Download Evaluation of Parathyroid Hormone and Preoperative Vitamin D as

Evaluation of Parathyroid Hormone and Preoperative Vitamin D as Predictive
Factors for Post-operative Hypocalcemia in Dogs with Primary
Hyperparathyroidism.
Master’s Thesis
Presented in Partial Fulfillment of the Requirements for the Degree of Masters of Science
in the Graduate School of The Ohio State University
By
Jennifer Song BS, DVM
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2016
Master’s Thesis Committee:
Kathleen Ham DVM, MS, DACVS - advisor
Dennis Chew DVM, MS, DACVIM
Chen Gilor DVM, PhD, DACVIM
Copyrighted by
Jennifer Song
2016
Abstract
Primary hyperparathyroidism (PHPTH) results in excessive secretion of
parathyroid hormone (PTH) causing hypercalcemia. Vitamin D also contributes to
calcium regulation. People with PHPTH and vitamin D deficiency have more severe
disease as seen with higher preoperative calcium and longer duration of postoperative
hypocalcemia. In people and dogs, a decrease of more than 50% of intraoperative PTH
concentration 10 minutes after parathyroidectomy is associated with complete surgical
resection of the autonomously functioning gland. Post-operative hypocalcemia after
parathyroidectomy necessitates immediate medical intervention and prolonged
hospitalization. Distinct pre-operative predictors of postoperative hypocalcemia have not
been identified in veterinary medicine.
The objective of this study was to prospectively monitor the trends in PTH
concentrations and vitamin D metabolites and determine predictive factors for the
development of postoperative hypocalcemia. Our hypothesis was that dogs with a greater
than 75% percent change between baseline and intraoperative PTH concentrations will be
more likely to develop hypocalcemia in the immediate post-operative period. A second
hypothesis is that dogs with low vitamin D status will be more likely to develop
postoperative hypocalcemia.
ii
Dogs that developed postoperative hypocalcemia had a significantly larger
decrease in PTH concentration than dogs that remained normocalcemic (p<0.008). All
dogs had low preoperative calcidiol concentration. This is the first prospective study
examining vitamin D and preoperative factors in dogs with PHPTH as predictors of
postoperative hypocalcemia.
iii
Acknowledgments
I would like to thank Dr. Kathleen Ham for her continued nurturing support and
mentorship throughout my residency. I would also like to thank Drs. Dennis Chew and
Chen Gilor for their curious minds, their dedication to this project and their sincere desire
to furthering veterinary research.
I am forever grateful to the owners and dogs that participated in this study. For
none of this would be possible without Dakota, Gabbi, Harley, Coz, Honey, Molly,
Nanook, Maggie Sue, Sassy and Dodger. Thank you.
Lastly, and certainly not the least, I would like to acknowledge Rachel Manson,
Shana Gilbert-Gregory, Katie McCool and Melissa Tropf for their ongoing love and
support.
iv
Vita
2006................................................................BS, Rutgers University – Cook College
2011................................................................DVM, The Ohio State University
2012................................................................Rotating Internship, University of
Pennsylvania
2013................................................................Surgery Internship, The Ohio State
University
2016................................................................Residency, Small Animal Surgery, The Ohio
State University
Publications
Song J, Drobatz K, Silverstein D. 2016. Evaluation of shortened PT or aPTT, TEG and
evidence of hypercoagulability in 25 dogs. J Vet Emerg Crit Care. 26(3): 398-405.
Song J, Sheehy J, Dyce J. 2013. En-block Femoral Cement Removal Technique after
Cemented Total Hip Replacement Failure in 2 dogs. Vet Comp Orthop and Traumatol.
26(2):130-134.
v
Fields of Study
Major Field: Comparative and Veterinary Medicine
vi
Table of Contents
Abstract ............................................................................................................................... ii
Acknowledgments.............................................................................................................. iv
Vita...................................................................................................................................... v
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
Chapter 1: Calcium and Vitamin D ................................................................................... 1
Chapter 2: Primary Hyperparathyroidism in Dogs ........................................................... 11
Chapter 3: Materials and Methods .................................................................................... 33
Chapter 4: Results ............................................................................................................. 40
Chapter 5: Discussion ....................................................................................................... 61
Chapter 6: Conclusion....................................................................................................... 72
References ......................................................................................................................... 74
vii
List of Tables
Table 1. Preoperative and postoperative data for dogs that remained normocalcemic and
developed hypocalcemic after parathyroidectomy ........................................................... 49
Table 2. Preoperative and postoperative data (1,25(OH)2D, 25(OH)D and PTH) for dogs
that remained normocalcemic and developed hypocalcemia after parathyroidectomy .... 50
Table 3. Percent PTH change, percent calcium change and vitamin D values ................ 54
viii
List of Figures
Figure 1. Trend of postoperative PTH concentration ...................................................... 51
Figure 2. Box-and-whiskers plot for PTH concentration at diagnosis and developing
postoperative hypocalcemia ............................................................................................. 52
Figure 3. Box-and-whiskers plot for preoperative PTH and developing postoperative
hypocalcemia .................................................................................................................... 53
Figure 4. Box-and-whiskers plot for percent PTH change (at diagnosis to intraoperative
sample) and the development of postoperative hypocalcemia ......................................... 55
Figure 5. Box-and-whiskers plot for percent PTH change (preoperative to intraoperative
PTH) and the development of postoperative hypocalcemia ............................................. 56
Figure 6. Trend of ionied calcium after parathyroidectomy ............................................ 57
Figure 7. Box-and-whiskers plot for percent change in ionized calcium (at diagnosis to
nadir sample) and the development of postoperative hypocalcemia ................................ 58
Figure 8. Box-and-whiskers plot for preoperative 25(OH)D and the development of
postoperative hypocalcemia ............................................................................................. 59
Figure 9. Box-and-whiskers plot for preoperative 1,25(OH)2D and the development of
postoperative hypocalcemia .............................................................................................. 60
ix
Chapter 1: Calcium and Vitamin D
1.1 – Introduction
The regulation of calcium is intricate and complex. The majority of calcium is
stored as hydroxyapatite within the skeleton. In the extracellular space, calcium circulates
in one of three fractions at approximately; 56% ionized, 34% protein-bound and 10%
complexed, with only ionized calcium exerting biological activity1. Intracellular calcium
is maintained in low concentrations, which allows rapid diffusion of ionized calcium
from the extracellular fluid into the cytoplasm through calcium sensitive channels. The
hormones involved in calcium homeostasis are parathyroid hormone (PTH), produced
from the parathyroid glands; calcitriol, the active metabolite of vitamin D; calcitonin
from the thyroid gland and calcium itself contributes to the feedback mechanism.
Based on the elimination half-life, PTH is responsible for minute-to-minute
adjustment of serum calcium while calcitriol maintains day-to-day control. The
concentration of calcium also contributes to calcium homeostasis. Calcium sensing
receptors (CSR) located in the parathyroid chief cells, C cells and renal epithelial cells
directly regulate intracellular ionized calcium concentration2. When low serum calcium is
detected, PTH secretion is stimulated while elevated serum calcium levels inhibit PTH
secretion, however, complete inhibition of PTH secretion does not
1
occur3. Calcitriol also has an intimate role in calcium homeostasis as it is both a product
of PTH stimulation and regulates PTH production. Activation of the renal calcium
sensing receptors during times of hypercalcemia causes increased calcium excretion
through urine whereas PTH increases tubular reabsorption of calcium4.
1.2 - Parathyroid Hormone
Parathyroid hormone (PTH), also known as parathormone or parathyrin, is the
only calcium-regulating hormone secreted by the chief cells of the parathyroid glands.
Release of PTH is in response to low systemic ionized calcium concentrations.
Hypocalcemia stimulates an increased rate of secretion while hypercalcemia and
calcitriol inhibits PTH synthesis and secretion5. Once released, PTH has a half-life of 3 to
5 minutes due to rapid cleavage by liver Kupffer cells6. For this reason a steady rate of
secretion occurs to maintain calcium homeostasis. The inverse relationship between PTH
and calcium ensure that a small change in calcium will cause a relatively large change in
PTH. Disease states such as primary hyperparathyroidism increases the PTH-calcium set
point to the right and thus increases the threshold of ionized calcium required to achieve
half the maximal PTH secretion7, 8.
PTH acts directly on bone and the kidneys through the parathyroid hormone 1
receptor to increase circulating calcium concentrations and also has an effect on the
central nervous system and pancreas via the parathyroid hormone 2 receptor in people9.
Regulation of PTH is complex with feedback systems involving calcium concentration,
calcitonin and calcitriol.
2
1.2.1 - Molecular structure
PTH is an 84 amino acid polypeptide chain that was first sequenced from the
bovine species in 197010, 11. Subsequently, the porcine PTH molecule and human PTH
form was sequenced shortly afterwards12. The hormone molecule is relatively well
preserved across species, with few amino acid substitutions. The human PTH molecule
contains asparagine at position 16 compared to the serine in the bovine and porcine
molecules13. Although this substitution is unique to the human molecular structure,
similarities are documented between the human and porcine PTH molecule that differ
from the bovine molecule13. The canine PTH molecule was successfully sequenced in the
mid 1990’s and was found to be 88% homologous to the human molecule14. There were
two substitutions in the (1-40) amino acid sequence at positions 7 and 16 that also occurs
in bovine and porcine PTH molecules3, 14.
These early extraction and purification studies identified PTH as the sole hormone
synthesized, stored and secreted from the parathyroid glands. It was also determined that
there was limited storage capacity for PTH, with estimates of approximately 0.004% PTH
per parathyroid gland weight, indicating a high turnover rate for biosynthesis and
secretion15. Further research has established that a prohormone molecule is synthesized
first by the parathyroid glands and then cleaved into the active PTH molecule. The
prohormone has an additional six amino acid sequence, Lys-Ser-Val-Lys-Lys-Arg, at the
amino terminus which are subsequently cleaved to form the active 84 amino acid
polypeptide for storage and secretion16. The entire 84 amino acid sequence is not
3
necessary for function as only the (1-34) amino acid sequence, is sufficient for biologic
activity13.
1.2.2 - Secretion rhythm
Numerous endogenous hormones are secreted in an episodic, cyclic or rhythmic
characteristic rather than continuously, in healthy individuals. Intermittent secretion is
known to exist for numerous hormones such as luteotropic and follicle-stimulating
hormones, cortisol, aldosterone, insulin and growth hormones17-21. The biologic rhythm
of PTH secretion is multifactorial with influences from both endogenous and exogenous
variables.
Circadian rhythms are known to influence hormone plasma concentrations due to
cyclical activity related to day-night, sleep cycle and activity-inactivity. Diurnal
variations in PTH secretion have been demonstrated in humans22 and in dogs23. Studies in
people reported one maximum PTH peak in the early morning hours leading to a nadir at
approximately 10:00 military time22, 24, 25. Due to the nocturnal PTH surge, sleep cycle
has also been implicated to affect PTH levels, in particular, PTH release was related to
sleep stages 3 and 426. However, other studies did not support this conclusion and did not
show a significant difference in nocturnal rise of PTH when healthy men were subjected
to forced wakefulness in constant environmental conditions24 and also if the timing of
sleep was shifted25 (25). Interestingly, calcium concentration also follows a diurnal
rhythm23, 27.
4
Under constant environmental factors with enforced wakefulness and set feeding
rations in men, the circadian rhythm of PTH remained relatively preserved, indicating a
large endogenous component of PTH secretion that is not affected by diet, posture or
sleep-wake cycles24. Other studies have documented a bimodal PTH pattern with a
similar primary peak in early morning and a secondary PTH peak in the afternoon28.
There is also a significant seasonal variation of PTH secretion in people, with a decline of
20% below the annual mean during summer seasons and 20% increase during winter
seasons29.
