Paediatrics and International Child Health

ISSN: 2046-9047 (Print) 2046-9055 (Online) Journal homepage: http://www.tandfonline.com/loi/ypch20

Three-monthly bolus vitamin D supplements

(1000 vs 400 IU/day) for prevention of bone
loss in children with difficult-to-treat nephrotic
syndrome: a randomised clinical trial

Dhurva Nandan Singh, Sriram Krishnamurthy, Sadish Kumar Kamalanathan,

K. T. Harichandrakumar & Palanisamy Sivamurukan

To cite this article: Dhurva Nandan Singh, Sriram Krishnamurthy, Sadish Kumar Kamalanathan,
K. T. Harichandrakumar & Palanisamy Sivamurukan (2018): Three-monthly bolus vitamin D
supplements (1000 vs 400 IU/day) for prevention of bone loss in children with difficult-to-treat
nephrotic syndrome: a randomised clinical trial, Paediatrics and International Child Health, DOI:
10.1080/20469047.2018.1505589

To link to this article: https://doi.org/10.1080/20469047.2018.1505589

Published online: 09 Aug 2018.

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PAEDIATRICS AND INTERNATIONAL CHILD HEALTH
https://doi.org/10.1080/20469047.2018.1505589

Three-monthly bolus vitamin D supplements (1000 vs 400 IU/day) for prevention

of bone loss in children with difficult-to-treat nephrotic syndrome: a randomised
clinical trial
Dhurva Nandan Singha, Sriram Krishnamurthya, Sadish Kumar Kamalanathanb, K. T. Harichandrakumarc
and Palanisamy Sivamurukana
a
Departments of Pediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India;
b
Endocrinology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India; cBiostatistics,
Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India

ABSTRACT ARTICLE HISTORY

Background: Nephrotic syndrome (NS) in children is one of the most common chronic Received 4 July 2017
diseases with a remitting and relapsing course. Glucocorticoids (prednisolone) are considered Accepted 24 July 2018
to be the treatment of choice but are associated with osteoporosis. There are no uniform KEYWORDS
consensus guidelines regarding the optimum dose of calcium and vitamin D for osteoprotec- Nephrotic syndrome;
tion. Some authorities suggest a daily dose of 1000 IU vitamin D for children for osteoprotec- prednisolone;
tion, while others suggest a daily dose of 400 IU. osteoprotection;
Objectives: To compare the efficacy of three-monthly bolus vitamin D supplementation (1000 calcium; vitamin D
vs 400 IU/day) to prevent bone loss in children with difficult-to-treat NS (DTNS).
Methods: In this parallel-group, open-label, randomised clinical trial, 60 children aged 1–18 years
with DTNS [37 with frequently relapsing NS (FRNS), 13 steroid-dependent NS (SDNS) and 10
steroid-resistant NS (SRNS)] were enrolled and block randomised in a 1:1 allocation ratio to
receive 1000 IU/day vitamin D (Group A, n = 30) or 400 IU/day (Group B, n = 30), administered
as three-monthly bolus supplemental doses. In Group A, vitamin D (cholecalciferol,
Calcirol®sachet) was administered as a stat dose of 90,000 IU every three months (calculated
for a period of three months at 1000 IU/day). In Group B, vitamin D (cholecalciferol) was
administered as a stat dose of 36,000 IU every three months (calculated for a period of three
months at 400 IU/day). The proportionate change in bone mineral content (BMC) was studied by
dual energy X-ray absorptiometry (DEXA) scan in both groups after vitamin D supplementation
by analysing the values of BMC obtained 12 months apart (baseline vs. after 12 months).
Results: Sixty children were randomised to receive vitamin D at a dose of either 1000 IU/day
(Group A) or 400 IU/day (Group B). The two groups were comparable in their baseline clinical and
laboratory parameters (including BMC and bone mineral density (BMD)). The distribution of the
three types of NS (FRNS, SDNS and SRNS) was also comparable in both groups. In Group A, there
were 19, 6 and 5 children with FRNS, SDNS and SRNS, respectively, and in Group B there were 18,
7 and 5 children with FRNS, SDNS and SRNS, respectively. The proportionate change in BMC was
not significantly different between the two groups (median proportionate change in BMC in
Group A 13.36% vs 11.59% in Group B, p = 0.22). Overall, BMC increased in both groups (96.7% in
each). Only one (3.3%) patient in each group exhibited bone loss. None of the patients had a
urinary calcium:creatinine ratio >0.2 at the end of the study.
Conclusion: Three-monthly bolus vitamin D dosing regimens administered either as 1000 or
400 IU/day prevent bone loss in children with DTNS who require long-term steroids. Overall,
three-monthly bolus supplemental prophylactic vitamin D, either 1000 or 400 IU/day, would
seem to be an effective strategy for preventing bone loss in children with DTNS, as evidenced
by the extremely low rates of bone loss (3.3% in each group), and is useful for delivering
optimal care to children with DTNS. However, since this study was designed as an equiva-
lence trial and not a superiority trial, further studies are required to demonstrate the super-
iority of the former regimen over the latter.

Abbreviations: BMC, bone mineral content; BMD, bone mineral density; DEXA, dual energy
X-ray absorptiometry; DTNS, difficult-to-treat nephrotic syndrome; FRNS, frequently relapsing
nephrotic syndrome; IFRNS, infrequently relapsing nephrotic syndrome; iPTH, intact parathyr-
oid hormone; NS, nephrotic syndrome; SDNS, steroid-dependent nephrotic syndrome; SRNS
steroid-resistant nephrotic syndrome.

