se values and the variance of the

coefficients.
Results were expressed as the mean ⫾ sd. P ⬍ 0.05 was considered
statistically significant and was not adjusted for multiple testing.
Results
Study subjects and baseline characteristics
Of the 179 subjects enrolled and randomly assigned to a
treatment group, 168 (94%) returned for follow-up BMD
scans and constituted the group on whom the intent to treat
analyses were based (Fig. 1); at study entry, 34 were pre-
menarcheal, and 134 were postmenarcheal. The baseline
406 J Clin Endocrinol Metab, February 2006, 91(2):405– 412 El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls
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characteristics of the subjects, including bone area and bone
density, serum 25(OH)D level, anthropometric and lifestyle-
related variables, were similar among the treatment groups
and in the menarcheal subgroups (Table 1).
At study entry, the mean serum 25(OH)D level was 14 ⫾
8 ng/ml. There were significant associations between base-
line serum 25(OH)D levels and spine BMD (r ⫽ 0.16; P ⫽
0.033), femoral neck BMD (r ⫽ 0.17; P ⫽ 0.028), radius BMD
(r ⫽ 0.24; P ⫽ 0.002). Similarly, there was a significant as-
sociation between baseline serum 25(OH)D levels and radius
BMC (r ⫽ 0.16; P ⫽ 0.033).
Response to treatment: serum 25(OH)D levels, lean mass,
BMC, and bone mineral mass
In subjects assigned to the high-dose vitamin D group,
25(OH)D levels reached a mean of 38 ⫾ 31 ng/ml in the overall
group and 28 ⫾ 9 ng/ml in the premenarcheal group (Table 2).
Conversely, levels remained in the low to midteens in the pla-
cebo and low-dose arms (Table 2). Serum 1,25(OH)
2
D levels
increased with the high dose (Table 2).
In the overall group of girls, there was a significant increase
in lean mass, a primary efficacy end point, but not in grip
strength (Table 2), in both vitamin D groups compared with the
placebo group. There was a trend for larger increments in BMC
in both treatment groups compared with placebo at several
skeletal sites; these increments were statistically significant for
total hip BMC (Table 2). There were significant negative cor-
relations between baseline serum 25(OH)D levels and percent
change spine BMD (r ⫽⫺0.16; P ⫽ 0.044) or percent change in
subtotal body BMD (r ⫽⫺0.20; P ⫽ 0.009; post hoc analyses).
Similarly, there were significant associations between baseline
serum 25(OH)D levels and percent change in spine BMC (r ⫽
⫺0.20; P ⫽ 0.010), percent change in femoral neck BMC (r ⫽
⫺0.16; P ⫽ 0.037), and percent change in radius BMC (r ⫽⫺0.17;
P ⫽ 0.029).
Exploratory subgroup analyses in premenarcheal girls re-
FIG. 1. Diagram outlining the flow of the
study subjects through all stages of the trial.
Of the 219 subjects who originally verbally
agreed to participate in the study, 40 were
excluded for the following reasons: two did
not meet the inclusion criteria, three had orig-
inally misstated their age, six had abnormal
baseline blood values, five were inaccessible,
and 24 changed their minds about participa-
tion.
El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls J Clin Endocrinol Metab, February 2006, 91(2):405– 412 407
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vealed a significant increase in lean mass, a primary efficacy
end point, but not in grip strength (Table 2), in both treatment
groups. Similarly, there was a consistent trend for increments
in BMC (Table 2) and BMD (data not shown) at several
skeletal sites in both treatment groups in a dose-dependant
pattern, reaching significance at trochanteric BMC (Table 2
and Fig. 2) and at the lumbar spine BMD, a primary end point
(P ⫽ 0.04), and almost reaching significance at the total hip
BMD (P ⫽ 0.06). There were no differences in changes in lean
mass, grip strength, BMD, or BMC among the three treat-
ment groups in postmenarcheal girls (data not shown).
