International Journal of Clinical Medicine, 2013, 4, 539-542
Published Online December 2013 (http://www.scirp.org/journal/ijcm)
http://dx.doi.org/10.4236/ijcm.2013.412093
Open Access IJCM
539
Severe Thyrotoxicosis Does Not Accelerate
1a-Hydroxylation of 25-Hydroxyvitamin D3 in Dogs.
Experimental Study
Christos Georgiou Velentzas1,2
1Toronto Western Hospital, Toronto, Canada; 2University of Athens, Athens, Greece.
Email: impression713@yahoo.gr
Received September 18th, 2013; revised October 17th, 2013; accepted November 14th, 2013
Copyright © 2013 Christos Georgiou Velentzas. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Two healthy dogs weighing 18 kg and 13 kg each received an intravenous injection of 7 μg/kg 25-hydroxyvitamin D3
(25OHD3). Subsequently, they were blood-sampled in order to determine the plasma levels of 25(OH)D3 over 4-hourly
time intervals and for a time period of 24 hours. After a period of 18 days since the last blood sampling, the animals
were brought to a hyperthyroid state and the intravenous injection of 7 μg/kg 25OHD3 was repeated. Blood sampling
was performed every 4 hours and over a time period of 24 hours in order to determine the levels of 25OHD3. The
graphic plotting of plasma levels of 25OHD3 in the euthyroid state did not differ from that in the hyperthyroid state.
This finding in dog animal experimentation is indicative that the increased levels of thyroid hormones did not affect the
activity of CYP27B1 and CYP24A1 enzymes that are related to the catabolism of 25OHD3 over a minimum of 24 hours
period.
Keywords: Thyrotoxicosis; Vitamin D; Dogs
1. Introduction
Thyrotoxicosis represents a morbid condition that gra-
dually deprives the body of Vitamin D. In thyrotoxicosis,
the plasma levels of 1,25-dihydroxyvitamin D (1,
25(OH)2D) are depressed. Moreover, hydroxylation of
Vitamin D on C25 position for the production of 25-hy-
droxyvitamin-D (25OHD) is accelerated resulting in low
plasma levels of Vitamin D [1-3]. The accelerated hy-
droxylation of Vitamin D on C25 position in cases of
thyrotoxicosis is due to the stimulation of liver micro-
somal enzymes (CYP27A1, CYP2J3, CYP2R1, CYP3A4)
by the thyroid hormones. In addition, the pathogenesis of
the attenuated levels of 1,25(OH)2D has not been fully
understood. Parathormone, serum calcium and phospho-
rus, CYP enzymes (CYP24A1, CYP27B1) VDR, steroid
and xenobiotic receptor (SXR) have all been reported as
being involved in the pathogenesis of the “depressed”
levels of 1,25(OH)2D in thyrotoxicosis [4].
In the following research study, we have attempted to
investigate the influence of thyroxine on the CYP27B1
enzyme, which through hydroxylation of 25OHD leads to
the production of 1,25(OH)2D.
2. Materials
The study involves two dogs weighing 18 kg (dog “W”)
and 13 kg (dog “B”), respectively. These dogs initially
were examined by a veterinary physician and were then
placed in a specially designated area for experimentation
(Vivarium) measuring 1 × 1 × 2 m with conditions of
20˚C - 24˚C temperature, good ventilation and lighting
systems and with excellent hygiene conditions. The ani-
mals received standardised food and water (adlibidum).
They were weighed daily and their pulse was monitored
and ranged between 90 - 100/min. The animals remained
in the designated areas for monitoring and for adaptation
to this new environment and personnel for a time period of
9 days. Table 1 shows the biochemical findings in the
blood of the animals.
3. Methods
The animals received an intravenous injection of 7 μg/kg
25OHD3 (approximately 5000 iu) and were subsequently
Severe Thyrotoxicosis Does Not Accelerate 1a-Hydroxylation of 25-Hydroxyvitamin D3 in Dogs. Experimental Study
540
blood sampled every 4 hours and for a total time period of
24 hours1. Eighteen days after the last blood sampling, the
dogs were administered thyroxine at a dose of 0.5 mg/kg
of body weight (IM-intramuscularly)2. Ten days later the
animals were in a severe hyperthyroid state, as confirmed
by physical examination (severe tachycardia that could
not be clinically monitored, aggression, tremor, muscle
weakness, constant barking, thirst, weight loss of 1 - 2 kg
etc) and mainly by the thyroid function tests that showed
low values prior to the start of the intervention and high
values at the end of the study (comparisons made with
human subject values). During the thyrotoxicosis phase
and due to the severe hyperstimulation the animals re-
ceived large doses of chlorpromazine, diazepam, nembu-
tale and b-blockers, since the animals showed great re-
sistance to these substances. At the hyperthyroid phase,
the animals had an intravenous injection of 7 μg/kg
25OHD3 and were blood sampled at 4-hourly intervals for
a time period of 24 hours.
