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J Musculoskelet Neuronal Interact 2019
Original Article
Effect of calcium and vitamin D supplementation with
and without collagen peptides on bone turnover in
postmenopausal women with osteopenia
Chrysoula Argyrou1*, Efthymia Karlafti1*, Kalliopi Lampropoulou-Adamidou1, Symeon Tournis1,
Konstantinos Makris2, George Trovas1, Ismene Dontas1, Ioannis K. Triantafyllopoulos1
1Laboratory for Research of the Musculoskeletal System “Th. Garofalidis”, Medical School, National and Kapodistrian University of
Athens, KAT General Hospital of Athens, Greece; 2Clinical Biochemistry Department, KAT General Hospital of Athens, Greece
* equal contribution
Introduction
Osteoporosis is undoubtedly one of the most common
diseases affecting older individuals with debilitating
consequences1. Osteopenia, defined as T-score between
-1 and -2.5, has also been associated with increased risk
of osteoporotic fractures and the associated morbidity and
mortality2. Prompt diagnosis, prevention and treatment of
both osteopenia and osteoporosis are essential in order to
minimize future fracture risk. The mainstay of treatment
of osteopenia and osteoporosis includes dietary changes,
regular weight-bearing exercises, calcium and vitamin D
supplementation and pharmacologic treatment mainly with
antiresorptive or anabolic agents2. Collagen peptides (CPs),
also called collagen hydrolysates produced by hydrolysis of
collagen , have also been shown to have h igh oral bioavailabilit y
and could have a place as a treatment option3-9.
Type I collagen comprises approximately 95% of the
entire collagen content of bone. Bone matrix, unlike other
connective tissues, possesses the unique ability to become
calcified. Spindle or plate-shaped crystals of hydroxyapatite
are found between and around collagen fibers, oriented in
the same direction as collagen fibers are10 -12 . Nowadays,
it is well-documented that type I collagen molecules are
involved in the mechanical properties of bone12,13. Collagen
Abstract
Objectives: Collagen peptides (CPs) seem to exert beneficial effects on bone and may have a role as a treatment option. In
the present randomized prospective study, we aimed to examine the efficacy, as expressed by changes in P1NP and CTX, and
the tolerability of 3-month supplementation of calcium, vitamin D with or without bioactive CPs in postmenopausal women
with osteopenia. Methods: Fifty-one female, postmenopausal women with osteopenia were allocated to two groups: Group
A received a sachet containing 5 mg CPs, 500 mg calcium lactate and 400 IU vitamin D3 and group B received a chewable
tablet containing 500 mg calcium carbonate and 400 IU vitamin D3 daily. Results: In group A, the P1NP levels significantly
decreased by 13.1% (p<0.001) and CTX levels decreased by 11.4% (p=0.058) within 3 months of supplementation. In
group B, P1NP and CTX did not change. Group A presented better compliance in comparison to group B and no adverse events
contrary to group B. Conclusions: These findings may reflect the reduction of the increased bone turnover in postmenopausal
women with the use of calcium, vitamin D and CPs supplements. The addition of CPs in a calcium and vitamin D supplement
may enhance its already known positive effect on bone metabolism. Clinical Trial ID: NCT03999775.
Keywords: Collagen Peptides, Bone Turnover Markers, Calcium Supplement, Osteopenia, Postmenopausal Women
The study was supported by VivaPharm. The necessary amount of
Colabone® sachets, containing 5 mg bioactive collagen peptides
(Fortibone®), 500 mg calcium lactate and 400 IU vitamin D3 for
the conduction of the study was also provided by VivaPharm. The
authors have nothing to declare.