1.2.3 – Regulation
The rate of PTH secretion is inversely proportional to the concentration of serum
calcium30. The major stimulus of PTH secretion is hypocalcemia, which triggers rapid
PTH secretion from stored reserves and concurrently inhibits intracellular degradation of
PTH. A rapid decrease in circulating iCa by 2-3% can trigger a 400% increase in PTH
secretion31. In states of prolonged hypocalcemia, up regulation of PTH gene expression
and the proliferation of chief cells occur to maximize PTH secretion. Measures used to
protect against hypercalcemia include up regulation of intracellular PTH degradation
within the parathyroid glands and inhibition of PTH gene transcription when calcitriol
activates the vitamin D receptor (VDR)3. Negative feedback by the arachidonic acid
pathway also contributes to rapidly decreasing PTH secretion4.
5
1.3 - PTH Target organs
PTH exerts direct effects on the skeleton and kidney and has indirect effects on
the intestines. The effect of PTH on the skeleton is biphasic with an immediate “rapid”
phase and a delayed “slow” phase. In the rapid phase called “osteolysis,” PTH is bound
to osteoblasts and osteocytes and results in increased calcium-phosphate salt absorption.
The slow phase of PTH’s effect on bone is caused by osteoblastic activation of
osteoclasts resulting in prolonged bone resorption and bone remodeling4. PTH increases
osteoclast formation to increase bone resorption and the release of calcium. In the
kidneys, PTH causes reabsorption of calcium in the distal tubules, collecting tubules and
to a lesser extent from the ascending loop of Henle. PTH also causes rapid loss of
phosphorus in the urine by decreasing reabsorption in the proximal tubules4. PTH
activates and induces synthesis of the enzyme 1α-hydroxylase in the renal epithelium of
the proximal convoluted tubules, which is responsible for the conversion of calcidiol (25hydroxycholecalciferol) to the active vitamin D metabolite 1,25-dihydroxycholecalciferol
(calcitriol). Calcitriol contributes to elevating circulating calcium concentration by acting
on enterocytes to increases intestinal absorption of calcium and phosphorus4.
1.4 - Vitamin D
Vitamin D is a fat-soluble prohormone. There are two main forms of Vitamin D,
ergocalciferol (vitamin D2), which is of plant origin or cholecalciferol (vitamin D3) that
can be synthesized by skin or acquired from other animal tissue. In mammals, Vitamin D
can be obtained from dietary intake or by dermal synthesis from sunlight. The skin of
6
humans, horses, pigs, rats, cattle and sheep contain adequate quantities of 7dehydrocholesterol which can be converted to cholecalciferol, however the skin of cats
and dogs do not contain a significant quantity of 7-dehydrocholesterol making dietary
intake in these species essential32.
Calcitriol, 1,25-dihydroxyvitamin D (1,25(OH)2D), is the most biologically active
form of vitamin D and is essential for maintenance of calcium and phosphate
homeostasis, normal bone formation, cellular growth control and differentiation of
various tissues33, 34. Cholecalciferol requires two enzyme conversions to become the
active molecule, calcitriol. In the liver, cholecalciferol is converted to calcidiol, 25hydroxyvitamin D (25(OH)D), by 25-hydroxylase and then by 1α-hydroxylase to
1,25(OH)2D in the kidney.
The major site of vitamin D metabolite regulation is through the activity of
renal1α-hydroxylase, which is up regulated by PTH35, 36. Extrarenal sites of 1αhydroxylase have also been reported in keratinocytes, testicles, brain, cultured bone cells,
macrophages, placenta, prostate cells, islet cells of the pancreas and the parathyroid
glands37-44. Extrarenal 1α-hydroxylase results in local production of 1,25(OH)2D for
autocrine or paracrine regulation compared to the endocrine function of renal 1αhydroxylase45.
Calcitriol circulates in the blood and binds to the vitamin D receptor (VDR) to
activate intracellular transcription factors and modulate gene expression33. Regulation of
calcium homeostasis is one of the major functions of 1,25(OH)2D mainly targeting the
intestines, kidneys, bone and the parathyroid glands.
7
1.5 - Impact of Vitamin D and calcium regulation
It is well known that the role of vitamin D has significant effect on calcium
regulation. Vitamin D and PTH secretion have an inverse relationship where decreased
vitamin D causes an increased synthesis of PTH46. Geographic and seasonal associations
in plasma vitamin D concentrations exist in people with higher 25-hydroxyvitamin D
levels in late summer seasons46, 47.
1.5.1 - Role of vitamin D on the intestines
Calcitriol is the main factor in regulating intestinal calcium absorption secondary
to dietary intake. Binding of 1,25(OH)2D to VDR activates transport of calcium either by
passive diffusion mechanism via paracellular pathway or actively through a transcellular
pathway. The paracellular pathway predominates when dietary levels of calcium are high
with calcium transport through tight junctions48. The transcellular pathway involves
transport of calcium from the brush border in the duodenum and jejunum through the
transient receptor potential vanillinoid 6 (TPRV6), followed by cystolic binding to the
calcium binding protein calbindin and then excretion through the plasma membrane
calcium ATPase on the basolateral membrane49. This pathway predominates when
dietary calcium levels are normal to low48, 49.
8
1.5.2 - Role of vitamin D on the kidneys
Regulation of calcium reabsorption in the kidneys occurs in the distal nephron.
Binding of 1,25(OH)2D to renal VDR stimulates an active calcium transcellular pathway
whereas the proximal tubule is responsible for passive resorption of a large part of the
filtered calcium50. The active transcellular resorption of calcium occurs in the distal
tubule into the cytoplasm through the TPRV-5, followed by coupling to calcium binding
proteins and then excretion through the plasma membrane calcium ATPase and sodiumcalcium exchanger on the basolateral membrane49. TRPV5 is the rate-limiting step of
renal calcium resorption is regulated by binding of the fibroblast growth factor-23 and
Klotho complex49, 51. Experimental Vdr null mice have significant calciuria despite
systemic hypocalcemia due to impaired renal calcium reabsorption52. The amount of
renal calcium resorption varies depending with the amount of intestinal calcium
absorption.
1.5.3 - Role of vitamin D on bone
One of the main roles of the vitamin D system is to maintain calcium homeostasis
even at the expense of bone mass and strength. The effect of calcitriol on bone is
dependent on the overall calcium balance such that during positive calcium balance,
calcitriol regulates bone metabolism53. The skeleton serves as a calcium reserve when
insufficient calcium is obtained from the intestines and kidneys. VDR null mice that are
calcium deficient develop osteopenia and spontaneous fractures53. During negative
9
calcium balance, calcitriol will prioritize maintaining normocalcemia by increasing bone
resorption and suppression of bone mineralization53.
1.5.4 - Role of vitamin D on parathyroid glands
Calcitriol directly plays an important role in the feedback regulation of
parathyroid function by causing suppression of PTH transcription and secretion.
Calcitriol is also known to inhibit parathyroid cell proliferation43. By decreasing PTH
secretion, calcitriol indirectly regulates the synthesis of 1,25(OH)2D by limiting the
activity of 1α-hydroxylase, a PTH-induced enzyme, activity in the kidney54. An
overexpression of 1α-hydroxylase was identified in parathyroid adenomas and in
hyperplastic parathyroid glands associated with secondary hyperparathyroidism
compared to normal parathyroid glands. It is believed that this increased expression of
1α-hydroxylase may lead to increased local production of 1,25(OH)2D in an effort to
control parathyroid cell proliferation43. Although the formation of 1,25(OH)2D may be
based on availability of circulating 25(OH)D, it was found that when 25(OH)D was
administered, individuals with PHPTH had a significantly higher circulating 1,25(OH)2D
compared to normal controls55. People with chronic vitamin D-deficiency may have
accelerated parathyroid adenoma growth and aggravate skeletal changes56, 57.
10
Chapter 2: Primary Hyperparathyroidism in Dogs
Hyperparathyroidism is a process associated with the excessive secretion of
parathyroid hormone (PTH) in circulating blood. Primary hyperparathyroidism (PHPTH)
indicates hyperfunctioning parathyroid glands independent of circulating ionized calcium
concentraton; secondary hyperparathyroidism occurs in response to prolonged states of
hypocalcemia as seen with chronic kidney failure or with vitamin D deficiencies. Tertiary
hyperparathyroidism is a condition reflecting the development of autonomous
parathyroid function following a period of secondary hyperparathyroidism resulting in
both elevated PTH and presence of ionized hypercalcemia58.
In health, elevated plasma circulating calcium levels inhibit PTH secretion while
low serum calcium levels stimulate PTH release from non-pathologic parathyroid glands.
Even in the presence of severe hypercalcemia, complete inhibition of PTH secretion does
not occur3, 14. People with PHPTH have altered physiologic calcium homeostasis that
increases the calcium set point and requires a greater concentration of calcium to inhibit
PTH secretion59.
PHPTH is usually clinically diagnosed following the discovery of increased
circulating ionized calcium. PTH is secreted by parathyroid chief cells and inversely
fluctuates with circulating ionized calcium concentrations. A diurnal peak of PTH
11
secretion has been reported in dogs, in which the morning PTH concentration was
approximately twice the lowest concentration during the day23. The short half-life of
circulating PTH necessitates a steady rate of secretion to maintain calcium homeostasis58.
The concept of normocalcemic primary hyperparathyroidism has been described in
people and is a disorder of mineral metabolism and excessive PTH secretion with normal
calcium levels60. As PTH and calcium have cyclic secretion patterns, diagnosis of
normocalcemic primary hyperparathyroidism require consistently normal albuminadjusted total serum calcium and normal ionized calcium with elevated22, 23, 60. In
addition, secondary causes of elevated PTH such as vitamin D deficiency, hypercalicuria,
concurrent gastrointesintal malabsorption conditions and PTH inducing medication
should also be ruled out60.
2.1 - PHPTH in dogs
PHPTH was first recognized in veterinary medicine in 1958 and has historically
been reported as uncommon in dogs and rare in cats61. Literature review over the past two
decades yielded few large case series with an abundance of smaller case series and case
reports62-71. Recent literature has suggested the incidence of PHPTH is more common
than historically reported72.
Approximately 90% of dogs and cats have been reported with one solitary
hyperfunctioning gland62, 64, 73, 74. In another study, 42% of dogs were found to have two
abnormal parathyroid glands67. The mean age of diagnosis is approximately 11 years old
and ranges from adult to geriatric (6-17 years old) dogs with no sex predilection62. An
12
autosomal dominant genetic inheritance pattern predisposes Keeshond dogs to PHPT
with odds ratio of 50.7 (75). Siamese cats may be at increased risk76.
2.2 - Anatomy of the parathyroid glands
In dogs, the parathyroid glands are ellipsoid discs measuring 2 to 5 mm in
diameter and 0.5 to 1 mm in width77. The four most regularly occurring parathyroid
glands are the external and internal parathyroid glands, also named parathyroid III and IV
respectively referring to the embryologic pharyngeal pouch78. The external parathyroid
gland is usually located at the cranial dorsolateral margin of the thyroid gland and the
internal parathyroid gland tends to be embedded within the caudal portion of the thyroid
gland. Variations in location to both the external and internal parathyroid gland have been
described78, 79.
Accessory or ectopic parathyroid glands are rare with a reported incidence of 36% in dogs and 35-50% in cats80, 81. Locations of ectopic parathyroid gland vary, but can
be visualized on serial sections of the thyroid gland82 or may migrate into the thorax with
the thymus78. Earlier publications have identified as many as 13 parathyroid glands in one
dog61, 83. Ectopic glandular tissues from the cervical to thoracic inlet has been described
and are related to the embryonic development from the third and fourth pharyngeal
pouch61.
