Dietary 25-hydroxyvitamin D 3 effects on vitamin D status,plasma metabolites and urine mineral excretion in adult cats

Abstract

Objectives

The study aimed to evaluate the safety and effectiveness of vitamin D supplementation with dietary 25-hydroxyvitamin D₃ (25[OH]D₃) in adult cats.

Methods

Three levels of dietary 25(OH)D₃ concentrations (4.9, 8.4, 11.8 µg/kg as fed) were received by five adult cats for 9 weeks, each in a randomized complete block design. Effects were determined on plasma or serum concentrations of 25(OH)D₃, 24,25-dihydroxyvitamin D₃, calcitriol, parathyroid hormone, ionized calcium, urinary excretions of phosphorus, calcium and magnesium, and clinical hematology and chemistry panels.

Results

The lowest concentration of dietary 25(OH)D₃ supported elevation of vitamin D status, with no adverse effects. Supplementation of 8.4 µg/kg 25(OH)D₃ had significant effects on the urinary magnesium: creatinine ratio. Increasing supplementation up to 11.8 µg/kg 25(OH)D₃ had significant effects on plasma concentrations of calcium and magnesium, and vitamin D metabolites.

Conclusions and relevance

Dietary supplementation with approximately 5.0 µg/kg of 25(OH)D₃ or the ingested equivalence of 0.09 µg of 25(OH)D₃ per metabolic body weight (kg^0.67) is a safe, potent and effective means for raising vitamin D status in cats. A higher dose with approximately 11.8 µg/kg of 25(OH)D₃ resulted in elevation in C-3 epimers of 25(OH)D₃ and slight elevation in plasma magnesium and calcium concentrations above their respective reference intervals.

Keywords

25-Hydroxyvitamin D 24,25-dihydroxyvitamin D C-3 epimer urine calcium phosphorus magnesium

Introduction

Continued research on vitamin D has developed our understanding of the vitamin beyond its role in mineral metabolism and skeletal health. Vitamin D receptors and 1-alpha-hydroxylase (CYP27B1), the enzyme that hydroxylates the main circulating form of vitamin D, calcidiol (25-hydroxyvitamin D), to the biologically most active form, calcitriol (1α,25-dihydroxyvitamin D), are now recognized to be present in multiple extrarenal tissues.^1,2 Low serum concentrations of calcidiol, a conventional indicator of vitamin D status, have been associated with immunologic, neurologic, gastrointestinal and cardiovascular disease in humans.¹ Cats with inflammatory bowel disease, intestinal small cell lymphoma, cholestatic liver disease, feline immunodeficiency virus and infections have been found to have low levels of serum vitamin D concentrations, as measured by serum calcidiol.^3–5 In a study looking at vitamin D status in cats with cardiomyopathy, researchers found that age played an important negative relationship with vitamin D status.⁶

Calcidiol, in the form of 25-hydroxycholecalciferol (25[OH]D₃), has been shown to be more potent and effective than cholecalciferol (D₃) in raising vitamin D status in humans when ingested.⁷ Oral 25(OH)D₃ is absorbed faster than D₃, and, like D₃, it has a high affinity to the vitamin D-binding protein (VDBP).⁷ Compared with D₃, 25(OH)D₃ is bound to VDBP immediately after intestinal absorption, rapidly making it part of the circulating 25(OH)D₃ pool. The impact of ingested D₃ on the circulating pool of 25(OH)D₃ is blunted and delayed by liver uptake and hydroxylation of D₃.

Ingested calcidiol is a suggested effective means of raising vitamin D status in cats with enteropathies and hepatopathies.⁸ However, vitamin D activity, as may be imparted by ingested calcidiol binding to intestinal epithelial vitamin D receptors, hypothetically may stimulate excessive intestinal phosphorus and calcium absorption and, in turn, stimulate undesired urinary excretion of phosphorus, calcium and magnesium.^9–12 We hypothesized that dietary supplementation with the vitamin D₃ form of calcidiol, 25(OH)D₃, may cause clinically significant increases in urinary mineral excretion by cats. This report describes the testing of this hypothesis in adult cats given a custom-formulated, dry-type diet supplemented with differing concentrations of 25(OH)D₃.

Materials and methods

Animals

All cats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care and Use Committee of the University of Missouri (protocol #9214).¹³ Five university-owned, purpose-bred, castrated male domestic shorthair cats aged 9–13 years were studied. The cats were determined to be healthy based on physical examination by a veterinarian and pretrial hematology and chemistry analysis. The cats weighed between 4.3 and 5.1 kg and had body condition scores between 5/9 and 6/9.¹⁴ The cats were group-housed except for daily periods of single housing for monitoring of food intake in an American Association for Laboratory Animal Science accredited, humidity, temperature and light cycle-controlled facility. Food was presented once daily; water was continuously available.