Correspond ing author: Kalliopi Lampropoulou-Adamidou, MD, MSc, PhD,
Orthopaedic Surgeon, Laboratory for Research of the Musculoskeletal
System “Th. Garof alidis”, Medical S chool, Natio nal & Kapodistri an University
of Athens, KAT General Hospital of Athens, Greece, 10 Athinas Str., Kifissia,
PC: 14561, Athens, Greece
E-mail: kilampropoulou@gmail.com
Edited by: P. Makras
Accepted 30 July 2019
Journal of Musculoskeletal
and Neuronal Interactions
Αccepted Article
2http://www.ismni.org
C. Argyrou et al.: Effect of collagen peptides on bone turnover
peptide compounds seem to exert their beneficial effect on
bone by affecting bone remodeling and mineralization of the
bone matrix, promoting the proliferation and differentiation
of pre-osteoblasts while reducing the maturation of
osteoclasts14-16 . Several preclinical studies performed in mice
and rats support this notion and also suggested that orally
administrated CPs increased bone mineral density (BMD), as
well as the compositional and the biodynamic characteristics
of vertebrae17-19. Human studies in postmenopausal women
have also yielded positive results with increased BMD
and blood biomarkers after 6 months and 1 year of oral
administration6,7.
In the present randomized prospective study, we aimed
to examine and compare the efficacy, as represented by the
changes in bone biomarkers procollagen type I N-terminal
propeptide (P1NP) and C-terminal telopeptide of collagen I
(CTX), and tolera bility of 3-month sup plementation of ca lcium,
vitamin D with and without bioactive CPs in postmenopausal
women with osteopenia.
Materials and methods
The study protocol was approved by the ethic committee
of the KAT General Hospital of Athens and written informed
consent was obtained from all participants. The study is
registered at ClinicalTrials.gov (NCT03999775). Subjects
could withdraw from the study at any time, either by their
personal decision or at the discretion of the investigator;
withdrawn subjects were not replaced.
Female, postmenopausal women with T-score in the
osteopenic range (-1.0 >T-score >-2.5) at either the lumbar
spine (LS) or femur as measured by dual energy X-ray
absorptiometry (DXA), were included in our study. Patients
with T-score in the osteoporotic range (T-score <-2.5) at
any site, patients receiving supplements of calcium and/or
vitamin D at that time or during the last year, or medications
known to positively or negatively affect bone turnover or BMD
at that time or during the last 3 years (e.g. antiresorptive
agents, oestrogens, systemic corticosteroids), or having a
known secondary cause of osteoporosis (e.g. alcohol abuse,
thyrotoxicosis etc) were excluded from the study. Patients
who did not attend to their follow-up appointment and
consequently had only the baseline measurements were also
excluded from the analysis.
On the basis of these criteria, 51 postmenopausal women
were recruited after carrying measurements of BMD at the
LS and the femur using DXA. All participants were assessed
at baseline, provided information on their demographics and
medical history and they completed a brief questionnaire
assessing their dietary calcium intake. The subjects received
the study’s supplementation and instructions at baseline and
the follow-up visit was scheduled. Participants were reviewed
after 3 months, when they were asked about their self-
reported compliance and persistence, any health issues or
side effects experienced and other medications. Compliance
was also estimated, at the follow-up appointment, when the
supplement or a prescription was given to the patients and
they were asked if they had any remnant of the supplement.
Patients were classified as non-compliant with 0-49% drug
intake, partially compliant with 50-74% drug intake and
compliant with 75-100% drug intake. A gentle reminder was
also performed for the continuous receipt of the medicine.
Participants were randomized into two groups. Group A
consisted of 24 subjects who received a sachet containing 5
mg bioactive collagen peptides (Fortibone®), 500 mg calcium
lactate and 400 IU vitamin D3 (Colabone®, VivaPharm)
per day, provided by us. Group B (control) consisted of 27
subjects who received a chewable tablet containing 500 mg
calcium carbonate and 400 IU vitamin D3 per day, which is
one of the commonest supplement combinations given in
everyday clinical practice, prescribed from us.