13
2.2.1 - Vascular supply
In the dog, blood supply, lymphatic drainage and innervation of the parathyroid
glands are related to the thyroid gland. The main blood supply is via the cranial and
caudal thyroid arteries. The cranial thyroid artery is the first major branch off the
common carotid artery and supplies the cranial pole of the thyroid gland. One smaller
vessel from the cranial thyroid artery supplies the external parathyroid gland. The caudal
thyroid artery arises from a branch of the brachiocephalic artery to supply the caudal pole
of the thyroid gland and anastomoses with the cranial thyroid artery. The internal
parathyroid gland is supplied by blood vessels that surround the thyroid parenchyma.
Venous drainage parallels arterial supply with the cranial and caudal thyroid vein
draining into the internal jugular vein. Lymphatic drainage of the parathyroid gland is to
the retropharyngeal lymph nodes. This is one of the few glands that have lymph drainage
cranial to the organ77.
2.2.2 - Nervous system
The thyroid nerve is formed from fibers from the cranial cervical ganglion and
cranial laryngeal nerve. The principle innervation to the parathyroid glands is derived
from the autonomic nervous system. The specific role of the autonomic nervous system
in relation to glandular secretion is not clearly understood, but is believed to have effects
on blood perfusion rates to the gland77.
14
2.3 - Impact of vitamin D deficiency and PHPTH
People with PHPTH with concurrent vitamin D deficiency are associated with
more severe disease as seen with higher calcium, higher alkaline phosphatase, higher
baseline PTH and larger parathyroid glands43, 46, 57, 84.
The definitions of vitamin D deficiency and insufficiency are highly controversial
in people with vitamin D deficiency as defined as 25-hydroxyvitamin D <20 ng/ml
whereas vitamin D insufficiency is 20-29 ng/ml and vitamin D sufficient identified as
>30 ng/ml in a population of PHPTH people46. Others suggest vitamin D insufficiency
between 10-20 ng/ml (25-50 nmol/L) and vitamin D deficiency below 10 ng/ml (25
nmol/L). Vitamin D levels between 4.8-10 ng/ml (12-25 nmol/L) will cause proximal
myopathy or increased bone turnover and levels below 4-4.8 ng/ml (10-12 nmol/L) were
associated with frank osteomalacia85, 86.
2.4 - Clinical signs of PHPTH
Clinical signs in dogs with PHPTH tend to be mild and include polydipsia,
polyuria, weakness, lethargy, gastrointestinal disease, stranguria, pollakuria, urethral
obstruction87, 88. Approximately 40% of dogs may be asymptomatic62. Polyuria and
polydipsia are often the most common presenting compliant62. Of 210 dogs with PHPTH,
31% of dogs had urolithiasis and 29% of dogs had urinary tract infections62.
Gastrointestinal signs are secondary to reduced excitability of gastrointestinal smooth
muscle4. To compensate for dehydration from obligatory polyuria, polydipsia ensures.
Due to impaired renal concentrating abilities, dogs are prone to urinary tract infections
15
with or without urinary stones. Calcium oxalate stones form secondary to hypercalciuria
and can cause urethral obstruction4, 87.
2.5 - Diagnostics
Investigation into a diagnosis of PHPTH is usually initiated after an elevated total
serum calcium level is reported on blood work. Dogs with PHPTH, especially compared
to dogs with other causes of hypercalcemia are generally healthy compared to other dogs
with hypercalcemia87.
2.5.1 - Blood work
In veterinary medicine, elevated total calcium concentration is the hallmark
abnormality detected prompting further investigation. Serum calcium exists in three
forms: 56% ionized (biologically active form), 10% complexed (bound to phosphate,
bicarbonate, sulfate, citrate and lactate) and 34% protein-bound4. Elevation in total
calcium may not reflect an elevation in the ionized fraction of calcium. Although changes
in albumin, globulin, protein content, presence of lipemia or hemoconcentration of
plasma or serum can alter total calcium results, the concentration of ionized calcium is
not usually affected87. Presence of acidemia decreases calcium binding to protein and
consequently increases the ionized calcium. Ionized calcium may be a more sensitivity
indicator of systemic calcium than total calcium. Reports in people have identified that
although patients with elevated total calcium also had elevated ionized calcium, a subset
of patients with normal total calcium had elevated ionized calcium levels89. This
16
diagnostic discordance has also been described in dogs31.
Serum phosphorus concentration in dogs with PHPTH is often reported to be
below the reference range or within the lower half of the reference range. This occurs
following PTH-induced renal inhibition of proximal tubular phosphorus resorption4.
Approximately 65% of dogs had low serum phosphorus concentration and 34% had
serum phosphorus concentration within reference ranges62. The presence of
hypophosphotemia or normophosphotemia rules out primary renal disease and secondary
hyperparathyroidism as causes for hypercalcemia. Alkaline phosphatase (ALP) may be
elevated in dogs with PHPTH due to increased bone turnover from osteoclastic activity63.
Utilizing duel-energy X-ray absorptiometry (DEXA) technology can quantify bone
density. These studies have not been performed in dogs with PHPTH. Increased ALP is
correlated with development of rapid and prolonged postoperative hypocalcemia and
hungry bone syndrome in people90.
Hemogram changes are minimal and associated with dehydration from polyuria
and polydipsia87. Common urinalysis findings include isosthenuria or hyposthenuria,
hematuria, pyuria and crystalluria secondary to polyuria and polydipsia, urinary tract
infections and/or uroliths87.
2.5.2 – PTH analysis
Diagnosis of PHPTH is based on normal or elevated serum PTH concentrations in
the face of ionized hypercalcemia. This is an inappropriate response since high
circulating ionized calcium levels should inhibit PTH synthesis and release87. In one
17
report, approximately 75% of dogs with PHPTH had a PTH concentration within the
reference range62.
PTH is an 84 amino acid polypeptide chain that can be analyzed by
immunoradiometric assay (RIA) or by chemiluminescent assay (CLA). The first 34
amino acid sequences, which is approximately 30-40% of the entire sequence, is
sufficient for biologic activity13. Many challenges with the accuracy of RIA have been
identified over the years. Early assays that rely on detection of the mid-region sequence
or C-terminus of PTH may be falsely elevated in certain disease conditions of delayed
renal clearance such as in chronic kidney disease91. Fine-tuning of the first generation
RIA have improved the sensitivity of PTH detection and the third generation tests
utilizing antibodies directed at the C-terminal region in conjunction with antibodies
directed at the first four amino acid of the sequence at the N-terminal to detect the intact
PTH molecule92. Intact PTH assays have been reported to overestimate the presence and
severity of parathyroid-mediated osseous abnormalities in uremic patients93. In addition,
there is discordance between different assay manufactures making comparing PTH values
challenging92. Compared to the RIA the CLA is a rapid in-house test that can be
performed at many specialty institutions. The CLA can also detect the biologically active
intact PTH molecule. Due to the quick turnaround of results, intraoperative PTH testing
has revolutionized the standard of care for parathyroidectomy in people94. As PTH is
relatively well preserved across species, human PTH assays have been validated for use
in dogs67, 68.
18
Although few larger specialty veterinary hospitals may have access to an in-house
CLA analyzer, the majority of general practitioner veterinarians ship serum to the
Michigan State University – Diagnostic Center for Population and Animal Health (MSUDCPAH) laboratory for RIA PTH analysis. The exact methodology of the MSU-DCPAH
for PTH analysis is proprietary and not available for review. Even though the PTH
molecule is relatively preserved across species, the variations between species and posttranslational modifications can alter the affinity of the animal PTH molecule for the antiPTH antibody used in the assay8. Other species-specific molecules or molecules
associated with certain pathologic conditions might interfere with PTH detection8. At this
institution, the older in-house model of the analyzer prohibits use of CLA technology for
PTH analysis and samples are shipped to MSU-DCPAH for analysis.
2.5.3 - Intraoperative PTH Assay
Rapid intraoperative PTH assay has high accuracy, approximately 95%95,
associated with surgical excision of all autonomously functioning parathyroid tissue in
people96. Circulating half-life of PTH in people with normal functioning kidneys ranges
from 1.68-21.5 minutes95. For intraoperative testing in people, baseline PTH
concentration is obtained prior to parathyroidectomy and compared to a blood sample
obtained at a set time point after the affected parathyroid gland has been removed97. A
decrease of more than 50% between post-excision and pre-excision samples was
indicative surgical cure97.
19
Since the PTH molecule is relatively conserved between species, a CLA PTH
assay has recently been validated in dogs67. In that study, all dogs with uniglandular
disease had more than 50% decrease in PTH concentration after parathyroidectomy,
while dogs with multiglandular disease required removal of both abnormal parathyroid
glands prior to the PTH concentration decreasing to more than 50% compared to preexcision67. Intraoperative PTH analysis has also identified a significant increase in PTH
concentration was associated with surgical manipulation of the affected gland in dogs68.
2.5.4 – Vitamin D analysis
One of the main roles of the vitamin D system is to maintain calcium homeostasis
even at the expense of bone mass and strength. The bone serves as a calcium reserve
when insufficient calcium is obtained from the intestines and kidneys. Vitamin D plays
an integral role in PTH synthesis and calcium regulation and should be evaluated during
workup of PHPTH.
Circulating concentrations of 1,25(OH)2D are tightly regulated by the body while
the major circulating form of vitamin D is 25-hydroxyvitamin D which is the best
indicator of available vitamin D in humans and animals47, 98. Similar to PTH analysis,
vitamin D is evaluated by either RIA or CLA. Variations in the recognized antibodies are
associated with IRIA methodology with some assays recognizing both 25OHD2 and
25OHD3, whereas others underestimate 25OHD299.
20
2.6 – Diagnostic imaging
Imaging of parathyroid glands can be challenging due to their small size and
superficial location. A gold standard parathyroid imaging modality in people has not been
recognized, however cervical ultrasonography, computed tomography (CT), magnetic
resonance imaging (MRI) and nuclear scintigraphy have all been used to localize affected
parathyroid glands100-106.
2.6.1 – Ultrasound
Ultrasonographic images of normal parathyroid glands in normocalcemic animals
are not commonly seen and when observed are usually less than 2-3 mm in length, round
or oval in shape and anechoic or hypoechoic compared to surrounding thyroid
parenchyma107-109. The most common structure misinterpreted for parathyroid glands
were thyroid lobules, thyroid cysts, parathyroid cysts, lymphoid tissue and vasculature
100, 109. Other causes of misidentification include ectopic location of parathyroid tissue,
parathyroid tissue deep to the trachea or parathyroid tissue that is isoechoic to the thyroid
glands or too small to distinguish109.
Neoplastic lesions, adenoma or adenocarcinoma of the parathyroid glands,
measure greater than 4 mm in length while hyperplastic parathyroid glands are between
3-4 mm in size107. Good correlation has been reported comparing ultrasound
measurements and gross parathyroid measurements107, 109. The success of ultrasound in
the identification of normal an abnormal parathyroid glands is user dependent. Without
21
cytology or histopathology, it is impossible to differentiate parathyroid nodule from other
thyroid pathologies.
2.6.2 - Nuclear scintigraphy
Nuclear scintigraphy is a commonly used imaging modality in people for
detection and localization of hyperfunctioning parathyroid tissue. Organs absorb
radioisotopes and emit gamma radiation that is captured with scintillation, or gamma
cameras. The most common radioisotopes used are thallium, sestamibi and
tetrofosmin101.
Subtraction technique requires two different radioisotopes to detect abnormal
parathyroid tissue106. The thyroid gland absorbs both Thallium (201TI) and pertechentate99
Tc radioisotopes while hyperfunctioning parathyroid glands only take up 201Tl but not
99m-Tc pertechnetate. Once acquired, the images are subtracted from each other to
identify the abnormal parathyroid tissue106.
