Evangelia Maniaki: Exploring the relationship between calcitonin, ionized calcium, and bone turnover in cats with and without naturally occurring hypercalcemia

Methods: Hypercalcemic cats (persistently increased iCa concentration [>1.40 mmol/l]) were identified retrospectively via a medical database search; additional hypercalcemic and normocalcemic cats were recruited prospectively. Data regarding routine biochemical and urine testing, diagnostic imaging and additional blood testing were obtained. Serum alkaline phosphatase (ALP) activity was used as a marker of bone turnover. Serum calcitonin concentration was analyzed using a previously validated immunoradiometric assay. Hypercalcemic cats with an increased calcitonin concentration (>0.9 ng/L) were termed responders. Group comparisons were performed using a Mann-Whitney test for continuous variables and a χ2 test for categorical variables. Spearman’s correlation coefficient was used to examine the relationships between calcitonin, iCa and ALP.

Results: Twenty-six hypercalcemic and 25 normocalcemic cats were recruited. Only 5/26 (19.2%) of the hypercalcemic cats were identified as responders, and all were diagnosed with idiopathic hypercalcemia. There was no significant correlation between the concentrations of calcitonin and iCa (p = 0.929), calcitonin and ALP (p = 0.917) or iCa and ALP (p = 0.678) in hypercalcemic cats, however, a significant negative correlation was observed between calcitonin and ALP (p = 0.037) when normocalcemic and hypercalcemic cats with an elevated calcitonin concentration were analyzed together.

Discussion: The expected increase in calcitonin concentration was present in only a small subset of hypercalcemic cats; no correlation was found between iCa and calcitonin concentration. The inverse relationship between calcitonin and ALP in cats with increased calcitonin concentrations suggests that the ability of calcitonin to correct hypercalcemia may be related to the degree of bone turnover.

1 Introduction

Calcium homeostasis is tightly regulated by interactions between parathyroid hormone (PTH), calcitriol (the active form of vitamin D) and calcitonin. Calcitonin is synthesized by the parafollicular or C cells in the thyroid gland and secreted in response to hypercalcemia. Calcitonin restores normocalcemia by predominantly inhibiting osteoclastic bone resorption (1–3), but also by decreasing renal tubular reabsorption of calcium (4). The ability of calcitonin to decrease the magnitude of hypercalcemia has been established both in azotemic and non-azotemic animal models (5–8), but it is not yet clear whether its significance is greater in one state compared to the other. Further, it has been suggested in some species that the ability of calcitonin to reduce circulating calcium concentration is related to the degree of bone turnover (9, 10). The role of calcitonin in calcium regulation has not been fully elucidated in most mammals and studies investigating the precise function of calcitonin in cats are lacking, however, its function is acknowledged to be conserved across species (11).

Idiopathic hypercalcemia is one of the most commonly reported causes of naturally occurring hypercalcemia, reported in 48–52% of hypercalcemic cats (12). Chronic kidney disease (CKD) is also common (13), reported in 25.4–35% (14, 15) of hypercalcemic cats. However, the relationship between CKD and hypercalcemia is complex as hypercalcemia can both cause renal damage and develop as a consequence of CKD (16). Hypercalcemia of malignancy accounts for 29.6–35% of hypercalcemia in cats (14, 15, 17). Other causes of hypercalcemia in cats include hypervitaminosis D, primary hyperparathyroidism, granulomatous disease, hypoadrenocorticism, and hyperthyroidism (14, 15, 17–24).

A previous study in cats with experimentally induced hypercalcemia identified a group of cats that failed to increase calcitonin production in response to hypercalcemia (25). Although C cells were also present in these cats, their numbers were significantly lower (total number of calcitonin-positive C cells and mean number of C cells per field), and their functional ability to produce calcitonin is unknown. These cats were termed “non-responders,” and this finding suggests that a subgroup of cats may not increase calcitonin concentration in response to ionized hypercalcemia. Similarly, in a recent study exploring calcitonin response to naturally occurring ionized hypercalcemia in cats with azotemic CKD (26), only a third of the cats had a measurable increase in calcitonin concentration.

