25-Hydroxyvitamin-D3 levels are positively related to subsequent cortical bone development in childhood: findings from a large prospective cohort study.

Abstract

In exploring relationships between vitamin D status in childhood and cortical bone, little relationship was observed with plasma concentrations of 25-hydroxyvitamin-D(2) [25(OH)D(2)], whereas 25-hydroxyvitamin-D(3) [25(OH)D(3)] was positively related to cortical bone mineral content (BMC(C)) and cortical thickness, suggesting D(3) exerts a beneficial effect on cortical bone development in contrast to D(2).
The study is aimed to determine whether vitamin D status in childhood is related to cortical bone development by examining prospective relationships between plasma concentrations of 25(OH)D(2) and 25(OH)D(3) at 7.6, 9.9 or 11.8 years and peripheral quantitative computed tomography (pQCT) measurements of the mid-tibia at age 15.5 years, in children from the Avon Longitudinal Study of Parents and Children.
Relationships between vitamin D status and pQCT outcomes were analysed by bootstrap linear regression, adjusted for age, sex, body composition, socioeconomic position and physical activity, in 2,247 subjects in whom all covariates were available. 25(OH)D(3) was also adjusted for season and 25(OH)D(2), and 25(OH)D(2) for 25(OH)D(3).
25(OH)D(3) was positively related to BMC(C) [0.066(0.009,0.122), P = 0.02], whereas no association was seen with 25(OH)D(2) [-0.008(-0.044,0.027), P = 0.7] [beta (with 95% CI) represents SD changes per doubling of vitamin D], P = 0.03 for difference in associations of 25(OH)D(2) and 25(OH)D(3) with BMC(C). There were also differences in associations with cortical geometry, since 25(OH)D(3) was positively related to cortical thickness [0.11(0.04, 0.19), P = 0.002], whereas no association was seen with 25(OH)D(2) [-0.04(-0.08,0.009), P = 0.1], P = 0.0005 for difference. These relationships translated into differences in biomechanical strength as reflected by buckling ratio, which was positively related to 25(OH)D(2) [0.06(0.01,0.11), P = 0.02] indicating less resistance to buckling, but inversely related to 25(OH)D(3) [-0.1(-0.19,-0.02), P = 0.03], P = 0.001 for difference.
In contrast to 25(OH)D(2), 25(OH)D(3) was positively related to subsequent cortical bone mass and predicted strength. In vitamin D-deficient children in whom supplementation is being considered, our results suggest that D(3) should be used in preference to D(2).

Full text

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ORIGINAL ARTICLE
25-Hydroxyvitamin-D3levels are positively related
to subsequent cortical bone development in childhood:
findings from a large prospective cohort study
A. Sayers & W. D. Fraser & D. A. Lawlor & J. H. Tobias
Received: 20 June 2011 /Accepted: 19 September 2011 /Published online: 12 November 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract
Summary InexploringrelationshipsbetweenvitaminDstatus
in childhood and cortical bone, little relationship was
observed with plasma concentrations of 25-hydroxyvitamin-
D2[25(OH)D2], whereas 25-hydroxyvitamin-D3[25(OH)D3]
was positively related to cortical bone mineral content
(BMCC) and cortical thickness, suggesting D3 exerts a
beneficial effect on cortical bone development in contrast
to D2.
Introduction The study is aimed to determine whether
vitamin D status in childhood is related to cortical bone
development by examining prospective relationships be-
tween plasma concentrations of 25(OH)D2and 25(OH)D3
at 7.6, 9.9 or 11.8 years and peripheral quantitative
computed tomography (pQCT) measurements of the mid-
tibia at age 15.5 years, in children from the Avon
Longitudinal Study of Parents and Children.
Methods Relationships between vitamin D status and
pQCT outcomes were analysed by bootstrap linear regres-
sion, adjusted for age, sex, body composition, socioeco-
nomic position and physical activity, in 2,247 subjects in
whom all covariates were available. 25(OH)D3was also
adjusted for season and 25(OH)D2, and 25(OH)D2 for
25(OH)D3.
Results 25(OH)D3was positively related to BMCC[0.066
(0.009,0.122), P=0.02], whereas no association was seen
with 25(OH)D2[−0.008(−0.044,0.027), P=0.7] [beta (with
95% CI) represents SD changes per doubling of vitamin D],
P=0.03 for difference in associations of 25(OH)D2and 25
(OH)D3 with BMCC. There were also differences in
associations with cortical geometry, since 25(OH)D3was
positively related to cortical thickness [0.11(0.04, 0.19), P=
0.002], whereas no association was seen with 25(OH)D2
[−0.04(−0.08,0.009), P=0.1], P=0.0005 for difference.
These relationships translated into differences in biome-
chanical strength as reflected by buckling ratio, which was
positively related to 25(OH)D2[0.06(0.01,0.11), P=0.02]
indicating less resistance to buckling, but inversely related
to 25(OH)D3 [−0.1(−0.19,-0.02), P=0.03], P=0.001 for
difference.
Conclusions In contrast to 25(OH)D2, 25(OH)D3 was
positively related to subsequent cortical bone mass and
predicted strength. In vitamin D-deficient children in whom
supplementation is being considered, our results suggest
that D3should be used in preference to D2.
Keywords Adolescence.ALSPAC.Cortical bone.pQCT.
Vitamin D
Electronic supplementary material The online version of this article
(doi:10.1007/s00198-011-1813-9) contains supplementary material,
which is available to authorized users.
A. Sayers:J. H. Tobias (*)
Musculoskeletal Research Unit, School of Clinical Sciences,
University of Bristol,
Bristol, UK
e-mail: jon.tobias@bristol.ac.uk
A. Sayers
e-mail: adrian.sayers@bristol.ac.uk
W. D. Fraser
Norwich Medical School, Faculty of Medicine and Health
Sciences, University of East Anglia,
Norwich, UK
D. A. Lawlor
MRC Centre for Causal Analyses in Translational Epidemiology,
School of Social and Community Medicine, University of Bristol,
Bristol, UK
A. Sayers
Musculoskeletal Research Unit, Avon Orthopaedic Centre,
Southmead Hospital,
Bristol BS10 5NB, UK
Osteoporos Int (2012) 23:2117–2128
DOI 10.1007/s00198-011-1813-9

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Introduction
Severe vitamin D deficiency, caused by reduced sun
exposure, leads to osteomalacia (adults)/rickets (children)
resulting from defective skeletal mineralisation. Milder
vitamin D deficiency, termed ‘insufficiency’, may also
affect skeletal health in the elderly by reducing bone
mineral density (BMD) and increasing fracture risk due to
secondary hyperparathyroidism, in the absence of mineralisa-
tion defects [1]. If also applicable in childhood, vitamin D
requirements in children would need to be set to prevent
insufficiency rather than vitamin D deficiency and rickets
[2]. Vitamin D insufficiency in children may be relatively
common. For example, in Maine, USA, 48% of girls aged 9–
11 had a total 25-hydroxyvitamin D (25(OH)D) level below
the 50 nmol l-1(20 ng ml-1) cutoff commonly used to
indicate D insufficiency at least once over 3 years [3].
