- Email: [email protected]
Parathyroid Hormone, Ionized Calcium, and 25-Hydroxyvitamin D Concentrations in the Domestic Ferret (Mustela putorius furo)
AEMV FORUM PARATHYROID HORMONE, IONIZED CALCIUM, AND 25-HYDROXYVITAMIN D CONCENTRATIONS IN THE DOMESTIC FERRET (MUSTELA PUTORIUS FURO) Sarah A. Cannizzo, VMD, Markus Rick, MedVet, PhD, Tara M. Harrison, DVM, MPVM, Dip. ACZM, Dip. ACVPM, and Craig A. Harms, DVM, PhD, Dip. ACZM
Abstract The objective of this study was to measure parathyroid hormone (PTH), ionized calcium, and 25-hydroxyvitamin D concentrations in healthy intact adult ferrets. Serum was collected from 16 clinically healthy adult ferrets (8 males and 8 females). Concentrations of PTH and 25-hydroxyvitamin D were measured via commercially available radioimmunoassays validated for humans, dogs, and cats (PTH) plus horses (25-hydroxyvitamin D). Concentrations of ionized calcium (at pH 7.4) were measured using an ion-specific electrode. Median (minimum, maximum) concentrations were as follows: PTH 8.7 (2.2, 24.4) pmol/L, ionized calcium 1.15 (1.09, 1.25) mmol/L, and 25-hydroxyvitamin D 94 (61, 138) nmol/L). Female ferrets had significantly higher concentrations of PTH than male ferrets (female median: 17.1 pmol/L; male median: 6.9 pmol/L). Associations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight were evaluated. There was a weak negative correlation between PTH concentration and total calcium concentration in female ferrets. As none of these assays have been validated for use in ferrets, results may provide a baseline for clinicians evaluating ferrets for disorders of calcium homeostasis, parathyroid glands, and paraneoplastic syndromes. Copyright 2017 Elsevier Inc. All rights reserved. Key words: ferret; mustelid; parathyroid hormone; ionized calcium; 25-hydroxyvitamin D
T
he parathyroid glands are endocrine organs that are closely associated with the thyroid. In ferrets, the parathyroid glands are discrete, pinkish discs usually found medially on the cranial pole of the thyroid gland at the level of the fourth or fifth tracheal rings.1 The parathyroid glands secrete parathyroid hormone (PTH). In its biologically active form, PTH is an 84-amino acid peptide that is highly conserved in mammals,2 and is secreted by the chief cells of the parathyroid in response to hypocalcemia or low concentrations of ionized calcium, the biologically active form of calcium.3 PTH is the primary regulatory hormone for calcium and phosphate metabolism, while its secretion is inhibited by increased serum concentrations of ionized calcium and vitamin D. PTH targets the bone, intestines, and the kidneys to increase serum concentrations of ionized calcium by mobilizing calcium and phosphate from bones and increasing gastrointestinal absorption of calcium in the presence of vitamin D. In the kidneys, PTH promotes calcium reabsorption in the ascending loop of Henle, distal tubule, and collecting tubule, and promotes reabsorption of phosphate in the proximal tubule.
From the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; the Environmental Medicine Consortium and Center for Marine Sciences and Technology, North Carolina State University, Morehead City, NC, USA; and the Department of Pathobiology and Diagnostic Investigation, Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI, USA. Address correspondence to: Craig A. Harms, DVM, PhD, Dip. ACZM, Center for Marine Sciences and Technology, North Carolina State University, 303 College Circle, Morehead City, NC 28557. E-mail: [email protected] Ó 2017 Elsevier Inc. All rights reserved. 1557-5063/17/2101-$30.00 http://dx.doi.org/10.1053/j.jepm.2017.07.004
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Journal of Exotic Pet Medicine 26 (2017), pp 294–299
Ionized calcium refers to the fraction of calcium that is unbound in plasma, highly controlled, hormonally regulated, and involved in pathologic conditions, such as disorders of the endocrine, renal, and skeletal systems, granulomatous diseases, and neoplasia.4 The calcium analyte in a serum or plasma chemistry is the total calcium, which includes free or ionized calcium, calcium bound to anionic proteins, and calcium bound to nonprotein anions, or complexed calcium.4 Total calcium measurements are influenced by the total protein concentration, particularly the albumin concentration, and therefore, hypoproteinemia or hypoalbuminemia can result in a hypocalcemia that does not reflect a calcium regulation dysfunction. To evaluate biologically active calcium without this influence, ionized calcium is measured. In humans and dogs, ionized calcium is a more sensitive indicator of primary hyperparathyroidism and a more reliable calcium measurement in critically ill patients.5-8 Vitamin D plays an important role in calcium homeostasis and it targets the same organs as PTH. The largest fraction of vitamin D in the blood is 25-hydroxyvitamin D.9 It is synthesized in the liver from Vitamin D3 (cholecalciferol). 25-hydroxyvitamin D is then hydroxylated by 1-αhydroxylase in the kidney to the active 1,25dihydroxyvitamin D (calcitriol). Synthesis of the renal 1-α-hydroxylase enzyme is stimulated by PTH. Vitamin D promotes calcium reabsorption by the renal tubules, calcium and phosphate absorption in the intestines, and osteolysis to liberate calcium and phosphate from bones.9 Vitamin D also provides negative feedback on the secretion of PTH, and therefore deficiencies in vitamin D can cause a secondary hyperparathyroidism. Chronic renal failure can lead to decreased conversion of 25-hydroxyvitamin D to the metabolically active 1,25-dihydroxyvitamin D, which inhibits PTH secretion. Vitamin D deficiencies can also result from all-meat diets in ferrets.10 There is a lack of published information regarding reference values for PTH, ionized calcium, and 25-hydroxyvitamin D in domestic ferrets and other mustelids. While these assays have not been validated for use in ferrets, results will expand the reference material available to clinicians, especially for consideration of endocrine, renal, or neoplastic diseases in a ferret patient.