The primary endpoint of the study was the change of P1NP
and CTX levels following the 3-month calcium, vitamin D and
CPs supplementation. The secondary endpoints were the
comparison of %-changes of P1NP and CTX levels between
the group of calcium, vitamin D and CPs and the group of
calcium and vitamin D supplementation, the comparison
of adverse effects (tolerability), and/or the adherence to
treatment between the two groups.
Biochemical measurements
All blood samples were collected at baseline and after
3 months after overnight fasting for the measurement of
common biochemical blood exams to exclude secondary
causes of osteoporosis and more specific blood exams
including total calcium, intact-parathyroid hormone (iPTH),
25-hydroxy-vitamin D [25(OH)D], N-MID-osteocalcin
(N-MID-OC), total procollagen type I N-terminal propeptide
(total-P1NP) and C-terminal telopeptide of collagen I (β-CTX).
All samples were centrifuged within one hour from collection
(at 3000 rpm for 10 min), aliquoted and stored at -80oC until
tested.
Total serum calcium levels were measured with a
colorimetric assay on Architect-8000 Chemistry analyzer
(Abbott, Chicago, Il, USA). The measurement range of this
assay is 2.0-24.0 mg/dL (or 0.50-6.00 mmol/L) the total
analytical imprecision of this assay in our laboratory is <1.0%.
Serum iPTH levels were measured by a second-generation
electrochemiluminescence immunoassay (ECLIA) on Cobas
e411 automated analyzer (Roche Diagnostics GmbH,
Mannheim, Germany) according to the manufacturer’s
instructions. The measurement range of this assay is 1.20-
5000 pg/mL or 0.127-530 pmol/L. The total analytical
imprecision of this assays in our laboratory is <4.0%.
Serum levels of total-P1NP (reference range 16.27-73.87
ng/mL) were determined with an ECLIA immunoassay on
Cobas e411. This method measures the total-P1NP. The
measurement range of this assay is 5-1200 ng/mL. The
total analytical imprecision of this assays in our laboratory
is <4.5%.
Serum levels of the β-CTX (reference range <1.008 ng/
mL) were determined with an ECLIA immunoassay on Cobas
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C. Argyrou et al.: Effect of collagen peptides on bone turnover
e411. The measurement range of this assay is 0.010-6.00
ng/mL or 10-6000 ng/L. The total analytical imprecision of
this assays in our laboratory is <3.5%.
Serum levels of total 25(OH)D levels were determined with
an ECLIA immunoassay on Cobas e411. The measurement
range of this assay is 3-70 ng/mL or 7.5-175 nmol/L. The
total analytical imprecision of this assays in our laboratory
is < 4.7% .
Serum levels of N-MID-OC were determined with an ECLIA
immunoassay on Cobas e411. This assay detects both the
N-MID fragment as well as the intact molecule of OC. The
measurement range of this assay is 0.5-300 ng/mL. The
total analytical imprecision of this assay in our lab is <3.5%.
Statistical analysis
Intention-to-treat (ITT) analysis was performed. The
Pearson’s chi-square test was used to evaluate differences
between categorical values, the Mann-Whitney U test to
evaluate differences between continuous and ordinal data
of the two groups, and the Wilcoxon signed-rank test to
evaluate differences between the not normally distributed
P1NP and CTX levels within the groups. All the continuous
data were presented as mean ± standard deviation (SD) for
the homogeneity of the presentation. All the statistical tests
were performed using SPSS 20 statistical software (SPSS
Inc, Chicago, IL). A value of p<0.05 was selected to indicate
statistical significance.
Results
After 3 months, 21 patients of group A and 22 patients
of group B were analysed. Demographics and baseline
characteristics were comparable between the two groups
(Table 1). All the patients had creatinine levels within the
normal range, with a mean glomerular filtration rate by the
MDRD Equation 83 ± 16.7 mL /min /1.73 m2. One patient of
Tabl e 1. Demographics and baseline characteristics of the two groups.
