Elevated 24,25-Dihydroxyvitamin D Serum Concentrations in Two Dogs with Cholecalciferol Toxicosis

Abstract

Vitamin D toxicosis poses a health threat to dogs, with cases often stemming from cholecalciferol rodenticide ingestion. This case report investigates two clinical cases of canine cholecalciferol toxicosis, shedding light on the persistent elevation of 25-hydroxyvitamin D (25(OH)D) and the adaptive response of 24,25-dihydroxyvitamin D (24,25(OH)₂D). Serum samples from affected dogs were analyzed over several months, revealing sustained increases in 25(OH)D concentrations. Notably, concurrent measurements of 24,25(OH)₂D unveiled a marked elevation, suggesting a compensatory mechanism to mitigate calcitriol excess and hypercalcemia. These findings highlight the potential role of upregulating 24-hydroxylase activity as a therapeutic target for managing cholecalciferol toxicosis. These cases underscore the importance of understanding vitamin D metabolism in canine toxicology and prompt further exploration into novel treatment strategies and other research areas.

1. Introduction

Vitamin D in the form of cholecalciferol (D₃) is an essential nutrient in dogs and cats, as they cannot synthesize vitamin D from ultraviolet light [1]. Vitamin D is converted to several metabolites, first to 25-hydroxyvitamin D (25(OH)D, i.e., calcidiol) by 25-hydroxylase in the liver, then to 1,25-dihydroxyvitamin D (1,25(OH)₂D, i.e., calcitriol) by 1a-hydroxylase (Figure 1A) primarily in the renal tubules [2]. The most bioactive vitamin D metabolite is calcitriol, which plays a vital role in calcium and phosphorus homeostasis by raising plasma calcium through several mechanisms [2]. Dogs and cats can tolerate a wide range of dietary cholecalciferol inclusions; however, excessive cholecalciferol can lead to hypercalcemia [3,4]. One mechanism that seemingly allows dogs and cats to tolerate higher amounts of cholecalciferol is increasing conversion of 25(OH)₂D to 24,25-dihydroxyvitamin D (24,25(OH)₂D) by 24-hydroxylase. Although 24,25(OH)₂D is a comparatively inert metabolite, it has roles in bone metabolism [5]. It also likely plays a role in intestinal calcium homeostasis [6], suppressing the activity of 1,25(OH)₂D and parathyroid hormone on calcium transport [7].

Cases of vitamin D toxicosis have been reported in dogs [3,8,9], cats [4,10], and both dogs and cats [11]. While persistent levels of 25-hydroxyvitamin D (25(OH)D) have been reported following vitamin D toxicosis in a dog [8], changes in circulating 24,25(OH)₂D in dogs with vitamin D toxicosis have not been described. This case report aims to report 24,25(OH)₂D concentrations in two dogs following vitamin D toxicosis. This new information provides mechanistic insight into the metabolic handling of vitamin D metabolites in states of excess exposure to exogenous cholecalciferol.

2. Detailed Case Description

Case #1: A 4-year-old female spayed mixed breed dog was referred to the Cornell University Hospital for Animals (CUHA) for evaluation of hyporexia, hypercalcemia, and azotemia. The dog lived on a farm and had potential access to rodenticides, but direct ingestion was never observed. The dog was fed an over-the-counter commercial diet with no known reported recalls for excessive vitamin D inclusions.

Her primary care veterinarian initially saw her for a 3-day history of decreased appetite and being slower to finish her food. An in-house chemistry panel showed total hypercalcemia (Ca 14.7 mg/dL, RR 7.9–12.0 mg/dL) and azotemia (creatinine 2.9 mg/dL, reference range [RR] 0.5–1.8 mg/dL; blood urea nitrogen [BUN] 45 mg/dL, RR 7–27 mg/dL). An in-house urinalysis showed an inappropriately dilute urine specific gravity of 1.019 with an inactive sediment.