MATERIALS AND METHODS Animals This project was approved by the North Carolina State University (NCSU) Institutional Care and Animal Use Committee. Sixteen ferrets (8 males and 8 females) were obtained from a commercial breeding facility (Marshall Bioresources, North Rose, NY, USA) for a course in ferret medicine and surgery offered to veterinary students biennially at the NCSU College of Veterinary Medicine. The age of the ferrets ranged from 0.6 to 1.4 years (median 1 year) and they weighed 0.5 to 1.6 kg (median 1.1 kg; females 0.5 to 1.0, median 0.8 kg; males 1.2 to 1.6, median 1.4 kg). There were no abnormalities detected in the physical examinations of the ferrets. The ferrets were housed individually in standard stainless steel cages within a temperature-controlled room with no other occupants. The light cycle provided 12 hours of light daily. The ferrets were fed a commercial ferret diet (Premium Marshall Ferret Diet, Marshall Pet Products, Inc., Wolcott, NY, USA). Venipuncture Venipuncture was performed on the same day at approximately the same time for all ferrets in early December 2015. The ferrets were sedated with 0.25 mg/kg detomidine gel administered transmucosally (Dormosedan Gel; Orion Corporation, Turku, Finland). Full physical examinations were performed on all ferrets. Blood was collected from the jugular vein or the cranial vena cava with a 22-gauge, 1.6 cm needle and 6 mL syringe. A maximum of 5 mL/kg of blood was collected. After venipuncture, detomidine gel was reversed with 0.25 mg/kg atipamezole (Antisedan; Zoetis, New York, NY, USA) administered intramuscularly. For each ferret, a subsample of whole blood was transferred to heparinized microhematocrit tubes for determining packed cell volumes (PCV) and total solids. The remaining blood was transferred to a 7.0 mL no additive serum tube (BD Vacutainer; Becton, Dickinson, Franklin Lakes, NJ, USA). Samples were allowed to clot for at least 30 minutes and then centrifuged at 563xg for 10 minutes to separate the serum. Serum was transferred to cryovials, which were submitted to the Clinical Pathology Laboratory at NCSU College of Veterinary Medicine for serum chemistries or the Diagnostic Center for
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TABLE 1. Median, minimum (min), and maximum (max) concentrations for PTH, ionized calcium, and 25-hydroxyvitamin D in clinically healthy adult ferrets Analytes Intact parathyroid hormone (pmol/L) Ionized calcium (mmol/L at pH 7.4)a 25-hydroxyvitamin D (nmol/L) a
Median Min Max 8.7
2.2
1.15
1.09
94
61
24.4 1.25 138
n ¼ 15.
Population and Animal Health (DCPAH) at Michigan State University (MSU) to determine PTH, ionized calcium, and 25-hydroxyvitamin D concentrations. Serum chemistries were performed using the Cobas c 501 (Roche Diagnostics, Indianapolis, IN, USA). Serum samples sent to MSU—DCPAH were refrigerated overnight and then sent by overnight courier on ice. PTH, ionized calcium, and 25-hydroxyvitamin D assays Intact PTH was measured via a commercially available radioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA) for humans. The manufacturer of this assay is confidential, but the assay is used for veterinary diagnostic purposes primarily in dogs and cats and has been validated for that use at the MSU—DCPAH. The interassay and intraassay coefficient of variation (CV) for the PTH assay in dogs are 10% and 3%, respectively. Ionized calcium was measured using an ionspecific electrode (Nova 8þ; NOVA Biomedical, Waltham, MA, USA). The canine interassay CV ranges from 2.5% to 6.6%. A commercially available RIA (DiaSorrin, Stillwater, MN, USA) for humans was used to measure 25-hydroxyvitamin D in ferret serum. The assay measured both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and has been validated for use in horses, cats, and dogs by MSU—DCPAH. The interassay CV for dogs ranged from 10.3% to 15.3%; the intraassay CV for dogs ranged from 4.2% to 5%. Statistical analysis A commercial program was used for statistical analysis (JMP Pro, Version 12.0.1, SAS, Cary, NC, USA). Data were tested for normality using the Shapiro-Wilk test (P o 0.05). Not all data were normally distributed, so nonparametric statistics were used. American Society for Veterinary Clinical 2 9 6
Pathology (ASVCP) guidelines for presenting data based on sample sizes less than 20 and greater than 10 were followed.11 Based on ASVCP guidelines for subclass sample sizes less than 40, results were not partitioned. Differences between the sexes were, nevertheless, compared by the Wilcoxon rank sum test. Associations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight were assessed with Kendall’s τ rank correlation. Statistical significance was set at P o 0.05. RESULTS Median, minimum, and maximum concentrations for PTH, ionized calcium, and 25-hydroxyvitamin D are listed in Table 1. There was insufficient sample volume to measure ionized calcium in 1 ferret. The distributions of results for PTH, ionized calcium, and 25-hydroxyvitamin D for male and female ferrets are depicted in Figure 1. Female ferrets had a significantly higher PTH concentration than male ferrets (median [min, max] 17.1 [4.0, 24.4] vs. 6.9 [2.2, 14.0] mg/dL, P ¼ 0.036). Male ferrets had a higher total calcium concentration than female ferrets (median [min, max] 9.1 [8.9, 9.4] vs. 8.6 [8.3, 9.0] mg/dL, P ¼ 0.006). There was a weak negative correlation between PTH concentration and total calcium concentration in female ferrets (τ ¼−0.5930, P ¼ 0.044). There was a positive correlation between magnesium and phosphorus (τ ¼ 0.4539, P ¼ 0.019). There were no other significant correlations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight for male or female ferrets. Fifty percent of ferrets (4 males and 4 females) were hyperglycemic (serum glucose 4200 mg/dL), as has been observed with the use of detomidine gel sedation.12 The PCV ranged from 34% to 50% (median 47%). All values for PCV/total solids and serum chemistries were within published reference intervals with the following exceptions: 3 male ferrets with GGT activities of 15, 31, and 34 U/L (reference interval for male ferrets: 0.2-14.0 U/L); 1 female ferret with a GGT activity of 21 U/L (reference interval for female ferrets: 7.9-20.3 U/L); and 1 female ferret with a total protein concentration of 4.6 g/dL (reference interval for female ferrets: 5.1-7.2 g/dL), and a globulin concentration of 2.0 g/dL (reference interval for female ferrets: 2.2-3.2 g/dL).13-15 The day after venipuncture, routine orchiectomies, and ovariohysterectomies were performed on all ferrets. One female ferret had enlarged lymph nodes. Excisional biopsies of the lymph nodes Journal of Exotic Pet Medicine 26 (2017), pp 294–299
FIGURE 1. Histogram of PTH (A), ionized calcium (B), and 25-hydroxyvitamin D (C) results (n ¼ 8 male and 8 female ferrets, except for 7 male ferrets in B).