Introduction relapsing course. Glucocorticoids (prednisolone) are the

treatment of choice [1]. Steroid-sensitive NS is sub-
Nephrotic syndrome (NS) is one of the most common
grouped on the basis of response to corticosteroids as
chronic diseases in children with a remitting and

CONTACT Sriram Krishnamurthy drsriramk@yahoo.com

© 2018 Informa UK Limited, trading as Taylor & Francis Group
2 D. N. SINGH ET AL.
infrequently relapsing NS (IFRNS), frequently relapsing NS
(FRNS) and steroid-dependent NS (SDNS). A relatively
smaller proportion of patients who do not respond to
steroids are classified as steroid-resistant NS (SRNS). On
the basis of disease burden, FRNS, SDNS and SRNS are
considered to be difficult-to-treat NS (DTNS).
Glucocorticoids have been associated with osteoporosis
[2–4]. Although their adverse effects on bone health have
been well described in children with NS [2–4], there are
no uniform consensus guidelines on the optimum dose
of calcium and vitamin D for preventing bone loss in this
patient population.
Some have suggested a daily dose of 1000 IU for
children to prevent bone loss, while others have sug-
gested a daily dose of 400 IU [5–7]. The 1000 IU dose has
been used recently in a randomised trial in 41 steroid-
naïve pre-pubertal children who received prednisolone
for 12 weeks [2]. Patients were randomised to the inter-
vention group (IG, 1000 IU/day vitamin D and elemental
calcium 500 mg/day) or the control group (CG). Bone
mineral content (BMC) and bone mineral density (BMD)
were estimated at baseline and at 12 weeks. Children in
the IG showed an increase of 11.2% in BMC versus an
8.9% decline in the CG (p < 0.0001). The 400 IU dose was
used in another clinical trial in 40 children with NS who
received prednisolone (2 mg/kg/day for 4 weeks then
on alternate days at the same dose for 4 weeks) [3]. The
patients were randomised to treatment (vitamin D 400
IU plus calcium 1 g daily) and non-treatment groups.
BMD, serum calcium, phosphorus, alkaline phosphatase
and urinary calcium and phosphorus excretions were
analysed at the outset and two months after treatment.
BMD was significantly decreased in both the treatment
group [0.54 (0.15) to 0.51 (0.1) g/cm2, p = 0.001] and
non-treatment group [0.52 (0.18) to 0.45 (0.16) g/cm2,
p < 0.001) group. However, the percentage of BMD loss
was significantly lower in the treatment group than in
the non-treatment group [4.6 (2.1)% vs 13.0 (4.0) %,
respectively, p < 0.001). Thus, there is uncertainty
about the optimal dose of vitamin D to prevent bone
loss in childhood NS.
A recent randomised clinical trial in India demon-
strated that bolus vitamin D at 1000 IU/day is marginally
more effective than bolus vitamin D at 400 IU/day for
osteoprotection in children with new-onset and IFRNS
who are known to require steroids for less than three
months [8]. However, no randomised controlled trials
are available which compare different doses of vitamin
D for osteoprotection in children with DTNS who are
receiving long-term steroids (>3 months). To aid plan-
ning strategies for osteoprotection in DTNS, studies of
BMC in these groups of patients are required.
This clinical trial was undertaken to compare the
efficacy of two vitamin D dosing regimens in chil-
dren with DTNS receiving steroids for more than
three months and to examine whether the differ-
ence between the two vitamin D doses is really
significant in terms of preventing bone loss. It was
hypothesised that 1000 is equivalent to 400 IU/day
vitamin D in preventing bone loss in children with
NS on long-term steroids for managing DTNS when
administered on a three-monthly basis as a bolus
supplemental dose.
Methods
This randomised, parallel-group, open-label, equiva-
lence clinical trial was conducted at the paediatric
nephrology outpatient clinic of the Jawaharlal
Institute of Postgraduate Medical Education and
Research, a tertiary-care hospital in Pondicherry,
south India from September 2015 through February
2017. Sixty children with DTNS were recruited and
allocated to one of the two treatment groups (1000
and 400 IU/day vitamin D) in a ratio of 1:1. The first
author (DNS) administered the vitamin D as a three-
monthly bolus (1000 IU/day and 400 IU/day for
Groups A and B, respectively) under the supervision
of the corresponding author (SK) over a 12-month
period. The trial was registered in the Clinical Trial
Registry of India (CTRI/2015/10/009984). Informed
consent was obtained from the parents before sub-
jects were enrolled into the study.
Objectives
The primary objective was to compare the propor-
tionate change in BMC after one year between chil-
dren with DTNS receiving 1000 IU/day vitamin D
versus those receiving 400 IU/day (in the form of
three-monthly bolus cumulative doses over a 12-
month period). The secondary objective was to com-
pare the proportionate change in BMD, serum cal-
cium, phosphate, alkaline phosphatase, intact
parathyroid hormone (iPTH) and 25-hydroxy vitamin
D levels between the two groups after one year.
Inclusion and exclusion criteria
Children aged 1–18 years with DTNS (FRNS, SDNS and
SRNS) considered to have DTNS were enrolled. Children
with new-onset NS, IFRNS and secondary NS (e.g. lupus
nephritis, IgA nephropathy, Henoch–Schönlein purpura
nephritis), those already receiving calcium or vitamin D
supplements or any drug interfering with vitamin D or
calcium metabolism (e.g. phenytoin, phenobarbitone,
methotrexate), those with a history of hypocalcaemic
seizures, tetany, rickets or fractures and children with
renal calculi, chronic renal failure or chronic liver disease
were excluded. Children already enrolled once in the
study were not enrolled again during a further relapse.
Infants with NS were not included in the study since
they have a high risk of having genetic mutations and
an increased likelihood of not responding to steroids [1].
 PAEDIATRICS AND INTERNATIONAL CHILD HEALTH 3