To ensure adequate vitamin D status, recommended
dietary allowances of vitamin D have recently been
proposed across different age groups including children
[4]. However, a recent Cochrane review concluded that
vitamin D supplementation in healthy children had limited
effects, but more trials are required to confirm the efficacy
of supplementation in deficient children [5]. Whereas three
studies in children reported modest improvements in bone
outcomes following treatment with cholecalciferol (D3) [6–
8], ergocalciferol (D2) was without effect in one study [9].
A possible explanation is that D2may be less potent than
D3, since D3and its metabolites have a higher affinity than
D2 for hepatic 25-hydroxylase and vitamin D receptors
[10]. Furthermore, in one such study, effects of D3
supplementation on BMD were suggested to be due to
changes in lean mass [6], consistent with observations that
levels of vitamin D metabolites and sunlight exposure are
related to height and body composition [11–13], which are
in turn strongly related to bone parameters [14].
Observational studies of the relationship between plasma
concentration of total 25(OH)D and bone outcomes in
childhood have yielded conflicting findings [15–17]. These
differences may have arisen from confounding, which is
difficult to adjust based on results of total 25(OH)D levels,
since D2and D3are derived from different sources. For
example, as the majority of D3 is derived from skin
synthesis following the action of UVR, 25(OH)D3levels
are affected by factors influencing sun exposure such as
outdoor physical activity which is known to affect bone
development [18]. Whereas dietary fish intake and fortifica-
tion of certain foods contribute to D3, D2is mainly derived
from fungi, plants and dietary supplements, implying that
dietary patterns affect levels of 25-hydroxyvitamin-D2[25
(OH)D2] and, to a lesser extent, 25-hydroxyvitamin-D3[25
(OH)D3]. This represents another source of confounding
since dietary patterns may affect bone development [19],
possibly through coassociation with socioeconomic position
(SEP) which is also related to bone development in
childhood [20].
We examined whether vitamin D status influences
cortical bone development in childhood, based on 25(OH)
D2and 25(OH)D3concentrations measured at age 7.6, 9.9
or 11.8, and results of peripheral quantitative computed
tomography (pQCT) scans of the mid-tibia performed at
age 15.5, in the Avon Longitudinal Study of Parents and
Children (ALSPAC). Specifically, we wished to determine
(i) whether vitamin D status is related to subsequent cortical
bone development, (ii) to what extent are its associations
which we find are independent of confounders including
physical activity and body composition (based on contem-
poraneous measures of fat and lean mass by DXA), and (iii)
whether there is any evidence that 25(OH)D3has stronger
associations with cortical bone development than 25(OH)
D2, suggesting supplementation regimes based on D3are
likely to be more effective compared to those using D2.
Methods
Study population
All pregnant women resident within a defined part of the
former county of Avon in South West England with an
expected date of delivery between April 1991 and December
1992 were eligible for recruitment, of whom 14,451 were
enrolled [21] (http://www.alspac.bristol.ac.uk). Written in-
formed consent was provided by the mothers, and informed
assent was obtained from the children at the time of
assessment. Ethical approval was obtained from the
ALSPAC Law and Ethics Committee (internal) and the
Central and South Bristol Research Ethics Committee
(external). Data in ALSPAC is collected by self-completion
postal questionnaires sent to main caregivers and the children
themselves, by abstraction from medical records, and from
examination of the children at research clinics. All children
with available data were included in the analyses.
Blood measurements
The primary exposures for this study were circulating
concentrations of 25(OH)D2and 25(OH)D3as measured on
nonfasting blood samples collected at the age 9.9 research
clinic. If no samples were available from the 9.9 clinic,
samples from the 11.8 clinic were used, or from the age
7.6 year clinic if neither the 9.9 or 11.8 were available.
Following collection samples were immediately spun,
frozen and stored at −80°C. Assays were performed in
2010 after a maximum of 12 years in storage with no
previous freeze–thaw cycles during this period. 25(OH)D2,
2118 Osteoporos Int (2012) 23:2117–2128

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25(OH)D3and deuterated internal standard were extracted
from serum samples, following protein precipitation, using
Isolute C18 solid phase extraction cartridges. Potential
interfering compounds were removed by initial elution with
50% methanol followed by elution of the vitamins using
10% tetrahydrofuran in acetonitrile. Dried extracts were
reconstituted prior to injection into a high performance
liquid chromatography tandem mass spectrometre in the
multiple reaction mode (MRM). The MRM transitions (m/z)
used were 413.2>395.3, 401.1>383.3 and 407.5>107.2 for
25(OH)D2, 25(OH)D3and hexa-deuterated(OH)D3, respec-
tively. Coefficients of variation (CVs) for the assay were
<10% across a working range of 1 to 250 ng ml-1for both 25
(OH)D2and 25(OH)D3. Intact parathyroid hormone [iPTH
(1–84)] [1] was measured by electrochemiluminescence
immunoassay on an Elecsys 2010 immunoanalyzer (Roche,
Lewes, UK). Inter-assay CV was less than 6% from 2 to 50
pmol l-1. The assay sensitivity (replicates of the zero
standard) was 1 pmol l-1.
pQCT variables
At the age 15.5 research clinic, pQCT scans at the 50%
mid-tibia were also performed using the Stratec XCT2000L
(Stratec, Pforzheim, Germany). Cortical bone area, cortical
bone mineral content (BMCC), cortical bone mineral
density (BMDC), periosteal circumference, endosteal cir-
cumference and cortical thickness were recorded. Strength
parametres comprised section modulus, cross-sectional
moment of inertia, strength strain index and buckling ratio.
A density of >650 mg cm-3was used to define cortical
bone. Endosteal and periosteal circumference were derived
using a circular ring model. 4502 pQCT scans were
performed, of which 88 were excluded due to major motion
artifacts. Coefficients of variation for pQCT scans, based on
139 subjects scanned a mean of 31 days apart, were 2.7%,
1.3% and 2.9% for BMCC, BMDCand cortical bone area,
respectively.
