Serum 25‐hydroxyvitamin D, vitamin D receptor, and vitamin D binding protein concentrations in dogs with acute pancreatitis compared to healthy control dogs

Abstract

Background

Previous studies have documented vitamin D imbalance in dogs with acute pancreatitis (AP), but no studies have investigated serum vitamin D receptor (VDR) and vitamin D‐binding protein (VDBP) concentrations.

Objectives

Compare serum 25‐hydroxyvitamin D (25[OH]D), VDR, and VDBP concentrations in healthy dogs and dogs with AP and identify correlations between these concentrations with ionized calcium, C‐reactive protein (CRP), and canine‐specific pancreatic lipase (Spec cPL) concentrations.

Animals

Twenty‐two dogs with AP and 20 healthy control dogs.

Methods

Prospective cross‐sectional study. Serum 25(OH)D concentrations were measured using a chemiluminescence immunoassay, and VDR and VDBP concentrations were measured using a ELISA kit designed for dogs.

Results

Serum concentrations of 25(OH)D were lower in dogs with AP (mean ± SD, 66.1 ± 39.2 ng/mL) than in controls (96.8 ± 30.4 ng/mL; P = .01), and VDR concentrations were lower in dogs with AP (5.3 ± 3.5 ng/mL) than in controls (7.4 ± 2.5 ng/mL; P = .03). No difference was observed in serum VDBP concentrations between the groups. Serum VDR concentrations differed between survivors (median [interquartile range] = 6.6 [4.3‐8.2] ng/mL) and nonsurvivors (2.7 [0.5‐3.5] ng/mL; P = .01). Negative correlations were observed among serum VDR, CRP (r _s  = −0.55), and Spec cPL (r _s  = −0.47) concentrations in dogs with AP.

Conclusions and Clinical Importance

Dogs with AP had lower serum 25(OH)D and VDR concentrations than controls. Additionally, our study suggests a potential role of VDR expression in the inflammatory process of AP in dogs.

Abbreviations

25(OH)D

25‐hydroxyvitamin D

AP

acute pancreatitis

CRP

C‐reactive protein

iCa

ionized calcium

IQR

interquartile range

PTH

parathyroid hormone

Spec cPL

specific canine pancreatic lipase

VDBP

vitamin D binding protein

VDR

vitamin D receptor

1. INTRODUCTION

Acute pancreatitis (AP) is a common pancreatic disease in dogs. ¹ , ² It is an acute inflammatory process in the pancreas and causes local and systemic inflammatory response. ¹ , ² Dogs with AP present with clinical signs such as anorexia, vomiting, abdominal pain, and diarrhea, ² , ³ and mortality for affected dogs is approximately 27% to 42%. ⁴ The etiology of AP in dogs is poorly understood, and most cases are considered idiopathic in origin. ¹ , ⁵ A previous study documented vitamin D imbalances in dogs with AP, and an association between serum 25‐hydroxyvitamin D (25[OH]D) concentrations and clinical outcome was reported. ⁶ Thus, alterations in vitamin D metabolism might be associated with the development or progression of AP.

Vitamin D represents a group of steroidal hormones that controls calcium homeostasis. ⁷ Additionally, it affects anti‐inflammatory processes and immune regulation, ⁸ , ⁹ highlighting the importance of vitamin D in inflammatory diseases. Cholecalciferol (vitamin D3) absorbed from the intestine is transported to the liver by binding to circulating vitamin D‐binding proteins (VDBP), ¹⁰ , ¹¹ and hydroxylated into 25(OH)D. ⁸ , ⁹ , ¹² In the kidneys, 25(OH)D is converted into 1,25‐dihydroxyvitamin D, the biologically active form. ⁸ , ⁹ , ¹² The active form of vitamin D binds to vitamin D receptors (VDR) expressed in various tissues, ¹³ and activated VDR regulates target genes, bringing about the biological effects of vitamin D. ¹²

Considering the important roles of VDR and VDBP in the functions of vitamin D, VDR expression and serum VDBP concentrations in inflammatory diseases are of interest. The association between VDR polymorphisms and increased inflammation has been reported in several inflammatory diseases, such as inflammatory bowel disease, asthma, and sepsis. ¹⁴ , ¹⁵ , ¹⁶ Additionally, decreased VDBP concentrations may exacerbate vitamin D insufficiency in critically ill humans. ¹⁷ However, no studies have investigated serum VDR and VDBP concentrations in dogs with AP.