Randomisation, allocation concealment In Group A, vitamin D (cholecalciferol, Calcirol®sachet)

was administered as a stat dose of 90,000 IU every three
Block randomisation using nine blocks of four sizes
months, calculated for a period of three months as 1000
(two, four, six and eight) was generated using random
IU/day. In Group B, vitamin D (cholecalciferol) was admi-
allocation software version 2.0 (Informer Technologies
nistered as a stat dose of 36,000 IU every three months,
Inc.) to allocate the subjects into two groups (Group A
calculated for a period of three months as 400 IU/day.
1000 IU/day vitamin D; Group B 400 IU/day vitamin D)
For appropriate dosing, each Calcirol® sachet (con-
in an allocation ratio of 1:1 by a person not directly
taining 60,000 IU cholecalciferol) was dissolved in
involved with data collection, analysis or interpreta-
10 mL of distilled water. This created a suspension
tion. The randomisation list was concealed from the
with a concentration of 1 mL = 6000 IU cholecalci-
investigators undertaking the study. The patients
ferol, and 15 mL of this suspension was administered
were not stratified at trial entry into different clinical
to Group A. The corresponding dose in Group B was
subgroups of DTNS or age. Allocation was concealed
6 mL. Generic calcium carbonate was also given to
by placing individual assignments (folded twice) in
both study groups in a tablet of 250 mg elemental
serially numbered, opaque-sealed envelopes by some-
calcium twice daily for 12 months.
one not involved in the trial.
Subjects who forgot to take the prednisolone in
Patients were assessed for eligibility and enrolled
the morning were advised to take the tablet by the
sequentially by DNS under the supervision of the corre-
evening to ensure the correct cumulative dose per
sponding author (SK). The envelopes were opened by
day. If it was missed for a full day, it was considered
DNS only after obtaining written, informed consent for
to be protocol violation. Taking a wrong dose of
enrolment after which the subjects were assigned by
prednisolone was also considered to be protocol vio-
DNS to the allocated intervention group. DNS and SK
lation. No changes were made to the trial design after
could not be blinded as they had to check the dose of
it was commenced. Although BMD was estimated,
vitamin D administered to subjects during enrolment.
BMC was used as the main marker of calcium content.
However, the statistician was blinded to the assigned
intervention until initial analysis and preparation of the
first draft of the manuscript. Laboratory investigations
The following investigations were performed in both
dosing groups on enrolment: DEXA scan, urine protein:
Interventions and management of nephrotic
creatinine ratio, serum albumin, creatinine, cholesterol,
syndrome
25-hydroxy cholecalciferol, iPTH, calcium, phosphate,
Nephrotic syndrome was diagnosed, classified and alkaline phosphatase, age, gender, blood pressure,
managed according to the guidelines of the Indian height, weight, body mass index and clinical phenotype
Paediatric Nephrology Group, Indian Academy of of NS and recorded at that time on a pre-designed
Paediatrics [1]. Children with FRNS, SDNS and SRNS proforma. Z-scores for height were recorded from the
received appropriate immunosuppressants (e.g. leva- following source: www.int/growthref/tools/en/
misole, cyclophosphamide, mycophenolate mofetil,
cyclosporin) and glucocorticoids, as per these guide-
Biochemical investigations
lines [1,9]
Five mL of intravenous blood was drawn from subjects
to estimate the serum 25-hydroxy-cholecalciferol, iPTH,
Study groups calcium, phosphate and alkaline phosphate at 0 month
(baseline) and at 12 months. Serum 25-hydroxy chole-
Group A were children with DTNS receiving elemental
calciferol levels were estimated by chemiluminescence.
calcium 500 mg per day with 1000 IU vitamin D per
day as three-monthly bolus supplemental doses over
a 12-month period. Group B were children with DTNS DEXA scan (dual energy x-ray absorptiometry)
receiving elemental calcium 500 mg per day with 400
The Department of Endocrinology estimated BMC and
IU vitamin D per day as three-monthly bolus supple-
BMD in the lumbar spine (L1–L4) in both groups at
mental doses over a 12-month period.
0 months (baseline) and after 12 months of vitamin D
supplementation. This was undertaken using a DEXA
Vitamin D dosing in the two groups scanner (bone densitometer, Discovery Wi, Hologic
Company, USA). The instrument was calibrated with
After confirming the diagnosis of DTNS, patients were phantoms supplied by the manufacturer and mea-
commenced on prednisolone (2 mg/kg/day) and/or surements were precise to within 1%. If BMC fell by
an alternative regimen and randomised into the more than 1% from baseline, it was considered to be
aforementioned study groups (1000 vs 400 IU/day). bone loss.
4 D. N. SINGH ET AL.
Follow-up
Parents were taught to perform and grade urine albu-
min with a dipstick (once a day, in a freshly voided early
morning urine specimen) and to keep a diary at home.
During enrolment, parents whose children were in
relapse were instructed on changing the steroid dose
to alternate days after attaining remission. Children
were followed up monthly for 12 months in the paedia-
tric nephrology clinic. At the end of the study, BMC,
BMD, serum calcium, phosphate, alkaline phosphatase
and the urine calcium:creatinine ratio were recorded in
both groups. Cumulative prednisolone received and the
proportionate change in BMC, BMD, serum 25-hydroxy
cholecalciferol, iPTH, calcium, phosphate and alkaline
phosphatase were also calculated at the end of the
study in both groups. No changes were made to the
trial’s intended aims after the trial was commenced.
Data on clinical, biochemical profile, BMC and BMD
values were recorded in a structured proforma.
Definitions [1,10].
Nephrotic syndrome. Proteinuria (urine protein:
creatinine ratio >2.0 or urine albumin 3+/4+ on dip-
stick) and hypo-albuminaemia (<2.5 g/dL), hypercho-
lesterolaemia (serum cholesterol >200 mg/dL).
Difficult-to-treat nephrotic syndrome (DTNS).
Steroid-dependent, frequently relapsing and steroid-
resistant clinical subtypes of idiopathic NS.
Frequently relapsing nephrotic syndrome (FRNS).
Two or more relapses in the first 6 months or more
than three relapses in any 12 months.
Steroid-dependent nephrotic syndrome (SDNS).
Two consecutive relapses while on alternate-day ster-
oids or within 14 days of its discontinuation.
Steroid-resistant nephrotic syndrome (SRNS). No
remission despite prednisolone at a dose of 2 mg/
kg/day for 4 weeks.
Remission. Urine albumin nil or trace (or protei-
nuria <4 mg/m2/h) for three consecutive early morn-
ing specimens.
Relapse. Urine albumin 3+ or 4+ (or proteinuria
>40 mg/m2/h) for thee consecutive early morning speci-
mens having previously been in remission.
Complete remission in SRNS. The presence of trace
or negative proteinuria (by dipstick test) or spot urine
protein to creatinine ratio (Up/Uc) < 0.2 mg/mg.
Partial remission in SRNS. 1+ to 2+ proteinuria (or
Up/Uc of 0.2–2), serum albumin >2.5 g/dL and no
oedema.
No response in SRNS. 3+ to 4+ proteinuria (or Up/
Uc > 2), serum albumin <2.5 g/dL or oedema.
Proportionate change in BMC
ðFollow  up BMCBaseline BMCÞ
¼ x100
Baseline BMC
Proportionate change in BMD
ðFollow  up BMDBaseline BMDÞ
¼ x100
Baseline BMD
All other proportionate changes [in calcium, phos-
phate, alkaline phosphatase, body mass index (BMI)]
between the two groups were calculated similarly.
Bone mineral content (BMC). The mineral quantity
measured in grams in a given projection of bone (L1–L4)
on DEXA scan.
Bone mineral density (BMD). Obtained by dividing
the BMC by the bone area (as per the values obtained
on DEXA scan); expressed as g/cm2.
Sample size
Sample size was estimated with an expected mean
proportionate change in BMC between the treatment
regimens as 10% with a standard deviation (SD) of
16.5% and 8.25% in each regimen [2,3] with a 5% level
of significance and 90% power. The minimum sample
size required for the study was 24 in each group and
was further increased as 30 per group with an
expected attrition rate of 20%.
Statistical analysis
Data were expressed as mean and SD or median and
interquartile range (IQR), as appropriate. Means and
SD were compared using Student’s t-test, and med-
ians and IQR were compared using the non-para-
metric Mann–Whitney U-test. Proportions were
compared using the χ2 test; p < 0.05 was considered
significant. Rosenthal’s formula [11] was used to cal-
culate the effect sizes (r values) for the non-parametric
Mann–Whitney U-test. The r value thus calculated was
interpreted as follows: 0.1–0.3 small effect, 0.31–0.5
medium effect, >0.5 large effect. Data were evaluated
using SPSS version 20.0 (SPSS Inc., Chicago). Intention-
to-treat analysis was undertaken to study the out-
come variables. Subgroup analysis for proportionate
change in BMC in FRNS, SDNS and SRNS was per-
formed separately.
Ethics approval
The study was approved by the Ethics Committee
(human studies) of JIPMER, Approval No. JIP/IEC/
2015/15/587). It was performed in accordance with
the ethics principles for medical research in human
subjects of the World Medical Association, Declaration
of Helsinki.
 PAEDIATRICS AND INTERNATIONAL CHILD HEALTH 5