Double-phase scintigraphy with a single agent uses Technectium-99m-sestamibi
(99mTc-sestamibi). Success of this procedure is based on the differential washout of
99m
Tc-sestamibi from thyroid and parathyroid tissue102. Hyperfunctioning parathyroid
glands will absorb the radioisotope in a similar fashion as thyroid tissue during the initial
phase, and will remain radioactive through the delayed phase102. Double phase
scintigraphy has a sensitivity of 83-95% for detection of parathyroid adenoma102-104 and a
37-62%103, 104 for parathyroid hyperplasia in people. The specificity of parathyroid
scintigraphy is difficult to assess since unaffected patients rarely undergo diagnostics. In
22
one scintigraphy study, a specificity of 75% was reported as three of four patients who
did not have parathyroid disease had negative scans103.
Limited reports of scintigraphy use for diagnosing and localizing abnormal
parathyroid glands are available in veterinary medicine110, 111. One case report with
scintigraphy correctly identified the presence and location of a parathyroid adenoma110.
In a larger study, the overall sensitivity of parathyroid scintigraphy was 11%, specificity
of 50% and overall accuracy of 27% with a sensitivity of 25% for localizing parathyroid
adenoma and 0% for parathyroid hyperplasia111.
Possible explanations for this discrepancy between success of parathyroid
scintigraphy in humans and dogs may be related to parathyroid size. Hyperfunctioning
parathyroid glands in dogs measured 0.3-2.0 cm111 compared to abnormal human
parathyroid glands ranging from 1.2-6.5 cm in diameter105. In another study, the smallest
adenoma detected by scintigraphy measured 0.7 cm in diameter in people112. It is also
possible that dogs with hyperfunctioning parathyroid glands have a faster washout time
than compared to abnormal human parathyroid glands necessitating different image
capture time. Other limitations of scintigraphy in veterinary medicine involve cost of
equipment, appropriate housing and disposal of waste for radioactive patients and trained
personnel.
2.6.3 - Computed Tomography/Magnetic Resonance Imaging
Advanced imaging with CT and MRI are usually reserved for localization of
suspected ectopic parathyroid tissue after failed initial treatment. Most functional
23
parathyroid adenomas are hyperenhancing on CT, which distinguishes them from lymph
nodes. In humans, MRI images are obtained from the hyoid bone to the sternal notch.
Parathyroid tissue has variable intensity on MRI, but typically shows intermediate-to-low
signal intensity on T1-weighted images and high signal intensity on T2-weight images101.
The sensitivity and specificity of CT/MRI to diagnose and localize functional parathyroid
glands in veterinary medicine is not reported. Limitations due to cost of procedure,
necessity of general anesthesia and availability of the equipment make CT and MRI less
commonly used in veterinary medicine.
2.7 - Pre-treatment
Definitive guidelines of when to initiate pre-treatment prior to parathyroidectomy
is lacking in veterinary medicine. Conflicting correlation of preoperative calcium levels
and developing postoperative hypocalcemia is reported in people113. The current
recommendation in people is to pretreat with vitamin D metabolites in individuals with
vitamin D deficiency prior to parathyroidectomy114.
If the level of hypercalcemia necessitates management prior to ablation procedure
or surgery, a variety of treatments are available such as saline diuresis, diuretic therapy,
glucocorticoids, bisphosphonates and calcitonin. The majority of treatment is aimed at
increasing calciuresis in the kidneys with saline diuresis or addition of furosemide.
Glucocorticoids will also reduce bone resorption of calcium and bisphosphonates and
calcitonin have inhibitory effects on osteoclastic bone resorption activity87. The goal of
pretreatment with an active vitamin D metabolite such as calcitriol is to lessen the
24
severity or prevent the onset of post-operative hypocalcemia, by increasing enterocyte
absorption of calcium into the circulation to counteract the acute decrease in PTH
production following removal of hyperactive parathyroid tissue. This topic remains
controversial to the type of medication used, dose and ideal duration prior to surgery.
Dogs with preoperative serum total calcium concentration less than 14 mg/dl have
a small risk of developing postoperative hypocalcemia in the opinion of some authors.
Consideration for prophylactic treatment with calcitriol for dogs with serum total calcium
greater than 15 mg/dl has been recommended87. In one study, dogs that did not
experience tetany after parathyroidectomy had preoperative mean total calcium of 13.7
mg/dl compared to dogs that experienced tetany that had mean total calcium of 15.4
mg/dl 63. In that study, 11 of 12 dogs that developed postoperative hypocalcemia were not
prophylactically treated preoperatively with calcium salts or calcitriol63. In another study,
dogs with mean preoperative total calcium 13.7 mg/dl did not develop hypocalcemia
compared to dogs with mean preoperative total calcium of 16.8 mg/dl that developed
postoperative hypocalcemia64. In one study, 13 of 19 dogs were pretreated with
alfacalcidiol, a hydroxylated vitamin D analogue, with or without calcium carbonate prior
to surgery64. The amount of pretreatment time varied from one day prior to surgery to
initiating pretreatment the day of surgery64.
The vitamin D metabolite of choice is calcitriol, compared to cholecalciferol and
ergocalciferol, due to its short time to achieve biological effect (1-4 days) and short
circulating half-life. The proposed dosing regimen and duration for calcitriol is 10-20
ng/kg orally every 12 hours starting 3 to 5 days prior to surgery for 2-3 days, then
25
decreasing the dose to 5 ng/kg orally every 12 hours until surgery115. This
recommendation has been controversial since it is not known whether this short course of
calcitriol would be sufficient time to prime the gastrointestinal system to adequately
increase calcium absorption.
2.8 - Treatment Options
Current mainstay treatment options in veterinary medicine are with ultrasoundguided heat ablation or parathyroidectomy.
2.8.1 - Ethanol ablation
Percutaneous ethanol ablation of the parathyroid glands for PHPTH in dogs was
first described in 199940. Ethanol causes coagulation necrosis and vascular thrombosis of
exposed tissue87. The volume of the pathological parathyroid gland is approximated with
ultrasonography and a matched volume of 96% ethanol is injected into the pathologic
gland with ultrasound guidance until the hypoechoic tissue becomes hyperechoic71.
Reports for the overall success rates for ethanol ablation have differed. The majority of
PHPTH dogs had resolution of hypercalcemia with one treatment, however additional
treatment may be needed66, 71. Complications are related to extravasation of ethanol to
affect the surrounding tissues and include coughing, dysphonia secondary to chemical
injury to the recurrent laryngeal nerve, and continued hypercalcemia66, 70, 71.
26
2.8.2 - Heat ablation
This technique utilizes radiofrequency to cause thermal necrosis. The abnormal
parathyroid gland has to be larger than 3 mm to ensure adequate needle placement71.
Unlike ethanol ablation, extravasation does not occur with heat ablation and tends to
spare the local blood supply116. This procedure requires general anesthesia and a skilled
ultrasonographer. In one study, 92% of dogs had resolution of hypercalcemia with one
treatment of percutaneous ultrasound-guided heat ablation71. Another study reported a
success rate of 73%70. Complications reported included cough, transient dysphonia,
Horner’s syndrome and failure to resolve hypercalcemia71.
2.8.3 - Parathyroidectomy
Surgical removal of the parathyroid glands has been the primary treatment for
PHPTH in dogs for decades63. Success rates, as defined by resolution of hypercalcemia,
of 94-100% have been reported63, 64, 67, 71. Since ademonas do not tend to extend beyond
the parathyroid capsule, the affected gland can be completely excised between the
external parathyroid gland and the thyroid capsule. If the internal parathyroid gland
cannot be isolated from the thyroid gland, a partial thyroidectomy with preservation of
the ipsilateral external parathyroid gland can be elected. In situations where carcinoma is
a more likely diagnosis, complete thyroparathyroidectomy is performed80.
When comparing all three procedures, a statistical significance for success was
noted comparing ethanol ablation to parathyroidectomy to treat PHPTH. Percutaneous
ultrasound-guided ethanol ablation was the least successful of the treatment options and
27
also associated with the most complications. Compared to ethanol ablation,
radiofrequency causes less damage to the surrounding tissues. Success rates of dogs
treated with heat ablation had similar outcomes to dogs treated with parathyroidectomy.
Time to hypercalcemia resolution after heat ablation treatment was significantly longer
compared to ethanol treated dogs71.
2.9 – Postoperative hypocalcemia
Postoperative hypocalcemia is the most common complication after
parathyroidectomy. Between 33-40% of dogs became hypocalcemic following surgical
parathyroidectomy and approximately 9.7-11% of the dogs developed clinical signs in
two reports69, 71. Calcium concentrations start to decrease within 12 hours after surgery
but can continue to decline over a period of 7 days63, 71, 87. The decrease of ionized
calcium occurs in a linear fashion in the first 24 hours after parathyroidectomy65. There
was no statistically significant predictive value of the slope of the decreased ionized
calcium level during the first 24 hours after surgery and the development of
hypocalcemia65.
It is expected that hypersecreting parathyroid glands would cause negative
feedback leading to inhibition and atrophy of the remaining non-pathologic parathyroid
glands. Parathyroidectomy of the pathologic gland can lead to an acute and severe
decrease in PTH levels resulting in hypocalcemia until the remaining parathyroid glands
increase the synthesis and secretion of PTH. Although calcitriol is a hallmark treatment
option of postoperative hypocalcemia, it causes direct inhibition to parathyroid cell
28
proliferation and suppression of PTH transcription and secretion43. Traditional treatment
for postoperative hypocalcemia is aimed at administering the end products of PTH action
such as administering calcium salts or calcitriol.
Common clinical signs and symptoms of hypocalcemia include muscle twitching,
muscle fasciculation, pawing or rubbing of the muzzle, weakness, lethargy, cardiac
arrhythmias, tetany and death87. Post-operative dogs should be kept under strict exercise
restriction to limit the amount of calcium required for muscle metabolism and have serial
ionized calcium measured at least every 12 hours for the first 2 to 4 days after surgery. In
one study, 63% of dogs had the largest drop in calcium within the first 24 hours
postoperatively and 30% of dogs had a decrease in calcium by 72 hours after surgery63.
Length of hospitalization is often clinician dependent, but recommendations for up to five
days have been made to allow close monitoring to detect clinically important low
concentrations of ionized calcium and immediate medical treatment if indicated115.
Emergency management of hypocalcemia includes administering intravenous
calcium gluconate 10% solution slowly at a dose of 0.5-1.5 ml/kg intravenously. If the
clinical hypocalcemia is relatively mild (ie: pawing at face, tremors, muscle
fasciculation), a constant rate infusion of calcium gluconate rather than a bolus can be
utilized. It is imperative heart rate and rhythm is monitored with electrocardiogram, as
bradycardia or disturbance to electrical conductance and death are possible with too rapid
administration. Calcium gluconate should never be administered subcutaneously, even
when diluted, as side effects include calcinosis cutis, severe pain, inflammation, abscess
and necrosis115. Once the dog is stable, oral supplementation of calcitriol and calcium
29
carbonate should be initiated. Calcitriol can be loaded at a dose of 10-15 ng/kg orally
every 12 hours for 3-4 days, then decreased to 5 ng/kg orally every 12 hours can be
administered to improve intestinal absorption of calcium and phosphorus. Oral
supplementation of 25 mg/kg of elemental calcium every 12 hours can also be
considered. Weekly evaluation of ionized calcium is necessary until steady state of serum
calcium concentration has been reached at which point gradual tapering of calcium
carbonate followed by tapering of calcitriol can be attempted. Target levels of ionized
calcium that are desired should be high enough to ameliorate clinical signs but not high
enough to blunt parathyroid gland activity which is just below the lower reference range
or in the lower half of the reference range. The entire tapering process can take 3-6
months and some dogs may require lifelong supplementation80. The administration of
PTH (1-84) has recently been approved for the treatment of hypoparathyroidism in
people117. But this treatment option has not yet been reported for use in dogs.