The aims of this study were (a) to compare serum calcitonin concentrations between normocalcemic and hypercalcemic cats, (b) to evaluate calcitonin response in cats with naturally occurring hypercalcemia, and (c) to investigate the relationship between calcitonin, ionized calcium (iCa) and bone turnover in cats. We hypothesized that there would be no difference in the serum calcitonin concentrations between normocalcemic and hypercalcemic cats.

2 Materials and methods

This case–control study was approved by the University of Bristol’s Animal Welfare and Ethical Review Body (VIN/15/050).

The medical database at Langford Vets’ Small Animal Hospital, University of Bristol, UK was searched to identify hypercalcemic cats referred to the Feline Centre between January 2011 and June 2015. Additional hypercalcemic and normocalcemic (control) cats were identified at the time of presentation to the Feline Centre between July 2015 and June 2016. Hypercalcemia was defined as persistently increased iCa concentration (>1.40 mmoL/L [reference interval (RI): 1.10–1.40 mmoL/L]) on two or more repeated samples. Normocalcemia was defined as an iCa concentration and/or a total calcium (tCa) concentration (RI: 2.30–2.50 mmoL/L) within the RI. iCa was measured in heparinized blood immediately following collection using an ion-selective electron analyzer (RAPIDPoint® 500). The control cat population included normocalcemic cats without disease or receiving medication known to affect calcium or calcitonin homeostasis.

Serum concentrations of tCa, creatinine, urea, phosphate, alkaline phosphatase (ALP) and urine specific gravity (USG) were measured at a commercial reference laboratory (Langford Diagnostic Laboratories, Langford Vets, Bristol). Urine was either voided voluntarily and collected using non–absorbent cat litter or collected via cystocentesis. Additional testing in individual hypercalcemic cases was undertaken according to the clinician’s decision (an ECVIM [European College of Veterinary Internal Medicine] diplomate or diplomat in training), and this was also recorded in the study. This included serum measurement of PTH, parathyroid hormone-related protein (PTHrP), calcitriol, basal cortisol, and findings from imaging modalities (radiography, ultrasound, computed tomography, magnetic resonance imaging). Clinical notes were reviewed by an ECVIM diplomate to obtain the clinician’s final diagnosis for both hypercalcemic (cases) and normocalcemic (controls) cats.

Serum biochemistry and measurement of PTH, PTHrP, and calcitriol was submitted to a commercial reference laboratory (Langford Vets Diagnostic Laboratories). Cats with a concentration below the lower limit of detection of the PTH assay (1 pmol/L) were assigned a value of 1 pmol/L. Serum ALP activity was used as a marker of bone turnover (27, 28). Residual serum samples that had been stored at –80°C for an average of 416 days (range: 90 to 742 days) were transported frozen to the University of Cordoba, Spain for measurement of calcitonin concentration.

Measurement of calcitonin concentration was undertaken using a human immunoradiometric assay (Scantibodies Laboratory Inc., Santee, California) previously validated for cats (25). Cats with calcitonin concentration below the lower limit of detection of the assay (0.9 ng/L or 0.9 pg/mL) were assigned a value of 0.9 ng/L. Hypercalcemic cats were further grouped into responders and non-responders based on whether they had a calcitonin concentration above the lower limit of detection.

Statistical analysis was conducted using SPSS (version 27.0, IBM Corporation, United States) and GraphPad Prism (version 8.1.2, GraphPad Software, United States). Data were assessed for normality using the Kolmogorov–Smirnov test and by visual inspection of graphical plots. Non-parametric statistical tests were applied to the data. Group comparisons were performed using a Mann–Whitney test for continuous variables and a χ2 test or Fisher’s exact test for categorical variables. The correlation between iCa and calcitonin, iCa and ALP activity, and calcitonin and ALP activity were examined using Spearman’s correlation coefficient (ρ). Statistical significance was set at p < 0.05, and an exact (2-tailed) significance is reported.

3 Results

Cats were recruited both retrospectively (medical database search between January 2011 and June 2015) and prospectively (recruitment between July 2015 and June 2016) as illustrated in Figure 1. Normocalcemic cats were only recruited prospectively. The total study population comprised of 51 cats: 26 hypercalcemic and 25 normocalcemic.

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