Results (96.7% in each group). Only one (3.3%) patient in

each treatment group exhibited bone loss. None of
Children were recruited and followed up from
the patients had a urinary calcium:creatinine ratio
September 2015 through February 2017. Seventy chil-
>0.2 at the end of the study.
dren with DTNS (FRNS/SDNS/SRNS) were assessed for
Table 3 shows the BMC, BMD, 25-OH vitamin D and
eligibility. Ten were excluded (Figure 1). Therefore, 60
other laboratory parameters measured 12 months
were randomised to receive either 1000 (Group A) or
apart. BMC, BMD and 25-OH vitamin D levels had
400 IU/day vitamin D (Group B). The two groups were
increased significantly by the end of the study.
comparable in terms of their baseline clinical and
Appendix Tables A1–A3 compare outcomes in both
laboratory parameters (including BMC and BMD)
groups for the FRNS, SDNS and SRNS cases. The pro-
(Table 1). None of the patients had hypercalciuria
portionate changes in BMC and BMD were not statis-
(urinary calcium:creatinine ratio >0.2). Two of the 60
tically different between the two groups.
children developed tetany, one took wrong doses of
calcium, three did not attend for the second DEXA
scan and one SRNS child died of spontaneous bacter-
Discussion
ial peritonitis (Figure 1). No patient failed to take the
medication. A total of 53 children completed the This randomised clinical trial evaluated the effects on
study. BMC of two dosing regimens of three-monthly prophy-
Table 2 compares outcome (intention to treat ana- lactic bolus vitamin D supplemental doses administered
lysis) in the two groups. The primary outcome vari- on a cumulative basis as 1000 or 400 IU/day during
able, the proportionate change in BMC, was not steroid therapy in children with DTNS. The baseline
significantly different between the two groups (med- characteristics in both groups were comparable, indicat-
ian proportionate change in BMC 13.36% vs 11.59%, ing satisfactory randomisation. The study shows that
p = 0.22). Overall, BMC increased in both groups both dosing regimens are effective in preventing bone

Figure 1. Inflow of patients into the study.