Effect of vitamin D supplementation on changes in height,
weight, and bone area (surrogates of changes in bone size)
There were no differences among the three treatment
groups in changes in weight in the overall group or by
menarcheal category (Table 2). There was a trend for an
increase in height with vitamin D supplementation that al-
most reached significance (P ⫽ 0.07; Table 2). There was an
effect of treatment on bone area at several skeletal sites where
a significant treatment effect on BMC was noted (Table 2).
Adjusted analyses for changes in bone area and lean mass
Regression analyses were conducted to further elucidate the
physiological pathway for the effect of vitamin D on BMC and
BMD. The introduction of percent change in area or percent
change in lean mass as covariates in the model caused a de-
crease in the strength of the treatment effect on BMC or BMD,
as reflected by a decrease in the

estimate (data not shown) and
an increase in the P value for the primary end point (lumbar
spine BMD) and the secondary end points (hip BMD, hip BMC,
and trochanter BMC; Table 3).
Compliance with study drug, adverse events, and safety
One hundred sixty-six subjects returned their study bot-
tles. The mean percent intake of the total dose given for
vitamin D was 98 ⫾ 3% in the placebo group, 98 ⫾ 3% in the
low-dose group, and 97 ⫾ 3% in the high-dose group.
The treatment was very well tolerated. Only two subjects
(1%) had serum calcium levels above the upper limit of normal
for children (10.7 mg/dl) (26) at 1 yr. The serum calcium values
were 10.8 and 11.1 mg/dl, and they were both in the placebo
group. Similarly, three subjects (1.5%) had high serum 25(OH)D
levels at the end of the study (103, 161, and 195 ng/ml); all were
in the high-dose group, but none had concomitant hypercalcemia.
Eleven girls (6.1%) dropped out of the study (Fig. 1). There
were no differences in dropout rates by treatment group. The
reasons for dropout included being afraid of needle sticks,
unable to make appointments, disliking the taste of the med-
ication, and changing their mind about the study. One girl
dropped out at 7 months because of the development of glo-
merulonephritis, documented by biopsy, and treated as post-
streptococcal glomerulonephritis. The treatment code was bro-
ken, and she was in the low-dose vitamin D treatment group.
The average number of sick days per year was the same for
all three treatment groups, averaging 2 d/yr. There was no
effect of treatment on self-reported incident fractures.
Discussion
Vitamin D supplementation for 1 yr resulted in substantial
increments in lean mass, bone area, and bone mass in girls
ages 10 –17 yr and was well tolerated. There was a trend for
the increments in bone mass to be larger at the high dose,