Group A (n=21) Group B (n=22) p-value
Age (years) 62 .1 ± 6.3 62 ± 7.6 0.96 4
BM I (kg /cm2)26.8 ± 5 .1 26.5 ± 4.6 0.842
Dietary calcium intake (mg/day) 799 ± 427 671 ± 358 0.3
Calcium (mmol/L) 2.35 ± 0.08 2.39 ± 0.12 0.289
25(OH)vitD3 (nmol/L) 66.55 ± 24.07 73.05 ± 27.17 0.412
PTH (pmol/L) 5.67 ± 1.54 5.57 ± 1.76 0.849
OC (ng/mL) 24 .9 ± 7.1 26.4 ± 9.3 0.578
LS BMD (g/cm2)1.024 ± 0.083 0.986 ± 0.057 0.094
LS T-score -1. 28 ± 0.757 -1. 61 ± 0.485 0.084
TH BMD (g/cm2)0.833 ± 0.081 0.824 ± 0.059 0.678
TH T-score -1. 4 ± 0.679 -1. 4 ± 0.494 0.767
BMI, body mass index; PTH, parathyroid hormone; OC, osteocalcin; LS, lumbar spine; TH, total hip; BMD, bone mineral density.
Tabl e 2. Mean values of bone turnover markers of the two groups at baseline and 3 months of supplementation and comparisons between
and within the groups.
Group A Group B Between groups
p-value
P1NP baseline (mean ± SD) 60.2 ± 15. 6 59 .1 ± 25.2 0.2 01
P1NP at 3 months (mean ± SD) 52 ± 15.1 57.1 ± 24.1 0.981
Within group p-value <0. 001 0.210
P1NP % change from baseline to 3rd month (mean ± SD) -13.1 ± 12. 3 -2 .1 ± 12.6 0 . 0 11
CTX baseline (mean ± SD) 384 ± 107 418 ± 189 0.773
CTX at 3 months (mean ± SD) 333 ± 112 421 ± 210 0.382
Within group p-value 0.058 0.922
CTX % change from baseline to 3rd month (mean ± SD) -11 . 4 ± 24 3.5 ± 29.9 0.079
P1NP, procollagen type I N-terminal propeptide; CTX, C-terminal telopeptide of collagen I. P1NP in ng/ml, CTX in pg /ml.
4http://www.ismni.org
C. Argyrou et al.: Effect of collagen peptides on bone turnover
group A withdrew her consent because of unrelated illness
(she was diagnosed with fibromyalgia). Two patients of group
B withdrew their consent because of unrelated illness (the
one was diagnosed with breast cancer and the second with
ankylosing spondylitis), and one patient because she had
attributed biliary colic to the supplement. Furthermore, 2
patients from group A and 2 from group B did not attend the
scheduled follow-up.
Ιn group A, P1NP levels significantly decreased by 13.1%
(p<0.001) and CTX levels decreased by 11.4% (p=0.058)
within 3 months of supplementation while in group B, P1NP
levels decreased by 2.1 % and CTX levels increased by 3.5%
(both p>0.05). Post-hoc power analysis indicated that our
study had 64% and 53% power of demonstrating a decrease
of 8 ng/ml and 50 pg/ml, for P1NP and CTX, respectively.
The % decrease of P1NP was significantly lower in group A
as compared with group B (-13.1 ± 12.3 % vs. -2.1 ± 12 .6 %,
p=0.011), while % change of CTX tended to be lower, albeit
not significantly, in group A as compared with group B (-11.4
± 24 % vs. 3.5 ± 29.9%, p=0.079). Post-hoc power analysis
indicated that our study had 81% power of demonstrating
a between groups difference of >10% in % change from
baseline for P1NP (Table 2).
In group A, all patients (100%) were compliant to the
supplement. In group B, 17 patients (77%) were compliant,
2 (9%) partially compliant, while 3 (14%) were non-
compliant (p=0.027). There were no reported serious
adverse events. Three patients (14%) reported minor
adverse events (constipation, indigestion and biliary colic)
in group B (p=0.083). The first patient remained in group
B with a change to a similar supplement, the second patient
was allocated to group A because she declined to change her
supplement to a similar one as she had the same experience
in the past, and the patient who attributed the biliary colic to
the initiation of the supplement withdrew her consent.