Diet

An extruded, dry-expanded custom diet (see Table 1 in the supplementary material) was used for this trial. The diet met with the Association of American Feed Control Officials cat food nutrient profiles for adult maintenance.¹⁵ Three batches of this diet were used, with each batch varying in only vitamin D₃ and 25(OH)D₃ content (Table 1). The supplemented source of 25(OH)D₃ in the diet batches was HyD, a product of DSM Nutritional Products. These diet batches were variably combined to create three targeted, as fed, dietary 25(OH)D₃ concentrations: 5.3, 10.7 or 16.0 µg/kg. Concentrations of D₃ in all batches were assessed by a commercial laboratory at the trial start (Eurofins Scientific). Concentrations of 25(OH)D₃ in all batches were determined in our laboratory using procedures described by Jakobsen et al,¹⁶ with minor modifications of the high performance liquid chromatography and liquid chromatography tandem mass spectrometry methods.¹⁷ Occurrence of the alpha (α)- and beta (β)-epimers of 25(OH)D₃ were not measured in the diets, but the total of 25(OH)D₃ in the diets before 25(OH)D₃ supplementation was low (~1.4 µg/kg). Proximate analysis and select mineral analyses were determined by the University of Missouri Agricultural Experiment Station Chemical Laboratories.

Table 1

Targeted and measured vitamin D₃ and 25-hydroxyvitamin D3 (25[OH]D₃) as fed concentrations of supplemented and unsupplemented diet and trial diet mixtures with low, medium and high 25(OH)D₃ supplementation

Diets	Moisture (%)	Vitamin D₃ (µg/kg)		25(OH)D₃ (µg/kg)
Diets	Moisture (%)	Targeted	Measured	Targeted	Measured
Pretrial (vitamin D₃ supplemented)	5.8	81	90.5	0	1.3
A (unsupplemented)	6.7	0	9.1	0	1.5
B (25[OH]D₃ supplemented)	5.5	0	10.4	16.0	11.8
Trial diet mixtures*
Low 25(OH)D₃ supplementation (2 parts diet A + 1 part diet B)	–	0	9.5	5.3	4.9
Medium 25(OH)D₃ supplementation (1 part diet A + 2 parts diet B)	–	0	10.0	10.7	8.4
High 25(OH)D₃ supplementation (diet B)	–	0	10.4	16.0	11.8

Concentrations of vitamin D₃ and 25(OH)D₃ estimated from the measured values for diets A and B

Design

Three periods of feeding were conducted using the five cats in a randomized complete block design. All cats were maintained on the pretrial diet for 13 weeks. During each period, the cats were given one of the three 25(OH)D₃ supplementation diet options for 9 weeks. There were no intervening periods of wash out, and an accumulation effect was not expected, as supported by an expected time of equilibration of 25(OH)D₃ in plasma of 4 weeks following the change in ingested dosage of 25(OH)D₃ (R Backus, unpublished data).

In the mornings, following overnight food withholding at the trial start and at the end of each 9-week period, body weight (BW) measurements were recorded. In addition, jugular venous blood (3–5 ml) was collected for clinical hematology and chemistry analyses and vitamin D metabolite, parathyroid hormone (PTH) and ionized calcium (iCa) concentration determinations.

At the start and at the end of each 9-week period, voided urine was collected for each of five consecutive days to quantify urine phosphorus, calcium and magnesium excretions. For this, the cats were individually housed and urine was collected in litter boxes using non-absorbent plastic bead litter.

Laboratory determinations

Serum concentrations of calcitriol, 1,25(OH)₂D₃, were determined with a commercially available radioimmunoassay kit (AA–54; Immunodiagnostic Systems). Plasma concentrations of 25(OH)D₃ and 24,25-dihydroxyvitamin D₃ (24,25[OH]₂D₃) and their C-3 α-epimers were determined by chromatographic methods.⁸ Blood and plasma aliquots were submitted to the University of Missouri Veterinary Medical Diagnostic Laboratory (VMDL) for clinical hematology and chemistry analyses. Serum was submitted for iCa and PTH determinations (Michigan State University Veterinary Diagnostic Laboratory, Lansing).

The concentrations of urine calcium, phosphorus, magnesium and creatinine were determined by the VMDL. Daily excreted amounts of each mineral were determined by multiplying urine mineral concentration by volume of collected urine.

