Evaluation of Parathyroid and Preoperative as Predictive Factors for Post-operative in Dogs with Primary .

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 (PTH) causing hypercalcemia. Vitamin D also contributes to regulation. People with PHPTH and 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 is associated with complete surgical resection of the autonomously functioning . 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 involved in calcium are parathyroid hormone (PTH), produced from the parathyroid ; , the active metabolite of vitamin D; from the 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 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, , , 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 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 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 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 resulting in prolonged and bone remodeling4. PTH increases 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 of the proximal convoluted tubules, which is responsible for the conversion of calcidiol (25- hydroxycholecalciferol) 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 , 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 7- dehydrocholesterol 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, 25- hydroxyvitamin 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 1α- hydroxylase have also been reported in keratinocytes, , brain, cultured bone cells, macrophages, , prostate cells, islet cells of the pancreas and the parathyroid

37-44 glands . Extrarenal 1α-hydroxylase results in local production of 1,25(OH)2D for autocrine or paracrine regulation compared to the endocrine function of renal 1α- hydroxylase45.

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 sodium- calcium 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 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 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 albumin- adjusted 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 - 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 3-

6% 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 . The cranial thyroid 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 draining into the . Lymphatic drainage of the parathyroid gland is to the retropharyngeal nodes. This is one of the few glands that have lymph drainage cranial to the organ77.

2.2.2 - Nervous system

The thyroid 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 (MSU-

DCPAH) 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 post- translational modifications can alter the affinity of the animal PTH molecule for the anti-

PTH 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 pre- excision67. 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

99 25OHD3, whereas others underestimate 25OHD2 .

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 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 pertechentate-

99Tc 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

99mTc-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 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 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 ultrasound- guided 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 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 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 University-

Diagnostic 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 25-

Vitamin 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 (pre- and post- operative) and the development of postoperative ionized hypocalcemia. Mann-

Whitney 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, 12.1-

20.4 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 1,25- dihydroxyvitamin D (1,25(OH)2D)concentration in dogs was 54.9-97.9 ng/ml and 168.9-

428 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 , 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 1,25- dihydroxyvitamin 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.

Preop Preop ALP Preop iMg Preop tCa Preop iCa Time to iCa nadir Preop iCa/iMg phosphorus (IU/mL) (mg/dl) (mg/dl) (mg/dl) iCa nadir (mg/dl) iCa/iMg ratio (mg/dl) (hr) ratio nadir Reference 3.2-8.1 15-120 1.04-1.46 9.3-11.6 4.9-5.8 - - - - p value - - - 0.095 0.056 - - 0.15 0.69 Normocalcemic dogs: 1 1.6 343 1.16 12.1 5.96 48 5.29 5.14 5.00 2 2.7 281 1.13 13.3 6.82 48 5.73 6.04 5.02 7 3.8 2038 1.37 13.5 6.16 48 5.04 4.50 4.29 9 2.5 40 1.01 13.3 6.45 96 5.27 6.39 5.67 10 2.5 1042 1.24 15.1 6.25 72 5.67 5.04 5.29

49 Median 2.5 343 1.16 13.3 6.25 48 5.29 5.14 5.02

Range 1.6-3.8 40-2038 1.01-1.37 12.1-15.1 5.96-6.82 48-96 5.04-5.73 4.50-6.39 4.29-5.67

Hypocalcemic dogs: 3 2.1 93 1.05 15.2 8.02 72 4.9 7.64 5.51 4 4.1 155 1.28 20.4 8.17 144 4.71 6.38 4.66 5 2.0 227 1.20 16 9.22 84 4.42 7.68 5.53 6 3.1 307 1.44 15.6 7.37 48 4.79 5.12 3.77 8 4.3 107 1.03 12.6 6.23 60 4.38 6.05 4.47 Median 3.1 155 1.20 15.6 8.02 72 4.71 6.38 4.66 Range 2.0-4.3 93-307 1.03-1.44 12.6-20.4 6.23-9.22 48-144 4.38-4.9 5.12-7.68 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 Preop 25(OH)D PTH at Preop PTH 1,25(OH)2D nadir 25(OH)D nadir diagnosis PTH nadir (pg/ml) (pg/ml) (ng/ml) (ng/ml) (pmol/L) (pmol/L) (pmol/L) Reference 168-428 - 54.9-97.9 - 9.3-11.6 9.3-11.6 - p value 0.42 - 0.69 - 0.016 0.032 - Normocalcemic dogs: 1 203.2 135.2 21.6 18.7 1.3 3.5 2 2 187.4 121.8 24 21.7 3.3 4.9 1.5 7 229.4 204.3 44.7 40.8 2.4 2.8 1.3 9 250.7 65.9 45.2 34.7 2.3 0.6 1.2 10 304.2 195.6 64.4 67.9 5.9 1.9 1.9 Median 229.4 135.2 44.7 34.7 2.4 2.8 1.5 Range 187.4-304.2 65.9-204.3 21.6-64.4 18.7-67.9 1.3-5.9 0.6-4.9 1.2-2.0