Other variables
At 15.5 years research clinics, standing height (mm) was
measured using the Harpenden Stadiometer (Holtain,
Crymych, Wales, UK), and weight using the Tanita Body
Fat Analyzer (model TBF 305; Tanita, Arlington Heights,
IL, USA). Whole body DXA scans were performed using a
Lunar Prodigy scanner with paediatric scanning software
(GE Lunar Prodigy, Madison, WI, USA), providing
measures of total body fat and lean mass. Maternal SEP
was recorded at 32 weeks gestation by questionnaire and
categorised according to the Office of Population Censuses
and Surveys. Maternal education was assessed at the same
time by questionnaire. Pubertal stage was assessed using a
Tanner stage (pubic hair domain) questionnaire completed
at age 14.7 years [22]. Moderate and vigorous physical
activity was assessed by actigraph accelerometre at age 11,
and subsequently found to be related to BMD in ALSPAC
[23]. Date of birth and sex was obtained from birth
notification, and date of the scan was recorded automati-
cally, allowing age at scan to be calculated.
Statistical analyses
Descriptive statistics show means, standard deviation (SD),
medians and lower and upper quartiles. Analyses were
performed using seasonally adjusted 25 (OH)D3, which was
modelled according to date of blood sampling using linear
regression with trigonometric sine and cosine functions. 25
(OH)D3was logetransformed to reduce heteroscedasticity.
The residual was used as the primary 25(OH)D3exposure
variable in subsequent regression analyses. All analyses
were performed on standardised variables, i.e. subtracting the
mean and dividing by the SD. To include all participants on
whom a 25(OH)D2was assayed, those with a value below
the detectable limit of the assay (0.5 ng ml-1) were assigned a
binary variable indicating whether an individual was at or
below the lower limit, which was used as a covariable in all
regression models. No individuals had 25(OH)D3below the
detectable limit of the assay. Models were checked for
linearity by adding higher-order terms into the linear
predictor and by comparing the likelihood of nested models.
Further analyses were performed using a nonparametric
bootstrap procedure in conjunction with OLS linear
regression, based on 5,000 replications. Beta (β) estimates
and standard errors were calculated from the mean and SD
of the bootstrap distribution, respectively. All P values were
calculated using bootstrap means and standard errors,
compared to a Z-distribution and 95% percentile confidence
intervals calculated. Beta estimates were multiplied by
loge(2) and interpreted as per doubling in 25(OH)D2or 25
(OH)D3. Estimates were calculated separately for males and
females, and the difference investigated using a bootstrap
Waldtest.Combined(maleandfemale)associationswerealso
investigated following adjustment for sex. The difference
between the effect of 25(OH)D2 and 25(OH)D3 was
calculated from the bootstrap replicate distribution, and the
P values using a Wald test.
Minimally adjusted analyses (model 1), which were based
on seasonally adjusted 25(OH)D3levels or 25(OH)D2, were
adjusted for sex, age at pQCT scan, and adjusted for 25(OH)
D2 and seasonally adjusted 25(OH)D3, respectively. In
model 2, we additionally adjusted for loge-transformed
fat mass, lean mass and height. In the final model (model 3),
we also adjusted for physical activity and social economic
factors (maternal or paternal social class, maternal education).
Analyses with endosteal circumference were adjusted for
Osteoporos Int (2012) 23:2117–21282119

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periosteal circumference throughout (endosteal adjusted for
periosteal circumference). Sensitivity analyses were per-
formed based on model 2 by: (a) adjusting for parathyroid
hormone (PTH); (b) restricting those with available puberty
information and then, in this subgroup, examining the impact
of adjusting for pubertal status (tanner stages IV/V versus
earlier stages); and (c) restricting those with 25(OH)D assays
collected at age 9.9 years. All analyses were conducted using
STATA 11.2(College Station, TX, USA), and data is assumed
to be missing at random.
Results
Descriptive analyses
There were 1,709 boys and 1,870 girls with pQCT scans
(age 15.5 years), and plasma 25(OH)D2and 25(OH)D3(age
7.6, 9.9 or 11.8 years; see Fig. 1). Those who were included
in the analysis were of higher maternal and paternal social
class compared to those who were not. Boys were taller,
heavier and had greater lean mass compared to girls,
whereas fat mass was higher in girls (Table 1). BMCC,
cortical bone area, periosteal circumference, endosteal
circumference and cortical thickness were greater in boys
compared to girls, whereas BMDCwas higher in girls. 25
(OH)D3levels were slightly higher in boys and 25(OH)D2
levels slightly higher in girls. PTH levels were slightly
higher in girls. There was evidence of weak inverse
associations between 25(OH)D2and height LM and FM,
which appeared somewhat stronger in girls compared to
boys, e.g. P=0.06 for gender interaction test for association
with height (Table 2). There was little association between
25(OH)D3and height, and LM P>0.75, and weak evidence
of an association with FM P=0.06. 25(OH)D3 was
inversely related to PTH, whereas no association was seen
for 25(OH)D2. There was a very weak association between
seasonally adjusted 25(OH)D3and 25(OH)D2, r=−0.0298
P=0.155, excluding those subjects in whom 25(OH)D2was
below the assay detection limit.
Live Births
(Singleton & Multiple)
N=14,062
Age 9.9 Vitamin D
N=5,095
Age 7.6 Vitamin D
N=1,283
Age 11.8 Vitamin D
N=1198
Vitamin D age 7, 9 or 11
N=7,560
No
Yes
No
Vitamin D Samples
Yes
Yes
pQCT and DXA
Confounding Variables:
Maternal education
Maternal/Paternal Social class
Physical Activity
Age 15.5 pQCT &
Anthropometry
N=3,579
Age 15.5 Fully AdjustedAnalyses
N=2,247
White Ethnicity
N=6,450
Recruited
N=14,541
Excluded (Non-White /Missing)
N=1129
Initial Recruitment
Fig. 1 Summary of data
collection
2120Osteoporos Int (2012) 23:2117–2128

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Associations between 25(OH)D concentrations and pQCT
variables
There was a weak inverse association between 25(OH)D2
and BMDC in both minimally and more fully adjusted
analyses, to a similar extent in boys and girls (Table 3). For
example, in boys and girls combined, in our most
completely adjusted model, a doubling in 25(OH)D2was
associated with a 0.05SD decrease in BMDC. Whereas 25
(OH)D2 was unrelated to periosteal circumference in
minimally adjusted analyses, there was a weak positive
association in more fully adjusted models, to a similar
degree, in boys and girls. In minimally adjusted analyses,
25(OH)D2 was inversely related to cortical bone area,
BMCC, endosteal adjusted for periosteal circumference and
cortical thickness in females, but this was not seen after
more complete adjustment. There was a positive association
between 25(OH)D2levels and buckling ratio in all models,
to a similar extent, in boys and girls (Table S1).