Our objectives were to (a) compare the serum 25(OH)D, VDR, and VDBP concentrations in healthy dogs and dogs with AP; (b) compare these concentrations between survivors and nonsurvivors; (c) identify correlations between serum 25(OH)D, VDR, and VDBP concentrations and ionized calcium (iCa) concentrations, C‐reactive protein (CRP), a biomarker reflecting the inflammatory status, and canine‐specific pancreatic lipase (Spec cPL), a biomarker reflecting pancreatic damage; and (d) compare serum 25(OH)D, VDR, and VDBP concentrations before and after treatment of AP in dogs.

2. MATERIALS AND METHODS

2.1. Study group

This study was approved by the Chungbuk National University Animal Care Committee (CBNUA‐1710‐22‐01). This prospective cross‐sectional study included 22 client‐owned dogs with AP, 13 client‐owned clinically healthy dogs, and 7 healthy beagle dogs. Healthy and client‐owned dogs with AP were examined at our institution between May 2019 and July 2022.

Client‐owned healthy dogs without any clinical signs visited our hospital for routine health examinations. Healthy controls were included based on unremarkable findings on physical examination, CBC, serum biochemical analysis, serum electrolyte concentrations, venous blood gas analysis, urinalysis, survey radiography, and abdominal ultrasonography.

A diagnosis of clinical AP was established if all the following enrollment criteria were met: (a) at least 2 of the following acute clinical signs (duration ≤3 days): abdominal pain, anorexia, vomiting, and diarrhea, ¹⁸ (b) ultrasonographic findings suggestive of AP, including hypoechoic and enlarged pancreatic parenchyma with irregular margins or irregular shape, hyperechoic mesentery, or localized free abdominal fluid, ¹⁹ and (c) increased serum Spec cPL concentration (>400 ug/L; reference interval, 0‐200 μg/L; IDEXX Reference Laboratory Inc, Westbrook, ME). ²⁰

Considering the potential effects of liver and kidney diseases on the concentration of circulating VDBP, ¹¹ dogs with nephrotic syndrome, acute renal failure, renal tubular injury (granular casts in urine sediment), and hepatic synthetic dysfunction (liver disease with hypoalbuminemia, decreased blood urea nitrogen concentration, or hypocholesterolemia) were excluded.

2.2. Grouping

Dogs with AP were divided into survivors and nonsurvivors. Survivors were defined as dogs discharged from the hospital that survived for at least 14 days. Nonsurvivors were defined as those that died during hospitalization despite treatment. Dogs that died within 14 days of discharge or those that were not treated were excluded.

2.3. After treatment assessment

All dogs with AP were treated using standardized treatment protocols. ⁵ , ²¹ , ²² For fluid therapy, lactated Ringer's solution was administered for rehydration and maintenance of fluid requirements. Maropitant was used as the first‐choice antiemetic, and ondansetron was added if vomiting persisted. As an antacid, esomeprazole was administered to dogs with hematochezia or melena, otherwise famotidine was administered. Abdominal pain was managed by administration of butorphanol tartrate, buprenorphine, lidocaine in combination with ketamine, or fentanyl, depending on pain severity. Enteral nutrition was initiated within the first 24‐48 hours of presentation. A low‐fat diet was provided as soon as vomiting subsided; if refused, a nasogastric tube was placed. Broad‐spectrum antibiotics (ampicillin‐sulbactam and metronidazole) were administered to dogs with fever or neutropenia with a left shift. ⁵ , ²¹ , ²² Dogs were discharged when clinical signs of AP resolved.

Serum samples were obtained at least 14 days after discharge, considering the time required for the serum concentrations of 25(OH)D, VDR, and VDBP to return to normal after treatment of AP. Serum concentrations of 25(OH)D, VDR, and VDBP were compared before and after treatment (n = 11).

2.4. Assays

Blood samples after 8 hours of fasting were collected from the jugular or cephalic vein in heparinized and plain tubes. After blood collection, heparinized blood samples were immediately analyzed for iCa using a blood gas analyzer (ABL80 Flex Co‐ox; Radiometer Medical, Copenhagen, Denmark). After the samples in plain tubes were centrifuged at 2000 × g for 10 minutes at room temperature, the serum was used for biochemical analysis and submitted to commercial laboratories to measure circulating Spec cPL, 25(OH)D, and parathyroid hormone (PTH) concentrations. The remaining serum was stored at −80°C within 1 hour of collection until serum VDR and VDBP concentrations were measured.