Discussion
As part of osteopenia prevention and treatment, the use of
supplemental calcium and vitamin D therapy has been shown
to suppress bone turnover, increase bone mass, and even
decrease fracture incidence20 -22. Interestingly, even in young
adults, calcium and vitamin D supplementation improves
bone health23. The favorable effects of calcium and vitamin D
supplements on blood biomarkers have been demonstrated
by several studies24 -26. Our results were consistent with
these studies. Rajatanavin et al found a significant decrease
of P1NP by 22.7% and of CTX by 32.5% after 1 year of 500
mg calcium supplementation24. Kruger et al presented a
decrease of P1NP by 18% and of CTX by 28% after 3 months
of fortified milk containing 900 mg calcium and 6.4 μg
vitamin D25 and Aloia et al found decrease of P1NP by 7.2%
and of CTX by 6.3% at 15 weeks of supplementation with
1200 mg calcium and 100 μg vitamin D26.
Despite the extensive research on calcium and vitamin D
supplementation, only a few clinical studies have evaluated
the efficacy of CPs so far. In the randomized, double-blind
placebo-controlled study, conducted by Elam et al, it was
shown that after a 6-month administration of calcium,
vitamin D and calcium-collagen chelate dietary supplement
in osteopenic postmenopausal women, biomarkers of bone
turnover were improved. Specifically, TRAP5b (tartrate-
resistant acid phosphatase isoform-5b) and sclerostin
were reduced and the bone specific alkaline phosphatase/
TRAP5b ratio was increased7. Significant increase of BMD
was also observed with smaller corresponding changes of
bone biomarkers in their 3-month preliminary study, which
is closer to the timeframe of the present study27. Recently,
another randomized, double-blinded, placebo-controlled
study performed by König et al also yielded similar results6.
This study evaluated the effects of CPs compared to placebo
(receiving maltodextrin) on BMD and biomarkers after 1
year. The biomarkers used were P1NP and CTX showing a
significant increase in P1N P by 11.6% in CPs group compared
to placebo and a significant increase in CTX by 17.6% in
placebo group compared to CPs group. However, unlike
Elam’s et al.7,27 and the present study, calcium or vitamin D
was not provided in either group albeit encouraged according
to patients’ needs.
On the other hand, no significant effect of dietary
supplementation with CPs on biochemical bone markers
was proved by Cuneo et al over a period of 6 months28.
They conducted a randomized double-blind clinical assay in
postmenopausal osteopenic women. A comparison of levels
of bone markers (CTX, osteocalcin and bone specific alkaline
phosphatase) between collagen hydrolysates and placebo
group demonstrated no differences. However, the authors
highlighted that the majority of the patients exhibited poor
calcium intake and increased body weight, parameters that
may have influenced their results28. In this study collagen
hydrolysates supplement was used without calcium and
vitamin D supplementation, as in the study of König et al.6.
This fact could be the reason why we cannot compare our
study’s results with these of the above studies.
In the present study, in comparisons within the groups,
there was a statistically significant decrease of P1NP by
13.1% and a trend of decrease of CTX levels by 11.4%
within 3 months of calcium, vitamin D and bioactive CPs
supplementation, but there was no significant change of
these bone biomarkers within 3 months of calcium and
vitamin D without CPs supplementation in osteopenic
postmenopausal women. In comparisons between the groups,
the supplementation of calcium, vitamin D and bioactive
CPs for 3 months had a statistically significant decrease of
P1NP levels in comparison with the change observed with the
supplementation of calcium, vitamin D without bioactive CPs.
Respectively, the supplementation of calcium, vitamin D and
bioactive CPs for 3 months showed a trend of decrease of
CTX levels in comparison with the change observed with the
supplementation of calcium, vitamin D without bioactive CPs.