Positive associations were observed between 25(OH)D3
and cortical bone area and BMCC in anthropometry
adjusted and fully adjusted analyses (Table 4). In all
models, 25(OH)D3 was positively related to cortical
thickness and inversely related to endosteal adjusted for
periosteal circumference. For example, in our most fully
adjusted model, a doubling in 25(OH)D3was associated
with a 0.11 SD increase in cortical thickness. There was
also an inverse association between 25(OH)D3and buck-
Table 1 Descriptive statistics
[N (male)=1,709; N (female)=1,870]
Mean(SD)25th 50th75th
Age [year] Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
15.5
15.5
174.3
164.8
63.5
58.8
20.8
21.6
10.8
18.6
49.8
37.1
1,074.2
1,124.6
329.1
275.1
353.8
309.3
76.2
69.5
40.9
37.0
(0.26)
(0.28)
(7.5)
(6.1)
(11.4)
(10.3)
(3.1)
(3.5)
(7.8)
(7.9)
(6.6)
(3.9)
(34.3)
(22.3)
(46.8)
(36.6)
(53.2)
(41.0)
(5.3)
(4.9)
(5.9)
(5.4)
(0.7)
(0.6)
(9.0)
(8.2)
(1.9)
(1.9)
(1.8)
(2.3)
15.3
15.3
169.7
160.7
56.0
51.9
18.8
19.3
5.7
13.2
45.7
34.5
1,053.1
1,111.2
297.1
250.0
318.8
281.1
72.8
66.3
37.1
33.6
15.4
15.4
174.5
164.7
61.9
57.0
20.2
21.0
8.3
17.1
49.9
36.8
1,077.1
1,126.3
329.3
273.6
353.7
308.0
76.1
69.2
40.4
36.5
15.6
15.6
179.4
168.6
69.3
63.9
22.2
23.2
12.9
22.1
54.1
39.5
1,099.2
1,139.8
359.6
298.7
388.3
335.9
79.6
72.6
44.1
39.7
AnthropometryHeight [cm]
Weight [kg]
BMI [kg m-2]
Fat mass-Total body [kg]
Lean mass-Total body [kg]
pQCTBMDC [mg cm-3]
BAC [mm2]
BMCC[mg]
PC [mm]
EC [mm]
CT [mm]5.63
5.17
24.1
22.8
1.80
1.89
4.53
5.11
5.2
4.8
18.1
17.1
0.5
0.5
3.2
3.5
5.7
5.2
23.0
22.1
1.2
1.4
4.2
4.6
6.1
5.6
28.5
27.4
2.6
2.7
5.5
6.1
Plasma measures 25(OH)D3[ng ml-1]
25(OH)D2[ng ml-1]
PTH [pmol l-1]
Table shows descriptive characteristics of anthropometric parametres, 50% tibia pQCT parametres, and plasma measures in males and females at
age 15.5 years. Statistics are presented as means, SDs, medians, and upper and lower quartiles
Osteoporos Int (2012) 23:2117–2128 2121

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ling ratio in both minimally and more fully adjusted
analyses (Table S2), suggesting a protective effect on the
skeleton since buckling ratio is inversely related to bone
strength. These associations tended to be stronger in boys,
in whom beta coefficients were two to three times higher
than in girls, and P values for gender-specific regression
equations were only below the P<0.05 significance
threshold in boys. However, formal gender interaction tests
were consistentlyP>=0.1, and so evidence that these
associations were stronger in boys compared to girls is
not compelling.
Subsequently, we compared associations between 25
(OH)D2and pQCT parametres as shown in Table 3, with
associations between 25(OH)3 and pQCT parametres as
shown in Table 4. P values for differences in these
associations are shown in Table 5, for minimally and more
fully adjusted models. In the case of BMDCand cortical
bone area, there was weak evidence of a difference between
25(OH)D2and 25(OH)D3in fully adjusted models, P=0.1
and P=0.07, respectively, boys and girls combined (Table 5).
For BMCC, there was moderate evidence of a difference
between 25(OH)D2and 25(OH)D3P<0.05 in all models,
boys and girls combined. There was strong evidence of
difference between 25(OH)D2 and 25(OH)D3 in CT,
endosteal adjusted for periosteal circumference and BR, P<
0.001 in minimal and more completely adjusted models,
boys and girls combined. Apart from weak evidence of a
difference in girls in our anthropometry-adjusted model (P=
0.04), there was no evidence of a difference between 25(OH)
D2and 25(OH)D3with respect to periosteal circumference.
No difference was observed for any model in respect of
associations between 25(OH)D2and 25(OH)D3and cross-
sectional moment of inertia, section modulus and strength
strain index (results not shown).
Sensitivity analyses and exploration of additional models
In view of the biological relationship between vitamin D
status and PTH concentrations, we examined whether
associations between pQCT parametres and 25(OH)D
which we observed were mediated by PTH, but repeating
the above analyses including additional adjustment for PTH
did not affect the results (see Table S3 for results for
buckling ratio, anthropometry-adjusted analyses). In the
case of associations between 25(OH)D2and buckling ratio,
β was attenuated by approximately 15% when restricting
analyses to those with complete puberty information, but no
further change was seen after adjusting for Tanner stage
within this subset. β for the association between 25(OH)D2
and buckling ratio increased by approximately 50% on
restricting analyses to subjects with blood samples at age
9.9, suggesting some associations may be strengthened
when vitamin D samples obtained a longer interval before
pQCT measurements are excluded. β values were very
similar across all groups for associations between 25(OH)
D3and buckling ratio. We found no evidence of nonline-
Table 2 Associations between plasma concentration of 25(OH)D2and 25(OH)D3and anthropometry variables
Vitamin 25(OH)D2
Vitamin 25(OH)D3
P value
(D2D3)
Minimally adjusted, N=3,579 (males=1,709) Minimally adjusted, N=3,579 (males=1,709)
Beta95% CIP value (sex)Beta95% CIP value (sex)
HeightMale
Female
ALL
Male
Female
ALL
Male
Female
ALL
Male
Female
ALL
−0.026
−0.070
−0.050
−0.021
−0.040
−0.030
−0.017
−0.040
−0.030
−0.010
−0.026
−0.019
(−0.072, 0.021)
(−0.107, -0.028)
(−0.085, -0.011)
(−0.059, 0.017)
(−0.073, -0.017)
(−0.063, -0.006)
(−0.066, 0.031)
(−0.081, -0.001)
(−0.069, 0.007)
(−0.064, 0.045)