were consistent with hyperplastic, reactive lymph nodes. This ferret had the second highest ionized calcium in the study (1.21 mmol/L). Another female had dysplasia of the left uterine horn. No histopathology was performed on the abnormal uterus. None of the biochemical or surgical findings was considered grounds for exclusion from the study. DISCUSSION This study provides baseline values for PTH, ionized calcium, and 25-hydroxyvitamin D concentrations in clinically healthy adult ferrets. These values can help clinicians increase the index of suspicion for diseases of the parathyroid16-18 and help identify paraneoplastic syndromes in ferrets with neoplasia (e.g., lymphoma).19-21 The PTH, ionized calcium, and 25-hydroxyvitamin D test methods used in this study have not been validated in the ferret. Insufficient sample volumes were available to perform validation experiments as part of this study, which would have been ideal. The clinical concordance demonstrated in this study may help support that these methods are performing acceptably and are worth further investigation. The immunoassay method of PTH measurement used
in this study has been validated in humans, dogs, and cats. Sequence alignment demonstrates a 90% identity between human and ferret and a 92% identity between ferret and canine PTH genetranslated regions.22 There is 100% identity among ferrets, humans, and dogs in the receptor-binding region.22 The degree to which sequence difference, post-translational modifications, or potential cross-reacting proteins in ferret serum affects the performance of this test methodology in the ferret is unknown. Analytic performance of this assay in ferrets should be considered as undetermined. The ion-specific electrode measurement of ionized calcium has been validated for and is commonly used in dogs and cats. Given this methodology and documented performance in other species, considerable performance difference would not be expected in ferret. The immunoassay method for measuring 25-hydroxyvitamin D used in this study has been validated in dogs and cats. Given the performance in other species and molecular identity of 25-hydroxyvitamin D in any species, considerable performance differences would not be expected in the ferret. However, as immunoassays are more susceptible to interspecies variation, analytic performance of this assay in ferrets should be considered as undetermined. Nevertheless, as evidenced by published case reports of hypoparathyroidism and pseudohypoparathyroidism in ferrets,16-18 clinicians are using these assays for ferrets despite the lack of published values for comparisons. Diseases of the parathyroids are rare in ferrets. There are 3 case reports of hypoparathyroidism and pseudohypoparathyroidism in ferrets.16-18 The case of pseudohypoparathyroidism reports a PTH concentration of 75.3 pmol/L in the ill ferret and 13 pmol/L in a reportedly healthy comparison ferret.16 This case also reports ionized calcium concentrations and vitamin D concentrations for the ill ferret (0.54 mmol/L, 144 nmol/L) and the reportedly healthy control (1.17 mmol/L, 128 nmol/L).16 The ill ferret had previously been treated with a vitamin D analog (dihydrotachysterol) orally but was not receiving it at the time of vitamin D testing. The PTH and vitamin D concentrations were measured by the same laboratory used by this current study. The second and third case reports describe 2 ferrets with suspected primary hypoparathyroidism.17,18 In the first hypoparathyroidism case report, the ferret presented for a history of seizures and had an ionized calcium of 0.49 mmol/L at initial presentation.17 Repeated ionized calcium concentrations (0.47 and 0.67 mmol/L) and PTH
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concentrations (2.3 and 3.4 pmol/L) were obtained. All PTH and ionized calcium measurements except for the presenting ionized calcium concentration were obtained from the same laboratory used in this current study. In the second case report of suspected primary hypoparathyriodism, PTH concentrations from 3 clinically healthy ferrets are used for comparison to the concentration measured in the ill ferret (0.67 pmol/L).18 The reportedly healthy ferrets had PTH concentrations ranging from 1.4 to 3.45 pmol/L. Those results were obtained using the ADVIA Centaur manual assay (ADVIA Centaur, Ready-Pack, Siemens Healthcare Diagnostics Inc., Erlangen, Germany).18 Hypercalcemia can be associated with endocrine disorders, renal disease, granulomatous disease, skeletal disease, increased endogenous or exogenous vitamin D, and neoplasia. In cats it can also be idiopathic. Hypercalcemia has been found in ferrets with lymphoma.19-21 In humans and domestic animals, paraneoplastic hypercalcemia can be caused by tumor-produced PTH-like proteins (e.g., PTH-related peptide [PTHrP]). These substances have not been specifically identified in ferrets, but it is likely that they exist. Human PTHrP was found to decrease production of osteodentin in ferret canine teeth, suggesting that PTHrP has biologic activity in ferrets.23 Hypercalcemia of malignancy can also occur in the absence of PTHrP.4 Measuring PTH can help clinicians rule out causes for hypercalcemia or hypocalcemia.24 An increased ionized calcium concentration paired with a normal to high PTH concentration is suggestive of hyperparathyroidism. Hyperparathryroidism can be primary or secondary to renal disease or nutritional imbalances. With chronic renal failure, ionized calcium can be low to low normal and the PTH concentration can be high, which is appropriate for the hypocalcemia. A low PTH concentration with a high ionized calcium concentration can be caused by neoplasia (i.e., hypercalcemia of malignancy). Low ionized calcium and low PTH concentrations indicate hypoparathyroidism. Concentrations of PTH were higher in female ferrets than male ferrets. Similar findings have been documented in humans and rats.25-27 In humans, PTH concentrations in response to vitamin D deficiency differ significantly between the sexes.27 Additionally, men and women have significantly different circadian patterns of PTH concentrations.25 The higher total calcium concentration in male ferrets is consistent with 2 9 8