6 D. N. SINGH ET AL.

Table 1. Demographic characteristics in the study subjects.

Variables Group A, 1000 IU vitamin D/day, n = 30 Group B, 400 IU vitamin D/day, n = 30 p-value
Age at enrolment, yrs 7.53 (3.59) 7.60 (4.30) 0.95
Age at onset of NS, yrs 4.92 (2.62) 4.70 (2.89) 0.76
Duration of NS, yrs 31.40 (17.80) 34.80 (22.80) 0.52
Male 17 (56.7%) 15 (50.0%) 0.79
Weight, kg 24.71 (10.44) 24.36 (12.35) 0.91
Height, cm 121.32 (19.91) 117.72 (19.91) 0.56
Height Z-score −0.32 (1.37) −1.17 (1.56) 0.03
Body mass index, kg/m2 15.93 (2.61) 16.68 (3.69) 0.37
Body surface area, m2 0.90 (0.26) 0.88 (0.32) 0.78
Clinical phenotype of NS (%)
FRNS 19 (63.3) 18 (60.0) 0.95
SDNS 6(20.0) 7 (23.3)
SRNS 5(16.7) 5 (16.7)
Immunosuppressants received (%)
Levamisole with prednisolone 13 (43.3) 14(46.7) 0.50
Cyclosporin with prednisolone 5 (16.7) 5(16.7)
Oral cyclophosphamide with prednisolone 4 (13.3) 4(13.3)
I/V cyclophosphamide with prednisolone 0 2(06.7)
MMF 8 (26.7) 4(13.3)
Rituximab followed by MMF 0 1(03.3)
Cumulative prednisolone dose per patient, mg/kg 194.14 (102.31) 187.76 (99.70) 0.67
Baseline bone mineral content, g 17.32 (6.99) 16.81 (9.67) 0.82
Baseline bone mineral density, g/cm2 0.54 (0.12) 0.51 (0.15) 0.39
Serum creatinine, µmol/L 56.58 (16.80) 55.69 (12.38) 0.90
eGFR (by Schwartz formula), mL/min/1.73 m 84.25 (24.53) 78.89 (16.48) 0.33
Serum albumin, g/L 36 (11.7) 36.9 (10.3) 0.76
Serum cholesterol, mmol/L 6.94 (3.44) 6.74 (2.61) 0.80
Serum calcium, mmol/L 2.38 (0.20) 2.35 (0.24) 0.56
Serum phosphate, mmol/L 1.48 (0.22) 1.53 (0.31) 0.51
Serum alkaline phosphatase, µkat/L 10.98 (3.35) 10.21 (3.07) 0.37
Serum 25-hydroxycholecalciferol, nmol/L 36.54 (16.5) 32.17 (14.63) 0.29
iPTH, ng/L 33.93 (21.98) 45.56 (38.41) 0.17
Urine protein:creatinine ratio 0.53 (2.56) 0.57 (2.55) 0.95
All values are mean (SD) or n (%); eGFR, estimated glomerular filtration rate; NS, nephrotic syndrome; FRNS, frequently relapsing NS; SDNS, steroid-
dependent nephrotic syndrome; SRNS, steroid-resistant nephrotic syndrome; MMF, mycophenolate mofetil; iPTH, intact parathyroid hormone.

Table 2. Comparison of proportionate change in outcome variables by intention-to-treat analysis.

Effect size for Mann–Whitney
Group A, 1000 IU vitamin D/day, Group B, 400 IU vitamin D/day, U-test p-
Characteristics (%) n = 30 n = 30 (r value) value
Bone mineral content 13.36 (6.88–20.34) 11.59 (8.47–17.43) 0.01 0.22
Bone mineral density 5.30 (3.93–9.25) 5.35 (2.29–11.70) <0.01 0.66
Serum 25-hydroxy 31.11 (5.35–63.55) 43.82 (21.12–144.15) 0.03 0.20
cholecalciferol
iPTH −22.63 (−51.62–37.63) −26.63 (−61.75–28.22) 0.01 0.22
Serum calcium −1.50 (−8.82–7.72) −2.04 (−8.41–6.83) <0.01 0.74
Serum phosphate 1.06 (−13.84–13.76) −2.04 (−10.04–15.87) <0.01 0.57
Serum alkaline phosphatase −13.07 (−34.53–6.26) −11.20 (−27.38–1.60) <0.01 0.63
BMI −3.52 (−10.43–1.66) −1.34 (−6.23–10.09) 0.02 0.76
All values median (interquartile range). iPTH, intact parathyroid hormone; a minus sign indicates the proportionate fall in the respective parameters.

loss, as evidenced by the positive proportionate change
in BMC over a one-year period in both treatment arms as
well as the extremely low rates of bone loss (3.3% in
each group). The proportionate changes in serum vita-
min D, calcium, phosphate and alkaline phosphatase
levels were also statistically equivalent in the two
groups. Both groups tolerated the vitamin D therapy
satisfactorily without adverse effects such as hypercal-
ciuria or hypercalcaemia. This study also provides impor-
tant insights into the longitudinal changes in BMC and
BMD in children with DTNS as well as the role of bolus
vitamin D and calcium supplements in preventing bone
loss in this patient population.
Glucocorticoids, the cornerstone of management of
childhood NS, are known to have negative effects on
BMD [12] by promoting apoptosis of osteoblasts, stimu-
lating osteoclasts and decreasing growth hormone
secretion, thereby decreasing bone mineralisation and
resulting in osteoporosis [13–15]. Insult to the growing
skeleton during infancy or childhood can be harmful,
predisposing to bone mineralisation defects and frac-
tures. It is, therefore, essential to develop optimal stra-
tegies for osteoprotection in these children.
In adult NS, prophylactic vitamin D doses of
1000–2000 IU/day have been proposed [16]. The
Indian Paediatric Nephrology group guidelines recom-
mends that calcium be administered (250–500 mg daily)
to a child with NS who is on prednisolone for more than
three months [1]. Interventional studies of vitamin D
supplementation in paediatric NS for osteoprotection

