Severe Thyrotoxicosis Does Not Accelerate 1a-Hydroxylation of 25-Hydroxyvitamin D3 in Dogs. Experimental Study

doi:10.4236/ijcm.2013.412093

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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: [email protected]

Received September 18th, 2013; revised October 17th, 2013; accepted November 14th, 2013

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

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

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

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

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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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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