(−0.076, 0.024)
(−0.064, 0.027)
0.06
−0.070
0.056
0.000
−0.027
0.034
0.007
−0.048
−0.070
−0.060
−0.260
−0.290
−0.270
(−0.169, 0.026)
(−0.016, 0.131)
(−0.061, 0.061)
(−0.112, 0.060)
(−0.012, 0.081)
(−0.040, 0.054)
(−0.160, 0.066)
(−0.140, -0.003)
(−0.124, 0.002)
(−0.367, -0.148)
(−0.392, -0.189)
(−0.346, -0.200)
0.04
0.42
0.01
0.17
0.90
0.01
0.14
0.61
0.44
0.40
0.01
0.01
0.01
Lean mass0.17 0.22
Fat mass0.30 0.72
Ln PTH0.550.65
Table shows associations between plasma concentration of 25(OH)D2and 25(OH)D3and height, total body lean mass, logefat mass and loge
parathyroid hormone (PTH), adjusted for sex, age at scan and 25(OH)D3and 25(OH)D2respectively, in 1709 males and 1870 females at age
15.5 years. Beta coefficients represent SD change in height, lean mass, logefat mass, and logePTH, per doubling of vitamin 25(OH)D2and 25
(OH)D3. 95% confidence intervals are presented for beta coefficients, P value (sex) shows the difference in associations between males and
females, and P value (D2D3) is the probability of a difference in associations between 25(OH)D2and 25(OH)D3
2122Osteoporos Int (2012) 23:2117–2128

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Table 3 Associations between plasma concentration of 25(OH)D2and pQCT parametres
Vitamin 25(OH)D2
Minimally adjusted, N=3,579
(males=1,709)
Anthropometry-adjusted, N=3,579
(males=1,709)
Anthropometry-, SES- and PA-adjusted,
N=2,247 (males=1,203)
Beta
95% CI
P value
(sex)
Beta
95% CI
P value
(sex)
Beta
95% CI
P value
(sex)
Cortical bone
mineral density
Male
−0.055
(−0.099, -0.010)
0.74
−0.048
(−0.091, -0.005)
0.89
−0.049
(−0.101, 0.004)
0.98
Female
−0.048
(−0.082, -0.014)
−0.046
(−0.078, -0.014)
−0.048
(−0.089, -0.008)
ALL
−0.051
(−0.084, -0.017)
−0.047
(−0.079, -0.015)
−0.048
(−0.087, -0.011)
Cortical bone area
Male
−0.006
(−0.055, 0.043)
0.04
0.013
(−0.021, 0.047)
0.32
0.006
(−0.038, 0.049)
0.90
Female
−0.054
(−0.095, -0.013)
−0.003
(−0.030, 0.025)
0.003
(−0.033, 0.039)
ALL
−0.033
(−0.071, 0.006)
0.004
(−0.022, 0.031)
0.004
(−0.029, 0.037)
Cortical bone
mineral content
Male
−0.021
(−0.074, 0.031)
0.05
0.000
(−0.035, 0.036)
0.37
−0.007
(−0.053, 0.039)
0.93
Female
−0.069
(−0.113, -0.026)
−0.015
(−0.044, 0.015)
−0.009
(−0.047, 0.028)
ALL
−0.048
(−0.089, -0.007)
−0.008
(−0.036, 0.020)
−0.008
(−0.044, 0.027)
Periosteal circumference
Male
0.018
(−0.026, 0.062)
0.08
0.035
(0.002, 0.067)
0.82
0.037
(−0.007, 0.080)
0.86
Female
−0.021
(−0.061, 0.021)
0.031
(0.000, 0.063)
0.041
(0.003, 0.079)
ALL
−0.004
(−0.040, 0.032)
0.033
(0.005, 0.060)
0.039
(0.006, 0.075)
Endosteal adjusted for
periosteal circumference
Male
0.026
(−0.012, 0.063)
0.14
0.01
(−0.024, 0.044)
0.22
0.017
(−0.025, 0.058)
0.71
Female
0.052
(0.021, 0.082)
0.029
(0.001, 0.057)
0.024
(−0.009, 0.059)
ALL
0.040
(0.011, 0.069)
0.021
(−0.007, 0.047)
0.021
(−0.010, 0.053)
Cortical thickness
Male
−0.031
(−0.085, 0.025)
0.07
−0.018
(−0.065, 0.030)
0.24
−0.029
(−0.088, 0.029)
0.73
Female
−0.078
(−0.123, -0.033)
−0.044
(−0.082, -0.006)
−0.039
(−0.089, 0.007)
ALL
−0.057
(−0.100, -0.014)
−0.032
(−0.069, 0.005)
−0.035
(−0.081, 0.009)
Table shows associations between plasma concentration of 25(OH)D2and 50% tibial pQCT parametres at age 15.5 years. Beta coefficients represent SD change in pQCT parametre per doubling of
vitamin 25(OH)D2. 95% Confidence intervals are presented with respect to the beta coefficients, P value (sex) shows the difference in associations between males and females. Results are also
shown for the following adjustments: minimally adjusted=sex and age at scan; anthropometry adjusted=minimally adjusted+height, logefat mass and lean mass; anthropometry, SES, PA
adjusted=anthropometry-adjusted+maternal and paternal social class, maternal education, and physical activity. All analyses were adjusted for vitamin 25(OH)D3
Osteoporos Int (2012) 23:2117–21282123

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Table 4 Associations between plasma concentration of 25(OH)D3and Pqct parametres
Vitamin 25(OH)D3
Minimally adjusted, N=3,579
(males=1,709)
Anthropometry-adjusted, N=3,579
(males=1,709)
Anthropometry-, SES- and PA-adjusted,
N=2,247 (males=1,203)
Beta
95% CI
P value
(sex)
Beta
95% CI
P value
(sex)
Beta
95% CI
P value
(sex)
Cortical bone
mineral density
Male
−0.028
(−0.124, 0.066)
0.52
−0.020
(−0.110, 0.070)
0.53
0.018
(−0.103, 0.137)
0.94
Female
0.010
(−0.054, 0.072)
0.015
(−0.047, 0.077)
0.013
(−0.065, 0.089)
ALL
−0.007
(−0.064, 0.047)
−0.001
(−0.054, 0.052)
0.016
(−0.054, 0.082)
Cortical bone area
Male
0.062
(−0.043, 0.163)
0.45
0.091
(0.023, 0.162)
0.05
0.100
(0.015, 0.191)
0.22
Female
0.013
(−0.064, 0.087)
0.006
(−0.047, 0.058)
0.031
(−0.034, 0.096)
ALL
0.036
(−0.028, 0.099)
0.045
(0.003, 0.087)
0.061
(0.008, 0.116)
Cortical bone
mineral content
Male
0.057
(−0.056, 0.170)
0.55
0.089
(0.019, 0.162)
0.08
0.105
(0.014, 0.198)
0.23
Female
0.015
(−0.067, 0.093)
0.008
(−0.049, 0.064)
0.034
(−0.036, 0.103)
ALL
0.035
(−0.034, 0.104)
0.045
(0.002, 0.090)
0.066
(0.009, 0.122)
Periosteal circumference
Male
0.002
(−0.095, 0.096)
0.65
0.034
(−0.039, 0.104)
0.13
0.004
(−0.090, 0.099)
0.96
Female
−0.027
(−0.111, 0.054)
−0.038
(−0.096, 0.021)
0.001
(−0.069, 0.068)
ALL
−0.013
(−0.077, 0.047)
−0.005
(−0.052, 0.039)
0.002
(−0.056, 0.059)
Endosteal adjusted for
periosteal circumference
Male
−0.083
(−0.161, -0.007)
0.43
−0.097
(−0.164, -0.031)
0.17
−0.127
(−0.214, -0.045)
0.13
Female
−0.044
(−0.100, 0.014)
−0.036
(−0.087, 0.018)
−0.043
(−0.106, 0.020)
ALL
−0.062
(−0.108, -0.015)
−0.064
(−0.105, -0.022)
−0.080
(−0.132, -0.029)
Cortical thickness
Male
0.117