Upon admission to CUHA, a thorough diagnostic work-up included a complete physical exam (including digital rectal exam), complete blood count, chemistry panel, urinalysis, point-of-care infectious disease testing (SNAP 4Dx Plus Test, IDEXX Laboratories, Inc., Westbrook, ME, USA), thoracic radiographs, abdominal ultrasound, cervical ultrasound, and bone marrow aspirate. Relevant clinicopathologic findings (

Table 1. Summary of clinicopathologic, vitamin D metabolite, and PTH measurements in a 4-year-old female spayed mixed breed dog (Case #1) with vitamin D toxicosis.

			Date
Analyte	Units	Reference Range/Values 1	9/15/20	9/17/20	9/18/20	9/23/20	10/26/20	12/9/20	2/10/21	3/10/21	3/19/21
Creatinine	mg/dL	0.6–1.4	2.7 (H)	1.5 (H)	1.6 (H)	1.3	0.9			0.8	0.8
Calcium (total)	mg/dL	9.4–11.1	14.6 (H)	12.8 (H)	12.3 (H)	11.2 (H)	10.6			10.3	10.6
Phosphate	mg/dL	2.7–5.4	4.8	4.4	5.1	2.3 (L)	3.5			2.8	3
Albumin	g/dL	3.2–4.1	4.2 (H)	3.9	4.2 (H)	4.2 (H)	4.4 (H)			4.2 (H)	4
iCa	mmol/L	1.16–1.48 2	1.64 (H) *	1.64 (H)	1.54 (H)	1.33	1.36				1.33
25(OH)D (RIA)	nmol/L	109–423			9994 ‡ (H)		1458 (H)	975 (H)	1745 #
PTH (CLIA)	pmol/L	1.1–10.6			0.4		2.1	1.5	2.7
iCa (ISE)	mmol/L	1.25–1.45			1.43		1.33	1.37	1.31
25(OH)D3 3	ng/mL	33, 26.6–39.3, 54.1									60.8 (↑↑)
1,25(OH)2D3 3	pg/mL	58.2, 51.0–72.3, 79.3									63
24,25(OH)2D3 3	ng/mL	17.4, 12.0–31.6, 34.9									63 (↑↑)

¹ Reference values: median, interquartile range (IQR), maximum healthy control dog (n = 9) values obtained in a previous study [12]. ² Ionized Ca measured by a Roche Cobas 2 in most cases unless the reported value is designated with an asterisk, where measured by a Gaslye Rapid Point with a reference range of 1.18–1.37. ³ Heartland assays: LC-MS/MS = liquid chromatography–tandem mass spectrometry. ^‡ Value was well above the standard curve and estimated. H = high, above reference range; L = low, below reference range; # = toxicity range; ↑↑ = value > max; iCa = ionized calcium; PTH = parathyroid hormone. Michigan State University Diagnostic Laboratory Tests: RIA = radioimmunoassay, CLIA = chemiluminescent immunoassay, ISE = ion-selective electrode. Abbreviations in vitamin D metabolite nomenclature are as follows: (OH) = hydroxyvitamin D, (OH)₂ = dihydroxyvitamin D, and D₃ = measurements from assays that distinguish cholecalciferol-derived metabolites.

) included total hypercalcemia (14.6 mg/dL, RR 9.4–11.1 mg/dL) and azotemia (creatinine 2.7 mg/dL, RR 0.6–1.4 mg/dL; BUN 55 mg/dL, RR 9–26 mg/dL), but no apparent cause for hypercalcemia was identified.

To evaluate potential causes for hypercalcemia, we submitted a panel measuring ionized calcium (iCa), parathyroid hormone (PTH), and 25(OH)D to the Michigan State University Veterinary Diagnostic Laboratory (MSU-VDL). Low PTH combined with excessive 25(OH)D concentrations (Table 1) were consistent with cholecalciferol toxicity, most likely from rodenticide exposure.

The patient was treated for cholecalciferol toxicity according to previously established protocols and discharged [8]. On a recheck exam five days (9/23/20) following discharge, venous blood gas showed a resolution of ionized hypercalcemia, and a chemistry panel showed a resolution of azotemia.

