Accepted: 13 January 2018 DOI: 10.1111/jmp.12334
ORIGINAL ARTICLE
25-OH-vitamin D, parathyroid hormone, and calcium serum levels in captive common marmosets (Callithrix jacchus): Reference values and effect of age, sex, season, and closure of long bone epiphyses Leslie Bosseler1
| Jaco Bakker2 | Luc Duchateau3 | Ed Remarque4 |
Jan A.M. Langermans2 | Pieter Cornillie5 | Koen Chiers1 1
Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 2
Animal Science Department, Biomedical Primate Research Centre, Rijswijk, The Netherlands 3
Biometrics Research Group, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 4
Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands 5
Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Correspondence Leslie Bosseler, Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Email:
[email protected]
Abstract Background: To date, reference values for 25-OH-vitamin D, parathyroid hormone (PTH), and calcium in serum of common marmosets (Callithrix jacchus) based on a large sample size are not available. Methods: Serum reference values for these parameters were determined and correlated with sex, age, season of sampling, and time of long bone epiphyseal closure in captive-housed marmosets. Results and Conclusions: The 90% reference range for serum 25-OH-vitamin D is 47.40-370.4 nmol/L, for PTH 2.10-30.51 pmol/L, and for calcium 2.08-2.63 mmol/L. Lower levels of vitamin D were measured in fall compared with the other seasons. Levels of PTH were higher in males than in females, and calcium levels were lower in younger animals compared with older marmosets. No other effects of age, sex, season, or timing of growth plate closure were found. KEYWORDS
blood biochemistry, bone growth, growth plate, new world monkey
1 | INTRODUCTION
in both vitamin D and PTH blood levels.5,6,8,9 To find a physiological explanation for this, we studied if this variation could be due to the
Common marmosets (Callithrix jacchus) are regularly used in biomed-
season of blood sampling, the sex, the age, or correlated to the closure
ical research and often serve as models for human diseases, such as
of long bone epiphyses. In addition, as no reference data regarding
multiple sclerosis,1 Parkinson’s disease,2,3 and Alzheimer’s disease.4
25-OH-vitamin D, PTH, or calcium serum levels exist for common
Despite their popularity as experimental animals, many questions
marmosets, a large number of common marmosets were sampled and
still remain about some aspects of their physiology, such as their vi-
reference ranges were calculated.
tamin D metabolism. Marmosets are known to have high circulating blood levels of vitamin D due to a relative end-organ resistance to this compound.5-7 Although reference values for vitamin D, parathyroid hormone (PTH), and calcium are lacking in common marmosets, a few studies on small subsets of marmosets reported high variations
2 | MATERIALS AND METHODS 2.1 | Humane care guidelines Common marmosets (Callithrix jacchus, age range 5-142 months)
All the animal experiments were performed at the Biomedical Primate Research Centre, Rijswijk, the Netherlands.
J Med Primatol. 2018;1–6.
originating from and housed in a breeding colony at the Biomedical Primate Research Centre (BPRC, Rijswijk, the Netherlands) were
wileyonlinelibrary.com/journal/jmp © 2018 John Wiley & Sons A/S. | 1 Published by John Wiley & Sons Ltd
|
BOSSELER et al.
2
used. Details about housing conditions and feeding have already 8,9
25-OH-vitamin D and PTH levels were measured using an au-
Briefly, all animals were group-housed in large
tomated chemiluminescence microparticle immunoassay (SSDZ
cages with free access to an inside and outside enclosure (each
Medical Lab, Delft, the Netherlands), and calcium levels were mea-
18 m3) and biofloors. Lighting in the indoor enclosures consisted
sured using a Cobas Integra 400 Plus (F Hoffmann-La Roche, Basel,
of full-spectrum fluorescent bulbs placed close to the cages. There
Switzerland). Frequency distribution histograms and correspond-
been described.
was a 12 hour:12 hour light:dark cycle with lights on at 7:00 am.
ing best-fitted Gaussian curves were made, and reference values
They were fed commercial monkey pellets (with a vitamin D con-
for 25-OH-vitamin D, PTH, and calcium were calculated based on
tent of 3000 IU/kg) (Ssniff, Soest, Germany) ad libitum and supple-
the 90% reference range. As PTH levels were right-hand skewed,
mented with vitamin D droplets (12 IU per marmoset) (Davitamon,
they were log-transformed. To test whether the large variations in
Omega Pharma, Rotterdam, the Netherlands), as well as limited
25-OH-vitamin D result in predictable physiological PTH-and cal-
amounts of Arabic gum, fresh fruit, and living insects. Ethics ap-
cium levels, linear regression analyses were performed. Differences
proval by the Animal Experiments Committee of the BPRC was
in the levels of 25-OH-vitamin D, PTH, and calcium related to age,
obtained prior to the commencement of the study (Dec 676). The
sex, season, or growth plate closure (see below) were analyzed
procedures performed in this study were in accordance with the
based on a linear mixed model with marmoset as random effect,
Dutch laws on animal experimentation, with the regulations for
sex, season, and growth plate closure as categorical fixed effects
animal handling as described in the EU Directive 63/2010 and with
and age as a continuous fixed effect. Also all two-way interactions
the Weatherall report (2006). BPRC is Association for Assessment
were included initially but removed from the final model when not
and Accreditation of Laboratory Animal Care International
significant. Testing was based on the F-test using Satterthwaite’s
(AAALAC) accredited.
approximation to determine the denominator degrees of freedom.