published reference values for ferrets.14 Higher serum calcium has been found in human boys compared to girls.28 The hyperglycemia observed in half of the ferrets can be attributed to the administration of detomidine gel. This finding is consistent with use of an α-2-receptor agonist and has been reported in ferrets sedated with detomidine gel.12 This study provides reference values for PTH, ionized calcium, and 25-hydroxyvitamin D concentrations in adult ferrets and fills a gap in the existing ferret and mustelid literature. This information will be valuable to clinicians treating the critically ill ferret as well as evaluating ferrets for endocrine disorders, diseases that disrupt calcium homeostasis, and neoplasias that can result in paraneoplastic syndromes. ACKNOWLEDGMENTS This project was funded in part by the North Carolina State University Support Fund for Aquatic Animal Medicine. The authors thank the following people for their assistance during the North Carolina State University College of Veterinary Medicine ferret medicine and surgery selective teaching laboratory: Heather Broadhurst, Kent Passingham, and Brianne Phillips. The authors also thank the North Carolina State University College of Veterinary Medicine students who participated in this course. REFERENCES 1. Evans H, An NQ: Anatomy, in Fox J (ed): Biology and Diseases of the Ferret. (ed 3). Philadelphia, PA, WileyBlackwell, pp 23-67, 2014 2. Bell O, Silver J, Naveh-Many T: Parathyroid hormone, from gene to protein, in Naveh-Many T (ed): Molecular Biology of the Parathyroid. Boston, MA, Springer US, pp 8-28, 2005 3. Felsenfeld AJ, Rodríguez M, Aguilera-Tejero E: Dynamics of parathyroid hormone secretion in health and secondary hyperparathyroidism. Clin J Am Soc Nephrol 2:1283-1305, 2007 4. Stockham SL, Scott MA: Fundamentals of Veterinary Clinical Pathology (ed 2). Ames, IA: Blackwell Publishing, 2008 5. Zaloga GP, Willey S, Tomasic P, et al: Free fatty acids alter calcium binding: a cause for misinterpretation of serum calcium values and hypocalcemia in critical illness. J Clin Endocrinol Metab 64:1010-1014, 1987 6. Slomp J, van der Voort PHJ, Gerritsen RT, et al: Albuminadjusted calcium is not suitable for diagnosis of hyperand hypocalcemia in the critically ill. Crit Care Med 31: 1389-1393, 2003 7. Sharp CR, Kerl ME, Mann FA: A comparison of total calcium, corrected calcium, and ionized calcium concentrations as indicators of calcium homeostasis among
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hypoalbuminemic dogs requiring intensive care. J Vet Emerg Crit Care 19:571-578, 2009 Tee MC, Holmes DT, Wiseman SM: Ionized vs serum calcium in the diagnosis and management of primary hyperparathyroidism: which is superior? Am J Surg 205: 591-596, 2013 Landry CS, Ruppe MD, Grubbs EG: Vitamin D receptors and parathyroid glands. Endocr Prac 17:63-68, 2011 Fox JG, Schultz CS, Vester Boler BM: Nutrition of the ferret, in Fox J (ed): Biology and Diseases of the Ferret. (ed 3). Philadelphia, PA, Wiley-Blackwell, pp 123-143, 2014 American Society for Veterinary Clinical Pathology (ASVCP). ASVCP Quality Assurance And Laboratory Standards Committee (QALS) guidelines for the determination of reference intervals in veterinary species and other related topics. http:// www.asvcp.org/pubs/qas. Accessed 15 September 2016 Phillips BE, Harms CA, Messenger KM: Oral transmucosal detomidine gel for the sedation of the domestic ferret (Mustela putorius furo). J Exot Pet Med 24:446-454, 2015 Hein J, Spreyer F, Sauter-Louis C, et al: Reference ranges for laboratory parameters in ferrets. Vet Rec 171:218-218, 2012 Morrisey JK: Ferrets, in Carpenter JW (ed): Exotic Animal Formulary. (ed 4). St. Louis, MO, Elsevier/Saunders, pp 560-594, 2013 Fox JG: Normal clinical and biological parameters, in Fox J (ed): Biology and Diseases of the Ferret. (ed 3). Philadelphia, PA, Wiley-Blackwell, pp 157-185, 2014 Wilson GH, Greene CE, Greenacre CB: Suspected pseudohypoparathyroidism in a domestic ferret. J Am Vet Med Assoc 222:1093-1096, 2003 de Matos RE, Connolly MJ, Starkey SR, et al: Suspected primary hypoparathyroidism in a domestic ferret (Mustela putorius furo). J Am Vet Med Assoc 245:419-424, 2014
18. Martínez C, Sabater M, Giner J, et al: Spontaneous primary hypoparathyroidism in a ferret (Mustela putorius furo). J Exot Pet Med 24:333-339, 2015 19. Batchelder MA, Erdman SE, Li X, et al: A cluster of cases of juvenile mediastinal lymphoma in a ferret colony. Lab Anim Sci 46:271-274, 1996 20. Erdman SE, Brown SA, Kawasaki TA, et al: Clinical and pathologic findings in ferrets with lymphoma: 60 cases (1982-1994). J Am Vet Med Assoc 208:1,285-1,289, 1996 21. Ammersbach M, Delay J, Caswell JL, et al: Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets. Vet Pathol 45:663-673, 2008 22. http://www.uniprot.org. Accessed 10 February 2017 23. Kline LW, Yu DC: Effects of calcitonin, calcitonin gene-related peptide, human recombinant bone morphogenetic protein-2, and parathyroid hormone-related protein on endodontically treated ferret canines. J Endod 35:866-869, 2009 24. Greco DS: Endocrine causes of calcium disorders. Top Companion Anim Med 27:150-155, 2012 25. Calvo MS, Eastell R, Offord KP, et al: Circadian variation in ionized calcium and intact parathyroid hormone: evidence for sex differences in calcium homeostasis. J Clin Endocrinol Metab 72:69-76, 1991 26. D’Amour P, Rousseau L, Hornyak S, et al: Rat parathyroid hormone (rPTH) ELISAs specific for regions (2–7), (22– 34) and (40–60) of the rat PTH structure: influence of sex and age. Gen Comp Endocrinol 168:312-317, 2010 27. Di Monaco M, Castiglioni C, Vallero F, et al: Parathyroid hormone response to severe vitamin D deficiency is sex associated: an observational study of 571 hip fracture inpatients. J Nutr Health Aging 17:180-184, 2013 28. Marwaha RK, Khadgawat R, Tandon N, et al: Reference intervals of serum calcium, ionized calcium, phosphate and alkaline phosphatase in healthy Indian school children and adolescents. Clin Biochem 43:1216-1219, 2010