Serum Spec cPL concentrations were measured by a commercial laboratory (IDEXX Laboratories, Inc., Westbrook, Maine, USA) using a quantitative immunoassay. Serum 25(OH)D concentrations were measured by a commercial laboratory (VDI laboratory, LLC., Simi Valley, California, USA) using a chemiluminescence immunoassay for quantitative determination with Liaison equipment (DiaSorin Inc., Stillwater, Minnesota, USA). The reference range of 25(OH)D in dogs was 100.0‐150.0 ng/mL, and the vitamin D status of each dog was categorized as sufficiency (100.0‐150.0 ng/mL), insufficiency (40.1‐99.9 ng/mL), or deficiency (≤40.0 ng/mL). Serum PTH concentrations were measured at the same laboratory (VDI Laboratory, LLC) using a chemiluminescence assay (DiaSorin Inc., Stillwater, Minnesota, USA) to measure intact PTH.

Serum VDR concentrations were measured by competitive enzyme immunoassay using a Vitamin D Receptor ELISA kit designed for dogs (Amsbio, Cambridge, Massachusetts, USA). The detection range of the kit was from 2.5 to 50 ng/mL, with a sensitivity of 0.1 ng/mL. The intra‐ and inter‐assay coefficients of variation were 4.4% and 5.6%, respectively, according to the manufacturer's information. Serum VDBP concentrations were measured using a quantitative sandwich immunoassay with the Canine Vitamin D Binding Protein ELISA Kit (MyBioSource, San Diego, California, USA). The detection range of the kit was 31.2‐1000 μg/mL, with a sensitivity of 5.0 μg/mL. Both the intra‐ and inter‐assay coefficients of variation were <15%. All processes were performed according to the manufacturer's instructions.

2.5. Statistical analysis

Statistical analyses were performed using Prism 6 software (GraphPad Software Inc., La Jolla, California, USA). The Shapiro‐Wilk test was conducted to determine normal distributions. The results are expressed as means ± SD when normally distributed or medians and interquartile ranges (IQR) when non‐normally distributed. Fisher's exact test was used to compare the proportion of patients with vitamin D deficiency between the 2 groups. Independent‐sample t‐tests or Mann‐Whitney U‐tests were used to compare the demographic characteristics and concentrations of circulating 25(OH)D, VDR, and VDBP between the 2 groups. The correlations between the serum VDR and VDBP concentrations with iCa, CRP and Spec cPL concentrations were assessed using the Pearson correlation test (when normally distributed) or Spearman's rank correlation analysis (when non‐normally distributed). A partial correlation analysis of iCa concentrations also was performed to control for the potential confounding effect of PTH. Paired t‐tests were performed to compare data before and after treatment in dogs with AP. For statistical analysis, CRP <5.0 and >500.0 mg/L was recorded as 2.5 and 500.1 mg/L, respectively, and Spec cPL <30 and >2000 ng/mL was recorded as 15 and 2001 ng/mL, respectively. For all analyses, P < .05 was considered significant.

3. RESULTS

3.1. Study group

Twenty‐two dogs with AP and 20 healthy dogs were included in the study. The dogs with AP consisted of 5 Maltese, 4 Miniature Poodles, 2 Chihuahuas, 2 Dachshunds, and 1 each of Pomeranian, Yorkshire Terrier, Shih Tzu, Miniature Pinscher, Miniature Schnauzer, Spitz, Jindo, Golden Retriever, and mixed‐breed dogs. The healthy dogs consisted of 7 Beagles, 5 mixed‐breed dogs, 3 Maltese, and 1 each of Miniature Poodle, Pomeranian, Spitz, Cavalier King Charles Spaniel, and Siberian Husky breeds. The demographic characteristics of the dogs are shown in Table 1. Significant differences were found in the mean age and median concentrations of serum iCa, CRP, and Spec cPL between healthy dogs and dogs with AP (P < .001). Of the 22 dogs with AP, 17 survived and were discharged from the hospital, and 5 died.

TABLE 1.

Demographics of dogs included in this study.

