Comparative Biochemistry and Physiology, Part B 165 (2013) 196–200

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Comparative Biochemistry and Physiology, Part B

journal homepage: www.elsevier.com/locate/cbpb

Blood vitamin D3 metabolite concentrations of adult female bearded

dragons (Pogona vitticeps) remain stable after ceasing UVb exposure
D.G.A.B. Oonincx a, b,⁎, M.D. van de Wal c, G. Bosch a, J.B.G. Stumpel c, A.C. Heijboer d,
J.P.T.M. van Leeuwen e, W.H. Hendriks a, c, M. Kik c
a
Animal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands
b
Laboratory of Entomology, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands
c
Department of Pathobiology, Pathology Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
d
Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
e
Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Vitamin D deficiency can lead to several health problems collectively called metabolic bone disease (MBD).
Received 7 March 2013 One commonly kept reptile species prone to develop MBD if managed incorrectly is the bearded dragon
Received in revised form 29 April 2013 (Pogona vitticeps). This study aimed to determine the extent to which adult female bearded dragons fed a
Accepted 29 April 2013
diet low in vitamin D can use stored vitamin D and its metabolites to maintain plasma 25(OH)D3 and
Available online 3 May 2013
1,25(OH)2D3 concentrations after discontinuing UVb exposure. Blood samples of healthy adult female beard-
Keywords:
ed dragons, exposed to UVb radiation for over 6 months were collected (day 0) after which UVb exposure
Bearded dragons was discontinued for 83 days and blood was collected. Blood plasma was analysed for concentrations of
Pogona vitticeps total Ca, total P, ionized Ca, uric acid, 25(OH)D3 and 1,25(OH)2D3. There was no significant change in plasma
Plasma concentrations 25(OH)D3 and 1,25(OH)2D3 concentrations during the study. While total Ca and P in whole blood was found
Vitamin D metabolites to significantly decrease over time (P b 0.0088 and 0.0016, respectively), values were within the reference
Ultraviolet light exposure range. Plasma ionized Ca tended (P = 0.0525) to decrease during the study. Adult female bearded dragons,
Depletion previously exposed to UVb, are able to maintain blood vitamin D metabolite concentrations when UVb expo-
Reptile
sure is discontinued for a period of up to 83 days.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction Vitamin D by itself is biologically inert and is either stored in the ad-
ipose tissue or hydroxylated in the liver to 25(OH)D3 (Holick, 1981;
A common health problem in captive reptiles is metabolic bone dis- Jones et al., 1998; Holick, 2007). Renal and intracellular hydroxylation
ease, a complex of diseases in which functional and morphological of 25(OH)D3 yields 1,25(OH)2D3, the biologically active form of vitamin
changes in bone occur caused by an imbalance of vitamin D, calcium D3 in the body (Holick, 1981, 2007). Although 1,25(OH)2 D3 is biologi-
(Ca) and/or phosphorus (P) (McWilliams and Leeson, 2001; Mader, cally the most active vitamin D3 metabolite, its levels in the blood-
2006). Vitamin D is an important regulator of Ca and P metabolism in stream are tightly controlled whereas concentrations of 25(OH)D3 in
mammals and reptiles (Holick, 1981; Dittmer and Thompson, 2011). It blood are indicative for vitamin D status (Laing and Fraser, 1999;
can either be exogenously supplied via the diet or endogenously synthe- Holick, 2007; Heaney et al., 2009). Further hydrolysis of 1,25(OH)2D3
sized through conversion of 7-dehydrocholesterol to pre-vitamin D3 by by D-24-hydroxylase leads to the biliary excretory product calcitroic
UVb exposure (290–320 nm) and a subsequent, thermally dependent, acid (Jones et al., 1998). The importance of dietary and UVb-mediated
isomerization to vitamin D3 (Holick, 1981; Jones et al., 1998; Dittmer endogenous synthesis of vitamin D, and as such vitamin D metabolism,
and Thompson, 2011). Prolonged exposure to sunlight can result in the appears to differ between reptile species (Allen et al., 1995; Ferguson et
photoconversion of pre-vitamin D3 to lumisterol and, to a lesser extent, al., 2009; Oonincx et al., 2010).
tachysterol (Holick, 1981; MacLaughlin et al., 1982; Holick, 2007). The If reptiles in captivity are not exposed to direct sunlight, artificial
latter has been suggested to serve as a system preventing vitamin D UVb lighting or a dietary supply of vitamin D is required to ensure
intoxication (Holick, 1981, 2007). adequate plasma vitamin D metabolite concentrations (Mader, 2006).
The importance of UVb lighting for bearded dragons held in captivity
is evident as recently it was shown that weekly oral vitamin D supple-
⁎ Corresponding author at: Laboratory of Entomology, Wageningen University, PO Box
mentation, even at high dosages, was ineffective in attaining plasma
8031, 6700 EH Wageningen, The Netherlands. Tel.: +31 317482019. 25(OH)D3 and 1,25(OH)2D3 concentrations similar to UVb exposed
E-mail address: [email protected] (D.G.A.B. Oonincx). bearded dragons (Oonincx et al., 2010). Also in other reptile species

