Effects of surgery on free and total 25 hydroxyvitamin D concentrations in dogs

Abstract Background It is unclear whether a low total 25(OH)D concentration is a cause or consequence of illnesses. To address this knowledge gap, studies measuring free and total 25(OH)D during the evolution and resolution of an inflammatory process are required. Objectives Serum total and free 25(OH)D concentrations would transiently decline after cruciate surgery in dogs. Animals Seventeen client‐owned dogs with a spontaneous cranial cruciate ligament rupture (CCLR). Methods A longitudinal cohort study involving the measurement of serum concentrations of total and free 25(OH)D, total calcium, creatinine, albumin, phosphate, C‐reactive protein and plasma ionized calcium, at 1 day before and a median time of 1 and 60 days after surgical treatment of CCLR. Results Median serum concentrations of total 25(OH)D before surgery (80.3 nmoL/L [range, 43.5‐137.3]) significantly declined immediately after surgery; (64.8 nmoL/L [range, 36.3‐116.5] 1 day after surgery, P < .005) before increasing to become nonsignificantly different from concentrations before surgery at day 60 after surgery (median 78.0 nmoL/L [range, 24.2‐115.8], P = .14). In contrast, median free 25(OH)D concentrations before surgery (7.6 pg/mL [range, 3.8‐12.2]) significantly increased immediately after surgery (9.2 pg/mL [range, 5.2‐15.7], P < .05) before declining to become nonsignificantly different from before surgery concentrations at day 60 after surgery (median 6.2 pg/mL [range, 4.0‐15.8], P = .37). Conclusion and Clinical Importance This study reveals the difficulties of assessing vitamin D status in dogs following elective surgery.


| INTRODUCTION
Vitamin D has a well-established role in the development and maintenance of skeletal health. 1,2 Over the past decade, interest in the nonskeletal effects of vitamin D in dogs has increased, partly due to a growing recognition that many nonskeletal tissues express the vitamin D receptor (VDR). 3  during an inflammatory process since dogs with a CCLR are typically clinically stable before surgery and frequently develop systemic inflammation shortly after surgery, which subsides in dogs that recover from the procedure. 16 Therefore, the objective of this study was to longitudinally track total and free 25(OH)D and C-reactive protein (CRP) concentrations, together with ionized calcium, total calcium, phosphate, albumin, and creatinine concentrations, in clinically stable dogs undergoing elective surgical treatment of their CCLR.

| METHODS
All dogs diagnosed with a CCLR at the Hospital for Small Animals, University of Edinburgh, between June 2015 and December 2017 were considered eligible for inclusion in the study. Entry criteria included a confirmed diagnosis of CCLR and owner consent to take a blood sample the day before surgery, 1 day after the surgical treatment of the CCLR, and at the 8 week after surgery re-examination.
The surgical treatment method undertaken was decided upon by the attending clinician and was either a tibial plateau leveling osteotomy (TPLO) or cranial closing wedge osteotomy (CCWO).
Blood samples collected at each timepoint were placed into plain, ethylenediaminetetraacetic acid (EDTA), and lithium heparin blood collection tubes, which were refrigerated after collection. Albumin, total calcium, ionized calcium, creatinine, and inorganic phosphate were all measured within 3 hours of collection. Serum and EDTA plasma were separated by centrifugation within 3 hours of collection and aliquoted for subsequent batch analysis of total and free 25(OH) D and parathyroid hormone (PTH), respectively, and stored frozen at −80 C until analysis.
Albumin, total calcium, creatinine, inorganic phosphate, and CRP concentrations were measured using an Au480 Chemistry Analyser (Beckman Coulter [UK] Ltd, High Wycombe, UK). The following assays were used for the measurement of the abovementioned analytes: bromocresol green for albumin, Arzenazo for total calcium, modified Jaffe for creatinine, phosphomolybdate complex for inorganic phosphate, and a species-specific immunoturbidimetric assay for CRP. Ion- For each metabolite, the 2 after surgery blood sample results were compared to the before surgery blood sample by a Wilcoxon matched pairs signed-rank test. Significance was set at P < .05.

The study was approved by the University of Edinburgh Animal
Welfare and Ethics Review Board.

| RESULTS
Seventeen dogs were enrolled in the study. The median age of the dogs was 6.7 years and ranged from 2.1 years to 11.6 years. There   However, this study longitudinally tracked 25(OH)D concentrations following significant hemodilution during cardiopulmonary bypass procedures and so did not mimic the clinical situation of isotonic fluid therapy associated with elective surgery in our study. The decline in total, but not ionized, calcium could reflect the decline in albumin concentration, which reduced the protein-bound calcium component. 28,29 Although the relationship between 25(OH)D and nonskeletal dis- Another limitation was that although no dogs had evidence of systemic disease other than a CCLR based on history and physical examination, the lack of extensive before surgery diagnostic investigations means that the presence of other concurrent, subclinical diseases cannot be definitively excluded.

ACKNOWLEDGMENTS
No funding was received for this study.