	Healthy dogs (n = 20)	Dogs with AP (n = 22)	Dogs with AP
			Survivors (n = 17)	Nonsurvivors (n = 5)
Age (years)	6.0 ± 2.8	10.7 ± 4.1 a	10.2 (7.2‐14.2)	11.0 (8.7‐13.5)
Body weight (kg)	6.5 (4.4‐10.9)	4.7 (3.2‐9.6)	3.6 (3.0‐8.2)	8.1 (6.6‐15.5) c
Sex (number) IF/SF/IM/CM	8/4/1/7	3/10/1/8	2/7/1/7	1/3/0/1
Breed (number)
Maltese	3	5	4	1
Miniature Poodle	1	4	4	0
Beagle	7	0	0	0
Mixed breeds	5	1	1	0
Others	4	12	8	4
iCa (mmol/L)	1.40 (1.37‐1.44)	1.27 (1.11‐1.34) b	1.28 (1.12‐1.33)	1.19 (1.00‐1.40)
CRP (mg/L)	4.1 (2.5‐6.8)	88.8 (36.2‐180.1) b	59.8 (28.8‐154.4)	170.0 (81.8‐324.8)
Spec cPL (ng/mL)	15 (15‐47)	885 (654‐1939) b	827.3 (486.1‐1000.0)	2001 (1419‐2001) d

Note: Data are expressed as mean ± SD, median (interquartile range), or number.

Abbreviations: AP, acute pancreatitis; CM, castrated male; CRP, C‐reactive protein; iCa, ionized calcium; IF, intact female; IM, intact male; SF, spayed female; Spec cPL, specific canine pancreatic lipase.

^a Statistically significant difference (P < .001) compared to healthy dogs using independent‐sample t‐tests.

^b Statistically significant difference (P < .001) compared to healthy dogs using Mann‐Whitney U‐test.

^c Statistically significant differences (P < .05) compared with survivors using the Mann‐Whitney U‐test.

^d Statistically significant differences (P < .01) compared with survivors using the Mann‐Whitney U‐test.

3.2. Comparison of serum 25(OH)D, VDR, and VDBP concentrations between healthy dogs and dogs with AP

None of the 20 healthy dogs had vitamin D deficiency, 11 dogs had vitamin D insufficiency (11/20, 55.0%), and 9 had vitamin D sufficiency (9/20, 45.0%). Of the 22 dogs with AP, 8, 9, and 5 had vitamin D deficiency (8/22, 36.4%), insufficiency (9/22, 40.9%), and sufficiency (5/22, 22.7%), respectively. The proportions of dogs with vitamin D deficiency significantly differed between healthy dogs and dogs with AP (P = .004). Serum 25(OH)D concentrations in dogs with AP (mean ± SD, 66.1 ± 39.2 ng/mL) were significantly lower than those in healthy dogs (96.8 ± 30.4 ng/mL; P = .01; Figure 1A). The serum VDR concentrations in dogs with AP (5.3 ± 3.5 ng/mL) were significantly lower than those in healthy dogs (7.4 ± 2.5 ng/mL; P = .03; Figure 1B). No significant difference was observed in serum VDBP concentrations in dogs with AP (81.7 ± 27.0 μg/mL) and healthy dogs (73.2 ± 31.2 μg/mL; P = .35; Figure 1C).

FIGURE 1.

Comparison of serum (A) 25(OH)D, (B) VDR, and (C) VDBP concentrations between healthy dogs (n = 20) and dogs with AP (n = 22). The horizontal bars show the means and standard deviations. Independent‐sample t‐tests. *P < .05, **P < .01. 25(OH)D, 25‐hydroxyvitamin D; AP, acute pancreatitis; VDBP, vitamin D binding protein; VDR, vitamin D receptor.

3.3. Comparison of serum 25(OH)D, VDR, and VDBP concentrations between survivors and nonsurvivors among dogs with AP

Significant differences were found in the median body weight (P = .04) and median concentrations of serum Spec cPL (P = .005) between survivors and nonsurvivors among dogs with AP. However, median age (P = 1.00), serum iCa concentrations (P = .88), and CRP concentrations (P = .1) were not significantly different (Table 1). Of the 17 survivors, 4, 9, and 4 dogs had vitamin D deficiency (4/17, 23.5%), insufficiency (9/17, 53.0%), and sufficiency (4/17, 23.5%), respectively. Of the 5 nonsurvivors, 4 had vitamin D deficiency (4/5, 80.0%), and 1 had vitamin D sufficiency (1/5, 20%). The proportion of vitamin D deficiency significantly differed between survivors and nonsurvivors (P = .04). No significant differences in serum 25(OH)D concentration were found between survivors (median, 75.8 ng/mL; IQR, 41.0‐99.6 ng/mL) and nonsurvivors (median, 33.8 ng/mL; IQR, 25.6‐90.3 ng/mL; P = .23; Figure 2A). However, a significant difference in serum VDR concentrations was found between survivors (median, 6.6 ng/mL; IQR, 4.3‐8.2 ng/mL) and nonsurvivors (median, 2.7 ng/mL; IQR, 0.5‐3.5 ng/mL; P = .01; Figure 2B). The VDBP concentrations were not different between survivors (median, 75.5 μg/mL; IQR, 59.2‐108.5 μg/mL) and nonsurvivors (median, 90.8 μg/mL; IQR, 55.4‐111.7 μg/mL; P = .82; Figure 2C).