Table 3. Comparison of BMC, BMD and laboratory parameters in the study groups before and after calcium and vitamin
supplements.
All subjects, n = 60
1-year fol-
Baseline low-up
Characteristics n = 60 n = 53
Bone mineral content, g 17.38 (8.55) 19.46 (9.26)
Bone mineral density, g/cm2 0.53 (0.13) 0.56 (0.14)
Serum 25-hydroxy 34.47 (15.70) 45.40 (14.43)
cholecalciferol, nmol/L
iPTH, ng/ml 40.48 (32.86) 30.29 (22.65)
Serum calcium, mmol/L 2.36 (0.21) 2.30 (0.18)
Serum phosphate, mmol/L 1.51 (0.24) 1.56 (0.55)
p-values in bold type are statistically significant.
have used various doses such as 200 IU/day [4], 400 IU/
day [3,8], 800 IU/day [17] and 1000 IU/day [2,8].
However, there are no randomised controlled trials
(RCT) which compare two different bolus supplemental
dosing regimens of vitamin D for osteoprotection in
children with DTNS on prolonged steroids.
Some interventional studies have evaluated the effi-
cacy of calcium and vitamin D supplementation in pre-
venting corticosteroid-induced bone loss in children
with NS [2–4,8,17], three of which were randomised
controlled trials which excluded children with DTNS. In
an RCT in Turkey, 40 children with new-onset and IFRNS
on prednisolone therapy were randomised into a treat-
ment group (vitamin D 400 IU plus calcium 1 g per day)
and a non-treatment group (no vitamin D or calcium
supplements) [3]. Bone mineral density decreased sig-
nificantly in both groups; however, the BMD decrease
was lower in the treatment group [4.6% (2.1) vs 13.0%
(4.0) %] than in the non-treatment group, and the dif-
ference was statistically significant. In an RCT in India, 41
children with new-onset NS were randomised into an
intervention group who received vitamin D 1000 IU plus
calcium 500 mg per day and a control group who did
not receive vitamin D or calcium [2]. A mean increase in
BMC of 11.2% was seen in the intervention group and in
the control group there was an 8.9% decrease in BMC, a
statistically significant difference.
In a recent RCT in India, 28 children with new-onset
NS and 64 with IFRNS were block-randomised in a 1:1
allocation ratio into two interventional groups. Group A
received 1000 IU/day vitamin D and Group B received
400 IU/day vitamin D [8]. Vitamin D was administered as
an initial single bolus supplemental dose. The propor-
tionate change in BMC was analysed after completion of
steroid therapy. There was a greater median proportion-
ate change in BMC in the children who received 1000 IU/
day vitamin D [3.25% (−1.2 to 12.4)] than in those who
received 400 IU/day vitamin D [1.2% (−2.5 to 3.8)]
(p = 0.048); it was concluded that 1000 IU/day dose is
marginally more effective than 400 IU/day in children
with new-onset and IFRNS.
In a prospective, non-randomised interventional
study of 88 Indian children with relapsing NS receiving
prednisolone therapy, calcium and vitamin D3 in doses
PAEDIATRICS AND INTERNATIONAL CHILD HEALTH 7
Group A, 1000 IU vitamin D/day, n = 30 Group B, 400 IU vitamin D/day, n = 30
p- Baseline 1-year follow-up p- Baseline 1-year follow-up p-
value n = 30 n = 28 value n = 30 n = 25 value
<0.01 17.32 (6.99) 19.78 (7.27) <0.01 16.81 (9.67) 19.12 (11.16) <0.01
<0.01 0.54 (0.12) 0.58 (0.11) <0.01 0.51 (0.15) 0.55 (0.16) <0.01
<0.01 36.54 (16.50) 44.55 (15.67) 0.02 32.17 (14.63) 46.35 (13.13) 0.02
0.05 33.93 (21.98) 30.76 (27.93) 0.60 45.56 (38.41) 29.76 (15.26) 0.04
0.11 2.38 (0.20) 2.31 (0.19) 0.17 2.35 (0.24) 2.29 (0.18) 0.37
0.43 1.48 (0.22) 1.57 (0.56) 0.44 1.53 (0.31) 1.56 (0.54) 0.79
of 500 mg/day and 200 IU/day, respectively, resulted in
an 8.19% increase in BMD in the lumbar spine 6 months
after administration. A non-randomised study in Poland
of 25 children aged 5–17 years with NS who received
prednisolone and vitamin D (800 IU/day), total body
BMD (TB-BMD) was assessed at commencement of the
study and after 1 year of therapy. A significant decrease
in the TB-BMD Z-score and 25-hydroxy vitamin D3 serum
levels was seen, despite the administration of vitamin D
and supplementation with vitamin D at a dose higher
than 800 IU/day, which has been recommended to pre-
vent osteopenia [17].
The above studies [2–4,8,17] were of doses of vita-
min D ranging from 200 IU/day to 1000 IU/day for
osteoprotection. In our study, vitamin D supplements
were administered at doses of 1000 IU/day and 400
IU/day, and there were median increases in BMC of
13.36% and 11.59% in Groups A and B, respectively.
The proportionate change in BMC was measured as
the primary outcome, not BMD, as in the previous
study [8]. Although used in the diagnosis of osteo-
porosis in adults, BMD is not a measure of true volu-
metric bone density in growing children since it is
obtained by dividing BMC by bone area and therefore
excludes the depth (girth) value. During glucocorti-
coid therapy, all three components of skeletal mineral
acquisition (length, breadth and depth) are affected.
BMC is considered to be an acute indicator of bone
mass in the paediatric population as it takes account
of all three and therefore more accurately reflects
skeletal mineral status than BMD [2,3,18,19]. This
approach has been adopted in previous studies [2,8]
Several cross-sectional studies of children with NS
demonstrate a prevalence of 25-hydroxy vitamin D
deficiency of 20–100% [20–24] owing to the excretion
of vitamin D-binding protein in urine. However, it is
notable that serum 25-hydroxy vitamin D levels do not
accurately represent vitamin D stores in nephrotic chil-
dren. It is well known that in NS, loss of protein-bound
25-hydroxy vitamin D causes low serum 25-hydroxy
vitamin D despite normal 1,25-dihydroxy vitamin D
levels. This can make it difficult to estimate serum 25-
hydroxy vitamin D in nephrotic children [2].
