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Domestic Animal Endocrinology 36 (2009) 197–201
Rapid calcitonin response to experimental hypercalcemia in healthy horses夽 K.M. Rourke a , C.W. Kohn a , A.L. Levine b , T.J. Rosol b , R.E. Toribio a,∗ a
b
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon Tharp Street, Columbus, OH 43210, USA Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA Received 1 October 2008; received in revised form 17 October 2008; accepted 6 November 2008
Abstract Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca2+ ) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5 mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca2+ concentrations increased from 6.2 ± 0.3 mg/dL to 9.9 ± 0.5 mg/dL (4 min; P < 0.01). Serum CT increased from 16.7 ± 8.0 pg/mL to 87.1 ± 55.8 pg/mL at 2 min, and 102.5 ± 51.1 pg/mL at 4 min (P < 0.01). Serum CT returned to baseline by 20 min, whereas serum Ca2+ returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca2+ or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca2+ concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT. © 2008 Elsevier Inc. All rights reserved. Keywords: Calcitonin; Calcium; Horse; Equine; Hypercalcemia
1. Introduction Calcium is one of the most abundant elements in nature. In vertebrates, most calcium is in the skeleton, 夽 Funded by the Ohio State University College of Veterinary Medicine Equine Research Funds. ∗ Corresponding author. Tel.: +1 614 292 3278; fax: +1 614 688 5642. E-mail address:
[email protected] (R.E. Toribio).
0739-7240/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2008.11.004
where it has structural functions to support the body against gravity, to protect internal organs, and to host blood-forming elements. As a regulatory ion, calcium also has important physiological functions such as muscle contraction, hormone secretion, enzyme activation, cell division, cell membrane stability, neuromuscular excitability, blood coagulation, and cell death. To maintain extracellular ionized calcium (Ca2+ ) within narrow limits, there is a homeostatic system that includes 3 hormones (parathyroid hormone [PTH], calcitonin [CT],
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and vitamin D [1,25-dihydroxyvitamin D]); 3 body systems (bone, intestine, and kidney); and a Ca2+ -sensing receptor [1,2]. Hypocalcemia induces PTH release, which in turn increases renal calcium reabsorption and bone resorption, and increases renal synthesis of 1,25dihydroxyvitamin D3. Calcitonin is a 32-amino-acid protein secreted by the C-cells of the thyroid gland in response to hypercalcemia to decrease osteoclastic bone resorption and serum Ca2+ concentrations [3,4]. Although the principles of calcium regulation are similar for all mammals, horses are unique regarding calcium metabolism, as they have high total and ionized calcium concentrations [2,5,6], a high calcium set-point [6], low serum concentrations of vitamin D metabolites [7], high intestinal calcium absorption [8], and high urinary calcium excretion [5,9] when compared to other species. Hypocalcemia and hypercalcemia are frequent findings in horses with various pathological conditions [2,5,10,11]. It has been proposed that increased CT concentrations may be involved in the pathogenesis of hypocalcemia in equine sepsis and endotoxemia [4,5]. We have made major progress in understanding parathyroid gland function in healthy and diseased horses [1,4–6,9,12]. This progress has been due in part to the availability of human PTH immunoassays that we have previously validated in horses [5]. When compared to PTH, limited information on CT is available in horses [4,13–15]. To further study the role of genes of the CT family in equine calcium homeostasis, we recently cloned the equine CT-(CALC-I) and calcitonin-gene-related peptide (CALC-II) genes and determined that equine CT has 90% homology with human CT (65% and 53% for canine and bovine, respectively) [4]. We predicted that human immunoassays will detect equine CT and be useful to study equine calcium homeostasis. The goal of this study was to assess the CT response to acute hypercalcemia in healthy horses and to validate a human-specific CT radioimmunoassay. This information will be useful for future studies on equine and comparative calcium and mineral regulation. 2. Materials and methods 2.1. Horses Fourteen healthy mares aged 3–14 (7.9 ± 3.0) years and weighing 465–565 kg (510 ± 18 kg) were selected from The Ohio State University College of Veterinary Medicine teaching herd. All horses were in good body condition, fed a diet of grass pasture and grass hay (0.5% calcium and 0.25% phosphorus) and alfalfa hay
(1.4% calcium and 0.25% phosphorus), had no history of illnesses, and had received no treatments for 1 month prior to the study. To ensure health, complete physical examinations were performed. Values for a complete blood cell count, serum chemistry profile, serum total calcium, ionized calcium (Ca2+ ), total magnesium, ionized magnesium (Mg2+ ), phosphate, and plasma fibrinogen concentrations were within the reference ranges for all horses. Food and water were withheld for the duration of all experiments. Physical examinations (heart rate, respiratory rate, capillary refill time, and temperature) were evaluated every 15 min for 2 h, at 6 h, 12 h, and 24 h. The Ohio State University Institutional Laboratory Animal Care and Use Committee approved this study, and animals were treated following the NIH Institutional Animal Care and Use guidelines. 2.2. Experimental protocol—induction of hypercalcemia To assess changes in serum CT concentrations, hypercalcemia was induced in 10 healthy mares. Elemental calcium (5 mg/kg) was administered over 4 min, which corresponded to ∼120 mL of a 23% calcium gluconate solution (Vedco Inc., St. Joseph, MO) for a 500-kg horse. A 23% calcium gluconate solution contains 21.4 mg of elemental Ca2+ /mL. This dose was based on previous studies demonstrating that administration of 5 mg/kg of elemental calcium to healthy horses increases serum Ca2+ concentrations from a baseline value of 6.0–7.0 mg/dL to approximately 10.0 mg/dL [6]. Four horses were infused with 120 mL of 0.9% NaCl over 4 min to serve as controls. Blood samples to measure serum Ca2+ and CT concentrations were collected for 60 min. 2.3. Sampling An intravenous catheter (BD Angiocath, Becton Dickinson, Sandy, UT) was placed aseptically in each jugular vein. The catheter in the left jugular vein was used for the infusion of calcium gluconate, and the catheter in the right vein was used for blood sample collection. Venous blood samples for CT and Ca2+ concentrations were collected under anaerobic conditions at 0, 2, 4, 6, 8, 10, 20, 30, 40, 50, and 60 min in tubes with no additives, allowed to clot for 1 h, and centrifuged at 1000 × g for 5 min at 4 ◦ C. Serum samples used for measuring Ca2+ concentrations were processed immediately, whereas samples for CT quantification were stored at −80 ◦ C until batch analysis.
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2.4. Laboratory methods Hemograms were performed by an automated system (Cell-Dyn 3500, Abbott Diagnostics, Santa Clara, CA). Serum chemistry profiles were determined using an automated analyzer (Boehringer Mannheim/Hitachi 911 system, Boehringer Mannheim Corp, Indianapolis, IN). Serum Ca2+ concentrations were measured using Ca2+ -selective electrodes (Nova 8, Nova Biomedical, Waltham, MA). 2.5. CT radioimmunoassay Serum CT concentrations were determined with a commercial human-specific CT radioimmunoassay (Calcitonin DSL-1200 Radioimmunoassay, DSL, Webster, TX). The intra-assay coefficient of variation was determined from 6 replicates of equine serum samples containing low, moderate, and high CT concentrations. The interassay coefficient of variation was determined from values obtained by repeating the analysis of duplicate samples with low, moderate, and high CT concentrations in 3 different assays. According to the manufacturer, this assay has a calculated limit of detection of 14 pg/mL. To establish the detection limit for equine CT, we performed repeated CT measurements (interassay and intra-assay) using equine samples with low CT concentrations (