Persistent and severe hypocalcemia after parathyroidectomy can be part of what
has been termed “hungry bone syndrome” (HBS) and is seen with hypocalcemia,
hypophosphatemia, hypomagnesemia and increased potassium levels. Occurrence of
HBS is related to the increased skeletal usage of calcium, followed by removal of the
hypersecreting parathyroid gland resulting in an immediate arrest of bone resoprtion in
the face of continued bone formation. The duration of HBS is the time required for
skeletal normalization and has been reported to last up to 9 months or longer in people118.
Pretreatment with bisphosphonates with or without calcitriol have shown promising
30
effects to either lessen the degree of HBS or completely attenuate postoperative
hypocalcemia118.
2.10 - Study Rationale
Distinct pre-operative markers to predict postoperative hypocalcemia have not
been identified in veterinary medicine. Post-operative hypocalcemia after
parathyroidectomy is sometimes a life threatening condition depending on the nadir of
circulating calcium that develops. Immediate intervention is necessary in some dogs to
prevent death. Prolonged hospitalization is required for some dogs to allow time to gain
sufficient control of circulating calcium at safe levels. Identifying predictive factors for
the development of post-operative hypocalcemia would enhance monitoring and
treatment recommendations in the post-operative period. Unlike previous PHPTH
research in dogs, this study evaluates the relationship of PTH and vitamin D metabolites
with the development of hypocalcemia after parathyroidectomy. To the authors’
knowledge, this is the first study to prospectively investigate the relationship of vitamin
D metabolites in dogs with PHPTH at the time of initial diagnosis and at the time of
calcium nadir
The goal of this study was to identify preoperative parameters that may be utilized
to predict development of postoperative hypocalcemia. We hypothesized that dogs with a
>75% change between baseline and intraoperative PTH concentrations will be more
likely to develop hypocalcemia in the immediate post-operative period. We also
hypothesized that dogs with low preoperative serum 25-vitamin D or low preoperative
31
serum 1,25-vitamin D will be more likely to develop post-operative hypocalcemia in the
immediate post-operative period.
32
Chapter 3: Materials and Methods
3.1 – Animals
The study protocol was approved by The Ohio State University-Institutional
Animal Use Care and Committee. Dogs with naturally occurring PHPTH that were
presented to The Ohio State University-Veterinary Medicine Center (OSU-VMC) were
enrolled into the study with informed owner consent. Conformation of PHPTH was
established following analysis of parathyroid hormone (PTH); parathyroid hormone
related polypeptide (PTHrP) and ionized calcium (iCa) concentrations measured at the
Michigan State University-Diagnostic Center for Population and Animal Health
(DCPAH). The values from this panel were documented as the “Initial PTH value.”
In addition to a PTH/PTHrP/iCa panel, each enrolled dog had thoracic radiographs,
serum biochemical profile, complete blood count and cervical ultrasound performed.
Dogs that were presented with diagnostic tests (thoracic radiographs, DCPAH PTH
results, complete blood count or biochemical profile) results from the referring
veterinarian were accepted providing the diagnostic test was performed within 30 days to
the time of surgery.
Overnight hospitalization prior to surgery with or without intravenous crystalloid
fluid therapy was at the discretion of the surgeon responsible for the case. One dog was
treated with two days of saline diuresis and initiation of calcitriol prior to undergoing
parathyroidectomy based on the high preoperative ionized calcium level.
33
3.2 - Sampling protocol
Six ml’s of whole blood were obtained from each dog the day prior to
parathyroidectomy. A portion of blood (0.7 ml) was used for ionized calcium and ionized
magnesium analysis. Other analytes (Na, K, Cl, Mg, iCa, BUN, creatinine) were
measured at the clinician’s discretion. The 0.7 ml of whole blood was transferred into a
commercial pre-heparinized syringe and analyzed within 10 minutes of collection. The
remaining volume was placed into a red top tube and allowed to clot. Centrifugation of
the clotted sample was at 7000 rpm for 5 minutes. If at the end of the 5 minutes the
sample necessitated more time in the centrifuge, an additional 5-minute spin at 7000 rpm
was performed. The serum was transferred as 1 ml aliquot samples into Eppendorfb tubes
and stored in -80°C freezer until analysis.
3.2.1 - Sampling timeline
Each dog was sampled at the time of enrollment (Pre_O1) and the morning prior
to parathyroidectomy (Pre_O2) from a peripheral vein. An intraoperative blood sample
(IO1) was obtained through the central line placed into a jugular catheter 10 minutes after
removal of the affected parathyroid. If more than one parathyroid gland was affected, an
additional blood sample (IO2) was obtained 10 minutes after further parathyroidectomy.
All intraoperative and post-operative blood samples were obtained through the central
34
venous catheter. The first post-operative blood sample (PO1) was obtained at 7pm the
evening of surgery. Serial samples were obtained every 12 hours (ie: PO2 – 7am sample
morning after surgery and PO3 – 7pm sample, etc) until the dog was discharged from the
hospital. One dog was medically boarded after parathyroidectomy and sampling was
discontinued at 14 days after parathyroidectomy.
3.2.2 – Electrolytes
All iCa and iMg samples were submitted to OSU-VMC clinical pathology
laboratory for measurement. Heparinized blood samples were submitted immediately
after collection and analyzed within 10 minutes of collection. Preoperative samples and
postoperative samples were always submitted for a minimum of iCa and iMg panel.
Other measurements that were performed include Na, K, Cl, BUN and creatinine on the
NOVAa machine.
3.2.3 – PTH
Batched frozen serum samples were submitted to Michigan State UniversityDiagnostic Center for Population and Animal Health (DCPAH) for radioimmunoassayc
PTH analysis. Frozen samples were shipped overnight in a Styrofoam shipping boxes
with ice packs. Pre_O, IO, even day PO (ie: PO2, PO4) samples, 14 day postoperative
samples and 90 days postoperative samples were submitted for analysis.
35
3.2.4 - Vitamin D Metabolites
Batched frozen serum samples were submitted to HeartLand Assays for 25Vitamin Dd and 1,25 Vitamin Dd analysis. Frozen samples were shipped by overnight
express on dry ice in a Styrofoam shipping box. Pre_O sample and the serum associated
with ionized calcium nadir while hospitalized, 14 day postoperative sample and 90 days
postoperative samples were submitted for analysis.
3.3 - General anesthesia and parathyroidectomy
Premedication was dosed at the discretion of the anesthesia service. A peripheral
intravenous catheter was placed into the right or left cephalic vein. All dogs were placed
under general anesthesia, which was maintained with isofluoranee or sevofluranef and
100% oxygen. All patients received crystalloid fluidg .
Dogs were positioned in dorsal recumbency and connected to a hemodynamic
monitorh and standard surgical thermal support with a Bair Huggeri. A standard ventral
midline approach to the cervical region was performed on all dogs. Cervical exploration
of both left and right thyroid and parathyroid glands were performed prior to
parathyroidectomy. Based on surgeon preference, the affected parathyroid was removed
with a combination of sharp and blunt dissection or LigaSurej device. Depending on
surgeon preference, either a parathyroidectomy without peri-thyroid tissue was removed,
or a thyroidectomy was performed with both the non-pathologic and non-pathologic
parathyroid gland. One dog had a unilateral thyroidectomy and removed both the internal
and external parathyroid gland on that side. One dog had three suspected parathyroid
nodules removed by individual parathyroidectomies. Each parathyroid gland was
36
weighed (grams) and measured (mm). All tissues removed were submitted for
histopathology.
A triple lumen central catheterk was placed intraoperatively using the modified
Seldinger technique. This catheter was placed immediately after parathyroidectomy and
used for the intraoperative (IO) blood collection 10 minutes after parathyroidectomy. For
the dog that had multiple (3) suspect parathyroid nodules removed, separate IO samples
were collected for analysis 10 minutes after each parathyroidectomy. Routine closure was
performed and each dog was admitted into the Intensive Care Unit after recovery from
general anesthesia.
3.4 - Postoperative monitoring and hospitalization
All dogs recovered in the Intensive Care Unit after surgery for monitoring,
nursing care, crystalloidl fluid therapy and fentanylm continuous rate infusion or
intermittent methadonen bolus for pain control. Food and water were offered once the dog
was sufficiently recovered from general anesthesia to allow swallowing.
Blood collection was through the central venous catheter based on the blood
collection schedule. Standard operating procedure for blood collection from central
venous catheter involved withdrawing 6 ml of blood into a heparinized syringe prior to
collection of 6ml of blood for sample analysis. The 6ml of pre-sample blood was
administered back to the dog through the central venous catheter followed by heparinized
flush.
37
3.4.1 - Immediate perioperative period
Heparinized blood samples were obtained every 12 hours from the central venous
catheter to monitor ionized calcium levels based on the previously described schedule
and blood collection protocol. Additional iCa measurements were obtained for dogs that
showed clinical signs of hypocalcemia between the planned sampling times.
3.4.2 - Two week and three month recheck
Dogs were returned 10-14 days after surgery for incision evaluation and suture
removal. Heparinized blood was sampled at both the two week and three month recheck.
3.5 - Statistical analysis
Descriptive statistics were computed on SPSS (SPSS program) and reported as
median (range). Non-parametric tests were used because of the small number of dogs in
this study. Chi-squared analyses were used to evaluate relationships between binary
variables such as hypomagnesemia (pre- and post- operative), hypovitaminosis D (preand post- operative) and the development of postoperative ionized hypocalcemia. MannWhitney U tests were used for analysis of continuous variables with binary variables such
as percent change in PTH concentrations, length of hospitalization, pre-operative total
and ionized calcium concentration, pre-operative vitamin D metabolite concentrations
and the development of post-operative hypocalcemia. Spearman rho correlation analyses
were used to evaluate length of hospitalization with percent PTH change.
All statistical analysis was performed with commercially available SPSS
computer software. For all analysis, p<0.05 was considered significant.
38
3.6 - Footnotes
a
pOHx STAT, Nova biomedical, Waltham, MA, USA
b
Eppendorf tubes, Eppendorf North America, Hauppauge, NY, USA
c
PTH radioimmunoassay , MSU-DCPAH, Lansing, MI, USA
d
25VitaminD and 1,25Vitamin D, HeartLand Assays, Ames, IA, USA
e
Fluriso, MWI, Boise, ID, USA
f Sevoflurane, Baxter, Deerfield, IL, USA
g
Lactate Plus, Nova Biomedical Corporation, Waltham, MA, USA
h
Cardell MAX-12 DUO HD Multiparameter Monitor, Midmark Corp, Versailles, OH,
USA
i
Bair Hugger, 3M, St. Paul, MN, USA
j
LigaSure Small Jaw Open Instrument, Medtronic, Minneapolis, MN, USA
k
Triple lumen catheter, Arrow International Inc., Reading, PA, USA
l PlasmaLyte, Medline, Mundelein, IL, USA
m
Fentanyl citrate injectable, Hospira Inj., Lake Forest, IL, USA
n
Methadone, Xanodyne, Newport, KY
o
SPSS, IBM SPSS Statistics software-version 21
39
Chapter 4: Results
Ten dogs with PHPTH were enrolled in the study. Median age of dogs with
PHPTH was 10 years (range, 6-13 years). There were four mixed breed dogs and the
remaining breeds consisted of Siberian husky, standard Schnauzer, Golden retriever,
Redbone Coonhound, Border collie and Lhasa Apso. Seven dogs were spayed females
and three were castrated male dogs. Median body weight was 28.6 kg (range, 4.7-46 kg).
4.1 - Clinical signs
Six dogs presented for polyuria and polydipsia. Of these six, two dogs also
presented for weakness, lethargy and gastrointestinal signs such as anorexia and
intermittent vomiting. One dog was presented for urethral obstruction associated with
calcium oxalate urolithiasis. One dog had a history of weight loss, intermittent anorexia
and tremors. Two dogs had no clinical signs and were diagnosed on routine blood work
for annual examination.