especially in the subgroup of premenarcheal girls.
A high prevalence of hypovitaminosis D has been reported
in children and adolescents worldwide (2–10). Its importance
TABLE 1. Baseline characteristics by treatment group in the overall group of girls and by menarcheal status
Variable
All Girls Premenarcheal Postmenarcheal
PBO
(n ⫽ 55)
Low
(n ⫽ 58)
High
(n ⫽ 55)
PBO
(n ⫽ 8)
Low
(n ⫽ 12)
High
(n ⫽ 14)
PBO
(n ⫽ 47)
Low
(n ⫽ 46)
High
(n ⫽ 41)
Siblings (n) 8 6 2 0008 62
Age (yr) 13.6 (2.1) 13.0 (2.1) 13.1 (2.2) 10.9 (0.6) 10.6 (0.6) 10.8 (1.1) 14.0 (1.9) 13.6 (2.0) 13.9 (1.8)
Height (cm) 154 (10) 152 (9) 152 (10) 142 (8) 141 (9) 140 (7) 156 (9) 154 (7) 156 (8)
Weight (kg) 48 (11) 47 (11) 47 (13) 37 (11) 35 (8) 36 (8) 50 (10) 50 (10) 51 (12)
Calcium intake (mg/d) 672 (323) 674 (364) 686 (411) 805 (430) 811 (383) 816 (570) 650 (301) 638 (354) 642 (338)
Exposure to sun (h/wk) 8.1 (6.0) 7.4 (5.9) 6.6 (4.2) 6.4 (4.6) 4.5 (3.6) 5.7 (3.5) 8.4 (6.2) 8.2 (6.1) 6.8 (4.4)
Exercise (h/wk) 4.1 (5.0) 3.4 (4.0) 4.0 (5.6) 0.7 (1.0) 5.7 (5.6) 2.5 (4.9) 4.7 (5.1) 2.8 (3.2) 4.4 (5.7)
Grip strength (psi) 11.5 (2.2) 11.0 (2.2) 10.9 (2.2) 9.1 (2.1) 9.0 (2.1) 8.9 (2.1) 12.0 (2.0) 11.5 (2.0) 11.6 (1.8)
Lumbar spine BMC (g) 41.1 (12.0) 37.6 (10.7) 39.2 (12.9) 25.2 (6.4) 25.0 (5.2) 27.4 (6.3) 43.8 (10.6) 40.9 (9.2) 43.2 (12.1)
1/3 radius BMC (g) 1.4 (0.2) 1.3 (0.2) 1.3 (0.3) 1.0 (0.1) 1.0 (0.2) 1.0 (0.2) 1.4 (0.2) 1.4 (0.2) 1.4 (0.3)
Total hip BMC (g) 24.3 (5.4) 22.8 (5.5) 22.6 (5.9) 17.2 (5.3) 15.8 (4.2) 17.0 (3.8) 25.5 (4.4) 24.7 (4.2) 24.4 (5.3)
Femoral neck BMC (g) 3.3 (0.7) 3.3 (0.7) 3.3 (0.7) 2.4 (0.7) 2.5 (0.5) 2.6 (0.5) 3.5 (0.6) 3.5 (0.5) 3.5 (0.7)
Trochanter BMC (g) 6.3 (1.5) 5.9 (1.7) 5.9 (1.7) 4.6 (1.3) 4.0 (1.4) 4.7 (1.5) 6.6 (1.4) 6.4 (1.4) 6.3 (1.6)
Total body BMC (kg) 1.2 (0.3) 1.1 (0.3) 1.1 (0.4) 0.8 (0.2) 0.7 (0.2) 0.8 (0.2) 1.3 (0.3) 1.2 (0.2) 1.2 (0.3)
Lean mass (kg) 30.7 (6.0) 29.3 (5.5) 29.5 (6.5) 23.5 (4.9) 22.9 (4.4) 23.0 (4.2) 31.9 (5.3) 31.1 (4.5) 31.7 (5.6)
% Fat mass 28 (6) 29 (7) 28 (7) 27 (11) 26 (7) 26 (8) 28 (5) 30 (7) 29 (7)
S-Ca (mg/dl) 10.0 (0.4) 9.9 (0.3) 9.9 (0.4) 9.9 (0.4) 9.9 (0.3) 10.0 (0.3) 10.0 (0.4) 9.9 (0.4) 9.8 (0.4)
S-PO
4
(mg/dl)
4.4 (0.5) 4.3 (0.5) 4.3 (0.7) 4.7 (0.4) 4.6 (0.6) 4.7 (0.4) 4.4 (0.5) 4.3 (0.5) 4.1 (0.8)
S-ALKP (IU/liter) 199 (116) 208 (112) 232 (139) 298 (92) 275 (65) 332 (75) 182 (112) 191 (116) 198 (141)
S-25(OH)D (ng/ml) 14 (7) 14 (9) 14 (8) 13 (7) 15 (6) 14 (5) 14 (8) 14 (10) 14 (8)
S-1,25(OH)
2
D (pg/ml)
86 (30) 78 (29) 83 (27) 84 (27) 87 (29) 90 (29) 84 (32) 74 (29) 82 (27)
Values are means (
SD). The biochemical assays are reported in metric units (SI). To convert from metric to SI units, multiply calcium by 0.25
(mmol/liter); phosphorus by 0.32 (mmol/liter); 25(OH)D by 2.496 (nmol/liter); and 1,25(OH)
2
D by 2.6 (pmol/liter). PBO, Placebo.