Hypocalcemic dogs: 3 265.9 72.6 28.6 26.3 24.6 21.2 2.7 4 324.7 177.3 31 31.8 15.9 36.9 0.8 5 235 96.7* 35.6 25.9** 14 19.8 1.05^ 6 376.1 271.2 45.4 32.5 3.8 3.3 1.7 8 24.6 95.2* 9.2 10.3** 12 24.6 5.9^ Median 265.9 96.7 31 29.1 14 21.1 1.7 Range 24.6-376.1 72.6-271.2 9.2-45.4 10.3-32.5 3.8-24.6 3.3-36.9 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 follow- up 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

Median Normocalcemia Hypocalcemia p value (Range) (Range) (Range) %PTH change (initial to 77.6% 70.8% 81.7% <0.008 intraoperative) (53.9-91.1%) (53.8-74.5%) (80.7-91.1%) %PTH change (preoperative to 81.2% 75.0% 89.6% 0.032 intraoperative (6.7-94.0%) (16.7-82.9%) (78.8-94.0%) %iCa-nadir 23.9% 15.9% 38.9% 0.008 (9.38-52.1%) (9.28-18.3%) (29.7-52.1%) Pre25(OH)D and iCa nadir 29 ng/ml - - 0.60 (10.3-67.9/ml)

Pre1,25(OH)2D and iCa nadir 128.5 pg/ml - - 0.803 (65.9-271.2 pg/ml)

54

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. 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 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 pre- operative 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 post- operative 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 post- operative 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

References:

1. Schenck PA, Chew DJ. 2005. Prediction of serum ionized calcium concentration by use of serum total calcium concentration in dogs. Am J Vet Res. 66(8):1330-6.

2. Hofer AM, Brown EM. 2003. Extracellular calcium sensing and signaling. Nat Rev Mol Cell Biol. 4(7):530-8.

3. Kronenberg HM, Bringhurst FR, Segre GV et al. 2001. Parathyroid hormone biosynthesis and metabolism. In: Bilezikian JP, Marcus R, Levine Ma, editors. The parathryoids. San Diego: Academic Press. 17-30.

4. Schenck PA, Chew DJ, Nagode LA, Rosol TJ. 2006. Disorders of calcium: hypercalcemia and hypocalcemia. In: DiBartola SP, editor. Fluid, electrolyte, and acid- base disorders in small animal practice. 3rd edn. Saunders Elsevier, Missouri, 2006;122– 94.

5. Aurbach GD, Potts JT, Chase LR, Melson GL. 1969. Polypeptide hormones and . Ann Intern Med. 70(6):1243-65.

6. Serge GV, Perkins AS, Witters LA, et al. 1981. Metabolism of parathyroid hormone by isolated rat Kupffer cells and hepatocytes. J Clin Invest. 67(2):449-57.

7. Malberti F, Surian M, Cosci P. 1992. Effect of chronic intravenous calcitriol on parathyroid function and set point of calcium in dialysis patients with refractory secondary hyperparathyroidism. Nephrol Dial Transplant. 7(8):822–8.

8. Parker VJ, Gilor C, Chew DJ. 2015. Primary and secondary hyperparathyroidism in cats: pathophysiology, diagnosis and treatment. J Fel Med Surg. 17(5):427-39.

9. Bago AG, Palkovits M, Usdin TB, et al. 2009. Evidence for the expression of parathyroid hormone 2 receptor in the human brainstem. Idegyogy Sz. 61(3):123-6.

10. Niall HD, Keutmann H, Sauer R, et al. 1970. The amino acid sequence of bovine parathyroid hormone I. Hoppe Seylers Z Physiol Chem. 351(12):1586-8.