2.2 | Anesthesia, blood sampling, and radiography
2.4 | Determination of serum 25-OH-vitamin D, PTH, and calcium concentrations during long bone growth
The animals were anesthetized using alphaxalone (12 mg/kg IM, Alfaxan, Jurox Limited, UK). Blood samples were obtained by venipuncture from the vena femoralis using serum tubes. Blood sample
Fourteen clinically healthy common marmosets, aged between
volumes were limited by body weight such that volume did not ex-
5 and 37 months, were used for repeated serum sampling for
ceed 1% per 4 weeks. Subsequent blood sampling intervals occurred
25- OH- vitamin D and PTH and radiographic follow- up of their
at a maximum frequency of 1x/month/animal. Radiographs were
growth. Ten of those animals were used for calcium analysis as well
taken with a portable X-ray generator Mobile type HF2 (Verachtert
(see Table S1). For practical and ethical reasons, blood collections
Digital NV, Deurne, Belgium). Ventrodorsal and lateral images were
and radiography were grouped on specific days (rather than spe-
taken (both 55 Kv, 5 mAs). Radiographs were subsequently scanned
cific ages). As a result, the individual ages at time of sampling and
with an EXAMION® CR Vita 25 (Vtrade, Fernelmont, Belgium) and
in the intervals between sampling varied between the animals. PTH,
interpreted using OsiriX® (Pixmeo, Switzerland). Growth plates of
25- OH- vitamin D, and calcium serum levels were compared be-
the bones of the appendicular skeleton were assessed for time of
tween animals with early growth plate closure (14.95 months) in the abovementioned mixed model.
2.3 | Determination of serum reference values of 25-OH-vitamin D, PTH levels, and calcium and the effect of age, sex, and season of sampling During a period of 5 years, 272 serum samples of 67 clinically healthy common marmosets (5-142 months old) were analyzed for 25-OH-vitamin D, 254 serum samples of 64 common marmosets
3 | RESULTS 3.1 | Reference values of 25-OH-vitamin D, PTH, and calcium Frequency distribution histograms and corresponding best- fitted
for PTH (5-142 months), and 206 serum samples from 36 com-
Gaussian curves for the 25-OH-vitamin D, PTH, and calcium serum
mon marmosets (5-37 months) were analyzed for calcium. Prior to
levels of our test population of common marmosets are depicted in
blood sampling, food was withheld for 16 hours, but water intake
Figure 1. 25-OH-vitamin D levels varied widely in the population
was never restricted. The animals were sampled in the morning.
(22.0-492.4 nmol/L with a mean of 207.08 nmol/L and a coefficient
Samples were taken at (on average) 1-3 monthly intervals, except
of variability (CV) of 49.98%), while PTH concentrations showed lit-
for 38 marmosets, where only 1 serum sample was obtained. The
tle variation for the majority of animals, although several high outliers
discrepancy between the numbers of analyses is explained by the
were present (1.5-136 pmol/L with a mean of 10.56 pmol/L and a CV
fact that not enough blood was always available to perform all
of 111.27%). Calcium values varied only slightly between individual
three analyses. Serum samples were frozen (−20°C) immediately
measurements (1.83-2.87 mmol/L with a mean of 2.38 mmol/L and
until analysis.
a CV of 6.72%). Reference values, defined as the values between the
BOSSELER et al.
F I G U R E 1 Frequency distribution histogram and best-fitted Gaussian curve of 25-OH-vitamin D, parathyroid hormone (PTH), and calcium (Ca) serum concentrations in a population of healthy common marmosets. The vertical dotted line depicts the mean concentration in the population
5% and 95% percentile, are 47.40-370.4 nmol/L for 25-OH-vitamin D, 2.10-30.51 pmol/L for PTH, and 2.08-2.63 mmol/L for calcium.
|
3
F I G U R E 2 A, Calcium serum levels of common marmosets plotted against the age of the animals. The solid line represents a linear regression fit with a slope of 0.007 ± 0.002. The dots represent individual measurements. B, C, Tukey boxplots of serum parathyroid hormone levels and 25(OH) vitamin D levels in common marmosets plotted against respectively the sex and the season of sampling. Horizontal lines in the box represent the median, the first, and the third quartile. The mean is represented by +. Horizontal bars outside the box represent the lowest and highest value that are not outliers. Outliers are shown by individual dots
The relationship between 25-OH-vitamin D and calcium serum values was tested using a linear regression analysis, which resulted in R2 = 0.03702 and a P-value of .007. For 25-OH-vitamin D and PTH, the R2 = 0.1122 and the P -value