408 J Clin Endocrinol Metab, February 2006, 91(2):405– 412 El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls
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was underscored at a recent conference organized by the United
States National Institutes of Health, during which “an alarming
prevalence of low circulating levels of vitamin D” was noted
(27). The most well-recognized function of vitamin D is to
increase dietary calcium and phosphate absorption (1, 16), but
the impact of hypovitaminosis D, as opposed to severe defi-
ciency, on musculoskeletal health in children and adolescents
is still unclear. A beneficial effect of vitamin D replacement in
this group had not been established, to our knowledge, before
this trial.
Preliminary results from a 1-yr, randomized, placebo-con-
trolled trial conducted in Danish girls (mean age, 11 yr) revealed
no significant effect of vitamin D, given in relatively low doses
of 200 and 400 IU/d, on whole-body and lumbar spine BMC
(28). The differences in treatment effect between that trial and
the current one may have been due to differences in the baseline
characteristics of the study subjects, including mean calcium
intake and severity of hypovitaminosis D, the differences in
doses, or a combination of both. The doses of vitamin D used
in the Danish trial were substantially lower than the high dose
used in our trial, the dose at which the most consistent treatment
effect was noted. The small increments in serum 25(OH)D levels
achieved in the Danish trial, averaging 3–4 ng/ml, may po-
tentially explain the failure to detect any impact of therapy on
BMC (28). Although we noted a comparably small serum
25(OH)D response in the subjects receiving the equivalent of
200 IU/d, the dose was taken weekly and would have resulted
in pulses of serum 25(OH)D that may have different effects
from taking a daily dose of 200 IU.
The beneficial treatment effect noted in the overall group of
girls was paralleled by even more substantial increments in
BMC in premenarcheal girls, whereas no effect was detected in
postmenarcheal girls. This is consistent with observations from
calcium and exercise trials demonstrating an impact of the
intervention when administered to younger girls (29 –31) and
defining the most substantial benefit to occur before or within
a narrow time window around menarche (29, 32, 33). It is also
possible that a putative protective effect of vitamin D on bone
may have been overshadowed by the powerful effects of pu-
berty on skeletal growth.
As anticipated, the most substantial increments in bone mass
were in subjects with the lowest vitamin D levels at entry for the
high-dose arm, but not the low-dose arm, at the primary end