(0.006, 0.228)
0.39
0.131
(0.039, 0.226)
0.21
0.180
(0.061, 0.306)
0.13
Female
0.054
(−0.029, 0.137)
0.054
(−0.021, 0.126)
0.061
(−0.029, 0.151)
ALL
0.084
(0.014, 0.152)
0.089
(0.031, 0.148)
0.114
(0.041, 0.190)
Table shows associations between plasma concentration of 25(OH)D3and 50% tibial pQCT parametres at age 15.5 years. Beta coefficients represent SD change in pQCT parametre per doubling of
vitamin 25(OH)D3. 95% Confidence intervals are presented with respect to the beta coefficients, P value (sex) shows the difference in associations between males and females. Results are also
shown for the following adjustments: minimally adjusted=sex, season of 25(OH)D3measurement and age at scan; anthropometry-adjusted=minimally adjusted+height, logefat mass and lean mass;
anthropometry-, SES- and PA-adjusted=anthropometry adjusted+maternal and paternal social class, maternal education, and physical activity. All analyses were adjusted for vitamin 25(OH)D2
2124Osteoporos Int (2012) 23:2117–2128

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arity of associations between either seasonally adjusted 25
(OH)D3or 25(OH)D2in any of the models fitted.
Discussion
We report by far the largest prospective cohort study of
relationships between vitamin D status in childhood and
subsequent cortical bone outcomes. 25(OH)D3was posi-
tively related to BMCC as measured by pQCT approxi-
mately 5 years later, which appeared to be secondary to an
increase in CT. This association between 25(OH)D3and
cortical thickness resulted from a decrease in endosteal
expansion, since 25(OH)D3showed an equivalent inverse
association with endosteal adjusted for periosteal circum-
ference. This relationship may also have led to greater
biomechanical strength, in view of the inverse association
observed between 25(OH)D3and buckling ratio. The latter
relationship may have implications in terms of reduced
fracture risk in later life, based on associations between
buckling ratio as measured at the hip in elderly populations,
and subsequent risk of hip fracture [24, 25]. The effect sizes
that we observed were similar in magnitude to that of other
important external influences on skeletal development such
as fat mass, which we have previously reported to influence
cortical bone development [14]. In further analyses, based
on the same study sample, we found that a doubling in fat
mass was associated with a 0.13 SD increase in cortical
thickness (analyses adjusted for age and height), which was
similar to that seen for 25(OH)D3, of which a doubling was
associated with a 0.11 SD increase in cortical thickness.
Identification of 25(OH)D concentrations in childhood
associated with optimal outcomes for bone and other health
outcomes, and how these might translate into public health
recommendations, is a matter of controversy [26]. Argu-
ably, the finding that a doubling in 25(OH)D3is associated
with a 0.11 SD increase in cortical thickness is not a strong
enough effect to justify widespread vitamin D supplemen-
tation in childhood. Since >25% of our study population
had insufficient total 25(OH)D based on the 20 ng ml-1
cutoff [26], this conclusion is likely to apply to other,
predominantly Caucasian, populations with a similarly high
prevalence of vitamin D insufficiency based on this
definition. This may represent a contrast with early life
exposure in utero, when vitamin D status has been
suggested to have major long-term influences on subse-
quent bone development including periosteal growth [27,
28]. On the other hand, 25(OH)D3may have a stronger
Table 5 Differences between
associations of plasma concen-
tration of 25(OH)D2and 25
(OH)D3with pQCT parametres
Table shows the P value for
differences between the associa-
tions of plasma concentration of
25(OH)D2and 25(OH)D3with
50% tibial pQCT parametres at
age 15.5 years (as shown in
Tables 3 and 4, respectively).
Results are also shown for the
following adjustments: mini-
mally adjusted=sex and age at
scan; anthropometry-
adjusted=minimally adjusted
+height, logefat mass and lean
mass; anthropometry-, SES- and
PA-adjusted= anthropometry-
adjusted+maternal and paternal
social class, maternal education,
and physical activity. All results
are adjusted for 25(OH)D2and
D3
Sex Minimally adjusted
P value
Anthropometry-
adjusted P value
Anthropometry-,
SES- and PA-
adjusted P value
N=2,247
(males=1,203)
N=3,579
(males=1,709)
N=3,579
(males=1,709)
BMDC
Male
Female
All
Male
Female
All
Male
Female
All
Male
Female
All
Male
Female
All
Male
Female
All
Male
Female
All
0.62
0.11
0.19
0.25
0.13
0.06
0.22
0.07
0.04
0.77
0.89
0.80
0.01
0.01
0.01
0.02
0.01
0.01
0.03
0.01
0.01
0.58
0.08
0.14
0.05
0.77
0.10
0.03
0.46
0.04
0.98
0.04
0.15
0.01
0.03
0.01
0.01
0.02
0.01
0.03
0.01
0.01
0.31
0.16
0.10
0.07
0.45
0.07
0.03
0.28
0.03
0.53
0.30
0.26
0.01
0.07
0.01
0.01
0.05
0.01
0.01
0.04
0.01
BAC
BMCC
PC
ECPC
CT
BR
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association with cortical outcomes in certain subgroups, in
whomsupplementation may bemorejustifiable.Forexample,
beta coefficients were generally higher in boys, in whom a
doubling in 25(OH)D3 was associated with a 0.18 SD
increase in CT. Moreover, the magnitude of effects that we
observed may have been tempered by aspects of the study
design (see ‘Limitations’ below). Furthermore, whereas
observational studies of this nature provide some information
as to the likely benefits of vitamin D supplementation in
childhood, evidence from randomized controlled trials is
required before definitive conclusions can be drawn.