11. Brewer HB Jr, Ronan R. 1970. Bovine parathyroid hormone: amino acid sequence. Proc. Nat. Acad. Sci. 67(4):1862-9.

74

12. Aurbach GD, Keutmann HT, Niall HD, et al. 1972. Structure, synthesis, and mechanism of action of parathyroid hormone. Recent Prog Horm Res. 28: 353–398.

13. Aurbach GD. 1974. Biosynthesis, secretion and mechanism of action of parathyroid hormone. Trans Am Clin Climatol Assoc. 85:78–99.

14. Rosol TJ, Nagode LA, Couto CG, et al. 1992. Parathyroid hormone (PTH)-related protein, PTH, and 1,25-dihydroxyvitamin D in dogs with cancer-associated hypercalcemia. ;131(3):1157-64.

15. Sherwood LM, Mayer GP, Ramberg CF, et al. 1968. Regulation of parathyroid hormone secretion: proportional control by calcium, lack of effect of phosphate. Endocrinology. 83(5):1043-51.

16. Potts Jr JT. 1976. Recent advances in the chemistry and physiology of parathyroid hormones. Clin Endocrinol (Oxf). 5:S307-15.

17. Finkelstein JW, Boyar RM, Roffwarg HP, et al. 1973. secretion in congenital adrenal hyperplasia. J Clin Endocrinol Metab. 36(1):121–4.

18. Hellman L, Nakada F, Curti J et al, 1970. Cortisol is secreted episodically by normal man. J Clin Endocrinol Metab. 30(4):411–22.

19. Katz FH, Romfh P, Smith JA. 1972. Episodic Secretion of Aldosterone in Supine Man: Relationship to Cortisol. J Clin Endocrinol Metab. 178(1):178–81.

20. Lang DA, Matthews DR, Peto J, Turner RC. 1979. Cyclic oscillations of basal plasma glucose and insulin concentrations in human beings. N Engl J Med. 301(19):1023–7.

21. Naftolin F, Judd HL, Yen SS. 1973. Pulsatile patterns of and in man: the effects of clomiphene, with and without testosterone added. J Clin Endocrinol Metab. 36(2):285–8.

22. Jubiz W, Canterbury JM, Reiss E, Tyler FH. 1972. Circadian rhythm in serum parathyroid hormone concentration in human subjects: correlation with serum calcium, phosphate, albumin, and growth hormone levels. J Clin Invest. 51(8):2040–6.

23. López I, Aguilera-Tejero E, Estepa JC, et al. 2005. Diurnal variations in the plasma concentration of parathyroid hormone in dogs. Vet Rec. 157(12):344–7.

24. el-Hajj Fuleihan G, Klerman EB, Brown EN, et al. 1997. The parathyroid hormone circadian rhythm is truly endogenous--a general clinical research center study. J Clin Endocrinol Metab. 82(1):281–6. 75

25. Logue FC, Fraser WD, O'Reilly DS, et al. 1992. Sleep shift dissociates the nocturnal peaks of parathyroid hormone (1-84), nephrogenous cyclic adenosine monophosphate, and in normal men. J Clin Endocrinol Metab. 75(1):217-21.

26. Kripke DF, Lavie P, Parker D, et al. 1978. Plasma parathyroid hormone and calcium are related to sleep stage cycles. J Clin Endocrinol Metab. 47(5):1021–7.

27. Kitamura N, Shigeno C, Shiomi K, et al. 1990. Episodic fluctuation in serum intact parathyroid hormone concentration in men. J Clin Endocrinol Metab. 70(1): 252–63.

28. Nielsen HK, Brixen K, Kassem M, et al. 1991. Diurnal rhythm in serum : relation with sleep, growth hormone, and PTH(1-84). Calcif Tissue Int. 49(6):373–7.

29. Woitge HW, Knothe A, Witte K, et al. 2000. Circannual rhythms and interactions of vitamin D metabolites, parathyroid hormone, and biochemical markers of skeletal homeostasis: a prospective study. J Bone Miner Res. 15(12):2443-50.

30. Habener JF, Rosenblatt M, Potts Jr JT. 1984. Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action and metabolism. Phyiol Rev. 64(3):985-1053.

31. Schenck PA, Chew DJ, Behrend EN. 2005. Updates on hypercalcemic disorders. In: August J, ed. Consultations in Feline Internal Medicine. Vol. 5. St. Louis, Missouri: Elsevier, 2005:157–68.