4. Results
Euthyroid phase: The intravenous infusion of 7 μg/kg of
body weight 25OHD3 to dog “W” gave the following
values: minimum of 103.3 ng/ml (20th hour), maximum
of 179.60 ng/ml (4th hour) and mean value of 123.9 ±
28.2. The intravenous infusion of 7 μg/kg of body weight
to dog “B” gave the following values: minimum of 98.8
ng/ml (4th hour), maximum of 118.6 (20th hour) and
mean value of 108.3 ± 6.7.
Hyperthyroid phase: The intravenous infusion of the
same dose of 25OHD3 per kg of body weight to dog “W”
gave the following values: minimum of 109 ng/ml (20th
hour), maximum of 150.5 ng/ml (12th hour) and mean
value of 125 ± 16.4. The intravenous infusion to dog “B”
gave the following values: minimum of 112.4 ng/ml (12th
hour), maximum of 119.0 ng/ml (20th hour), and mean
value of 116 ± 2.4. The values of 25OHD3 in both animals
and measured 4-hourly in an euthyroid state and in a hy-
perthyroid state are demonstrated in Table 2 and Figure 1.
There was no statistically significant difference between
them (p = NS). The thyroxine values at the euthyroid
phase and the rest of the findings that are related to the
thyroxine metabolism are displayed in Table 3. The
health of the animals had completely recovered one month
after the last blood sampling.
5. Discussion
The plasma levels of 25OHD3 during the hyperthyroid
phase of the animals showed no difference in comparison
to those levels in the euthyroid phase. This indirectly
shows that the values of 25OHD3 were not deranged to the
hydroxylated products (1,25(OH)2D3 and 24,25(OH)2D3)
under the effect of the increased levels of thyroid hor-
mones.
The thyrotoxicosis of the animals was indisputable as
shown from the thyroid function tests (Table 3) and the
clinical presentation of animals.
1The substance 25OHD3 that was intravenously injected to the dogs is
manufactured by “Upjohn” company; a Standard graph is plotted fo
r
the determination of blood levels. A solution was prepared with this
substance mixed in propylene-glycol containing 100 μg/ml. The solu-
tion was sterilised through infiltration with millipore paper.
2The induction of hyperthyroidism in dogs is extremely difficult and
laborious because animals are significantly tolerant and resistant to
thyroxine (half-life time of thyroxine in dogs is 6.9 hours) [5]. The way
to induce hyperthyroidism in dogs can be found in reports and publica-
tions over previous decades, such as these of De Bodo and Sinkof
f
1953 [6], Brewster et al. 1956 [7], Piatnek and Olson 1961 [5], Pitanek-
Lennsen and Olson 1967 [8], Frey HMM 1967 [9]). The oral admini-
stration of thyroxine to the dogs and at a daily dose of 0.4 - 5. Omg was
ineffective. Subsequently, we applied the method of De Bodo and Sin-
koff 1953 [5] as it is considered to be the most rapid and effective
method.
In special plastic sterile tubes (Cooke Provial Cooke Laboratory
Products) with a sealing cap, we put 10 mg of thyroxine [L-thyroxin
sodium salt 1 gr BDH Biochemicals C6H2(OH)]C6H2CH(NH2)
COONa), Specific rotation (a) 20˚C + 16˚C to 20˚C 2% in acidified
ethane. Loss on drying at 105˚C.
The tubes were then sealed with a cap and were placed in the freezer.
Whenever needed to perform an IM injection of thyroxine, 2.5 ml o
f
alcohol was injected in a tube via a syringe (Absolut alcohol-Abort
Laboratories North Chicago 25 ampl 2 ml sterilized). Thyroxine pow-
der was rapidly dissolved in the alcohol. For further dilution, 1.5 - 2 ml
of water for injection was aspirated.
According to the properties of the substance, thyroxine is dissolvable
in alcohol at 1:250 and in water at 1:600. This thyroxine solution is
unsta
b
le and therefore the IM injection was done without delay and
under aseptic conditions to the animal’s thigh. In order to bring the
dogs to a hyperthyroid state it was necessary to administer thyroxine at
a dose of 0.25 - 0.52 mg/kg.
It is possible that the significantly raised levels of the
Figure 1. Plasma levels of 25OHD3 in both dogs in the
Euthyroid phase (E) and the Thyrotoxic phase (T) after the
ntravenous infusion of 7 μg/kg 25OHD3. i
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Severe Thyrotoxicosis Does Not Accelerate 1a-Hydroxylation of 25-Hydroxyvitamin D3 in Dogs. Experimental Study
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541
Table 1. Biochemical findings of dogs in the euthyroid phase (E) and the thyrotoxic phase (T).