Serial serum samples to measure iCa, PTH, and 25(OH)D were submitted to the MSU-VDL at one month, three months, and five months following discharge (Table 1). Due to the increase in 25(OH)D at the 5-month recheck, the owner was thoroughly questioned for possible re-exposure to cholecalciferol, and none was identified.

At six months following cholecalciferol rodenticide toxicity, the owner reported no signs of illness. A CBC, chemistry panel, and urinalysis were performed, and they were clinically normal with no relapse of azotemia or total hypercalcemia. A serum sample was submitted to a lab (Heartland Assays, Ames, IA, USA) accredited for vitamin D metabolite analysis to further characterize vitamin D metabolism by measuring 25(OH)D₃, 1,25(OH)₂D, and 24,25(OH)₂D (Table 1). The patient was subsequently lost to follow-up.

Case #1 highlights an acute case of vitamin D toxicity following presumptive cholecalciferol rodenticide ingestion. There was a persistent elevation in 25(OH)D for up to 6 months following an acute cholecalciferol rodenticide toxicity. While there was an approximately 90% reduction (9994 nmol/L to 975 nmol/L) in serum 25(OH)D serum concentrations three months following ingestion, at five months, there was an unexplained increase in 25(OH)D. Additional vitamin D metabolite measurements showed a normal level of 1,25(OH)₂D and a marked increase in 24,25(OH)₂D.

Case #2: A 2-year-old male neutered Chesapeake Bay Retriever was presented to the Cornell University Hospital for Animals for a 2-week history of lethargy, hyporexia, progressive vomiting, and worsening of historic polyuria/polydipsia. He had a history of inflammatory bowel disease and cutaneous adverse food reactions. Both conditions were managed on a commercially available elemental diet (ProPlan EL Elemental Canine Formula, Nestle Purina, St. Louis, MO, USA) for the previous 3 weeks.

Initial diagnostic work-up included a physical exam (including digital rectal exam), CBC, chemistry panel, and urinalysis; this identified ionized hypercalcemia (1.7 mEq/L, 1.18–1.37), renal azotemia (creatinine 1.7 mg/dL, 0.6–1.4; BUN 25 mg/dL, 9–26), hyperphosphatemia (7.2 mg/dL, 2.7–5.4), and a urine specific gravity of 1.006. Additional diagnostic testing, including an ACTH stimulation test, thoracic radiographs, abdominal ultrasound, and cervical ultrasound, did not identify a cause for hypercalcemia. Measurements of iCa, PTH, and 1,25(OH)₂D through the MSU-VDL were consistent with vitamin D toxicosis. It was subsequently discovered that the patient’s elemental diet was recalled for excessive vitamin D inclusion.

The patient was treated for cholecalciferol toxicity according to previously established protocols and discharged [8]. On a recheck exam three days following discharge, venous blood gas showed a resolution of ionized hypercalcemia, and a chemistry panel showed a resolution of azotemia (

Table 2. Summary of clinicopathologic, vitamin D metabolite, and PTH measurements in a 2-year-old male neutered Chesapeake Bay Retriever (Case #2) with vitamin D toxicosis.

			Date
Analyte	Units	Reference Range/Values 1	2/8/23	2/9/23	2/10/23	2/11/23	2/13/23	2/14/23	2/17/23	7/31/23
Creatinine	mg/dL	0.6–1.4	1.7 (H)	1.5 (H)	1.5 (H)	1.4	1.4	1.5 (H)	1.1
Calcium (total)	mg/dL	9.4–11.1	13.7 (H)	12.4 (H)	14.6 (H)	12.6 (H)	12.1 (H)	11.8 (H)	9.9
Phosphate	mg/dL	2.7–5.4	7.2 (H)	4.2	6.0 (H)	4.5	5.1	4.5	1.7 (L)
Albumin	g/dL	3.2–4.1	4.1	3.5	3.8	3.6	3.9	4	4
iCa	mmol/L	1.16–1.48		1.64 (H)	1.83 (H)	1.59 (H)	1.52 (H)	1.46
25(OH)D (RIA)	nmol/L	109–423								530 (H)
1,25(OH)2D (RIA)	pmol/L	164–523		612 (H)
PTH (CLIA)	pmol/L	1.1–10.6		1.0 (L)						3.7
iCa (ISE)	mmol/L	1.25–1.45		1.62 (H)						1.37
24,25(OH)2D3 2	ng/mL	17.4, 12.0–31.6, 34.9						56.9 (↑↑)		32.9 (↑)