4.2 - Parathyroid hormone
Five dogs had preoperative PTH concentrations above the reference range and
five dogs had preoperative PTH concentrations within the reference range (reference
range, 0.5-5.8 pmol/L). The median preoperative PTH concentration was 4.85 pmol/L
(range, 1.3-24.6 pmol/L). Four of the five dogs that developed postoperative
40
hypocalcemia had elevated PTH concentrations at the time of diagnosis (p=0.016).
(TABLE 1).
All PTH concentrations decreased into the normal ranges by 24 hours after
parathyroidectomy and continued to remain normal during the perioperative
hospitalization period (FIGURE 1). A statistical significance was found between the
PTH level at the time of diagnosis and prediction of postoperative hypocalcemia
(p=0.016; normocalcemic dogs: median, range 2.4 pmol/L,1.3-5.9 pmol/L; hypocalcemic
dogs: median, range 14 pmol/L, 3.8-24.6 pmol/L) (FIGURE 2). A statistical significance
was found between the preoperative PTH concentration and prediction of postoperative
hypocalcemia (p=0.032; normocalcemic dogs: median, range 2.8 pmol/L, 0.6-4.9 pmol/L;
hypocalcemic dogs: median, range 21.1 pmol/L, 3.3-36.9 pmol/L) (TABLE 1) (FIGURE
3).
The percent change of circulating PTH concentration between the PTH level at
diagnosis and intraoperative sample after parathyroidectomy was calculated. The median
PTH percent change in all dogs’ was 77.6% (range, 53.8%-91.1%). Dogs that developed
postoperative hypocalcemia had a significantly larger decrease in PTH concentration than
dogs that did not develop hypocalcemia (p<0.008; normocalcemic dogs: median, range
70.8%, 53.8%-74.5%; hypocalcemic dogs: median, range 81.7%, 80.7%-91.1%)
(TABLE 3) (FIGURE 4). A statistically significant difference was identified when
evaluating the percent PTH change between preoperative and intraoperative samples and
41
the development of hypocalcemia. The median PTH percent change (preoperative to
intraoperative) in all dogs was 81.2% (range, 16.7%-94.0%). Dogs that developed
postoperative hypocalcemia had a significantly larger decrease in PTH concentration than
dogs that did not develop hypocalcemia (p=0.032; normocalcemic dogs: median, range
75.0%, 16.7%-82.9%; hypocalcemic dogs: median, range 89.6%, 78.8%-94.0%)
(TABLE 3) (FIGURE 5).
The percent change in PTH from the time of diagnosis to the intraoperative
sample was significantly correlated to the post-operative ionized calcium nadir value
(p=0.02, postoperative ionized calcium nadir median, range 4.97 mg/dl, 4.38-5.73 mg/dl).
The percent change of PTH concentration was also significantly related to the need to
prescribe post-operative 1,25-dihydroxyvitamin D and calcium supplementation
(p<0.08). There was not a significant correlation between length of hospitalization after
surgery and percent change in PTH concentration (p=0.10).
4.3 – Calcium
The median preoperative total calcium concentration was 14.3 mg/dl (range, 12.120.4 mg/dl). Female dogs (n=7) in this study had higher median preoperative serum total
calcium of 15.5 mg/dl compared to male dogs (n=3) of 12.9 mg/dl. Median pre-operative
total calcium in dogs that remained normocalcemic postoperatively was not significantly
different than dogs that developed postoperative hypocalcemia (p=0.095; dogs that
remained normocalcemic: median, range 13.3 mg/dl, 12.1-15.1 mg/dl; dogs that
developed hypocalcemia: median, range: 15.6 mg/dl, 12.6-20.4 mg/dl) (TABLE 1).
42
Hypocalcemia was defined in this study as a concentration less than the reference range
of ionized calcium.
The median preoperative ionized calcium concentration was 6.64 mg/dl (range,
5.96-9.22 mg/dl). The median pre-operative ionized calcium between dogs that
developed and remained normocalcemic postoperatively and dogs that developed
postoperative hypocalcemia approached significance (p=0.056; dogs that remained
normocalcemic postoperative: median, range 6.25 mg/dl, 5.96-6.82 mg/dl; dogs that
developed postoperative hypocalcemia: median, range: 8.02 mg/dl, 6.23-9.22 mg/dl).
(TABLE 1).
All ionized calcium levels started to decrease starting the evening of surgery
(FIGURE 6). Ionized calcium concentration returned to normal within 12 hours (two
dogs), 24 hours (six dogs), and 36 hours (two dogs) after parathyroidectomy. Five dogs
developed hypocalcemia during the immediate postoperative period; three of the five
dogs developed subclinical hypocalcemia and two dogs developed clinical signs of
hypocalcemia. The immediate postoperative period in this study is defined as the time the
dog was hospitalized after parathyroidectomy. Calcium nadir values were reached within
48 hours postoperatively in four dogs, within 72 hours in three dogs, within 96 hours in
two dogs and at 144 hours in one dog.
The percent change of ionized calcium from the preoperative value to the
postoperative nadir was statistically significant (p = 0.008; dogs that remained
normocalcemic dogs: median, range 15.9%, 9.28-18.3%; hypocalcemic dogs: median,
range: 38.9%, 29.7-52.1%) (TABLE 3) (FIGURE 7).
43
4.4 – Phosphorus
Seven dogs had hypophosphatemia and three dogs had normal phosphorus values
at the time of presentation. Normal phosphorus values were designated to be 3.2-8.1
mg/dl. The median preoperative phosphorus concentration was 2.6 mg/dl (range, 1.6-4.3
mg/dl). (TABLE 1).
4.5 – Magnesium
Two dogs had serum ionized magnesium levels below the reference range and
eight dogs had serum magnesium concentrations within the reference range prior to
surgery. Median preoperative serum ionized magnesium concentration was 1.18 mg/dl
(range, 1.01-1.44 mg/dl). Median preoperative calcium:magnesium ratio for ten dogs was
6.04 (range 4.50-7.68). There was not a significant association between preoperative
calcium:magnesium ratio and development of post-operative hypocalcemia (p=0.15).
There was no association between calcium:magnesium ratio at nadir and the development
of post-operative hypocalcemia (p=0.69). (TABLE 1).
4.6 - Alkaline phosphatase
Six dogs had elevated alkaline phosphatase (ALP) concentrations above the
reference range, three dogs had normal pre-operative ALP and one dog had low ALP
values. Normal ALP reference ranges 15-120 IU/ml. The median ALP concentration was
44
254 IU/ml (range, 40-2038 IU/ml). (TABLE 1). No dogs were treated with oral or
injectable steroids at the time of surgery consultation or during the study period.
4.7 - Vitamin D
The reference range for 25-hydroxyvitamin D (25(OH)D) and 1,25dihydroxyvitamin D (1,25(OH)2D)concentration in dogs was 54.9-97.9 ng/ml and 168.9428 pg/ml respectively129. The median preoperative 25(OH)D concentration was 33.3
ng/ml (range, 9.2-64.4 ng/ml) and the median 1,25(OH)2D concentration was 242.85
pg/ml (range, 24.6-376.1 pg/ml). (TABLE 2). No significant difference was found
between the preoperative 25(OH)D or preoperative 1,25(OH)2D and the development of
postoperative hypocalcemia (p=0.69 and p=0.42, respectively) (FIGURE 8 and
FIGURE 9).
No statistical significance was found between preoperative 25(OH)D levels and
postoperative calcium nadir (p=0.60; median, range: 29 ng/ml, range 10.3-67.9 ng/ml).
No statistical significance was found between preoperative 1,25(OH)2D levels and
postoperative calcium nadir (p=0.803; median, range: 128.5 pmol/ml, 65.9-271.2
pmol/ml) (TABLE 3). No statistical significance was found between preoperative
vitamin D levels and postoperative 25(OH)D nadir (p=0.42) and 1,25(OH)2D nadir
(p=0.84).
45
4.8 - Cervical ultrasonography
All dogs had cervical ultrasounds performed. Fifteen presumed enlarged
parathyroid glands were detected preoperatively in ten dogs. Seven dogs had uniglandular
disease with three localized to the left parathyroid glands and four localized to the right
side. One dog had multiglandular enlargement of all parathyroid glands, one dog had
bilaterally enlarged hypoechoic parathyroid nodules with a larger right hypoechoic
nodule within the parenchyma of the right thyroid gland. Another dog had two
hypoechoic nodules within the parenchyma of the right thyroid gland.
4.9 - Surgical findings
Seven dogs had a parathyroidectomy of the enlarged parathyroid gland and three
dogs had the enlarged parathyroid gland removed by unilateral thyroidectomy. Not
including the normal sized parathyroid gland in the dogs that had a thyroidectomy, a total
of 12 enlarged suspect parathyroid glands were removed in 10 dogs. Four of 12 were left
parathyroid glands and 8 were from the right side. One dog had three abnormal
hypoechoic nodules identified on ultrasound and all three were removed during surgery.
4.10 – Histopathology
Ten of the 12 presumed parathyroid glands were confirmed to be parathyroid
glands with the remaining two being consistent with nodular thyroid hyperplasia. Two of
the 10 parathyroid glands were consistent with hyperplasia (20%), six were parathyroid
adenomas (60%) and the remaining two glands were carcinoma (20%). In one dog with
46
three hypoechoic nodules identified on ultrasound, one was parathyroid carcinoma, one
was thyroid carcinoma and the last gland was thyroid nodular C-cell hyperplasia.
4.11 – Postoperative hypocalcemia treatment
All dogs that developed clinical hypocalcemia (n=5) were immediately treated
with a bolus of intravenous calcium gluconate solution and continued on 1,25dihydroxyvitamin D and oral calcium carbonate. One dog that developed clinical ionized
hypocalcemia initially presented with an ionized calcium concentration of 8.17 mg/dl,
total calcium 20.4 mg/dl, and was treated for two days prior to parathyroidectomy with
intravenous saline diuresis and oral 1,25-dihydroxyvitamin D twice a day. This dog
developed neurologic signs that lead to seizure activity when the ionized calcium had
decreased post operatively but was still above the normal reference range. The other dog
that developed clinical hypocalcemia had a preoperative ionized hypercalcemia of 9.22
mg/dl, total calcium of 16 mg/dl, and was seen to paw the face when the ionized calcium
was 4.42 mg/dl. All dogs that developed postoperative hypocalcemia regardless of lack of
clinical signs received oral supplementation of calcitriol or calcium carbonate. The exact
decision as whether one or both oral supplementation was used was based on the primary
surgeons discretion.
4.12 - Length of hospitalization
The median length of hospitalization after surgery was 8 days (range, 1.5-9.5
days). One dog was discharged two days postoperatively, three days after surgery (four
47
dogs), four days after surgery (three dogs), nine days after surgery (one dog) and one dog
was discharged 10 days after surgery. The two dogs that were hospitalized for 9 and 10
days after parathyroidectomy both developed clinical hypocalcemia.
48
Table 1 – Preoperative and postoperative data values for dogs that remained normaocalcemia and developed hypocalcemia after
parathyroidectomy.