FIGURE 2.

Comparison of serum (A) 25(OH)D, (B) VDR, and (C) VDBP concentrations between survivors (n = 17) and nonsurvivors (n = 5) in dogs with AP. The horizontal bars show the medians and interquartile ranges from the first to the third quartile. The Mann‐Whitney U test. **P < .01. 25(OH)D, 25‐hydroxyvitamin D; AP, acute pancreatitis; VDBP, vitamin D binding protein; VDR, vitamin D receptor.

3.4. Correlation between serum 25(OH)D, VDR, and VDBP concentrations and iCa, CRP, and Spec cPL concentrations in dogs with AP

Significant negative correlations were found between serum VDR concentrations and CRP (r _s  = −0.55, P = .01) and Spec cPL (r _s  = −0.47, P = .03) concentrations. No simple correlation was observed between serum VDR concentrations and iCa concentrations (r = 0.15, P = .5), and no partial correlation, adjusted for serum PTH concentrations, was observed (partial r = 0.04, P = .85).

No correlations were found between serum 25(OH)D and iCa concentrations (r = 0.31, P = .16), iCa concentrations adjusted for serum PTH concentrations (partial r = 0.29, P = .2), CRP concentrations (r _s  = 0.03, P = .89), or Spec cPL concentrations (r _s  = −0.15, P = .5). No correlations were observed between serum VDBP and iCa concentrations (r = −0.24, P = .28), iCa concentrations adjusted for serum PTH (partial r = −0.22, P = .33), CRP (r _s  = −0.21, P = .34), and Spec cPL concentrations (r _s  = −0.09, P = .68; Table 2). No correlations were found between serum VDBP concentrations and 25(OH)D (r = −0.01, P = .96) or serum albumin concentrations (r = 0.02, P = .93).

TABLE 2.

Correlation between serum 25(OH)D, VDR, and VDBP concentrations with the iCa levels, CRP, and Spec cPL concentrations in dogs with AP.

	Correlation variables
	iCa		iCa adjusted for PTH concentrations		CRP		Spec cPL
	r (95% CI)	P	Partial r (95% CI)	P	r s (95% CI)	P	r s (95% CI)	P
25(OH)D	0.31 (−0.13 to −0.65)	.16	0.29 (−0.24 to 0.7)	.2	0.03 (−0.41 to 0.46)	.89	−0.15 (−0.55 to 0.30)	.5
VDR	0.15 (−0.29 to 0.54)	.5	0.04 (−0.38 to 0.46)	.85	−0.55 (−0.79 to −0.15)	.01	−0.47 (−0.75 to −0.05)	.03
VDBP	−0.24 (−0.60 to 0.20)	.28	−0.22 (−0.61 to 0.3)	.33	−0.21 (−0.59 to 0.24)	.34	−0.09 (−0.50 to 0.35)	.68

Note: Table 2 shows the coefficients and P‐value for the Pearson correlation test (iCa), partial correlation analysis (iCa adjusted for PTH levels), and Spearman's rank correlation analysis (CRP and Spec cPL).

Abbreviations: AP, acute pancreatitis; CI, confidence interval; CRP, C‐reactive protein; iCa, ionized calcium; PTH, parathyroid hormone; serum 25(OH)D, 25‐hydroxyvitamin D; Spec cPL, specific canine pancreatic lipase; VDBP, vitamin D binding protein; VDR, vitamin D receptor.

3.5. Comparison between before and after treatment concentrations of serum 25(OH)D, VDR, and VDBP in dogs with AP

Samples were obtained a median of 31 days (IQR, 20‐45 days) after discharge in 11 dogs. No significant differences in serum 25(OH)D (P = .24; Figure 3A), VDR (P = .57; Figure 3B), or VDBP (P = .07; Figure 3C) concentrations were found before and after treatment.