1096-4959/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.cbpb.2013.04.006
 D.G.A.B. Oonincx et al. / Comparative Biochemistry and Physiology, Part B 165 (2013) 196–200 197

UVb exposure results in elevated concentrations of 25(OH)D3 (Acierno 1.5 mL were obtained from the ventral tail vein of each animal using a
et al., 2006, 2008). 2 mL heparinized syringe (PICO syringe, Radiometer America, London,
It seems likely that during periods without UVb exposure (e.g. lack ON, Canada), 80 IU electrolyte balanced heparine (PICO50 radiometer,
of sunlight or ineffective artificial lamps), captive bearded dragons, Medical ApS, Brønshøj, Denmark) and a 25 gauge needle (BD
rely on vitamin D and 25(OH)D3 reserves to maintain an adequate Microlance 3 25G 0.5 × 16 mm ref 300600). Upon collection, the
vitamin D status. The extent of the storage capacity of 25(OH)D3 and blood was divided in two equal parts with one part being centrifuged
the time it takes for plasma concentrations to decrease is important (750 g for 10 min at 10 ºC) and stored (−80 °C) within 1 h after extrac-
information in maintaining the health of bearded dragons and other tion pending further analysis. The other half was stored in the syringe
reptiles held in captivity. and directly sent for analysis of Ca, P and uric acid. The BM of each ani-
The aim of the current study was to determine the extent to which mal was determined on the same day that blood was obtained.
adult bearded dragons fed a diet low in vitamin D can use stored vitamin
D and its metabolites to maintain plasma 25(OH)D3 and 1,25(OH)2D3 2.3. Chemical analyses
concentrations after discontinuation of UVb exposure.
Total Ca, P, and uric acid concentrations were determined by means
2. Material and methods of the Unicel DXC-600 (Beckman Coulter, Woerden, The Netherlands).
Ionized Ca concentration was determined by a blood gas analyser
2.1. Animals and diet (Rapidlab 1265, Siemens Nederland B.V., The Hague, the Netherlands).
Concentrations of 25(OH)D3 were determined at the Endocrine Labora-
Twenty-two healthy adult female bearded dragons (Pogona vitticeps) tory of the VU University Medical Center (Amsterdam, The Netherlands)
with a mean (± SD) body mass (BM) of 390 ± 74 g (range 310 – 481 g) by ID-XLC-MS/MS as described by Heijboer et al. (2012), with some
were used. The animals originated from an earlier experiment conducted modifications to optimize the method for bearded dragons. In short,
at our facilities in which the gender was determined by visual inspection deuterated internal standard [25(OH)D3-d6] was added to a 50 μL sam-
of the cloaca (Oonincx et al., 2010). Throughout the 83-day study, ple and 25(OH)D3 was released from its binding proteins with acetoni-
animals were provided ad libitum with a diet consisting of house crickets trile. Samples were extracted and analysed by XLC-MS/MS [a Symbiosis
(Acheta domesticus), grasshoppers (Locusta migratoria and Schistocerca online SPE system (Spark Holland, Emmen, The Netherlands)] coupled
gregaria) and mealworms (Tenebrio molitor) (Starfood, Barneveld, The to a Quattro Premier XE tandem mass spectrometer (Waters Corp.,
Netherlands). The insects were dusted with calcium carbonate Milford, MA, USA). A Kinetex 2.6u PFP column (Phenomenex, Utrecht,
(Ankerpoort NV, Maastricht, The Netherlands) before they were offered The Netherlands) was used for optimal separation. Total run time was
to the bearded dragons. Furthermore, one type of vegetable was provided 12 min. The limit of quantification was 8.0 nmol/L; intra-assay variation
daily, which was either bell peppers (Capsicum annuum), endive was b7.5% for concentrations between 50 and 300 nmol/L. The IDS
(Cichorium endivia) or Chinese cabbage (Brassica pekinensis). Vitamin D3 Gamma-B kit (Immunodiagnostic Systems, Tyne and Wear, UK)
concentrations of the diet provided, have been shown to be insufficient was used to determine concentrations of 1,25(OH)2D3 by immuno-