Statistical methods

Statistical analyses were performed using statistical software (SAS Version 9.4; SAS Institute). Observations were accepted as normal if both skew and kurtosis statistics were >−1.0 and <1.0. Repeated-measures, mixed-model ANOVA was used to determine the significance of the effect of diet on normal observations. Fixed and random effects were D vitamer supplementation (type or level) and individual subject, respectively. Tukey adjustments were used in multiple comparisons to identify significant between-diet differences. For non-normal observations, the significance of the effect of diet was determined using Friedman analyses, and sign-rank tests were used to identify significance for between-diet multiple comparisons. For normal and non-normal observations, central tendencies were expressed as mean and median, respectively. Variances were expressed as minimum and maximum values. Differences were considered significant if P <0.05, except for when sign-rank tests were used in Friedman test post-hoc analyses. In these cases, P ⩽0.0625 indicated significance.

Results

Animals

During the pretrial urine-collection period, struvite urolithiasis excluded 1/6 cats from the study. During the first of the three trial blocks, hyperthyroidism was identified in 1/5 remaining cats. This cat restarted the study 6 months later, following curative radioactive iodine therapy. In another cat, plasma concentrations of urea nitrogen (36 mg/dl) and creatinine (2.2 mg/dl) were in slight excess of reference interval (RI) upper limits (35 mg/dl and 2.0 mg/dl, respectively). The plasma values indicated the cat had International Renal Interest Society stage 2 chronic kidney disease at the trial start.¹⁸ Plasma concentrations of urea nitrogen and creatinine in the cat did not substantially change after consumption of any of the diets supplemented with 25(OH)D₃.

BW was stable for four cats throughout the trial. One cat gained 2% BW per week following treatment for hyperthyroidism. Therefore, an 18% caloric reduction was applied and BW stabilized. Mean daily intake in metabolizable energy among the cats was 213 kcal (range 195–332) or 891 kJ (range 815–1389), which on a metabolic BW basis (kg^0.67) was 72 kcal/kg^0.67 (range 63–110), a value less than a reported energy requirement of 100 kcal/kg^0.67 for lean cats.¹⁹

Diet analyses

While proximate and mineral contents of the trial diets were similar, measured concentrations of D₃ and 25(OH)D₃ in the diet batches slightly to moderately differed from the targeted values (Table 1). Moving forward, the measured 25(OH)D₃ concentrations (4.9, 8.4 and 11.8 µg/kg) will be referred to rather than the original target values (5.3, 10.7 and 16.0 µg/kg).

Vitamin D metabolites

At the end of the pretrial period, concentrations of the most abundant C-3 epimer of 25(OH)D₃, the β-epimer, and the concentrations of the α- and β-epimers together (Table 2) were within the laboratory RI (26–68 ng/ml).²⁰ In considering the measured D₃ concentration of the pretrial diet, the range of daily consumption of D₃ in the diet among the cats was 1.6–2.7 µg/kg^0.67. This amount was about 10 times greater than the recommended allowance for feline maintenance (0.17 µg/kg^0.67).¹⁹ The range of 25(OH)D₃ consumed per day when the diet containing the lowest 25(OH)D₃ supplementation (4.9 µg/kg) was only 0.08–0.10 µg/kg^0.67. This intake supported plasma concentrations of 25(OH)D₃ β-epimer and total of α- and β-epimers that were similar to those when the pretrial diet was given.

Plasma 25(OH)D₃ concentrations were increased (P <0.05) with dietary supplementations of 8.4 and 11.8 µg/kg of 25(OH)D₃ (Table 2), which resulted 1/5 cats having a β-epimer concentration of 25(OH)D₃ in excess of the aforementioned upper limit of the RI (>68 ng/ml).²⁰ The increased 25(OH)D₃ supplementations only slightly affected the α-epimer 25(OH)D₃ concentrations.

For 24,25(OH)₂D₃, plasma concentration of the β-epimer of 24,25(OH)₂D₃ significantly increased (P <0.05) with consumption of 25(OH)D₃ (Table 2). However, the α-epimer of 24,25(OH)₂D₃ remained low in concentration (<1.0 ng/ml) and did not significantly change with the increased supplementation. The 1,25(OH)₂D₃ concentrations did not significantly differ with D vitamer supplementation type or amounts.