49
Preop
phosphorus
(mg/dl)
Reference
3.2-8.1
p value
Normocalcemic dogs:
1.6
1
2.7
2
3.8
7
2.5
9
2.5
10
Median
2.5
Range
1.6-3.8
Hypocalcemic dogs:
3
4
5
6
8
Median
Range
2.1
4.1
2.0
3.1
4.3
3.1
2.0-4.3
Preop ALP
(IU/mL)
Preop iMg
(mg/dl)
Preop tCa
(mg/dl)
Preop iCa
(mg/dl)
15-120
-
1.04-1.46
-
9.3-11.6
0.095
4.9-5.8
0.056
Time to
iCa nadir
(hr)
-
iCa nadir
(mg/dl)
-
Preop
iCa/iMg
ratio
0.15
iCa/iMg
ratio
nadir
0.69
343
281
2038
40
1042
343
40-2038
1.16
1.13
1.37
1.01
1.24
1.16
1.01-1.37
12.1
13.3
13.5
13.3
15.1
13.3
12.1-15.1
5.96
6.82
6.16
6.45
6.25
6.25
5.96-6.82
48
48
48
96
72
48
48-96
5.29
5.73
5.04
5.27
5.67
5.29
5.04-5.73
5.14
6.04
4.50
6.39
5.04
5.14
4.50-6.39
5.00
5.02
4.29
5.67
5.29
5.02
4.29-5.67
93
155
227
307
107
155
93-307
1.05
1.28
1.20
1.44
1.03
1.20
1.03-1.44
15.2
20.4
16
15.6
12.6
15.6
12.6-20.4
8.02
8.17
9.22
7.37
6.23
8.02
6.23-9.22
72
144
84
48
60
72
48-144
4.9
4.71
4.42
4.79
4.38
4.71
4.38-4.9
7.64
6.38
7.68
5.12
6.05
6.38
5.12-7.68
5.51
4.66
5.53
3.77
4.47
4.66
3.77-5.53
49
Table 2 - Preoperative and postoperative data (1,25(OH)D, 25(OH)D and PTH) values
for dogs that remained normaocalcemia and developed hypocalcemia after
parathyroidectomy.
Preop
1,25(OH)2D
(pg/ml)
Reference
168-428
p value
0.42
Normocalcemic dogs:
203.2
1
187.4
2
229.4
7
250.7
9
304.2
10
Median
229.4
Range
187.4-304.2
Hypocalcemic dogs:
265.9
3
324.7
4
235
5
376.1
6
24.6
8
Median
265.9
Range
24.6-376.1
1,25(OH)2D
nadir
(pg/ml)
-
Preop
25(OH)D
(ng/ml)
54.9-97.9
0.69
25(OH)D
nadir
(ng/ml)
-
PTH at
diagnosis
(pmol/L)
9.3-11.6
0.016
Preop
PTH
(pmol/L)
9.3-11.6
0.032
PTH
nadir
(pmol/L)
-
135.2
121.8
204.3
65.9
195.6
135.2
65.9-204.3
21.6
24
44.7
45.2
64.4
44.7
21.6-64.4
18.7
21.7
40.8
34.7
67.9
34.7
18.7-67.9
1.3
3.3
2.4
2.3
5.9
2.4
1.3-5.9
3.5
4.9
2.8
0.6
1.9
2.8
0.6-4.9
2
1.5
1.3
1.2
1.9
1.5
1.2-2.0
72.6
177.3
96.7*
271.2
95.2*
96.7
72.6-271.2
28.6
31
35.6
45.4
9.2
31
9.2-45.4
26.3
31.8
25.9**
32.5
10.3**
29.1
10.3-32.5
24.6
15.9
14
3.8
12
14
3.8-24.6
21.2
36.9
19.8
3.3
24.6
21.1
3.3-36.9
2.7
0.8
1.05^
1.7
5.9^
1.7
0.8-5.9
*1,25VD values from 12 hours prior to calcium nadir
**25VD values from 12 hours prior to calcium nadir
^PTH was not submitted at that time point. The recorded value is an average of the PTH
concentration from 12 hours prior and 12 hours after.
50
Figure 1: Trend of PTH (pmol/L) concentration per dog while hospitalized and followup at 10-14 days (Day 14) and 90 days (Day 90) after parathyroidectomy. Day 0 =
Morning of surgery prior to parathyroidectomy.
51
Figure 2: PTH (pmol/L) level at the time of diagnosis and postoperative hypocalcemia.
Normocalcemic dogs: median, range 2.4 pmol/L,1.3-5.9 pmol/L; hypocalcemic dogs:
median, range 14 pmol/L, 3.8-24.6 pmol/L. (p = 0.016).
52
Figure 3: Preoperative PTH (pmol/L) level and postoperative hypocalcemia.
Normocalcemic dogs: median, range 2.8 pmol/L, 0.6-4.9 pmol/L; hypocalcemic dogs:
median, range 21.1 pmol/L, 3.3-36.9 pmol/L. (p = 0.032).
53
Table 3 – Percent PTH change, percent calcium change and vitamin D data values
%PTH change (initial to
intraoperative)
%PTH change (preoperative to
intraoperative
%iCa-nadir
Pre25(OH)D and iCa nadir
Pre1,25(OH)2D and iCa nadir
Median
(Range)
77.6%
(53.9-91.1%)
81.2%
(6.7-94.0%)
23.9%
(9.38-52.1%)
29 ng/ml
(10.3-67.9/ml)
128.5 pg/ml
(65.9-271.2 pg/ml)
54
Normocalcemia
(Range)
70.8%
(53.8-74.5%)
75.0%
(16.7-82.9%)
15.9%
(9.28-18.3%)
-
Hypocalcemia
(Range)
81.7%
(80.7-91.1%)
89.6%
(78.8-94.0%)
38.9%
(29.7-52.1%)
-
p value
-
-
0.803
<0.008
0.032
0.008
0.60
Figure 4: Percent PTH change (initial PTH at diagnosis and intraoperative PTH) and the
development of hypocalcemia. The median PTH percent change was 77.6% (range,
53.8%-91.1%). Normocalcemic dogs: median, range 70.8%, 53.8%-74.5%; hypocalcemic
dogs: median, range 81.7%, 80.7%-91.1%. (p < 0.008).
55
Figure 5: Percent PTH change (preoperative PTH and intraoperative PTH) and the
development of hypocalcemia. The median PTH percent change was 81.2% (range,
16.7%-94.0%). Normocalcemic dogs: median, range 75.0%, 16.7%-82.9%; hypocalcemic
dogs: median, range 89.6%, 78.8%-94.0%. (p = 0.032).
56
Figure 6: Trend of ionized calcium during the immediate postoperative period. Day 0 =
Morning of surgery prior to parathyroidectomy. Ionized calcium concentration returned
to normal ranges within 12 hours (n=2), 24 hours (n=6), and 36 hours (n=2) after
parathyroidectomy.
57
Figure 7: Percent change in ionized calcium (initial iCa to nadir iCa) and the
development of postoperative hypocalcemia. Normocalcemic dogs: median, range 15.9%,
9.28-18.3%; hypocalcemic dogs: median, range: 38.9%, 29.7-52.1%. (p = 0.008).
58
Figure 8: Preoperative 25(OH)D and development of postoperative hypocalcemia.
Normocalcemic dogs: median, range 44.7 ng/ml, 21.6-64.4 ng/ml; hypocalcemic dogs:
median, range 31 ng/ml, 9.2-45.4 ng/ml. (p = 0.69).
59
Figure 9: Preoperative 1,25(OH)2D and development of postoperative hypocalcemia.
Normocalcemic dogs: median, range 229.4 pg/ml, 187.4-304.2 pg/ml; hypocalcemic
dogs: median, range 265.9 pg/ml, 24.6-376.1 pg/ml. (p = 0.42).
60
Chapter 5: Discussion
Primary hyperparathyroidism in dogs can be associated with both subclinical and
life threatening hypocalcemia following surgical excision. The current literatures on
postoperative hypocalcemia are retrospective in nature with various conclusions. This is
the first prospective study accruing data in the perioperative and postoperative time
setting, including vitamin D metabolite levels with conclusions that are applicable in a
clinical setting.
The age and weight distribution of the dogs in this study were consistent with
previous reports indicating PHPTH tends to be a disease of older, medium-large sized
dogs62, 64, 67, 69. An autosomal dominant inheritance pattern has been identified in the
Keeshonden dog breed with an odds ratio of developing PHPTH of 50.763, 75. Results of
other studies have supported the genetic link that Keeshonden dogs are predisposed62, 63,
71
, however, there were no Keeshonden dogs enrolled in this study which may be
reflective of a lack of breed popularity within the general public, small sample size or
regional variation in populations of dogs.
PHPTH in people has a higher sex predisposition for women with a ratio of
approximately 2.8:1 to 3.3:1, occurring in post-menopausal women119. Estrogen has a
known protective effect against PTH on bone resorption
61
which may be why men were more commonly asymptomatic or symptomatic for kidney
stones whereas women more commonly had bone related diseases such as
osteoporosis119. Men were also noted to have significantly elevated preoperative serum
total calcium and parathyroid hormone concentrations119. No sex predisposition has been
reported in dogs62, 64, 67, 69. A greater number of female dogs than male dogs were reported
in this study with a higher median preoperative serum total calcium 15.5 mg/dl and 12.9
mg/dl, respectively, and the six highest PTH levels at diagnosis. The significance of this
is unknown and should be evaluated further in future studies with higher case numbers.
The clinical signs reported in this study were consistent with previous veterinary
PHPTH publications where the most common presenting sign was polyuria and
polydipsia62, 65. The percentage of dogs diagnosed with concurrent cystic calculi (20%) in
this study was similar to other publications that reported 24% of dogs to have cystic
calculi at the time of referral62, with one dog being presented on emergency for urethral
obstruction. The cystolith composition was calcium oxalate in both cases. The other
clinical signs that were seen in this study are categorized into gastrointestinal or
neurologic signs and tend to be less commonly reported by owners than polyuria and
polydipsia62, 64, 65. Twenty percent of dogs in this study were asymptomatic for PHPTH,
which is less compared to other publications62, 64, 65.
Dogs with PHPTH tend to have decreased serum phosphorus that is attributable to
PTH-induced inhibition of renal tubular phosphorus resorption62. Seven (70%) dogs had
hypophosphatemia and three (30%) dogs had normal phosphorus levels in this study.
62
This was a similar proportion to the finding of a larger retrospective study62 but not
consistent with the findings of another study63. Interestingly, the dog with the highest (4.3
mg/dl) preoperative phosphorus value was mildly azotemic and developed severe
azotemia at the three-month postoperative recheck. This was similar to three of the
PHPTH dogs reported in a previous study that had higher phosphorus levels and renal
insufficiency63. A correlation with preoperative phosphorus level and development of
postoperative hypocalcemia has not been documented.
Alkaline phosphatase may be elevated in dogs with PHPTH due to increased
activity of the bone isoenzyme relating to bone turnover. PTH has effects on osteoclasts
to resorb bone in order to mobilize and increase circulating calcium. Previously, an
elevated alkaline phosphatase had been documented in some dogs with PHPTH63. Seven
(70%) dogs had elevated alkaline phosphatase and three (30%) dogs had alkaline
phosphatase within normal ranges. None of the dogs were treated with steroids at the
time of surgery consultation or during the study period. PHPTH is a disease of older
dogs, however, and it is possible that concurrent hepatic, endocrinopathies or
administration of medications have contributed to elevation of alkaline phosphatase. It
has been reported in people that excess PTH causes increased alkaline phosphatase and is
also associated with radiographic evidence of bone resorption120. A duel-energy X-ray
absorptiometry (DEXA) scan would be necessary to evaluate for bone mineral density. In
addition, there were no correlations between preoperative alkaline phosphatase and PTH
concentrations in the current study.
63
Postoperative hypocalcemia is the most common complication after
parathyroidectomy. In previous studies, 33-40% of dogs became hypocalcemic following
surgical parathyroidectomy and approximately 9.7-11% of the dogs developed clinical
signs69, 71. The percentage of dogs that developed hypocalcemia in this study was higher
than previously reported. The mechanism for postoperative hypocalcemia after
parathyroidectomy is multifactorial and not fully understood. It likely involves
suppression of the normal parathyroid glands, sudden cessation of PTH mediated bone
resorption and increased uptake of calcium into remineralizing bone, decreased intestinal
absorption of calcium through decreased conversion of 25(OH)D to 1,25(OH)2D. Other
causes include surgical hypoparathyroidism, which is typically transient but may be
related to disruption of the blood supply to the remaining parathyroid glands.