32. How KL, Hazewinkle HAW, Mol JA. 1994. Dietary vitamin D dependence of cat and dog due to inadequate cutaneous synthesis of vitamin D. Gen Comp Endocrinol. 96(1):12-18.

33. Brumbaugh PF, Haussler MR. 1975. Specific binding of 1alpha,25- dihydroxycholecalciferol to nuclear components of chick intestine. J Biol Chem. 250(4):1588-94.

34. Jones G, Strugnell SA, DeLuca HF. 1999. Current understanding of the molecular actions of vitamin D. Physiol Rev. 78(4):1193-231.

35. Gray R, Boyle I, DeLuca HF. 1971. Vitamin D metabolism: the role of the kidney tissue. Science. 172(3989):1232-4.

36. Garabedian M, Holik MF, DeLuca HF, Boyle IT. 1972. Control of 25- hydroxycholecalciferol metabolism by the parathyroid glands. Proc Natl Acad Sci. 69(7):1673-6.

37. Fu GK, Lin D, Zhang MY, et al. 1997. Cloning of human 25-hydroxyvitamin D-1 76 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol. 11(13): 1961-70

38. Bikle DD, Nemanic MK, Whitney JO, Elias PW. 1986. Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry. 25(7):1545-8.

39. Diaz L, Sanchez I, Avila E, et al. 2000. Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells. J Clin Endocrinol Metab. 85(7):2545-9.

40. Delvin EE, Arabian A. 1997. Kinetics and regulation of 25-hydroxycholecalciferol 1- alpha-hydroxylase from cells isolated from cells isolated from human term decidua. Eur J Biochem. 163(3):659-62.

41. Monkawa T, Yoshida T, Hayashi M, Saruta T. 2000. Identification of 25- hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages. Kidney Int. 58(2):559-68.

42. Zehnder D, Bland R, Williams MC, et al. 2001. Extrarenal expression of 25- hydroxyvitamin-D3-1 alpha-hydroxylase. J Clin Endocrinol Metab. 86(2):888-94.

43. Segersten U, Correa P, Hewison M, et al. 2002. 25-Hydroxyvitamin D3-1α- Hydroxylase expression in normal and pathological parathyroid glands. J Clin Endocrinol Metab. 87(6):2967-72.

44. Schwartz GG, Whitlatch LW, Chen TC, et al. 1998. Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Cancer Epidemiol Biomarkers Prev. 7(5):391-5.

45. Hewison M, Bland R, Williams MC, et al. 2000. 1alpha-hydroxylase and the action of vitamin D. J Mol Endocrinol. 25(2):141-8.

46. Moosegaard B, Vestergaard P, Heickendorfft L, et al. 2005. Vitamin D status, seasonal variations, parathyroid adenoma weight and bone mineral density in primary hyperparathyroidism. Clin Endocrinol 63(5):506-13.

47. Lund B, Sorensen OH. 1979. Measurements of 25-hydroxyvitamin D in serum and its relation to sunshine, age and vitamin D in the Danish population. Scan J Clin Lab Invest. 39(1):23-30.

48. Bouillon R, Carmeliet G, Verlinden L, et al. 2008. Vitamin D and human health: lessons from vitamin D receptor null mice. Endocr Rev. 29(6):726-76.

77

49. Carmeliet G, Dermauw W, Buillon R. 2015. Vitamin D signaling in calcium and bone homeostasis: a delicate balance. Best Pract Res Clin Endocrinol Metab. 29(4):621-31.

50. Blaine J, Chonchol M, Levi M. 2015. Renal control of calcium, phosphate, and magnesium homeostasis. Clin J Am Soc Nephrol. 10(7):1257-72.

51. Andrukhova O, Smorodchenko A, Egerbacher M, et al. 2014. FGF23 promotes renal calcium reabsorption through the TRPV5 channel. EMBO J. 33(3):229-46.

52. Li YC, Bolt MJ, Cao LP, et al. 2001. Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism. Am J Physiol Endocrinol Metab. 281(3):E558-64.

53. Lieben L, Masuyama R, Torrekens S, et al. 2012. Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization. J Clin Invest. 122(5):1803-15.

54. Silver J, Yalcindag C, Sela-Brown A, et al. 1999. Regulation of the parathyroid hormone gene by vitamin D, calcium and phosphate. Kidney Int Suppl. 73:S2-7.