¹ Reference values: median, interquartile range, maximum healthy control dog (n = 9) values obtained in a previous study [12]. ² Measured at Heartland Assays by liquid chromatography–tandem mass spectrometry. H = high, above reference range; L = low, below reference range; ↑ = value > IQR; ↑↑ = value > max; iCa = ionized calcium; PTH = parathyroid hormone. Michigan State University Diagnostic Laboratory Tests: RIA = radioimmunoassay, CLIA = chemiluminescent immunoassay, ISE = ion-selective electrode. Abbreviations in vitamin D metabolite nomenclature are as follows: (OH) = hydroxyvitamin D, (OH)₂ = dihydroxyvitamin D, and D₃ = measurements from assays that distinguish cholecalciferol-derived metabolites.

).

Serum samples for the measurement of 24,25(OH)₂D₃ were collected at the initial diagnosis and the 5-month recheck (Table 2). An additional serum sample to measure iCa, PTH, and 25(OH)D was also submitted to the MSU-VDL at the 5-month recheck (Table 2).

Case #2 highlights a case of vitamin D toxicosis following repeated ingestion of a diet with excessive vitamin D supplementation for at least three weeks. This case also showed a persistent elevation in 25(OH)D for up to 5 months following diagnosis. Serial measurements of serum 24,25(OH)₂D₃ also showed a persistent increase during this period. While 1,25(OH)₂D was not measured contemporaneously, normal values for PTH and iCa suggest it was likely normal.

3. Discussion

These two cases of vitamin D toxicosis illustrated elevations in serum 25(OH)D for at least five months and showcase a possible mechanism of upregulating the activity of 24-hydroxylase to increase conversion of 25(OH)D to 24,25(OH)₂D and maintain normal levels of 1,25(OH)₂D. Previous literature has also shown persistent elevations in 25(OH)D following cholecalciferol rodenticide toxicity [8], but this is the first published case report measuring 24,25(OH)₂D.

Few studies have evaluated changes in circulating 24,25(OH)₂D concentrations in dogs compared to dietary intakes. One study in growing dogs demonstrated higher plasma concentrations of 24,25(OH)₂D and increased renal 24-hydroxylase gene expression when fed a diet containing 54,000 IU vs. 470 IU of vitamin D₃ on a per kg diet basis [13]. Another study found that 25(OH)D₃ supplementation was associated with increases in serum 24,25(OH)₂D₃ concentrations, but not when supplemented with cholecalciferol [14]. In one case of cholecalciferol toxicosis, 25(OH)D serum concentrations remained persistently increased for nearly a year [8]. In that case and ours, this observation is consistent with the highly lipid-soluble nature of 25(OH)D [8,15,16]. In this case, ionized hypercalcemia was never present after the patient was discharged, despite 25(OH)D concentrations that at one time were over one order of magnitude above the reference range. This information and the results in our cases suggest a key role for increasing 24,25(OH)₂D concentrations to mitigate the deleterious effects of cholecalciferol excess.

To the authors’ knowledge, only two published reports have used the same lab to measure 24,25(OH)₂D₃ in dogs [12,17]. As a reference group, we used data obtained from nine healthy dogs in a previous study by one of the authors [12]. The other previous study included serum 24,25(OH)₂D₃ concentrations obtained from 10 healthy dogs [17]. In that study, the upper range (excluding one outlier) was approximately 50 ng/mL for healthy control dogs. While this is higher than the reference values used for these cases, all 24,25(OH)₂D₃ concentrations measured, except for the last measurement in Case #2, were greater than 50 ng/mL. The decreased concentration of 24,25(OH)₂D₃ in Case #2, when measured after resolution of hypercalcemia, further suggests that the previous measurement reflected an increased serum concentration for this dog. Thus, while there is no reference range for serum 24,25(OH)₂D₃ in dogs established by the lab used, this supports the assertion of elevated 24,25(OH)₂D₃ associated with cholecalciferol toxicosis in these two cases.