to raise plasma levels of 25(OH)D3 above 10 nmol/L in juvenile bearded extraction followed by a 125I RIA for quantification; intra-assay variation
dragons without providing UVb exposure (Oonincx et al., 2010). Each ter- was b12%.
rarium contained a Petri dish with water, which was refreshed daily.
2.4. Statistical analyses
2.2. Housing and study design
Differences in plasma 25(OH)D3, 1,25(OH)2D3, Ca, P and ionized Ca
The bearded dragons were housed in pairs in terraria measuring between blood sampling groups at the start of the study were identified
124 × 50 × 40 cm (L × W × H) containing a 50 W halogen spot (E27, using Proc GLM in SAS (version 9.2, SAS Institute, Cary, NC, USA). Data of
Namiba Terra, Kempen, Germany) at 27 cm above ground level in the individual animals were subjected to least square linear regression
center of the cage. Ground coverage consisted of masonry sand. All analysis with 25(OH)D3, 1,25(OH)2D3,total Ca, total P, ionized Ca and
terraria were located in the same room with fluorescent lighting tubes uric acid as the dependent variable and time as the independent vari-
fixed to the ceiling. No UVb light from these tubes could enter the able. The slopes were averaged per terrarium (i.e. the experimental
terraria. Direct sunlight could not enter the room. No UVb light was unit) and tested for difference from zero, i.e. no change over time,
provided during the study. Animals housed together were selected using a t-test. Change over time with a probability of P b 0.05 was
based on having a similar BM. Before the start of UVb deprivation, accepted as statistically significant.
ultraviolet light was provided by means of compact fluorescent lamps
(26 watt Reptisun 5.0 UVb, Zoomed Laboratories, Inc., San Luis Obispo, 3. Results
CA, USA) placed horizontally in the cages at a distance of 29 cm above
ground level in the top corner of the terrarium, controlled by a timer Two animals did not complete the study: one was treated for con-
and providing 12 h of UV each day for a period in excess of 6 months. junctivitis and the second was euthanized due to a tail infection at day
Ultraviolet output of the lamps was measured by means of a UV meter 57 of the study. Two other animals had high plasma uric acid concentra-
(Solarmeter model 6.2, Solartech Inc., Harrison Township, MI, USA). tions (>1600 μmol/L) halfway into the study. All data of these four
Before the start of the experiment they provided an intensity of animals were excluded from the data analyses. Blood sampling was
20.0 ± 3.6 μW/cm2 (range: 15–26 μW/cm2) at a distance of 19 cm successful in 93.3% of the cases (six cases were unsuccessful).
directly underneath the middle part of the lamp. At the start of the study, there was no significant difference in BM
On day 0, all animals were sampled for blood after which UVb lamps (P = 0.1042) and in the concentrations of plasma 25(OH)D3 (P =
were switched off. Then, nine pairs were randomly divided into three 0.0900), plasma 1,25(OH)2D3 (P = 0.3576) and plasma total Ca
blood sampling groups by means of a randomization procedure in (P = 0.6348), ionized Ca (P = 0.7987) and uric acid (P = 0.2905)
Microsoft Excel (3–4 terraria per group), in order to reduce the frequen- between blood sampling groups. Differences were observed for plas-
cy of sampling and minimize interference on the measured parameters. ma concentration of total P (P = 0.0357).
Group 1 was sampled on day 8, 32, 69 and 83, group 2 on day 14, 41, 83 Although not significant (P = 0.1129), the BM of the animals de-
and group 3 on day 2, 23, 55 and 83. Four animals remained unallocated creased by 0.35 g/d over time (Table 1). The plasma 25(OH)D3 concen-
and were used in case blood could not be collected at a specific time tration remained stable throughout the 83 day study (Fig. 1); the slope
from one or more of the 18 animals. Blood samples of approximately of the overall regression line, combining all the data across terraria, did
198 D.G.A.B. Oonincx et al. / Comparative Biochemistry and Physiology, Part B 165 (2013) 196–200