55. LoCascio V., Adami S., Galvanini G., et al. 1985. Substrate-product relation of 1- hydroxylase activity in primary hyperparathyroidism. N Engl J Med. 313(18):1123-5.

56. Lumb GA, Stanbury SW. 1974. Parathyroidfunctioninhuman vitamin D deficiency and vitamin D deficiency in primary hyperparathyroidism. Am J Med. 56(5): 833–9.

57. Rao DS, Honasoge M, Devine GW, et al. 2000. Effect of vitamin D nutrition on parathyroid adenoma weight: pathogenetic and clinical implications. J Clin Endocrinol Metab. 88(3):1054-8.

58. Mihai R, Farndon JR. 2000. Parathyroid disease and calcium metabolism. Br J Anaesth. 85(1):29-43.

59. Cetani F, Picone A, Cerrai P, et al. 2000. Parathyroid expression of calcium-sensing receptor protein and in vivo parathyroid hormone-Ca(2+) set-point in patients with priamry hyperparathytroidism. J Clin Endocrinol Metab. 85(12):4789-94.

60. Cusano NE, Maalouf NM, Wang PY, et al. 2013. NOrmocalcemic hyperparathyroidims and hypoparathyroidism in two community-based nonreferral populations. J Clin Endrocrinol Metab. 98(7):2734-41.

61. Krook L. 1957. Spontaneous hyperparathyroidism in the dog. A pathological- anatomical study. Acta path. microbiol. scand. 122:1-88.

78

62. Feldman EC, Hoar B, Pollard J, et al. 2005. Pretreatment clinical and laboratory findings in dogs with primary hyperparathyroidism: 210 cases (1987-2004). J Am Vet Med Assoc. 227(5):756-61.

63. Berger B, Feldman EC. 1987. Primary hypercalcemia in dogs: 21 cases (1976-1986). J Am Vet Med Assoc. 191(3):350-6.

64. Gear RN, Neigher R, Skelly BJ, et al. 2005. Primary hyperparathyroidism in 29 dogs: diagnosis, treatment, outcome and associated renal failure. J Small Anim Pract. 46(1):10– 6

65. Arbaugh M, Smeak D, Monnet E. 2012. Evaluation of preoperative serum concentrations of ionized calcium and parathyroid hormone as predictors of hypocalcemia following parathyroidectomy in dogs with primary hyperparathyroidism: 17 cases (2001-2009). J Am Vet Med Assoc. 241(2):233-6.

66. Long CD, Goldstein RE, Hornof WJ, et al. 1999. Percutaneous ultrasound-guided chemical parathyroid ablation for treatment of primary hyperparathyroidism in dogs. J Am Vet Med Assoc. 215(2):217-21.

67. Ham KL, Greenfield CL, Barger A, et al. 2009. Validation of a rapid parathyroid hormone assay and intraoperative measurement of parathyroid hormone in dogs with benign natuerally occurring primary hyperparathyroidism. Vet Surg, 38(1): 122-32.

68. Graham KJ, Wilkinson M, Culvenor J, et al. 2012. Intraoperative parathyroid hormone concentration to confirm removal of ypersecretory parathyroid tissue and time to postoperative normocalcemia in nine dogs with primary hyperparathyroidism. Aust Vet J. 90(6):203-9.

69. Milovancev M, Schmiedt CW. 2013. Preoperative factors associated with postoperative hypocalcemia in dogs with primary hyperparathyroidism that underwent parathyroidectomy: 62 cases (2004-2009). J Am Vet Med Assoc. 242(4):507-15.

70. Pollard RE, Long CD, Nelson RW, et al. 2001. Percutaneous ultrasonographically guided radiofrequency heat ablation for treatment of primary hyperparathyroidism in dogs. J Am Vet Med Assoc. 218(7):1106-10.

71. Rasor L, Pollard R, Feldman EC. 2007. Retrosepctive evaluation of three treatment methods for primary hyperparathyroidism in dogs. J Am Anim Hosp Assoc. 43(2):70-77.

72. Refsal KR, Provencher-Bolliger AL, Graham PA, Nachreiner RF. 2001. Update on the diagnosis and treatment of disorders of calcium regulation. Vet Clin North Am Small Anim Pract. 31(5):1043-62.