Prolonged exposure of circulating PTH may alter the set-point mechanism
resulting in tissue resistance to PTH. This has been reported in people with chronic
kidney disease and primary hyperparathyroidism121, 122. People with chronically elevated
PTH concentration may have decreased sensitivity to 1,25(OH)2D as evident by
decreased density of the vitamin D receptor123, 124. In the postoperative period, this
alteration of PTH-calcium set-point may necessitate additional PTH to generate a
response from the target tissues and may lead to postoperative secondary
hyperparathyroidism7, 125.
Preoperative factors previously reported in association with postoperative calcium
nadir include a history of weakness, PTH concentration higher than reference range and
high serum BUN concentration69. That study also found a significant negative correlation
64
between age and post-operative calcium nadir, old dogs had lower serum total calcium
nadir concentrations when compared to young dogs69. The negative feedback caused by
chronic hypercalcemia the result of hypersecreting parathyroid glands, leads to atrophy of
the non-pathologic parathyroid glands. In this situation, following parathyroidectomy of
the pathologic gland, a transient hypoparathyroid state would occur, leading to continued
decrease of calcium levels, but studies have not routinely reported PTH concentrations
following parathyroidectomy.
Feldman et al73 suggested that PHPTH dogs with a higher preoperative serum
total calcium level (>14 mg/dl) are at a greater risk of developing postoperative
hypocalcemia. The results of our study did not show a statistical difference between
preoperative total calcium concentration and developing postoperative hypocalcemia.
The total calcium value is influenced by circulating protein concentration and albumin
concentration that were not assessed in this study. It is also known that total calcium is a
less sensitive parameter than ionized calcium as an assessment of circulating calcium
levels1. We did not find a significant correlation between preoperative ionized calcium
and preoperative PTH level in this study as expected in animals with parathyroid tumors
in which down regulation by calcium is lacking or absent. Preoperative ionized calcium
was not significantly correlated with developing postoperative hypocalcemia in a
retrospective study65. Our data showed a trend (p=0.056) towards higher pre-operative
ionized calcium in dogs that developed hypocalcemia post-operatively compared to dogs
that did not. The two dogs that developed clinical hypocalcemia had the highest preoperative total and ionized calcium levels. This may reflect prolonged atrophy of the
65
remaining parathyroid glands, recruitment of calcium for bone remineralization, or
continued polyuria with renal calcium excretion. When evaluating the percent change of
calcium between preoperative ionized calcium and the ionized calcium nadir after
surgery, a significant association (p=0.008) was found in dogs that became hypocalcemic.
Ionized calcium concentrations started to decrease in all dogs on the evening post
surgery. In eight of 10 dogs ionized calcium normalized within 24 hours after surgery and
in two dogs ionized calcium normalized by 36 hours after surgery. The decrease of
ionized calcium occurred in a linear fashion in the first 24 hours after parathyroidectomy
in our study as was reported in another study65. The rate of decline in the first 24 hours
did not predict the development of hypocalcemia in the same study65. The time to ionized
calcium nadir was variable in our study. Four dogs reached nadir 48 hours after surgery,
three dogs within 72 hours after surgery, two in 96 hours after surgery and the longest
time until nadir was 144 hours. Dogs in our study were discharged at varying days after
surgery based on the primary clinician discretion. It is possible that dogs that were
discharged relatively soon after surgery had reached a new nadir at home.
All elevated PTH concentrations decreased into the normal by 24 hours after
parathyroidectomy and all PTH concentrations continued to remain in the normal range
during the time of hospitalization after surgery. Other studies have also demonstrated that
PTH concentrations decrease within the first 24 hours after surgery and reach a plateau at
varying times afterwards70, 71. Unlike other reports64, 65 that did not find a correlation
between preoperative PTH concentration and prediction of postoperative hypocalcemia,
dogs that developed postoperative hypocalcemia had higher PTH concentrations at
66
diagnosis compared to dogs that did not develop hypocalcemia postoperatively. One
study found similar results with higher than reference range PTH to be associated with
low postoperative total calcium69.
The introduction of intraoperative parathyroid hormone assay has altered surgical
options and improved the success of parathyroidectomy in people with PHPTH. The
assay can measure PTH level in plasma and since the hormone has a short-half life, it
decreases rapidly when all hypersecreting tissue is excised6. A decrease in PTH
concentration of >50%, from the pre-surgical sample ten minutes after parathyroidectomy
is indicative of successful removal of all autonomously functioning parathyroid tissue in
people94, 126. Focused cervical surgery with minimally invasive approaches has improved
postoperative cosmesis in people requiring cervical interventions by allowing discrete
incisions126. Although cosmesis is not usually a significant concern in dogs,
intraoperative PTH assay has been used to confirm successful parathyroidectomy in
dogs67, 68.
In our study, the percent change of PTH between the sample at the time of
diagnosis and the intraoperative sample was correlated to development of hypocalcemia
after parathyroidectomy. Dogs with a percent change of PTH greater than 75% were
significantly more likely to develop postoperative hypocalcemia compared to dogs that
did not. The degree of normal parathyroid gland atrophy may be related to duration of
PHPTH or perhaps related to the severity of disease. The only stimulus of PTH
transcription and secretion is hypocalcemia. This suggests that individuals with
parathyroid gland atrophy would require a period of hypocalcemia in order to return a
67
non-functional parathyroid gland to secreting PTH. Unfortunately, significant
hypocalcemia is detrimental and can be associated with fatality. Interestingly,
1,25(OH)2D, which is the most active natural metabolite of vitamin D and a commonly
used medication in postoperative PHPTH individuals, inhibits parathyroid cell
proliferation and thus inhibits PTH secretion43. A correlation between parathyroid gland
size and amount of PTH secretion has been established in people127, 128. Literature about
the relationship between parathyroid gland size and amount of PTH produced has not
been reported in the veterinary medicine. Further evaluation of parathyroid gland mass,
PTH concentration, and severity of disease should be evaluated in future studies.
Cervical ultrasonography is an efficient and non-invasive method to examine
parathyroid gland size and determination of the number of glands affected. Previous
reports have identified ultrasonography as having good correlation with surgical findings
in dogs64, 65, 108. All dogs of this study received cervical ultrasonography evaluations by
board certified radiologists, or under the direct supervision of a board certified
radiologist, between two specialty practices. In terms of laterality, nine of the 10 dogs
had ultrasound reports that identified the correct location of the largest hypoechoic.
Previous reports identified an agreement of 75.9% between ultrasonographic results
regarding laterality of the abnormal parathyroid gland and surgical findings69. This
further supports the notion that success of ultrasonography is subjective and operator
dependent.
Clinical information about vitamin D metabolites in veterinary medicine has
largely focused on dogs and cats with chronic kidney disease129, 130, 131 and in dogs with
68
intestinal disease and hypocalcemia associated with hypovitaminosis D. Neither
25(OH)D or 1,25(OH)2D concentrations prior to surgery predicted the development of
post operative hypocalcemia. Also, the concentrations of 25(OH)D and 1,25(OH)2D at
the time of calcium nadir failed to predict those that developed hypocalcemia from those
that did not. The concentrations of 25(OH)D and 1,25(OH)2D did not differ between
preoperative measurement and measurements at the time of calcium nadir. It is
interesting to note that 25(OH)D concentrations were lower than the reference range at
the time of diagnosis even though it did not predict the development of hypocalcemia.
Regulation of calcium homeostasis is one of the major functions of 1,25(OH)2D,
which targets the intestines, kidneys, bone and the parathyroid glands. It is well known
that people with PHPTH and concurrent 25(OH)D deficiencies are associated with more
severe PHPTH disease as seen with higher calcium, higher alkaline phosphatase, higher
baseline PTH and larger parathyroid glands57, 84. In this study, a significant correlation
was not found comparing preoperative 25(OH)D or 1,25(OH)2D levels and developing
postoperative hypocalcemia. However, all 10 dogs in the study had low preoperative
25(OH)D levels and nine dogs had normal preoperative 1,25(OH)2D and one dog had low
preoperative 1,25(OH)2D levels. Possible reasons for low 25(OH)D in PHPTH can be
related to increased conversion of 25(OH)D to 1,25(OH)2D, decreased vitamin D
absorption, decreased conversion to 25(OH)D in the liver, increased 25(OH)D catabolism
or increased storage in adipose tissue46, 57.
Alternatively, chronic hypovitaminosis D may contribute to development of
PHPTH by inducing stimulation of PTH secretion to increase circulating 1,25(OH)2D. It
69
has also been suggested that chronic vitamin D deficiency may accelerate parathyroid
gland growth57. It is known that dogs with vitamin D deficiency have difficulties
increasing the amount of circulating calcium even in the presence of elevated PTH
production132. A type II statistical error cannot be ruled out and additional studies with
larger cohorts of dogs should be performed. The reference ranges established for vitamin
D metabolites in dogs is variable133, 134, 135. The reference range selected for use in this
study was based on that for 10 healthy companion dogs measured at the same lab and
recently reported in the veterinary literature129. All dogs in our study were fed a
commercial dog food that was not detailed as to nutritional D (cholecalciferol) intake. A
difference in vitamin D absorption may occur based on sex and breed of dog134 resulting
in varying concentrations of 25(OH)D following absorption. Additional research to better
understand vitamin D metabolism is warranted for dogs with PHPTH. A specific goal
should be to determine if low 25(OH)D concentrations as noted in our pre-operative dogs
are associated with the development of PHPTH and its severity.
This is the first prospective study analyzing PTH, 25(OH)D and 1,25(OH)2D
concentrations dogs with PHPTH undergoing surgical treatment. The pitfalls of using
different analyzers and non-standardized sample handling protocols used in previous
studies were avoided in the present study. All ionized calcium samples in this study were
handled based on a strict protocol and all measurements were performed on the same
machine. PTH as well as 25(OH)D and 1,25(OH)2D were measured by one laboratory
specializing in these measurements. This study provided time matched plasma ionized
70
calcium and serum PTH samples and time matched vitamin D metabolites which many
retrospective studies.
71
Chapter 6: Conclusion
Post-operative hypocalcemia after parathyroidectomy can be life threatening with
significant financial responsibilities. Identifying predictive factors for postoperative
hypocalcemia would improve patient care. In this study, hypocalcemia developed in five
of 10 dogs in our study, but only two of those five dogs developed recognizable clinical
signs. Based on the results of this study, dogs with a 75% or greater decrease of PTH
between the diagnostic sample or greater than 80% change in PTH from the pre-operative
to the intra-operative PTH sample had a significantly higher risk of developing postoperative hypocalcemia. The magnitude of the hypercalcemia based on total calcium
before surgery did not predict which dogs would develop hypocalcemia or clinical signs
of hypocalcemia. The magnitude of ionized hypercalcemia approached but did not
achieve statistical significance for the development of postoperative hypocaclemia.
Neither 25(OH)D or 1,25(OH)2D concentrations predicted which dogs developed postoperative hypocalcemia. Reasons for the low preoperative 25D concentrations are not
apparent.
Continued research is necessary to better understand the disease of PHPTH and
causes of prolonged postoperative hypocalcemia. In the clinical patient, the gold standard
of PHPTH monitoring should involve measuring intraoperative PTH to confirm excision
72
of hyperfunctioning parathyroid glands and predict the likelihood of postoperative
hypocalcemia. Vitamin D panels should also be evaluated prior to parathyroidectomy as
dogs with PHTPH may have low calcidiol values that may benefit from pretreatment
prior to parathyroidectomy.
73
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