not significantly (P = 0.5160) differ from zero. Similarly, the slope

(− 0.0015) of the regression line for plasma 1,25(OH)2D3 concentra-
tion was not significantly different (P = 0.6825) from zero (Fig. 2).
The mean concentrations for whole blood total Ca, ionized Ca,
total P and uric acid during 83 day of UVb deprivation are presented
in Table 1. Significant effects of deprivation over time were ob-
served for total Ca, and total P. Plasma total Ca decreased while ion-
ized Ca tended to increase during the UVb deprivation period. On
day 14 and 23, high plasma total Ca concentrations were observed
(12.82 and 13.14 mmol/L, respectively). Plasma total P decreased
(0.0402 mmol/L/d) to a similar extent as total Ca (0.0377 mmol/L/d)
over the 83 days. Uric acid concentration significantly (P b 0.0001)
increased by 1.523 μmol/L/d during the 83 days study.
Fig. 1. Mean and standard error of the mean for plasma 25(OH)D3 concentrations in
4. Discussion adult bearded dragons (Pogona vitticeps) during UVb deprivation. Slope of the regres-
sion line is not different from zero (P = 0.5160).

The present study shows that plasma 25(OH)D3 and 1,25(OH)2D3

concentrations of adult female bearded dragons remain stable over an et al. (1998) showed that orally administered cholecalciferol rapidly
83 day period even when dietary vitamin D was low and UVb exposure accumulates in adipose tissue and that adipose tissue acts as a buffer to
was absent. The dietary vitamin D concentration of the house crickets, maintain circulating 25(OH)D3 concentrations. Half-lives of plasma
grasshoppers, and mealworms fed to the bearded dragons contained cholecalciferol and 25(OH)D3 in rats are 1.4 and 22.5 days, respectively,
b36.3 IU/MJ gross energy (Oonincx et al., 2010). This concentration is while the half-life of cholecalciferol in perirenal and subcutaneous
similar to the recommendation of 35 IU vitamin D3/MJ gross energy adipose tissue were estimated to be 97.5 and 80.9 days, respectively
consumed (between 260 and1800 IU/kg diet, depending on insect (Brouwer et al., 1998). For growing pigs, the vitamin D storage capacity
species) to prevent vitamin D deficiency symptoms as recommended has been estimated to last approximately 7 days (Heaney et al., 2009),
by Kik and Beynen (2003). In a recent study, Oonincx et al. (2010) which is similar to a reported sharp decrease in plasma 25(OH)D3
reported low (~10 and 0.18 nmol/L, respectively) plasma 25(OH)D3 concentrations after 8 days when growing pigs were provided a vitamin
and 1,25(OH)2D3 concentrations for 3 and 6 month old growing bearded D free diet (Denis et al., 2000). However, in growing cats, a species
dragons fed the same diet as used in the present study and receiving no unable to convert 7-dehydrocholesterol to vitamin D by UVb exposure
UVb. In addition, the latter authors reported that weekly oral supple- (Morris, 1999), Rivers et al. (1979) were unable to induce vitamin D
mentation of vitamin D had no significant effect on plasma 25(OH)D3 deficiency after feeding a vitamin D free diet for about 275 days. The
and 1,25(OH)2D3 concentrations. UVb exposure on the other hand was latter authors calculated the half-life of 25(OH)D3 to be 57 days in cats.
found to maintain plasma 25(OH)D3 at ~190 nmol/L and p1asma In a study where adult men were deprived of UVb radiation and provided