79

73. Feldman EC, Nelson RW. 2004. Hypercalcemia and primary hyperparathyroidism. In: Feldman EC, Nelson RW, eds. Canine and feline endocrinology and reproduction. 3rd ed. St Louis: Elsevier Science, 660–715.

74. DeVries SE, Feldman EC, Nelson RW, et al. 1993. Primary parathyroid gland hyperplasia in dogs: six cases (1982–1991). J Am Vet Med Assoc. 202(7):1132–6.

75. Goldstein RE, Atwater DZ, Cazolli DM et al. 2007. Inheritance, mode of inheritance, and candidate for primary hyperparathyroidism in Keeshonden. J Vet Intern Med, 21:199–203.

76. Bailey DB, Page RL. 2007. Tumors of the . In Withrow SJ, Vail DM, editors: Withrow and MacEwen’s small animal clinical oncology, ed 4, Philadelphia, 2007, Saunders, 583–609.

77. Hullinger RL. 1993. The endocrine system. In Miller ME, Evans HE. editors: Miller’s anatomy of the dog, ed 3, Philadelphia, Saunders 574-9.

78. Godwin MC. 1937. The development of the parathyroids in the dog with emphasis upon the origin of accessory glands. Anat. Rec. 68:305-25.

79. Vicari EM. 1937. Observations on the nature of the parafollicular cells in the thyroid gland of the dog. Anat Rec. 68:281-5.

80. Seguin B, Brownlee L. 2012. Thyroid and Parathyroid Glands. In: Tobias KM, Johnston SA editors. Veterinary Surgery: Small Animal. Saunders Elsevier, St Louis. 2043-58.

81. Flanders JA, Neth S, Erb HN, et al. 1991. Functional analysis of ectopic parathyroid activity in cats. Am. J. Vet. Res. 52(8):1336–40.

82. Godwin MC. 1937. Complex IV in the dog with special emphasis on the relation of the ultimobranchial body to interfollicular cells in the postnatal thyroid gland. Am J Anat. 60:299-330.

83. Mulligan RM, Francis KC. 1951. Weights of thyroid and parathyroid glands of normal male dogs. Anat Rec. 110(2):139-43.

84. Untch BR, Barfield ME, Dar M, et al. 2007. Impact of 25-Hydroxyvitamin D deficiency on perioperative parathyroid hormone kinetics and results in patients with primary hyperparathyroidism. Surgery. 142(6):1022-6.

80

85. , P. 2001. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences of bone loss and fractures and therapeutic implications. Endocr Rev. 22(4). 477-501.

86. Parfitt AM. 1998. Osteomalacia and related disorders: In: L.V.Avioli, S.M. Krane efs. Metabolic Bone Diseases and Clinically Related Disorders. Academic Press, San Diego, 327-86.

87. Feldman EC. 2005. Disorders of the parathyroid glands. In: Ettinger SJ, Feldman EC, editors. Textbook of veterinary internal medicine. 6th ed. Saunders Elsevier, St Louis. 1508-29.

88. Feldman EC. 1989. Canine primary hyperparathyroidism. In: Kirk RW, Bonagura JD (eds): Current Veterinary Therapy X. Philadelphia, WB Saunders Co. 985-95.

89. Frizel D, Malleson A, Marks V. 1967. Plasma levels of ionized calcium and magnesium i n . Lancet. 1(7504):1360-1.

90. Brasier AR, Nussbaum SR. 1988. Hungry bone syndrome: clinical and biochemical predictors of its occurrence after parathyroid surgery. Am J Med. 84(4):654-00.

91. Martin KJ, Hruska KA, Freitag JJ, et al. 1979. The peripheral metabolism of parathyroid hormone. N Engl J Med. 301(20):1092-8.

92. Gao P, Scheibel S, D'Amour P, et al. 2001. Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone: 1-84: implications for improvement of accurate assessment of parathyroid function. J Bone Miner Res. 16(4):605-14.

93. Quarles LD, Lobaugh B, Murphy G. 1992. Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab. 75(1):145-50.

94. Irvin GL, Solorzano CC, Carneiro DM. 2004. Quick intraoperative parathyroid hormone assay: surgical adjunct to allow limited parathyroidectomy, improve success rate and predict outcome. 28(12):1287-92.

95. Harrison BJ, Triponez F. 2009. Intraoperative adjuncts in surgery for primary hyperparathyroidism. Langenbecks Arch Surg. 394(5):799-809.