points, spine BMD and subtotal body BMC. The beneficial effect
of vitamin D on bone mass in girls may be mediated though one
or more physiological pathways. Although intestinal calcium
TABLE 2. Serum levels of vitamin D metabolites and percent change in BMC, lean mass, bone area, grip strength, height, and weight at
1 yr by treatment group, in the overall group of girls and in premenarcheal girls
PBO Low High P overall
P value
PBO vs. Low PBO vs. High Low vs. High
Overall
a
S-25 (OH)D (ng/ml) 16 (8) 17 (6) 38 (31) ⬍0.001 NS ⬍0.001 ⬍0.001
S-1,25(OH)
2
D (pg/ml)
76 (30) 78 (30) 105 (33) ⬍0.001 NS ⬍0.001 ⬍0.001
LS BMC (%) 10.8 (8.5) 14.5 (12.0) 12.9 (10.4) 0.20 NS NS NS
LS area (%) 4.0 (4.6) 5.0 (6.3) 4.3 (5.4) 0.61 NS NS NS
Total hip BMC (%) 7.8 (7.7) 11.2 (9.3) 12.8 (10.5) 0.02 0.05 0.005 NS
Total hip area (%) 2.4 (4.5) 4.0 (4.6) 5.7 (5.8) 0.003 NS 0.001 NS
FN BMC (%) 3.9 (7.2) 4.4 (7.8) 5.2 (8.0) 0.70 NS NS NS
FN area (%) 0.7 (4.9) 0.03 (4.8) 0.8 (5.4) 0.67 NS NS NS
Trochanter BMC (%) 9.4 (13.3) 13.6 (14.8) 14.2 (17.2) 0.20 NS NS NS
Trochanter area (%) 4.7 (8.6) 6.8 (9.2) 7.8 (11.5) 0.24 NS NS NS
Sub total body BMC (%) 8.7 (8.8) 11.3 (10.4) 12.0 (11.3) 0.20 NS NS NS
Total body area (%) 5.0 (4.7) 6.1 (5.7) 6.2 (5.8) 0.43 NS NS NS
Sub total body lean mass (%) 5.7 (6.6) 8.7 (8.0) 9.0 (8.3) 0.05 0.042 0.027 NS
Grip strength (%) 13.5 (18.5) 20.1 (19.7) 17.4 (16.0) 0.16 NS NS NS
Height (%) 2.0 (1.8) 2.7 (2.3) 2.8 (2.4) 0.10 NS NS NS
Weight (%) 8.6 (7.8) 8.8 (8.3) 9.7 (8.5) 0.74 NS NS NS
Premenarche
b
S-25 (OH)D (ng/ml) 11 (6) 16 (5) 28 (9) ⬍0.001 NS ⬍0.001 ⬍0.001
S-1,25(OH)
2
D (pg/ml)
73 (20) 79 (32) 113 (38) 0.02 NS 0.014 0.016
LS BMC (%) 12.0 (9.9) 18.8 (13.0) 17.2 (10.2) 0.40 NS NS NS
LS area (%) 3.4 (7.0) 3.2 (9.5) 4.4 (7.7) 0.93 NS NS NS
Total hip BMC (%) 12.3 (12.4) 18.4 (9.1) 23.2 (11.0) 0.08 NS NS NS
Total hip area (%) 7.4 (7.5) 8.0 (4.4) 12.3 (6.5) 0.11 NS NS NS
FN BMC (%) 7.4 (4.5) 9.3 (9.3) 11.4 (7.9) 0.50 NS NS NS
FN area (%) 5.0 (3.2) 2.7 (5.5) 4.9 (5.2) 0.45 NS NS NS
Trochanter BMC (%) 12.5 (11.3) 32.2 (15.9) 25.7 (20.8) 0.05 0.018 NS NS
Trochanter area (%) 5.7 (8.1) 18.4 (8.9) 14.2 (15.0) 0.08 0.024 NS NS
Sub total body BMC (%) 15.4 (8.0) 19.9 (7.1) 21.8 (9.4) 0.20 NS NS NS
Total body area (%) 7.4 (3.9) 11.4 (4.0) 11.6 (4.2) 0.06 NS NS NS
Sub total body lean mass (%) 10.7 (5.2) 16.8 (6.6) 18.1 (6.7) 0.04 0.046 0.013 NS
Grip strength (%) 25.1 (30.6) 26.5 (17.8) 21.2 (16.9) 0.81 NS NS NS
Height (%) 3.8 (1.5) 5.0 (1.4) 5.6 (1.3) 0.07 NS NS NS
Weight (%) 14.9 (5.2) 15.3 (4.7) 18.4 (6.2) 0.25 NS NS NS
Values are means (
SD). LS, Lumbar spine; FN, femoral neck; PBO, placebo; NS, not significant.
a
Overall P value and post hoc P values between subgroups by ANOVA.
b
Overall P value by nonparametric test due to small sample size.