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Abstract The objective of this study was to measure parathyroid hormone (PTH), ionized calcium, and 25-hydroxyvitamin D concentrations in healthy intact adult ferrets. Serum was collected from 16 clinically healthy adult ferrets (8 males and 8 females). Concentrations of PTH and 25-hydroxyvitamin D were measured via commercially available radioimmunoassays validated for humans, dogs, and cats (PTH) plus horses (25-hydroxyvitamin D). Concentrations of ionized calcium (at pH 7.4) were measured using an ion-specific electrode. Median (minimum, maximum) concentrations were as follows: PTH 8.7 (2.2, 24.4) pmol/L, ionized calcium 1.15 (1.09, 1.25) mmol/L, and 25-hydroxyvitamin D 94 (61, 138) nmol/L). Female ferrets had significantly higher concentrations of PTH than male ferrets (female median: 17.1 pmol/L; male median: 6.9 pmol/L). Associations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight were evaluated. There was a weak negative correlation between PTH concentration and total calcium concentration in female ferrets. As none of these assays have been validated for use in ferrets, results may provide a baseline for clinicians evaluating ferrets for disorders of calcium homeostasis, parathyroid glands, and paraneoplastic syndromes. Copyright 2017 Elsevier Inc. All rights reserved. Key words: ferret; mustelid; parathyroid hormone; ionized calcium; 25-hydroxyvitamin D
T
he parathyroid glands are endocrine organs that are closely associated with the thyroid. In ferrets, the parathyroid glands are discrete, pinkish discs usually found medially on the cranial pole of the thyroid gland at the level of the fourth or fifth tracheal rings.1 The parathyroid glands secrete parathyroid hormone (PTH). In its biologically active form, PTH is an 84-amino acid peptide that is highly conserved in mammals,2 and is secreted by the chief cells of the parathyroid in response to hypocalcemia or low concentrations of ionized calcium, the biologically active form of calcium.3 PTH is the primary regulatory hormone for calcium and phosphate metabolism, while its secretion is inhibited by increased serum concentrations of ionized calcium and vitamin D. PTH targets the bone, intestines, and the kidneys to increase serum concentrations of ionized calcium by mobilizing calcium and phosphate from bones and increasing gastrointestinal absorption of calcium in the presence of vitamin D. In the kidneys, PTH promotes calcium reabsorption in the ascending loop of Henle, distal tubule, and collecting tubule, and promotes reabsorption of phosphate in the proximal tubule.
From the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; the Environmental Medicine Consortium and Center for Marine Sciences and Technology, North Carolina State University, Morehead City, NC, USA; and the Department of Pathobiology and Diagnostic Investigation, Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI, USA. Address correspondence to: Craig A. Harms, DVM, PhD, Dip. ACZM, Center for Marine Sciences and Technology, North Carolina State University, 303 College Circle, Morehead City, NC 28557. E-mail: [email protected] Ó 2017 Elsevier Inc. All rights reserved. 1557-5063/17/2101-$30.00 http://dx.doi.org/10.1053/j.jepm.2017.07.004
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Journal of Exotic Pet Medicine 26 (2017), pp 294–299
Ionized calcium refers to the fraction of calcium that is unbound in plasma, highly controlled, hormonally regulated, and involved in pathologic conditions, such as disorders of the endocrine, renal, and skeletal systems, granulomatous diseases, and neoplasia.4 The calcium analyte in a serum or plasma chemistry is the total calcium, which includes free or ionized calcium, calcium bound to anionic proteins, and calcium bound to nonprotein anions, or complexed calcium.4 Total calcium measurements are influenced by the total protein concentration, particularly the albumin concentration, and therefore, hypoproteinemia or hypoalbuminemia can result in a hypocalcemia that does not reflect a calcium regulation dysfunction. To evaluate biologically active calcium without this influence, ionized calcium is measured. In humans and dogs, ionized calcium is a more sensitive indicator of primary hyperparathyroidism and a more reliable calcium measurement in critically ill patients.5-8 Vitamin D plays an important role in calcium homeostasis and it targets the same organs as PTH. The largest fraction of vitamin D in the blood is 25-hydroxyvitamin D.9 It is synthesized in the liver from Vitamin D3 (cholecalciferol). 25-hydroxyvitamin D is then hydroxylated by 1-αhydroxylase in the kidney to the active 1,25dihydroxyvitamin D (calcitriol). Synthesis of the renal 1-α-hydroxylase enzyme is stimulated by PTH. Vitamin D promotes calcium reabsorption by the renal tubules, calcium and phosphate absorption in the intestines, and osteolysis to liberate calcium and phosphate from bones.9 Vitamin D also provides negative feedback on the secretion of PTH, and therefore deficiencies in vitamin D can cause a secondary hyperparathyroidism. Chronic renal failure can lead to decreased conversion of 25-hydroxyvitamin D to the metabolically active 1,25-dihydroxyvitamin D, which inhibits PTH secretion. Vitamin D deficiencies can also result from all-meat diets in ferrets.10 There is a lack of published information regarding reference values for PTH, ionized calcium, and 25-hydroxyvitamin D in domestic ferrets and other mustelids. While these assays have not been validated for use in ferrets, results will expand the reference material available to clinicians, especially for consideration of endocrine, renal, or neoplastic diseases in a ferret patient.