96. Vignali E, Picone A, Materazzi G et al. 2002. A quick intraoperative parathyroid hormone assay in the surgical management of patients with primary hyperparathyroidism: a study of 206 consecutive cases. Eur J Endocrinol. 146(6):783-8.

81

97. Agarwal G, Barakate MS, Robinson B et al. 2001. Intraoperative quick parathyroid hormone versus same-day parathyroid hormone testing for minimally invasive parathyroidectomy: a cost effectiveness study. Surgery. 130(6):963-70.

98. Hazewinkel HA, Tryfonidou MA. 2002. Vitamin D metabolism in dogs. Mol Cell Endocrin. 197(1):23–33.

99. Hollis BW. 2000. Comparison of commercially available 125I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. Clin Chem. 46(10):1657–61. 100. Frasoldati A, Valcavi R. 2004. Challenges in ultrasonography: lymphadenopathy and parathyroid glands. Endocr Pract 10(3):261-8.

101. Piciucchi A, Barone D, Gavelli G, et al. 2012. Primary Hyperparathyroidism: Imaging to Pathology. J Clin Imaging Sci. 2(59):Epublish.

102. Taillefer R, Boucher Y, Potvin C, et al. 1992. Localization of parathyroid adenomas in patients with hyperparathyroidism using a single radionuclide imaging procedure with Technetium-99m-sestamibi (Double-phase study). J Nucl Med. 33(10):1801-7.

103. Klieger P, O’Mara R. 1998. The diagnostic utility of dual phase Tc-99m-sestamibi parathyroid imaging. Clin Nucl Med. 23(4):208-11.

104. Lee VS, Wilkinson RH, Leight GS, et al. 1995. Hyperparathyroidism in high-risk surgical patients: Evaluation with double-phase technetium-99m-sestamibi imaging. Radiology. 197(3):627-33.

105. Khan A, Samtani S, Varma V, et al. 1994. Preoperative parathyroid localization: Prospective evaluation of technetium 99m sestamibi. Otolaryngol Head Neck Surg. 111(4):467-72.

106. Ferlin G, Borsato N, Camerani M, et al. 2002. New perspectives in localizing enlarged parathyroids by technetium-thallium subtraction scan. J Nucl Med. 24(5):438- 41.

107. Wisner ER, Penninck D, Biller DS, et al. 1997. High-resolution parathyroid sonography. Vet Radiol Ultrasound. 38(6):462-6.

108. Wisner ER, Nyland TG. 1998. Ultrasonography of the thyroid and parathyroid glands. Vet Clin N Am: Small Anim Pract. 28(4):973-91.

82

109. Liles SR, Linder KE, Cain B et al. 2010. Ultrasonography of histologically normal parathyroid glands and thyroid lobules in normocalcemic dogs. Vet Radiol Ultrasound 51(4):447-52.

110. Matwichuk CL, Taylor SM, Wilkinson AA, et al. 1996. Use of technetium Tc 99m sestamibi for detection of a parathyroid adenoma in a dog with primary hyperparathyroidism. J Am Vet Med Assoc. 209(10):1733-6.

111. Matwichuk CL, Taylor SM, Daniel GB, et al. 2000. Double-phase parathyroid scintigraphy in dogs using Technetium-99m-sestamibi. Vet Radiol Ultrasound. 41(5):461-9.

112. Caixas A, Berna L, Piera J, et al. 1995. Utility of 99m-Tc sestamibi scintigraphy as a first-line imaging procedure in the preoperative evaluation of hyperparathyroidism. Clinical Endocrinology. 43(5):525-30.

113. Heath DA, Van’t HW, Barnes AD et al. 1979. Value of 1-alpha-hydroxy vitamin D3 in treatment of primary hyperparathyroidism before parathyroidectomy. Br Med J. 1(6161): 450-2.

114. Bilezikian JP, Brandi ML, Eastell R, et al. 2014. Guidelines for the management of asymptomactic primary hyperparathyroidism: summary statement from the Fourth International Workshop. J Clin Endocrinol Metab. 99(10):3561-9

115. Galvao JF, Chew DJ. 2011. Metabolic complications of endocrine surgery in companion animals. Vet Clin Small Anim. 41(5):847-68.

116. Cosman ER, Nashold BS, Ovelman-Levitt J. 1984. Theoretical aspects of radiofrequency lesions in the dorsal root entry zone. Neurosurgery. 15(6):945-50.