El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls J Clin Endocrinol Metab, February 2006, 91(2):405– 412 409
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absorption was not assessed in this trial, this effect of vitamin
D is unequivocal (34). The relationship among vitamin D, mus-
cle function, and body weight are well established in the elderly
(12), but we are unaware of any such observations in young
adolescents. The increments in lean mass noted in this trial are
consistent with a direct effect of vitamin D on muscle, in part
mediating its beneficial effect on BMD and BMC. Our group
and others have observed close correlations between lean mass
or muscle mass and bone mass (35–37). The lack of a detectable
effect of treatment on grip strength could be explained by the
low sensitivity of that measurement (38). To our knowledge,
there are no studies relating vitamin D supplementation to
changes in bone size. Previous studies evaluating the effect of
calcium or calcium and vitamin D have reported increases in
bone area or height (30, 31, 39, 40), suggesting an effect of
calcium on bone modeling (31). An increased intake of protein
and an increase in growth factors could explain the anabolic
effect of milk intervention on bone (31, 40). The presence of
treatment differences in bone area at cortical sites and the trend
for treatment differences in height are consistent with an effect
of vitamin D on modeling and bone growth. This may be
explained by a direct effect of vitamin D on periosteal apposi-
tion or indirectly through lean mass/muscle mass, thus exert-
ing an anabolic effect on long bones. The decrements in the
magnitude of the

estimates relating the impact of vitamin D
to changes in BMD and BMC when adjusting for lean mass,
bone area, or both, further underscore the roles of these pre-
dictors in the causal pathway between vitamin D and bone
mass. The improved bone health may have been due to in-
creases in 1,25(OH)
2
D levels, 25(OH)D levels, or both.
1,25(OH)
2
D can be produced locally, thus rendering it impos
-
sible to dissect the above possibilities based on the observed
changes in serum levels of these substances (41).
The positive skeletal response to vitamin D replacement in
girls contrasts with the lack of any positive response in boys
(20). The sexual dimorphism in response to vitamin D supple-
FIG. 2. Box plots showing the median and interquartile range of the percent change in lean mass (A), the percent change in hip trochanteric
BMC (B), and the percent change in total hip BMC (C) by treatment group in premenarcheal girls. P values displayed represent results from
post hoc t testing on ANOVA. There was a significant effect of treatment on changes in lean mass and changes in trochanteric BMC at both
doses. There was a trend for a significant effect of treatment on percent changes in total hip BMC in premenarcheal girls.
410 J Clin Endocrinol Metab, February 2006, 91(2):405– 412 El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls
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mentation may have several explanations. Boys had a higher
calcium intake and exercised more than girls. There were also
gender differences in the severity of hypovitaminosis D at base-
line, differences in the serum 1,25(OH)
2
D levels achieved, and
the lack of an increase in lean mass and bone area in boys,
contrary to what was observed in girls (20). Furthermore, sex
differences in the hormonal profiles achieved during puberty
could explain the differences in the relationship between mus-
cle and bone in boys and girls (18, 37).
Treatment was well tolerated overall. Compliance, as esti-
mated by the volume of drug left in the returned bottles, was
excellent. It is possible that subjects may have manipulated the
volume returned, discarding the study drug, thus leading to an
overestimation of the compliance. This is unlikely due to the fact
that subjects were contacted every 2 wk to remind them to take
the study drug. Although a few subjects had high serum
25(OH)D levels, there was no evidence of vitamin D toxicity. In
the three subjects with high levels of vitamin D, not a single one
experienced concomitant hypercalcemia.