In those children in whom vitamin D supplementation is
being considered, an important question which follows is
which form of vitamin D is the most effective. In contrast to
the positive associations between 25(OH)D3and cortical
bone outcomes described above, relationships with 25(OH)
D2were null in the case of BMCCand cortical thickness.
Whereas a weak positive association was present between
25(OH)D2and periosteal circumference, there was a weak
inverse association with BMDC, as well as a weak positive
association with buckling ratio suggesting reduced resis-
tance to buckling. Taken together, these findings suggest
that, in contrast to 25(OH)D3, 25(OH)D2, at these concen-
trations, is not associated with an overall benefit in terms of
future cortical bone development, which may have impor-
tant implications in terms of the choice of vitamin D
supplementation in childhood.
To our knowledge, no previous study has examined the
separate relationships between 25(OH)D2, 25(OH)D3and
bone outcomes in childhood. Since 25(OH)D3makes the
major contribution to total 25(OH)D, it is relevant to
compare our findings with those from these previous
studies based on total 25(OH)D. In a prospective study of
171 girls aged 9–15 years, total 25(OH)D was positively
associated with gains in femoral neck BMD over the
following 3 years which may have reflected an influence of
25(OH)D3on cortical thickness as we observed [16]. On
the other hand, our findings contrast with those of a
previous study in which total 25(OH)D was found to be
positively related to BMDCof the radius and tibia in a
cross-sectional study based on 193 10- to 12-year-old girls
[15]. In terms of previous interventional studies, in a recent
study in 20 pairs of peripubertal female twins, D3supple-
ments for 6 months led to an increase in tibial cortical bone
area due to reduced endosteal expansion as assessed by
pQCT [7]. In contrast, in a recent D2supplementation trial
in 73 girls aged 12–14 years, no effect was observed on
pQCT parametres [9]. Although these findings are consis-
tent with our observation of an inverse association between
endosteal adjusted for periosteal circumference and 25(OH)
D3, but not 25(OH)D2, to our knowledge, no previous study
has directly compared the effect of administering these two
forms of vitamin D on cortical bone.
In terms of biological explanations for possible distinct
effects of 25(OH)D2and 25(OH)D3on bone, as suggested
by our results, indirect pathways via PTH may be involved.
Whereas 25(OH)D3 levels are known to be inversely
related to PTH, as confirmed here, an equivalent relation-
ship was not seen for 25(OH)D2, which is consistent with a
previous finding that a large dose of D3decreased PTH in
the elderly, whereas D2 was without effect [29]. Any
tendency for 25(OH)D2and 25(OH)D3to differ in respect
of their relationships with PTH may be partly due to the
fact that D2is less potent than D3: D3and its metabolites
have a higher affinity than D2for hepatic 25-hydroxylase
and vitamin D receptors; D3is not directly metabolised to
24(OH)D as is D2; 25(OH)D2 has a lower affinity for
vitamin D binding protein compared to 25(OH)D3, leading
to faster metabolism and a shorter half life [10]. However,
adjusting our analyses for PTH did not attenuate the
observed association between 25(OH)D3 and endosteal
adjusted for periosteal circumference, suggesting that
differing relationships with PTH are unlikely to explain
the distinct associations between 25(OH)D2, 25(OH)D3and
cortical bone parametres which we observed.
Alternatively, due to the observational nature of this
study, these different associations with 25(OH)D2and 25
(OH)D3may have arisen from confounding. For example,
25(OH)D3levels are determined by sun exposure and diet
that may be affected by a range of factors including SEP
and outdoor physical activity, which may confound
relationships with bone outcomes. Although the association
between 25(OH)D3and endosteal adjusted for periosteal
circumference was unaffected by adjusting for observed
measurements of these additional factors, unmeasured
confounders may be important. For example, D2intake is
related to consumption of fruits and vegetables, which is
positively associated with childhood BMD as measured by
DXA [30]. In addition, fruit and vegetable intake is related
to a ‘prudent’ or ‘healthy’ diet [31], of which intake in
pregnancy is positively associated with BMD in subsequent
childhood [19].
Limitations
In terms of limitations of this study, our pQCT measure-
ments comprised a single slice, namely, the 50% mid-tibia,
which is unable to provide any information about trabecular
bone. In the study of 171 girls aged 9–15 years described
above, the relationship between baseline total 25(OH)D and
subsequent gain in BMD across puberty was particularly
strong at the lumbar spine [16] which is rich in trabecular
bone. Whereas the present study suggests that 25(OH)D
status has minimal effects on cortical bone, it may be that
stronger effects exist for trabecular bone which we were
unable to evaluate here. A further limitation is the relatively
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long interval between measurement of 25(OH)D and mea-
surement of cortical bone from pQCTscans, which may have
reduced the strength of associations observed between these
sets of parametres. Finally, the generalisability of our findings
is limited by the fact that the subset of 3,579 subjects forming
the basis of the present study is likely to differ in important
ways from the original cohort drawn from the general
population. For example, maternal social class in the subset
on which this paper is based was higher compared with those
who were not included (P=0.0001).
In conclusion, we found that in contrast to 25(OH)D2, 25
(OH)D3, as measured in childhood, was positively related
to BMCC, cortical thickness and resistance to buckling as
assessed 5 years later. These different associations suggest
that supplementation with vitamin D3in childhood is likely
to prove more beneficial for subsequent cortical bone
development compared to vitamin D2, presumably reflecting
important differences between the actions of these two
isoforms on bone, the basis of which is currently unclear.
Interventional studies are justified in which effects of these
two forms of vitamin D are directly compared in the same
population, in order to test the conclusions from this
observational study, given that we are unable to exclude
confounding as a possible explanation for our findings.
Acknowledgements
who took part in this study, the midwives for their help in recruiting
them and the whole ALSPAC team, which includes interviewers,
computer and laboratory technicians, clerical workers, research
scientists, volunteers, managers, receptionists and nurses.
The work presented here is funded by a UK Medical Research
Council grant (G0701603). The UK Medical Research Council, the
Wellcome Trust and the University of Bristol provide core support for
ALSPAC. Salary support for AS is provided by Wellcome Trust grant
ref. 079960, which also funded the pQCT scans. DAL works in a
centre that receives core funds from the UK Medical Research Council
(G G0600705) and University of Bristol. No funding body directed
the study or interfered with its conduct and interpretation of results;
the views presented here are those of the authors and not necessarily
any funding body. This publication is the work of the authors who
serve as guarantors for the contents of this paper.
We are extremely grateful to all the families
Conflicts of interest
None.