MATERIALS AND METHODS Animals This project was approved by the North Carolina State University (NCSU) Institutional Care and Animal Use Committee. Sixteen ferrets (8 males and 8 females) were obtained from a commercial breeding facility (Marshall Bioresources, North Rose, NY, USA) for a course in ferret medicine and surgery offered to veterinary students biennially at the NCSU College of Veterinary Medicine. The age of the ferrets ranged from 0.6 to 1.4 years (median 1 year) and they weighed 0.5 to 1.6 kg (median 1.1 kg; females 0.5 to 1.0, median 0.8 kg; males 1.2 to 1.6, median 1.4 kg). There were no abnormalities detected in the physical examinations of the ferrets. The ferrets were housed individually in standard stainless steel cages within a temperature-controlled room with no other occupants. The light cycle provided 12 hours of light daily. The ferrets were fed a commercial ferret diet (Premium Marshall Ferret Diet, Marshall Pet Products, Inc., Wolcott, NY, USA). Venipuncture Venipuncture was performed on the same day at approximately the same time for all ferrets in early December 2015. The ferrets were sedated with 0.25 mg/kg detomidine gel administered transmucosally (Dormosedan Gel; Orion Corporation, Turku, Finland). Full physical examinations were performed on all ferrets. Blood was collected from the jugular vein or the cranial vena cava with a 22-gauge, 1.6 cm needle and 6 mL syringe. A maximum of 5 mL/kg of blood was collected. After venipuncture, detomidine gel was reversed with 0.25 mg/kg atipamezole (Antisedan; Zoetis, New York, NY, USA) administered intramuscularly. For each ferret, a subsample of whole blood was transferred to heparinized microhematocrit tubes for determining packed cell volumes (PCV) and total solids. The remaining blood was transferred to a 7.0 mL no additive serum tube (BD Vacutainer; Becton, Dickinson, Franklin Lakes, NJ, USA). Samples were allowed to clot for at least 30 minutes and then centrifuged at 563xg for 10 minutes to separate the serum. Serum was transferred to cryovials, which were submitted to the Clinical Pathology Laboratory at NCSU College of Veterinary Medicine for serum chemistries or the Diagnostic Center for
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TABLE 1. Median, minimum (min), and maximum (max) concentrations for PTH, ionized calcium, and 25-hydroxyvitamin D in clinically healthy adult ferrets Analytes Intact parathyroid hormone (pmol/L) Ionized calcium (mmol/L at pH 7.4)a 25-hydroxyvitamin D (nmol/L) a
Median Min Max 8.7
2.2
1.15
1.09
94
61
24.4 1.25 138
n ¼ 15.
Population and Animal Health (DCPAH) at Michigan State University (MSU) to determine PTH, ionized calcium, and 25-hydroxyvitamin D concentrations. Serum chemistries were performed using the Cobas c 501 (Roche Diagnostics, Indianapolis, IN, USA). Serum samples sent to MSU—DCPAH were refrigerated overnight and then sent by overnight courier on ice. PTH, ionized calcium, and 25-hydroxyvitamin D assays Intact PTH was measured via a commercially available radioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA) for humans. The manufacturer of this assay is confidential, but the assay is used for veterinary diagnostic purposes primarily in dogs and cats and has been validated for that use at the MSU—DCPAH. The interassay and intraassay coefficient of variation (CV) for the PTH assay in dogs are 10% and 3%, respectively. Ionized calcium was measured using an ionspecific electrode (Nova 8þ; NOVA Biomedical, Waltham, MA, USA). The canine interassay CV ranges from 2.5% to 6.6%. A commercially available RIA (DiaSorrin, Stillwater, MN, USA) for humans was used to measure 25-hydroxyvitamin D in ferret serum. The assay measured both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and has been validated for use in horses, cats, and dogs by MSU—DCPAH. The interassay CV for dogs ranged from 10.3% to 15.3%; the intraassay CV for dogs ranged from 4.2% to 5%. Statistical analysis A commercial program was used for statistical analysis (JMP Pro, Version 12.0.1, SAS, Cary, NC, USA). Data were tested for normality using the Shapiro-Wilk test (P o 0.05). Not all data were normally distributed, so nonparametric statistics were used. American Society for Veterinary Clinical 2 9 6
Pathology (ASVCP) guidelines for presenting data based on sample sizes less than 20 and greater than 10 were followed.11 Based on ASVCP guidelines for subclass sample sizes less than 40, results were not partitioned. Differences between the sexes were, nevertheless, compared by the Wilcoxon rank sum test. Associations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight were assessed with Kendall’s τ rank correlation. Statistical significance was set at P o 0.05. RESULTS Median, minimum, and maximum concentrations for PTH, ionized calcium, and 25-hydroxyvitamin D are listed in Table 1. There was insufficient sample volume to measure ionized calcium in 1 ferret. The distributions of results for PTH, ionized calcium, and 25-hydroxyvitamin D for male and female ferrets are depicted in Figure 1. Female ferrets had a significantly higher PTH concentration than male ferrets (median [min, max] 17.1 [4.0, 24.4] vs. 6.9 [2.2, 14.0] mg/dL, P ¼ 0.036). Male ferrets had a higher total calcium concentration than female ferrets (median [min, max] 9.1 [8.9, 9.4] vs. 8.6 [8.3, 9.0] mg/dL, P ¼ 0.006). There was a weak negative correlation between PTH concentration and total calcium concentration in female ferrets (τ ¼−0.5930, P ¼ 0.044). There was a positive correlation between magnesium and phosphorus (τ ¼ 0.4539, P ¼ 0.019). There were no other significant correlations among PTH, ionized calcium, 25-hydroxyvitamin D, calcium, phosphorus, magnesium, and weight for male or female ferrets. Fifty percent of ferrets (4 males and 4 females) were hyperglycemic (serum glucose 4200 mg/dL), as has been observed with the use of detomidine gel sedation.12 The PCV ranged from 34% to 50% (median 47%). All values for PCV/total solids and serum chemistries were within published reference intervals with the following exceptions: 3 male ferrets with GGT activities of 15, 31, and 34 U/L (reference interval for male ferrets: 0.2-14.0 U/L); 1 female ferret with a GGT activity of 21 U/L (reference interval for female ferrets: 7.9-20.3 U/L); and 1 female ferret with a total protein concentration of 4.6 g/dL (reference interval for female ferrets: 5.1-7.2 g/dL), and a globulin concentration of 2.0 g/dL (reference interval for female ferrets: 2.2-3.2 g/dL).13-15 The day after venipuncture, routine orchiectomies, and ovariohysterectomies were performed on all ferrets. One female ferret had enlarged lymph nodes. Excisional biopsies of the lymph nodes Journal of Exotic Pet Medicine 26 (2017), pp 294–299
FIGURE 1. Histogram of PTH (A), ionized calcium (B), and 25-hydroxyvitamin D (C) results (n ¼ 8 male and 8 female ferrets, except for 7 male ferrets in B).