1,25(OH)2D3 concentrations at ~1.0 nmol/L (Oonincx et al., 2010). At with a normal diet, 25(OH)D3 levels decreased by 20% between day 14
the start of the present study, plasma 25(OH)D3 and 1,25(OH)2D3 con- and 46 of the study, and after 65 days 25(OH)D3 levels had decreased
centrations were 181.4 ± 21.1 (range 48.0 to 267.5) and 2.57 ± 0.17 by 53% (Davies, 1985). In another study involving people during winter
(range 1.32 to 2.98) nmol/L, respectively. The plasma 25(OH)D3 concen- when cutaneous vitamin D production would be expected to be negligi-
tration was similar to the value reported for 6 month old bearded ble, oral dosages of vitamin D were provided in order to determine the
dragons (178.4 ± 9.0 nmol/L) while the plasma 1,25(OH)2D3 concen- proportion of daily requirements that was met by reserves in body tissue
tration was higher (1.21 ± 0.10 nmol/L) (Oonincx et al., 2010). (Heaney et al., 2003). In this study over 80% of the requirements was met
The importance of dietary vitamin D and vitamin D synthesized by by body reserves accumulated during summer. During the present
UVb as well as the storage capacity of vitamin D and its metabolites, 83 days study, vitamin D metabolite concentrations remained stable. A
can be expected to differ depending on species specific evolutionary similar study on male bearded dragons, in which serum 25(OH)D3 levels
factors such as dietary habit, daily activity and solar intensity. Presum- were determined 14 and 28 days after ceasing UVb, also found metabo-
ably, like mammals, adult bearded dragons are capable of storing vitamin lite concentrations to remain stable (Dralle et al., 2010). However,
D (cholecalciferol and ergocalciferol) and its metabolite 25(OH)D3 in juvenile monitor lizards showed a decrease in plasma 25(OH)D3 levels
adipose, muscle and liver tissue (Rivers et al., 1979; Brouwer et al., of 25–35% over a similar period of time (87 days) when fed a diet low
1998; Denis et al., 2000; Heaney et al., 2009). In subadult rats, Brouwer in vitamin D and receiving no UVb radiation (Ferguson et al., 2009). In

Table 1
Mean body mass and concentrations of total calcium, ionized calcium, total phosphorus and uric acid in whole blood of adult bearded dragons (Pogona vitticeps) during 83 day UVb
deprivation.

Day Body mass (g) Total Ca (mmol/L) Ionized Ca (mmol/L) Total P (mmol/L) Uric acid (μmol/L)

Mean SEM n Mean SEM n Mean SEM n Mean SEM n Mean SEM n

0 410.3 16.4 10 4.43 1.00 8 1.30 0.10 4 6.16 0.70 8 181.3 21.9 9
2 451.5 21.8 3 5.54 2.41 3 1.38 0.14 2 3.88 0.73 3 87.5 12.7 3
8 414.8 19.8 4 5.97 2.53 4 1.41 0.06 4 4.27 0.52 4 179.6 42.5 4
14 365.3 25.5 3 12.82 1.74 3 1.40 0.16 3 5.93 1.17 3 246.5 41.0 3
23 434.2 24.8 3 13.14 3.16 3 1.27 0.22 3 7.97 2.16 3 92.3 20.9 3
32 385.9 12.2 4 3.71 0.20 4 1.33 0.08 4 3.54 0.53 4 261.6 64.7 4
41 357.7 20.4 3 4.11 0.43 3 1.58 0.06 3 2.13 0.11 3 413.7 127.5 3
55 402.6 18.6 3 8.40 2.90 3 1.45 0.08 3 4.66 1.12 3 214.2 56.9 3
69 382.6 8.4 4 3.61 0.23 4 1.48 0.03 4 2.01 0.21 4 272.8 69.4 4
83 384.3 8.2 10 3.47 0.09 10 1.48 0.04 10 2.41 0.46 10 284.5 31.2 10
Slope −0.3492 0.1988 10 −0.0377 0.0113 10 0.0028 0.0012 10 −0.0402 0.0091 10 1.523 0.216 10
P-value 0.1129 0.0088 0.0525 0.0016 b0.0001
 D.G.A.B. Oonincx et al. / Comparative Biochemistry and Physiology, Part B 165 (2013) 196–200 199