Na Κ CI CO2 Prot Ca Ρ Uric acid Cr. Bilir. LDH
WE 138 4.9 99 19.5 6.7 9.3 2.8 5.5 0.9 0.8 288
WT 144 4.1 113 11.0 5.8 9.1 8.2 1.3 0.8 0.1 535
BE 152 4.6 114 15.5 5.9 10.4 4.3 1.0 0.9 0.3 430
BT 152 4.8 114 15.0 5.4 9.9 9.3 1.5 1.1 0.3 433
Table 2. Mean values of 25OHD3 of both animals on a 4-hourly basis in the euthyroid and hyperthyroid phase. There was no
statistically significant difference between the values in the euthyroid and hyperthyroid phase.
Euthyroid phase
4 hours 8 12 16 20 24
139.2 119.9 116.3 106.9 110.9 117.0
±57.1 ±10.5 ±5.5 ±3.1 ±10.8 ±7.0
Hyperthyroid phase
126.5 114.0 131.4 122.1 114.0 136.3
±17.1 ±3.5 ±26.9 ±8.8 ±7.0 ±2.7
Table 3. Thyroid gland function values in the euthyroid phase (E) and the thyrotoxic phase (T).
RiaT4 RiaT3 RT3U yGT ALP Chol.
μg% ng% % U/L mu/ml mg%
WE 1.0 50 61.0 1 52 -
WT >24 >800 65.7 8 208 119
BE 2.4 60 60.1 0 75 178
BT >24 >800 62.6 3 111 127
thyroid hormones in comparison to their levels during the
euthyroid phase (2 μg%), and also the suppressive medi-
cation given during the hyperthyroid phase, may have
affected in a negative way the enzyme activity or may
have almost completely suppressed their function. The
dose of 7 μg/kg 25OHD3 (~3600 - 5000) is most likely
excessive for animals weighing 13 and 18 kg. Neverthe-
less, it was taken into consideration that the plasma levels
of 25OHD3 in dogs has a wide variation ranging from 5 to
70 ng/ml depending on the respective reference range and
also on the type of food being received [10]. This dose
gave high plasma levels of 25OHD3 in the animals both
during the euthyroid and hyperthyroid phase. These levels
never exceeded the toxic levels which for humans are
defined as above the limit of 150, 206, 490, 690 and 468
ng/ml [11], and the serum calcium was similar in the
euthyroid and hyperthyroid state.
Due to the lack of data about the physiology of vitamin
D in dogs, it was not possible to evaluate the role of thy-
roid hormones on the metabolites and final products of
vitamin D.
This study focused on the function of CYP27B1 cyto-
chrome, which contains the 1a-hydroxylase enzyme
which hydroxylates 25OHD3 in the a-position.
6. Conclusions
The significantly increased levels of thyroid hormones did
not affect the increased plasma levels of 25OHD3 in the
dogs that were tested 4-hourly and for a time period of 24
hours. These findings lead us to the following assump-
tions:
1) The excessive increase in the plasma levels of thy-
roid hormones inhibited the activity of the hydroxylating
enzymes of 25OHD3 in the dogs, in contrast to the
stimulation of the liver microsomal enzymes (CYP27A1,
CYP3A4, CYP2J3, CYP2R1 which in humans induce the
rapid hydroxylation of vitamin D) in position 25.
2) It is necessary to allow for more time beyond 24
hours to have a more definite result.
Severe Thyrotoxicosis Does Not Accelerate 1a-Hydroxylation of 25-Hydroxyvitamin D3 in Dogs. Experimental Study
542
3) The significant increase in supply of 25OHD3 to the
CYP27B1 enzyme inhibited or slowed down the enzyme
activity.
4) The thyroid hormones have no effect on the hy-
droxylating enzymes of 25OHD in dogs. FGF23-cIotho
reduces the blood levels of 1,25(OH)2D3 [12]. There is a
lack of data in literature for the correlation of thyroxine
and the axis of osteoblasts-osteocytes-PTH-FGF23-clotho
and 1,25(OH)2D.
7. Addendum
In 1633 Galileo Galilei quoted the historical phrase “Ep-
pure si muove (still it moves, and yet it moves) when he
was forced from “Holy Inquisition” to recant his belief
that the earth moves around the sun (The event was first
reported in English print in 1757 by Giuseppe Baretti)
[13]. A minor yet similar event occurred to me as well. In
my case, the “Holy Inquisition” was my research study
that forced me to state that the increased levels of thyroid
hormones in dogs do not influence the metabolism of
25OHD3. On the contrary, I believe that the increased
blood levels of thyroid hormones in the body accelerate
the catabolism of vitamin D to give the end products of
calcitroic acid and lactones [14]. In favour of this theory
are: 1) the accelerated hydroxylation of Vitamin D on
C25 position in thyrotoxicosis, 2) the depressed levels of
1,25(OH)2D3 again in thyrotoxicosis, 3) the increased
1,25(OH)2D3 blood levels in hypothyroidism [15]. I be-
lieve that a more detailed investigation in human subjects
will clarify these issues in the future.
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