Table 2

Plasma concentrations of vitamin D metabolites for castrated male adult cats (n = 5) during dietary supplementations with vitamin D₃ and 25-hydroxyvitamin D3 (25[OH]D₃)

25(OH)D₃ concentration (µg/kg)	25(OH)D₃ (ng/ml)			24,25(OH)₂D₃ (ng/ml)		1,25(OH)₂D₃ (pg/ml)
25(OH)D₃ concentration (µg/kg)	β-epimer*	α-epimer*	Total (β + α)*	β-epimer^†	α-epimer	1,25(OH)₂D₃ (pg/ml)
0 (pretrial)	32 ± 3^a (26–43)	11 ± 2^a (8–16)	44 ± 4^a (34–58)	12 ± 2^a (5–18)	0.7 ± 0.3 (0.0–1.5)	136 ± 26(63–197)
4.9	39 ± 3^a (33–50)	7 ± 2^b (2–13)	47 ± 5^a (36–29)	19 ± 4^b (9–28)	1.4 ± 0.7 (0.0–3.6)	173 ± 30 (82–262)
8.4	51 ± 6^b (42–73)	9 ± 2^a (6–14)	60 ± 7^b (49–87)	21 ± 4^b (10–31)	1.4 ± 0.4 (0.0–2.4)	168 ± 34 (119–296)
11.8	55 ± 6^b (42–74)	10 ± 1^a (7–13)	65 ± 7^b (49–86)	23 ± 4^b (11–34)	1.2 ± 0.4 (0.0–2.7)	193 ± 49 (95–361)
Reported adult healthy cat observations	26–68 ng/ml^‡ 30–61 ng/ml 15–61 ng/ml 75 ± 35 ng/ml 81 ± 11 ng/ml 35 ± 13 ng/ml 72 ± 27 ng/ml 30–61 ng/ml (α-epimer, 3–30 ng/ml) 23–83 ng/ml 46–60 ng/ml (α-epimer, 9–12 ng/ml)			26 ± 13 ng/ml^§ 10–14 ng/ml		20–40 pg/ml^¶ 740 ± 350 pg/ml 24–78 pg/ml 80–132 pg/ml 37–142 pg/ml 10–16 pg/ml
P value	<0.001	0.001	<0.001	0.001	0.362	0.277

Data are mean ± SEM (range)

Where letter annotations are not the same, differences between supplementation amounts were significant (P <0.05 or P ⩽0.0625 when Friedman analysis was used)

†

Non-normal observations are expressed as median (range); all other observations are expressed as mean (range)

‡

Schenck et al,²⁰ Lalor et al,³ Titmarsh et al,⁵ Girard et al,²¹ Pineda et al,²² Reiter et al,²³ Paßlack et al,²⁴ Lalor et al,²⁵ Alexander et al²⁶ and Ware et al⁶

Pineda et al²² and Alexander et al²⁶

Schenck et al,²⁰ Pineda et al,²² Hostutler et al,²⁷ Crossley et al,²⁸ Van den Broek et al²⁹ and Alexander et al²⁶

24,25(OH)2D3 = 24,25-dihydroxyvitamin D3; 1,25(OH)2D3 = calcitriol

Clinical hematology and chemistry results

A significant effect of 25(OH)D₃ supplementation was found for two of the chemistry variables – plasma concentrations of calcium and magnesium (Table 3). The calcium and magnesium concentrations were modestly increased when the highest amount of dietary 25(OH)D₃ concentration was consumed. Other clinical chemistry observations (Table 3) and the hematology findings (see Table 2 in the supplementary material) were not significantly changed by supplementation amount. There was no significant effect (P = 0.1) of D vitamer supplementation type or level on PTH concentrations (Table 3). There was a slight but significant effect of supplementation type on serum iCa levels. The iCa concentrations pretrial, when D₃ supplementation was used, were less than the iCa concentrations when 25(OH)D₃ supplementation was used (P = 0.03).

Table 3

Plasma concentrations of clinical chemistry analytes, parathyroid hormone (PTH) and ionized calcium (iCa) for five castrated male adult cats after feeding a diet supplemented with vitamin D₃ (81 µg/kg) for 13 weeks and diet supplemented with 25-hydroxyvitamin D3 (25[OH]D₃) for 9 weeks at measured concentrations of either 4.9, 8.4 or 11.8 µg/kg