2011). An increased concentration relative to basal concentrations

was observed after 14 and 23 days, coinciding with the peak in total
Ca concentrations. One female deposited eggs on day 35 and again on
day 49. The total Ca concentration of this animal was elevated on day
23 (17.2 mmol/L) and day 55 (13.6 mmol/L). Several other females
had similar concentrations on blood sampling days but did not deposit
eggs. Possibly in those animals the ova may have been reabsorbed.
These fluctuations of total Ca and P would be expected if calcium was
mobilized from bone due to follicular development under the influence
of parathyroid hormone. Follicular development can lead to a fourfold
increase in calcium concentrations in lizards (Knotkova et al., 2005;
Mader, 2006; Tamukai et al., 2011). Whether the administration of
toltrazuril can also affect blood Ca or P concentrations in bearded
Fig. 2. Mean and standard error of the mean for plasma 1,25(OH)2D3 concentrations of dragons is unknown.
adult bearded dragons (Pogona vitticeps) during UVb deprivation. Slope of the regres- Although uric acid concentrations (Table 1) changed significantly
sion line was not different from zero (P = 0.6825).
during the study, these concentrations can be considered low to nor-
mal when compared to reference values of 173–595 μmol/L reported
the latter study, a large decline (73%) in vitamin D concentrations was for bearded dragons (Diethelm and Stein, 2006), and 119–565 μmol/L
also observed, indicating that vitamin D levels decline before 25(OH)D3. for female bearded dragons in summer (Tamukai et al., 2011).
In humans, if vitamin D intake is high, large amounts are stored as In conclusion, UVb deprivation combined with a diet low in vita-
vitamin D, presumably in the body fat, and conversion to 25(OH)D3 is min D for a period of 83 days did not significantly decrease plasma
slow (Heaney et al., 2008). In a recent study on previously sun- 25(OH)D3 and 1,25(OH)2D3 concentrations in adult female bearded
exposed Hermann's tortoises, the effects on plasma 25(OH)D3 concen- dragons, indicating a large endogenous storage capacity of vitamin
trations of artificial UVb were compared to those of sun exposure D and 25(OH)D3.
(Selleri and Di Girolamo, 2012). Within a period of 35 days, blood
25(OH)D3 concentration decreased by more than 50% in the groups Acknowledgments
exposed to artificial UVb. In the present study, vitamin D stores of the
bearded dragons were sufficiently large to maintain blood 25(OH)D3 The authors would like to thank the staff at the animal research
and 1,25(OH)2D3 concentrations for 83 days. This indicates that the facility “De Haar” at Wageningen University for their assistance in the
vitamin D storage capacity can differ greatly between reptile species. care of the animals. Also, Dr. Frances Baines is kindly thanked for her
Brouwer et al. (1998) showed that during fasting, preferential loss of careful review of the manuscript.
triacylglycerols from adipose tissue occurs, as opposed to cholecalciferol. This study was approved by the Committee for the Care and Use of
In the present study, a number of animals lost, while others gained Animals of Wageningen University (Wageningen, The Netherlands)
weight; on average the bearded dragons lost 6.3% of BM. At 42 days and started on the 15 th of September 2009.
into the study, faecal samples of several animals were found to contain
coccidia. All animals were treated with toltrazuril (Produlab Pharma, References
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