were consistent with hyperplastic, reactive lymph nodes. This ferret had the second highest ionized calcium in the study (1.21 mmol/L). Another female had dysplasia of the left uterine horn. No histopathology was performed on the abnormal uterus. None of the biochemical or surgical findings was considered grounds for exclusion from the study. DISCUSSION This study provides baseline values for PTH, ionized calcium, and 25-hydroxyvitamin D concentrations in clinically healthy adult ferrets. These values can help clinicians increase the index of suspicion for diseases of the parathyroid16-18 and help identify paraneoplastic syndromes in ferrets with neoplasia (e.g., lymphoma).19-21 The PTH, ionized calcium, and 25-hydroxyvitamin D test methods used in this study have not been validated in the ferret. Insufficient sample volumes were available to perform validation experiments as part of this study, which would have been ideal. The clinical concordance demonstrated in this study may help support that these methods are performing acceptably and are worth further investigation. The immunoassay method of PTH measurement used
in this study has been validated in humans, dogs, and cats. Sequence alignment demonstrates a 90% identity between human and ferret and a 92% identity between ferret and canine PTH genetranslated regions.22 There is 100% identity among ferrets, humans, and dogs in the receptor-binding region.22 The degree to which sequence difference, post-translational modifications, or potential cross-reacting proteins in ferret serum affects the performance of this test methodology in the ferret is unknown. Analytic performance of this assay in ferrets should be considered as undetermined. The ion-specific electrode measurement of ionized calcium has been validated for and is commonly used in dogs and cats. Given this methodology and documented performance in other species, considerable performance difference would not be expected in ferret. The immunoassay method for measuring 25-hydroxyvitamin D used in this study has been validated in dogs and cats. Given the performance in other species and molecular identity of 25-hydroxyvitamin D in any species, considerable performance differences would not be expected in the ferret. However, as immunoassays are more susceptible to interspecies variation, analytic performance of this assay in ferrets should be considered as undetermined. Nevertheless, as evidenced by published case reports of hypoparathyroidism and pseudohypoparathyroidism in ferrets,16-18 clinicians are using these assays for ferrets despite the lack of published values for comparisons. Diseases of the parathyroids are rare in ferrets. There are 3 case reports of hypoparathyroidism and pseudohypoparathyroidism in ferrets.16-18 The case of pseudohypoparathyroidism reports a PTH concentration of 75.3 pmol/L in the ill ferret and 13 pmol/L in a reportedly healthy comparison ferret.16 This case also reports ionized calcium concentrations and vitamin D concentrations for the ill ferret (0.54 mmol/L, 144 nmol/L) and the reportedly healthy control (1.17 mmol/L, 128 nmol/L).16 The ill ferret had previously been treated with a vitamin D analog (dihydrotachysterol) orally but was not receiving it at the time of vitamin D testing. The PTH and vitamin D concentrations were measured by the same laboratory used by this current study. The second and third case reports describe 2 ferrets with suspected primary hypoparathyroidism.17,18 In the first hypoparathyroidism case report, the ferret presented for a history of seizures and had an ionized calcium of 0.49 mmol/L at initial presentation.17 Repeated ionized calcium concentrations (0.47 and 0.67 mmol/L) and PTH
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concentrations (2.3 and 3.4 pmol/L) were obtained. All PTH and ionized calcium measurements except for the presenting ionized calcium concentration were obtained from the same laboratory used in this current study. In the second case report of suspected primary hypoparathyriodism, PTH concentrations from 3 clinically healthy ferrets are used for comparison to the concentration measured in the ill ferret (0.67 pmol/L).18 The reportedly healthy ferrets had PTH concentrations ranging from 1.4 to 3.45 pmol/L. Those results were obtained using the ADVIA Centaur manual assay (ADVIA Centaur, Ready-Pack, Siemens Healthcare Diagnostics Inc., Erlangen, Germany).18 Hypercalcemia can be associated with endocrine disorders, renal disease, granulomatous disease, skeletal disease, increased endogenous or exogenous vitamin D, and neoplasia. In cats it can also be idiopathic. Hypercalcemia has been found in ferrets with lymphoma.19-21 In humans and domestic animals, paraneoplastic hypercalcemia can be caused by tumor-produced PTH-like proteins (e.g., PTH-related peptide [PTHrP]). These substances have not been specifically identified in ferrets, but it is likely that they exist. Human PTHrP was found to decrease production of osteodentin in ferret canine teeth, suggesting that PTHrP has biologic activity in ferrets.23 Hypercalcemia of malignancy can also occur in the absence of PTHrP.4 Measuring PTH can help clinicians rule out causes for hypercalcemia or hypocalcemia.24 An increased ionized calcium concentration paired with a normal to high PTH