Nevertheless, at the end of this study, vitamin D levels
8 D. N. SINGH ET AL.
had increased in both groups. There was a non-signifi-
cant fall in serum calcium, probably because the study
was not sufficiently powered to examine this aspect.
Vitamin D was administered in a single bolus dose
instead of daily. A cost-effective method of administering
vitamin D is to give doses of up to 1,000,000 IU every two-
to-three months [25]. This regimen is safe because of the
wide therapeutic margin and its known safety profile.
Adherence to daily doses has been reported to be poor
in many large clinical trials [26]. Poor adherence has been
associated with difficulty in swallowing a number of
concurrent medications and the patient’s attitude to vita-
min D supplementation [27]. Vitamin D in a large bolus
dose has been associated with greater adherence than
daily and monthly dosing regimens and has the potential
to lead to sustained improvements in serum vitamin D
and parathyroid hormone (PTH) levels [28]. The sustained
effect of high-dose vitamin D has been attributed to its
long half-life. Upon ingestion, vitamin D is either con-
verted into 25(OH)D or redistributed into fat from which
it is slowly released. A study in Israeli adults estimated
that daily, weekly and monthly vitamin D would produce
the same circulating levels of vitamin D over an equiva-
lent period of time [29]. In the present study, vitamin D
doses of up to 1000 IU/day were administered since this
would not be expected to increase vitamin D2 to toxic
levels of >100 ng/mL, and no adverse effects were seen.
Such a methodology has been adopted earlier [2].
This is the first RCT to compare two doses of vitamin D
for osteoprotection in children with DTNS. Unlike some
previous studies [3,4,17], BMC was used instead of BMD
to assess bone mass because BMC is a more sensitive
marker for skeletal mineral derangement than BMD in
growing children [2]. The study also provides important
insights into longitudinal changes in BMC and BMD in
children with DTNS and the role of bolus vitamin D and
calcium supplements in preventing bone loss in these
patients.
The limitations of the study are as follows. Firstly, the
study enrolled three groups of DTNS (FRNS, SDNS and
SRNS). Nevertheless, their representation in both treat-
ment arms was statistically equivalent, thereby allowing
rational comparison of data. Secondly, the study was
designed as an equivalence trial, not a superiority trial,
and was not sufficiently powered to examine the differ-
ences in proportionate change in BMC on subgroup
analysis for the type of NS. Thirdly, serum 1,25 dihy-
droxycholecalciferol levels were not measured which
would have been a better predictor of vitamin D status
in nephrotic children than 25-hydroxy-vitamin D levels.
Fourthly, DEXA scans were undertaken only at 12-
months intervals. In our previous report [8], BMC in
new-onset and IFRNS decreased in one-third of cases
when BMC after steroid therapy was compared with
BMC before steroid therapy. If DEXA scans had been
performed at more frequent intervals, data regarding
the trend of BMC (rise or fall) could have been obtained.
Finally, it must be acknowledged that it is difficult to
ascertain the extent to which BMC increased over one
year owing to vitamin D supplementation since some
increase is expected because of normal growth.
Three-monthly bolus vitamin D doses, either 1000
IU/day or 400 IU/day, help prevent bone loss in children
with DTNS who require long-term steroids. Overall, the
administration of three-monthly bolus supplemental
prophylactic vitamin D of 1000 IU/day or 400 IU/day
seems to be an effective strategy for osteoprotection in
children with DTNS, as evidenced by the extremely low
rates of bone loss (3.3% in each group) and is useful for
delivering optimal care to children with DTNS.
However, since this study was designed as an equiva-
lence trial, not a superiority trial, further studies of larger
samples are required to demonstrate the superiority of
the former regimen over the latter.
Protocol accessible at Clinical Trial Registry of India (CTRI)
www.ctri.nic.in (Registration No. CTRI/2015/10/009984). Trial
registered prospectively as ‘Comparison of the efficacy of
two dosing regimens of vitamin D supplementation for
osteoprotection in children with difficult nephrotic syn-
drome: an open-label randomized clinical trial.’
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The study was funded by an intramural grant from the
authors’ institution to Sriram Krishnamurthy, which is grate-
fully acknowledged.
Notes on contributors
Dhurva Nandan Singh is a Resident in the Department of
Pediatrics, JIPMER, Pondicherry, India. His areas of interest
include pediatric nephrology and academic general
pediatrics.
Sriram Krishnamurthy is an Additional Professor of
Pediatrics at JIPMER, Pondicherry, India. He is a pediatric
nephrologist who looks after a significant number of chil-
dren with renal ailments including nephrotic syndrome,
glomerulopathies, renal tubulopathies, nephrolithiasis,
acute kidney injury, congenital anomalies of the kidney
and urinary tract and chronic kidney disease. He has
authored many papers in national and international
journals.
Sadish Kumar Kamalanathan is an Additional Professor of
Endocrinology at JIPMER, Pondicherry. His research interests
include vitamin D metabolism and Bone Densitometry.
KT Harichandrakumar is a biostatistician at JIPMER,
Pondicherry who contributed significantly to the study
results.
Palanisamy Sivamurukan is a Senior Resident in the
Department of Pediatrics, JIPMER, Pondicherry, India. His
areas of interest include pediatric nephrology and academic
general pediatrics.