Our study has several limitations. Dual-energy x-ray absorp-
tiometry was used to evaluate the effect of the intervention on
areal BMD and BMC, measures affected by bone size and
growth (42). There is currently no consensus on how to best
adjust for bone size when measuring bone mass using dual-
energy x-ray absorptiometry, but suggestions have included
adjustments in height, bone area, lean mass, pubertal stage, and
bone age (42). Although it is clear that these adjustments are
essential to evaluating data from studies of pathological con-
ditions in children, they are less crucial in randomized trials of
healthy children. Indeed, we studied normal subjects whose
baseline characteristics, including height, weight, lean mass,
bone mass, bone area, and pubertal stages, were all matched at
baseline. Adjustments in changes in lean mass and bone area in
response to the intervention were made only to gain insight into
the possible mechanisms underlining the beneficial effect of
vitamin D on bone. The increments in lean mass and bone mass
could have been more robust had the subjects received con-
comitant calcium, in light of their suboptimal intake (40). How-
ever, the aim of the trial was to ascertain the impact of vitamin
D per se, rather than calcium and vitamin D, on musculoskeletal
health. Other limitations include the lack of assessment of di-
etary vitamin D intake, the relatively short duration of the trial,
precluding conclusions regarding the sustainability of the ben-
efit, and the low power to demonstrate beneficial effect at all
skeletal sites in the subgroup analyses by pubertal stage. Nev-
ertheless, this trial demonstrates the importance of vitamin D
for musculoskeletal health in girls during a critical time for
growth. This has important implications in terms of public
health intervention measures.
Acknowledgments
We thank the administrators, school nurses, parents, and students
from the American Community School, the International College, the
Amlieh School, and the Ashbal Al Sahel School for their support in
TABLE 3. Multivariate analyses relating the effect of vitamin D treatment on BMD/BMC without and then after adjusting for bone area,
lean mass, and both by treatment assignment
Skeletal site Predictor P value
All girls
LS BMD (primary efficacy endpoint) Low dose 0.04
Low dose ⫹ % area 0.57
Low dose ⫹ % lean mass 0.40
Low dose ⫹ % area % lean mass 0.40
Hip BMC (secondary efficacy endpoint) Low dose 0.04
Low dose ⫹ % area 0.30
Low dose ⫹ % lean mass 0.50
Low dose ⫹ % area, % lean mass 0.70
High dose 0.005
High dose ⫹ % area 0.007
High dose ⫹ % lean mass 0.07
High dose ⫹ % area, % lean mass 0.40
Premenarcheal girls: exploratory preplanned analyses
LS BMD (primary efficacy endpoint) Low dose 0.007
Low dose ⫹ % area 0.009
Low dose ⫹ % lean mass 0.20
Low dose ⫹ % area, % lean mass 0.20
Hip BMC (secondary efficacy endpoint) High dose 0.045
High dose ⫹ % area 0.09
High dose ⫹ % lean mass 0.40
High dose ⫹ % area, % lean mass 0.50
Hip BMD (secondary efficacy endpoint) Low dose 0.02
Low dose ⫹ % area 0.005
Low dose ⫹ % lean mass 0.30
Low dose ⫹ % area, % lean mass 0.10
High dose 0.02
High dose ⫹ % area 0.09
High dose ⫹ % lean mass 0.40
High dose ⫹ % area, % lean mass 0.60
Trochanter BMC (secondary efficacy endpoint) Low dose 0.003
Low dose ⫹ % area 0.50
Low dose ⫹ % lean mass 0.07
Low dose ⫹ % area, % lean mass 0.80
El-Hajj Fuleihan et al. • Vitamin D Replacement in Adolescent Girls J Clin Endocrinol Metab, February 2006, 91(2):405– 412 411
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making this study possible. We thank Mrs. U. Usta for her assistance
with preparing the vitamin D solutions and implementing the random-
ization protocol, Mrs. S. Mroueh for her expert technical assistance with
the acquisition and analyses of the BMD scans, and Mrs. C. Hajj Shahine
for her tireless efforts in running the hormonal assays.
Received June 29, 2005. Accepted October 28, 2005.
Address all correspondence and requests for reprints to: Dr. Ghada
El-Hajj Fuleihan, Calcium Metabolism and Osteoporosis Program, Amer-
ican University of Beirut-Medical Center, Bliss Street, 113-6044 Beirut, Leb-
anon. E-mail: gf01@aub.edu.lb.
This work was supported in large part by an educational grant from the
Nestle Foundation and a grant from Merck KGaA.
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