117. Mannstadt M, Clarke BL, Vokes T, et al. 2013. Efficacy and safety of recombinant human parathyroid hormone (1– 84) in hypoparathyroidism (REPLACE): a double-blind, placebo-controlled, randomised, phase 3 study. Lancet Diabetes Endocrinol. 1(4):275– 83.

118. Witteveen JE, van Thiel S, Romijin JA, Hamdy NAT. 2013. Hungry bone syndrome: still a challenge in the post-operative management of primary hyperparathyroidism: a systematic review of the literature. Eur J Endocrinol. 168(3):R45–53.

119. Mazeh H, Sippel RS, Chen H. 2012. The role of gender in primary hyperparathyroidism: same disease, different presentation. Ann Surg Oncol. 19(9):2958- 62.

83

120. Scholz, DA. 1981 Asymptomatic primary hyperparathyroidism. 10-year prospective study. Mayo Clin Proc. 56(8):473-8.

121. McElduff A, LIssner D, Wilkinson M, Posen S. 1990. Parathyroid hormone sensitivity in primary hyperparathyroidism and idiopathic hypercalciuria: effects on postadenylate cyclase parameters. J Clin Endocrinol Metab. 70(5):1457-61.

122. Park-Sigal J, Don BR, Porzig A, et al. 2013. Severe hypercalcemic hyperparathyroidism developing in a patient with hyperaldosteronism and renal resistance to parathyroid hormone. J Bone Miner Res. 28(3):700-8.

123. Katoh N, Nakayama M, Shigematsu T, et al. 2000. Presence of sonographically detectable parathyroid glands can predict resistance to oral pulsed-dose calcitriol treatment of secondary hyperparathyroidism. Am J Kidney Dis. 35(3):465-8

124. Fukuda N, Tanaka H, Tominaga Y, et al. 1993. Decreased 1,25-dihydroxyvitamin D3 receptor density associated with severe forms of parathyroid hyperplasia in chronic uremic patients. J Clin Invest. 92(3):1436-43.

125. Cardinal H, Brossard JH, Roy L, et al. 1998. The set point of parathyroid hormone stimulation by calcium is normal in progressive renal failure. J Clin Endocrinol Metab. 83(11):3839-44.

126. Noureldine SI, Gooi Z, Tufano RP. 2015. Minimally invasive parathyroid surgery. Gland Surg. 4(5):410-9.

127. McCarron DA, Muther RS, Lenfesty B, Bennett WM. 1982. Parathyroid function in persistent hyperparathyroidism: relaionship to gland size. Kidney Int. 22(6):662-70.

128. Ichii M, Ishimura E, Okuno S, et al. 2010. Decreases in parathyroid gland volume after treatment in hemodialysis patients with secondary hyperparathyroidism. Nephron Clin Pract. 115(3):195-202.

129. Parker VJ, Harjes LM, Dembek K, et al. 2016. Vitamin D metabolites and their association with PTH, FGF-23, calcium and phosphorus concentration in dogs with various stages of chronic kidney disease. J Vet Intern Med. Ahead of print.

130. Cortadellas O, Fernandez del Palacio MJ, Bayon A. 2010. Calcium and phosphorus homeostasis in dogs with spontaneous chronic kidney disease at different stages of severity. J Vet Intern Med. 24(1):73-9.

131. Barber PJ, Elliott J. Feline chronic renal failure: calcium homeostasis in 80 cases diagnosed between 1992 and 1995. J Small Anim Pract. 39(3):108-16. 84

132. Hendy GN, Stotland MA, Grunbaum D, et al. 1989. Characteristics of secondary hyperparathyroidism in vitamin D-deficient dogs. Am J Phyiol. (256(6):E65-72.

133. Selting KA, Sharp CR, Ringold R, et al. 2014. Serum 25-hydroxyvitamin D concentrations in dogs – correlation with health and cancer risk. Vet Comp Oncol. doi:10.1111/vco.12052.

134. Sharp CR, Selting KA, Ringold R. 2015. The effect of diet on serum 25- hydroxyvitamin D concentrations in dogs. BMC Research Notes, 1–6.

135. Gerber B, Hassig M, Reusch CE. 2003. Serum concentrations of 1,25- dihydroxycholecalciferol and 25-hydroxycholecalciferol in clinically normal dogs and dogs with acute and chronic renal failure. Am J Vet Res. 64(9):1161-6

85