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10 D. N. SINGH ET AL.

Appendix

Table A1. Subgroup analysis comparing the proportionate change in outcome variables in the two study groups in children with
frequently relapsing nephrotic syndrome.
Group A, 1000 IU vitamin D/ Group B, 400 IU vitamin D/ Effect size for Mann–Whitney
Characteristics day, n = 18 day, n = 16 U-test (r value) p-value
BMC, % 13.36 (7.27–20.81) 11.99 (8.61–19.44) <0.01 0.44
BMD, % 5.30 (3.40–10.55) 6.94 (2.23–14.72) <0.01 0.63
Serum 25-hydroxy cholecalciferol, % 42.11 (10.76–82.24) 50.33 (19.87–186.64) 0.03 0.16
iPTH, % −20.98 (−54.47 to 141.58) −28.89 (−56.73 to 16.97) 0.03 0.12
Serum calcium, % −0.98 (−8.07 to 8.13) −2.13 (−7.09 to 6.21) <0.01 0.99
Serum phosphate, % 3.49 (−11.01 to 12.72) −0.31 (−16.42 to 18.77) <0.01 0.49
Serum alkaline phosphatase, % −8.58 (−33.12 to 21.17) −12.09 (−17.18 to 2.65) <0.01 0.61
BMI, % −6.67 (−10.38 to −0.26) −1.37 (−3.18 to 5.29) 0.08 0.17
All values are expressed as median (interquartile range). BMC, bone mineral content; BMD, bone mineral density; BMI, body mass index; iPTH, intact
parathyroid hormone. A minus sign indicates a proportionate fall in the respective parameters.

Table A2. Subgroup analysis comparing the proportionate change in outcome variables in the two study groups in children with
steroid-dependent nephrotic syndrome.
Group A 1000 IU vitamin D/ Group B 400 IU vitamin D/ Effect size for Mann–Whitney
Characteristics day, n = 6 day, n = 5 U-test (r value) p-value
BMC, % 13.35 (3.84–18.20) 9.38 (2.49–13.59) 0.05 0.53
BMD, % 5.93 (3.80–7.89) 3.90 (1.62–7.24) 0.08 0.37
Serum 25-hydroxy cholecalciferol, % 30.19 (−6.91 to 43.26) 43.82 (30.83–63.17) 0.21 0.12
iPTH, % −14.35 (−35.58 to 12.86) −19.25 (−71.09 to 14.86) 0.12 0.22
Serum calcium, % −8.49 (−19.06 to 0.50) −12.00 (−20.99 to 4.15) 0.01 0.99
Serum phosphate, % −20.10 (−33.54 to 1.04) −2.04 (−7.66 to 20.47) 0.33 0.26
Serum alkaline phosphatase, % −30.60 (−52.30 to 10.02) −21.80 (−54.08 to 15.16) 0.01 0.66
BMI, % −6.44 (−10.64 to 8.61) 10.06 (−10.00 to 12.36) 0.03 0.74
All values are expressed as median (inter quartile range); BMC, bone mineral content; BMD, bone mineral density; BMI, body mass index. A minus sign
indicates a proportionate fall in the respective parameters.

Table A3. Comparison of proportionate change in outcome variables in the two study groups in children with steroid-resistant
nephrotic syndrome.
Group A, 1000 IU vitamin D/ Group B, 400 IU vitamin D/ Effect size for Mann–Whitney
Characteristics day, n = 4 day, n = 4 U-test (r value) p-value
BMC, % 19.65 (5.49–27.16) 11.55 (7.85–15.01) 0.05 0.37
BMD, % 5.51 (−1.52,14.37) 4.47 (2.16–17.40) 0.14 0.77
Serum 25-hydroxy cholecalciferol, % 5.99 (−29.99,184.38) 14.52 (−66.14 to 121.44) 0.14 0.72
iPTH, % −36.73 (−61.00 to −1.46) 55.12 (−53.32 to 177.32) 0.01 0.19
Serum calcium, % 0.05 (−5.55 to 6.73) 4.66 (−0.99 to 13.53) 0.02 0.33
Serum phosphate, % 21.53 (2.21 to 47.66) −0.24 (−8.59 to 12.99) 0.00 0.16
Serum alkaline phosphatase, % 1.86 (−18.88 to 4.26) −4.99 (−43.23 to 8.27) 0.05 0.59
BMI, % 4.08 (1.67 to 24.82) 2.19 (−40.71 to 10.63) 0.05 0.28
All values are expressed as median (interquartile range). BMC, bone mineral content; BMD, bone mineral density; BMI, body mass index; iPTH, intact
parathyroid hormone. A minus sign indicates a proportionate fall in the respective parameters.