FIGURE 3.

Comparison of concentrations of circulating (A) 25(OH)D, (B) VDR, and (C) VDBP before and after treatment in dogs with AP (n = 11). There was no significant difference in the concentrations of circulating 25(OH)D (P = .24), VDR (P = .57), and VDBP (P = .07) before and after treatment. Paired t‐tests. 25(OH)D, 25‐hydroxyvitamin D; AP, acute pancreatitis; VDBP, vitamin D binding protein; VDR, vitamin D receptor.

4. DISCUSSION

We investigated serum 25(OH)D, VDR, and VDBP concentrations in dogs with AP. In our study, dogs with AP had significantly lower serum 25(OH)D and VDR concentrations than did healthy dogs. The VDR is expressed most in the duodenum, ileum, skin, and kidney of dogs, ²³ and also is expressed in the exocrine and endocrine pancreas, ²⁴ and obtaining these tissue samples from dogs with AP is unnecessarily invasive. Therefore, we evaluated VDR expression in serum. The results showed that lower VDR concentrations were associated with higher CRP and Spec cPL concentrations, which suggests that the VDR plays a role in regulating inflammatory processes in dogs with AP.

A previous study documented vitamin D imbalances in dogs with AP, ⁶ and our study showed similar results. We found that serum 25(OH)D concentrations were significantly lower in dogs with AP than in healthy dogs and that the prevalence of vitamin D deficiency was higher in nonsurvivors than in survivors. Whether vitamin D imbalance causes or arises from AP remains unknown. In humans with pancreatitis, decreased intestinal uptake of vitamin D associated with anorexia has been proposed as the cause of vitamin D deficiency. ²⁵ Dogs cannot synthesize vitamin D in the skin, and thus depend on their diet to obtain adequate amounts of vitamin D. ²⁶ Therefore, anorexia or gastrointestinal disorders can induce vitamin D deficiency in dogs. ²⁷ However, given that all dogs with AP in our study showed clinical signs within 3 days before presentation and serum 25(OH)D has a long half‐life of approximately 15 days, ²⁸ it is reasonable to expect that vitamin D deficiency would be the cause of AP rather than the result of anorexia in dogs with AP. Furthermore, serum 25(OH)D concentrations did not increase after resolution of clinical signs, which supports our assumption.

It has been argued that VDBP concentrations have limited impact on the biological activity of vitamin D status because VDBP concentrations do not affect the pool of biologically active free hormones. ²⁹ However, VDBP plays an important role in preventing rapid vitamin D deficiency by creating a large pool of circulating 25(OH)D. ³⁰ Decreased VDBP concentrations in critically ill human patients with sepsis have been reported, further exacerbating vitamin D insufficiency. ¹⁷ We examined VDBP concentrations to determine whether they affect the outcome and vitamin D status in dogs with AP. Serum VDBP concentrations did not affect clinical outcomes or 25(OH)D concentrations, suggesting little clinical relevance of serum VDBP concentrations and AP in dogs.

It has been reported that VDR gene polymorphisms are more frequent in human patients with AP than in controls. ³¹ Our study showed that serum VDR concentrations are significantly lower in dogs with AP than in healthy dogs and significantly different between nonsurvivors and survivors. Previous reports and our findings suggest that VDR expression may impact the development of AP in dogs. Treatment targeting VDR using calcipotriol, a vitamin D analog that controls VDR induction, decreased fibrosis and inflammation in acute and chronic pancreatitis mouse models. ³² Our study suggests that VDR could be a potential therapeutic target in dogs with AP.

In dogs with AP, lower VDR concentrations were associated with higher CRP and Spec cPL concentrations, suggesting that the VDR plays an important role in the inflammatory process of AP in dogs. Deficiency of VDR promotes inflammation by mediating nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) in mice. ³³ It is expressed in several inflammatory cells, including monocytes, neutrophils, and lymphocytes. ³⁴ It also is activated when damaged cells release intracellular contents and trigger the inflammatory response by activating caspase‐1. ³⁵ In mice and humans with AP, a link between NLRP3 activation and inflammation was demonstrated. ³⁶ , ³⁷ Our results suggest that VDR deficiency could promote inflammation in dogs with AP and additional studies to identify the link between VDR deficiency and NLRP3 activation would contribute to understanding the pathophysiology of the inflammatory process in dogs with AP.