concentration is suggestive of hyperparathyroidism. Hyperparathryroidism can be primary or secondary to renal disease or nutritional imbalances. With chronic renal failure, ionized calcium can be low to low normal and the PTH concentration can be high, which is appropriate for the hypocalcemia. A low PTH concentration with a high ionized calcium concentration can be caused by neoplasia (i.e., hypercalcemia of malignancy). Low ionized calcium and low PTH concentrations indicate hypoparathyroidism. Concentrations of PTH were higher in female ferrets than male ferrets. Similar findings have been documented in humans and rats.25-27 In humans, PTH concentrations in response to vitamin D deficiency differ significantly between the sexes.27 Additionally, men and women have significantly different circadian patterns of PTH concentrations.25 The higher total calcium concentration in male ferrets is consistent with 2 9 8
published reference values for ferrets.14 Higher serum calcium has been found in human boys compared to girls.28 The hyperglycemia observed in half of the ferrets can be attributed to the administration of detomidine gel. This finding is consistent with use of an α-2-receptor agonist and has been reported in ferrets sedated with detomidine gel.12 This study provides reference values for PTH, ionized calcium, and 25-hydroxyvitamin D concentrations in adult ferrets and fills a gap in the existing ferret and mustelid literature. This information will be valuable to clinicians treating the critically ill ferret as well as evaluating ferrets for endocrine disorders, diseases that disrupt calcium homeostasis, and neoplasias that can result in paraneoplastic syndromes. ACKNOWLEDGMENTS This project was funded in part by the North Carolina State University Support Fund for Aquatic Animal Medicine. The authors thank the following people for their assistance during the North Carolina State University College of Veterinary Medicine ferret medicine and surgery selective teaching laboratory: Heather Broadhurst, Kent Passingham, and Brianne Phillips. The authors also thank the North Carolina State University College of Veterinary Medicine students who participated in this course. REFERENCES 1. Evans H, An NQ: Anatomy, in Fox J (ed): Biology and Diseases of the Ferret. (ed 3). Philadelphia, PA, WileyBlackwell, pp 23-67, 2014 2. Bell O, Silver J, Naveh-Many T: Parathyroid hormone, from gene to protein, in Naveh-Many T (ed): Molecular Biology of the Parathyroid. Boston, MA, Springer US, pp 8-28, 2005 3. Felsenfeld AJ, Rodríguez M, Aguilera-Tejero E: Dynamics of parathyroid hormone secretion in health and secondary hyperparathyroidism. Clin J Am Soc Nephrol 2:1283-1305, 2007 4. Stockham SL, Scott MA: Fundamentals of Veterinary Clinical Pathology (ed 2). Ames, IA: Blackwell Publishing, 2008 5. Zaloga GP, Willey S, Tomasic P, et al: Free fatty acids alter calcium binding: a cause for misinterpretation of serum calcium values and hypocalcemia in critical illness. J Clin Endocrinol Metab 64:1010-1014, 1987 6. Slomp J, van der Voort PHJ, Gerritsen RT, et al: Albuminadjusted calcium is not suitable for diagnosis of hyperand hypocalcemia in the critically ill. Crit Care Med 31: 1389-1393, 2003 7. Sharp CR, Kerl ME, Mann FA: A comparison of total calcium, corrected calcium, and ionized calcium concentrations as indicators of calcium homeostasis among
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18. Martínez C, Sabater M, Giner J, et al: Spontaneous primary hypoparathyroidism in a ferret (Mustela putorius furo). J Exot Pet Med 24:333-339, 2015 19. Batchelder MA, Erdman SE, Li X, et al: A cluster of cases of juvenile mediastinal lymphoma in a ferret colony. Lab Anim Sci 46:271-274, 1996 20. Erdman SE, Brown SA, Kawasaki TA, et al: Clinical and pathologic findings in ferrets with lymphoma: 60 cases (1982-1994). J Am Vet Med Assoc 208:1,285-1,289, 1996 21. Ammersbach M, Delay J, Caswell JL, et al: Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets. Vet Pathol 45:663-673, 2008 22. http://www.uniprot.org. Accessed 10 February 2017 23. Kline LW, Yu DC: Effects of calcitonin, calcitonin gene-related peptide, human recombinant bone morphogenetic protein-2, and parathyroid hormone-related protein on endodontically treated ferret canines. J Endod 35:866-869, 2009 24. Greco DS: Endocrine causes of calcium disorders. Top Companion Anim Med 27:150-155, 2012 25. Calvo MS, Eastell R, Offord KP, et al: Circadian variation in ionized calcium and intact parathyroid hormone: evidence for sex differences in calcium homeostasis. J Clin Endocrinol Metab 72:69-76, 1991 26. D’Amour P, Rousseau L, Hornyak S, et al: Rat parathyroid hormone (rPTH) ELISAs specific for regions (2–7), (22– 34) and (40–60) of the rat PTH structure: influence of sex and age. Gen Comp Endocrinol 168:312-317, 2010 27. Di Monaco M, Castiglioni C, Vallero F, et al: Parathyroid hormone response to severe vitamin D deficiency is sex associated: an observational study of 571 hip fracture inpatients. J Nutr Health Aging 17:180-184, 2013 28. Marwaha RK, Khadgawat R, Tandon N, et al: Reference intervals of serum calcium, ionized calcium, phosphate and alkaline phosphatase in healthy Indian school children and adolescents. Clin Biochem 43:1216-1219, 2010
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