As these were clinical cases and were not part of a prospective study design, the consideration to measure 24,25(OH)₂D₃ occurred after case management began. Therefore, longitudinal data were variable, and some inferences must be made. For example, 25(OH)D concentrations were presumably increased at the onset for Case #2, given the patient’s hypercalcemia, exposure to excess cholecalciferol, and increased serum 1,25(OH)₂D concentrations. The decision not to measure 25(OH)D was clinically rational, as it would not have strengthened the diagnosis nor altered treatment. Additionally, two laboratories were used in both cases to measure vitamin D metabolites at various times. These laboratories used different methodologies, measuring different metabolite forms (total metabolite concentrations vs. cholecalciferol forms). Therefore, direct quantitative comparisons between methodologies should not be made. Indeed, this is why unit conversions were not performed for reporting herein. Finally, the relatively small number of time points measuring serum concentrations did not provide sufficient information to predict metabolite elimination kinetics.

Combining information from both cases suggests that early in vitamin D toxicosis, hypercalcemia occurs in the context of both increased 1,25(OH)₂D₃ and 24,25(OH)₂D₃ (Figure 1). However, as toxicosis transitions to a more chronic phase, presumably by the induction of 24-hydroxylase, 1,25(OH)₂D concentrations normalize, and increased concentrations of 24,25(OH)₂D₃ concentrations persist (Figure 1C). Increases in serum 24,25(OH)₂D concentrations could protect against hypercalcemia in two ways. The first is that by inducing 24-hydroxylase, the conversion of 25(OH)D to 1,25(OH)₂D is controlled to help maintain normal 1,25(OH)₂D concentrations. Additionally, although 24,25(OH)₂D is often considered a relatively inert metabolite, there are data supporting that it can oppose calcitriol’s effects at intestinal calcium absorption [7]. Another metabolite produced by 24-hydroxylase is 1,24,25-trihydroxy vitamin D. Although not measured in these cases, it is logical to assume that this metabolite is also increased to deactivate 1,25(OH)2D effectively.

Several molecules increase 24-hydroxylase activity, and some could be explored as adjunctive treatments to attenuate hypercalcemia in cholecalciferol toxicosis. Phorbol esters, in addition to 1,25(OH)₂D, increase gene expression of 24-hydroxylase in rat kidney cells [18]. Similarly, insulin in the presence of 1,25(OH)₂D increased 24-hydroxylase mRNA concentrations in rat osteoblastic cells [19]. Two vitamin D receptor agonists, known for minimal hypercalcemic effects, 22-oxacalcitriol and paricalcitol, could also be considered for modifying 24-hydroxylase activity. In mouse kidney and duodenal cells, 22-oxacalcitriol induced 24-hydroxylase gene activity and was slightly more potent in this effect than 1,25(OH)₂D [20]. Finally, in human patients with chronic kidney disease, paricalcitol administration was associated with lower 1,25(OH)₂D and 25(OH)D serum concentrations and increased 24,25(OH)₂D levels [21]. Paricalcitol is readily available and inexpensive. Therefore, it may be a realistic consideration for future trials in veterinary medicine. However, this would likely require preliminary pharmacodynamic studies to determine if there is an optimal dose that increases 24,25(OH)₂D₃ without causing hypercalcemia.

4. Conclusions

These cases illustrate for the first time increased serum 24,25(OH)₂D₃ concentrations in cholecalciferol toxicosis in dogs, representing an adaptive physiologic response to excess vitamin D intake. Strategies to accelerate the conversion of 25(OH)D to 24,25(OH)₂D through the upregulation of 24-hydroxylase may represent a novel therapeutic target for the medical management of cholecalciferol toxicosis, and warrant future exploration.