In our study, dogs with AP showed a correlation between serum VDR and Spec cPL concentrations, a serum biomarker reflecting pancreatic acinar cell damage. ³⁸ It is unclear whether the decreased serum VDR concentrations made pancreatic tissue more vulnerable to damage or whether more pancreatic damage leads to lower serum VDR concentrations. Decreased VDR expression in the colon has been reported in human patients with ulcerative colitis, ³⁹ , ⁴⁰ supporting that inflammation disrupts VDR expression in the affected tissues. During AP, injured pancreatic acinar cells might express less VDR than normal pancreatic cells, ²⁴ which may have contributed to the decreased serum VDR concentration. Additional studies comparing VDR expression in normal and inflamed pancreatic tissue would be beneficial to identify the tissues that contribute to decreased serum VDR concentrations.

Blood calcium concentration is tightly controlled by a complex process, which includes hormonal control of calcium fluxes by PTH, calcitonin, and vitamin D. ⁴¹ One of the major hormones regulating calcium is vitamin D, which stimulates intestinal absorption and renal tubular reabsorption of calcium by activating VDR. ¹³ , ⁴¹ , ⁴² A previous study showed that blood iCa concentrations were correlated with 25(OH)D concentrations in dogs with AP, suggesting vitamin D imbalance as a mechanism for hypocalcemia. ⁶ In our study, dogs with AP had lower blood iCa concentrations than normal dogs, but no correlation of blood iCa concentrations with serum 25(OH)D and VDR concentrations was observed, even after adjusting for serum PTH concentrations. Therefore, the decreased blood iCa concentrations in dogs with AP cannot be explained by vitamin D imbalance or decreased VDR expression alone, and various factors may have been involved. The proposed mechanisms of hypocalcemia in AP include the formation of calcium salts caused by auto‐digestion of mesenteric fat by leaking pancreatic enzymes, hypomagnesemia, and transient hypoparathyroidism. ⁴³ , ⁴⁴ , ⁴⁵ These factors, except for serum PTH concentrations, were not evaluated in our study and might have affected the results.

Our study had some limitations. First, the sample size of each group was small, which may have led to false‐negative results (type II statistical error). The sample size of the nonsurvivors was especially small (only 5 dogs), and thus the finding for the comparison of serum 25(OH)D concentrations between survivors and non‐survivors could be a false‐negative result. Second, dogs with AP were older than the healthy dogs. Previous studies have shown that age is not associated with serum 25(OH)D concentrations in dogs ⁴⁶ nor with serum VDBP concentration in humans. ⁴⁷ In our study, age was not correlated with serum 25(OH)D and VDBP concentrations in healthy dogs (data not shown), and it is unlikely that the results were affected by age. Although the influence of age on VDR expression in the parathyroid glands of humans ⁴⁸ and mammary glands in female dogs has been reported, ⁴⁹ age and serum VDR concentrations in healthy dogs were not correlated in our study (data not shown). Third, we could not evaluate some factors that regulate calcium homeostasis, such as calcitonin and magnesium. Therefore, additional studies are needed to fully understand the association between serum VDR and VDBP concentrations and blood calcium concentrations in dogs with AP. Finally, although all dogs enrolled in our study consumed commercial diets, each dog was fed a wide variety of diets, and information about the dietary intake of vitamin D was limited. Additional studies with large sample sizes and dogs for which information on dietary vitamin D intake is available will be needed to better understand the vitamin D status of dogs with AP.

In conclusion, we investigated serum 25(OH)D, VDR, and VDBP concentrations in dogs with AP. We found that dogs with AP had significantly lower serum VDR concentrations than did healthy dogs. Additionally, serum VDR concentrations correlated with CRP and Spec cPL concentrations, suggesting a potential role of VDR expression in the inflammatory process of AP in dogs.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF‐LABEL ANTIMICROBIAL DECLARATION

Authors declare no off‐label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

This study was approved by the Chungbuk National University Animal Care Committee and carried out according to the Guide for Care and Use of Animals (Chungbuk National University Animal Care Committee, CBNUA‐1710‐22‐01).

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.

Lee D, Koo Y, Chae Y, et al. Serum 25‐hydroxyvitamin D, vitamin D receptor, and vitamin D binding protein concentrations in dogs with acute pancreatitis compared to healthy control dogs. J Vet Intern Med. 2023;37(5):1694‐1702. doi: 10.1111/jvim.16809