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
References
1. Mosekilde L (2005) Vitamin D and the elderly. Clin Endocrinol
(Oxf) 62(3):265–281
2. Weaver CM (2007) Vitamin D, calcium homeostasis, and skeleton
accretion in children. J Bone Miner Res 22(Suppl 2):V45–V49
3. Sullivan SS, Rosen CJ, Halteman WA, Chen TC, Holick MF
(2005) Adolescent girls in Maine are at risk for vitamin D
insufficiency. J Am Dietetic Assoc 105(6):971–974
4. Ross AC, Taylor LC, Yaktine AL, Del Valle HB (2010)
Committee to review dietary reference intakes for vitamin D and
calcium. Institute of Medicine Institute of Medicine
5. Winzenberg TM, Powell S, Shaw KA, Jones G (2010) Vitamin D
supplementation for improving bone mineral density in children.
Cochrane Database Syst Rev 10:CD006944
6. El-Hajj Fuleihan G, Nabulsi M, Tamim H et al (2006) Effect of
vitamin D replacement on musculoskeletal parameters in school
children: a randomized controlled trial. J Clin Endocrinol Metab
91(2):405–412
7. Greene DA, Naughton GA (2010) Calcium and vitamin-D
supplementation on bone structural properties in peripubertal
female identical twins: a randomised controlled trial. Osteoporos
Int. Jun 11
8. Viljakainen HT, Natri AM, Karkkainen M et al (2006) A positive
dose–response effect of vitamin D supplementation on site-
specific bone mineral augmentation in adolescent girls: a
double-blinded randomized placebo-controlled 1-year interven-
tion. J Bone Miner Res 21(6):836–844
9. Ward KA, Das G, Roberts SA, et al (2010) A randomized,
controlled trial of vitamin D supplementation upon musculoskeletal
health in postmenarchal females. J Clin Endocrinol Metab. Jul 14
10. Houghton LA, Vieth R (2006) The case against ergocalciferol
(vitamin D2) as a vitamin supplement. Am J Clin Nutr 84(4):694–
697
11. Arunabh S, Pollack S, Yeh J, Aloia JF (2003) Body fat content
and 25-hydroxyvitamin D levels in healthy women. J Clin
Endocrinol Metab 88(1):157–161
12. Parikh SJ, Edelman M, Uwaifo GI et al (2004) The relationship
between obesity and serum 1,25-dihydroxy vitamin D concen-
trations in healthy adults. J Clin Endocrinol Metab 89(3):1196–
1199
13. Waldie KE, Poulton R, Kirk IJ, Silva PA (2000) The effects of
pre- and post-natal sunlight exposure on human growth: evidence
from the southern hemisphere. Early Hum Dev 60(1):35–42
14. Sayers A, Tobias JH (2010) Fat mass exerts a greater effect on
cortical bone mass in girls than boys. J Clin Endocrinol Metab 95
(2):699–706
15. Cheng S, Tylavsky F, Kroger H et al (2003) Association of low
25-hydroxyvitamin D concentrations with elevated parathyroid
hormone concentrations and low cortical bone density in early
pubertal and prepubertal Finnish girls. Am J Clin Nutr 78(3):485–
492
16. Lehtonen-Veromaa MK, Mottonen TT, Nuotio IO, Irjala KM,
Leino AE, Viikari JS (2002) Vitamin D and attainment of peak
bone mass among peripubertal Finnish girls: a 3-y prospective
study. Am J Clin Nutr 76(6):1446–1453
17. Tylavsky FA, Ryder KM, Li R et al (2007) Preliminary findings:
25(OH)D levels and PTH are indicators of rapid bone accrual in
pubertal children. J Am Coll Nutr 26(5):462–470
18. McKay HA, MacLean L, Petit M et al (2005) “Bounce at the
bell”: a novel program of short bouts of exercise improves
proximal femur bone mass in early pubertal children. Br J Sports
Med 39(8):521–526
19. Cole ZA, Gale CR, Javaid MK et al (2009) Maternal dietary
patterns during pregnancy and childhood bone mass: a longitudi-
nal study. J Bone Miner Res 24(4):663–668
20. Clark EM, Ness A, Tobias JH (2005) Social position affects bone
mass in childhood through opposing actions on height and weight.
J Bone Miner Res 20:2082–2089
21. Golding J, Pembrey M, Jones R (2001) ALSPAC — the Avon
Longitudinal Study of Parents and Children: 1. Study methodol-
ogy. Paediatr Perinat Epidemiol 15:74–87
Osteoporos Int (2012) 23:2117–2128 2127

Page 12

22. Morris N, Udrey J (1980) Validation of a self-administered
instrument to assess stage of adolescent development. J Youth
Adolesc 9:271–280
23. Tobias JH, Steer CD, Mattocks C, Riddoch C, Ness AR (2007)
Habitual levels of physical activity influence bone mass in
11 year-old children from the UK: findings from a large
population-based cohort. J Bone Miner Res 22:101–109
24. Kaptoge S, Beck TJ, Reeve J et al (2008) Prediction of incident
hip fracture risk by femur geometry variables measured by hip
structural analysis in the study of osteoporotic fractures. J Bone
Miner Res 23(12):1892–1904
25. Rivadeneira F, Zillikens MC, De Laet CE et al (2007) Femoral neck
BMDisastrongpredictorofhipfracturesusceptibilityinelderlymen
andwomenbecauseitdetectscorticalboneinstability:theRotterdam
study. J Bone Miner Res 22(11):1781–1790
26. Prentice A (2008) Vitamin D deficiency: a global perspective.
Nutr Rev 66(10 Suppl 2):S153–S164
27. Javaid MK, Crozier SR, Harvey NC et al (2006) Maternal vitamin
D status during pregnancy and childhood bone mass at age
9 years: a longitudinal study. Lancet 367(9504):36–43
28. Sayers A, Tobias JH (2009) Estimated maternal ultraviolet B
exposure levels in pregnancy influence skeletal development of
the child. J Clin Endocrinol Metab 94(3):765–771
29. Romagnoli E, Mascia ML, Cipriani C et al (2008) Short and long-
term variations in serum calciotropic hormones after a single very
large dose of ergocalciferol (vitamin D2) or cholecalciferol (vitamin
D3) in the elderly. J Clin Endocrinol Metab 93(8):3015–3020
30. McGartland CP, Robson PJ, Murray LJ et al (2004) Fruit and
vegetable consumption and bone mineral density: the Northern
Ireland Young Hearts Project. Am J Clin Nutr 80(4):1019–
1023
31. Crozier SR, Robinson SM, Borland SE, Inskip HM (2006)
Dietary patterns in the Southampton women’s survey. Eur J Clin
Nutr 60(12):1391–1399
2128Osteoporos Int (2012) 23:2117–2128