The decrease of P1NP and CTX after CPs supplementation
was in line with previous experimental studies14,15 and may
reflect the reduction of the already increased bone turnover
5http://www.ismni.org
C. Argyrou et al.: Effect of collagen peptides on bone turnover
in postmenopausal women15. However, this study did not
achieve to show any positive effect on bone biomarkers of
calcium and vitamin D supplementation as similar clinical
studies did24 -26. Thus, we could conclude that the addition of
CPs in a calcium and vitamin D supplement may enhance its
already known positive effect on bone metabolism.
As already mentioned, CPs are products of collagen
hydrolysis with high oral bioavailability4,5. To better
document the bioavailability of hydroxyprolyl-glycine (Hyp-
Gly), Sugihara et al quantified the ratio of Hyp-Gly and
prolyl-hydroxyproline (Pro-Hyp) in the peripheral blood
after oral administration, which was found to be increased29.
The results were in accord with those of Shigemura et al,
which showed that serum hydroxyproline peptide levels,
especially hydroxyproline-glycine, increased in a dose-
dependent manner and reached their peak after an hour of
oral administration of collagen hydrolysates3.
The mechanism leading to the beneficial effects of CPs
remain unclear. Kim et al observed that collagen hydrolysate
enhanced osteoblastic differentiation in human cells via
the expression of the COL1A1 gene and involved the ERK/
MAPK signaling pathway15. Moreover, Liu J et al. used
MC3T3-E1 pre-osteoblasts and concluded that bovine CPs
increased osteoblast proliferation, and played valuable role
in osteoblast differentiation and mineralization of the bone
matrix30. However, our results and previous experimental
studies measuring P1NP as a biomarker of bone formation
presented reduction of P1NP14,15 similarly with that observed
with the use of calcium and vitamin D24-26.
A limitation of this study may be considered the small
number of patients and thus of moderate power to detect
significant effects. Nonetheless, to our knowledge there are
only three published studies examining the effect of CPs
supplem entation on bone m etabolism and t his is the first study
that shows positive effects on the two reference markers of
bone turnover, CTX and P1NP31. Another limitation could be
the fact that although the decrease of PINP was statistically
significa nt, it did not exceed the least si gnificant chan ge of 30-
40% to prove a therapeutic effect28. However, this change of
P1NP with the use of calcium and vitamin D with or without
CP supplements was expected31 and was in accordance with
the relative clinical studies24 -26. Furthermore, the different
form of calcium provided in the two study groups may lead to
the difference in the minor adverse events (14% vs 0%) with
the calcium lactate being better tolerated than the calcium
carbonate32. However, we decided to approach the everyday
clinical practice and compare supplements that already exist
in the market. A third limitation is related to the compliance
of the patients. The supplement with CPs was provided by the
investigators directly to the patients without additional cost,
whereas the supplement of calcium, vitamin D without CPs
was prescribed and then the patient had to purchase it from
the pharmacy.
The present study shows the decrease of P1NP and
CTX levels within 3 months of supplementation of
calcium, vitamin D with bioactive CPs and no alteration
of bone markers after supplementation of calcium,
vitamin D without CPs for the same period in osteopenic
postmenopausal women. This finding may reflect the
decrease of bone turnover with the use of calcium, vitamin
D and CPs supplements and that the addition of CPs in a
calcium and vitamin D supplement may enhance its already
known positive effect on bone metabolism. Nevertheless,
the elucidation of the appropriate dosage and the effects
of the long-term treatment that is usually required in the
setting of osteopenia and osteoporosis are of utmost
importance and should be addressed in future studies.
Acknowledgements
The study was supported by VivaPharm. The necessary amount
of Colabone® sachets, containing 5 mg bioactive collagen peptides
(Fortibone®), 500 mg calcium lactate and 400 IU vitamin D3 for the
conduction of the study was also provided by VivaPharm.
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