Analyte	D₃ (µg/kg)	25(OH)D₃ (µg/kg)			P value	RI
Analyte	81.0	4.9	8.4	11.8	P value	RI
Glucose (mg/dl)	77 (75–101)	80 (76–106)	86 (71–111)	79 (77–130)	0.593	80–155
Urea nitrogen (mg/dl)	28 (23–36)	30 (22–37)	27 (23–27)	28 (21–45)	0.887	19–35
Creatinine (mg/dl)	1.7 (0.8–2.2)	1.8 (1.5–2.3)	1.6 (1.5–2.2)	1.6 (1.6–2.8)	0.405	0.9–2.0
Sodium (mEq/l)	153 (152–153)	151 (150–154)	153 (148–154)	153 (150–182)	0.432	148–154
Potassium (mEq/l)	4.0 (3.7–4.2)	4.0 (3.6–4.5)	4.1 (3.8–4.3)	4.0 (3.7–5.4)	0.182	3.1–4.6
Chloride (mEq/l)	119 (118–121)	118 (117–120)	119 (115–120)	118 (118–142)	0.392	112–123
Bicarbonate (mEq/l)*	16 (13–18)	16 (14–18)	16 (13–17)	16 (15–18)	0.807	13–23
Anion gap (mEq/l)*	22 (18–25)	22 (17–27)	22 (20–25)	23 (19–27)	0.832	13–26
Total protein (g/dl)	6.8 (6.4–7.5)	7.0 (6.5–7.1)	6.9 (6.6–7.3)	7.0 (6.6–8.7)	0.271	6.1–8.1
Albumin (g/dl)*	3.2 (3.1–3.3)	3.2 (2.9–3.4)	3.2 (2.9–3.4)	3.3 (3.1–3.7)	0.342	2.8–3.7
Globulin (g/dl)	3.7 (3.1–4.2)	3.7 (3.6–3.9)	3.7 (3.5–4.0)	3.8 (3.5–5.0)	0.744	2.5–5.2
Calcium^† (mg/dl)	9.7 (9.1–10.3)^a	9.8 (9.0–10.5)^a	9.7 (9.0–10.5)^a	10.1 (9.2–11.8)^b	0.018	8.6–10.4
Phosphorus (mg/dl)	4.2 (3.6–4.4)	4.3 (3.6–4.4)	4.2 (3.7–4.9)	4.1 (3.3–5.3)	0.884	2.3–5.0
Magnesium^† (mg/dl)	2.1 (2.0–2.2)^a	2.2 (2.1–2.2)^a	2.2 (2.1–2.3)^a	2.3 (2.1–2.6)^b	0.043	1.7–2.6
Cholesterol (mg/dl)*	212 (176–252)	209 (156–260)	205 (163–253)	229 (158–311)	0.392	80–258
Total bilirubin (mg/dl)	0.1 (0.1–0.1)	0.1 (0.1–0.1)	0.1 (0.1–0.1)	0.1 (0.1–0.2)	0.392	0.1–0.2
Enzyme activity (U/l)
ALT*	66 (43–108)	53 (5–97)	62 (44–86)	72 (46–106)	0.108	28–84
ALP	23 (19–41)	21 (18–33)	21 (17–31)	23 (21–34)	0.108	13–50
GGT	<3	<3	< 3	<3	–	0–3
CK	135 (65–434)	103 (65–188)	179 (80–190)	229 (88–261)	0.323	65–574
PTH (pmol/l)	0.27 (0.00–0.50)	0.92 (0.00–1.60)	1.00 (0.40–2.20)	0.98 (0.40–2.20)	0.102	0.40–2.50
iCa*^† (mmol/l)	1.10 (1.09–1.11)^a	1.20 (1.10–1.33)^b	1.19 (1.13–1.32)^b	1.20 (1.12–1.40)^b	0.030	1.00–1.40

Data are mean (range) unless otherwise indicated

Normal observations are expressed as mean (range); all other observations are expressed as median (range)

†

Where letter annotations are not the same, differences between supplementation amount were significant (P <0.05 or P ⩽0.0625 when Friedman analysis was used)

RI = reference interval; ALT = alanine transaminase; ALP = alkaline phosphatase; GGT = gamma-glutamyl transferase; CK = creatinine kinase; PTH = parathyroid hormone; iCa = ionized calcium

Urine mineral excretions

Several methods of expressing urine mineral excretion were evaluated (Table 4). There was no significant difference of effect between the pretrial D₃ and the low (4.9 µg/kg) 25(OH)D₃ supplementations on urine calcium, magnesium and phosphorus excretions. When supplemented with 8.4 µg/kg of 25(OH)D₃, the urine magnesium:creatinine ratio was slightly increased above that of the pretrial when D₃ was the D vitamer supplementation source. However, supplementing with a greater concentration of 25(OH)D₃ (11.8 µg/kg) vs pretrial D₃ supplementation did not significantly increase the urine magnesium:creatinine ratio. The D vitamer supplementation type or amount did not significantly affect urine ratios of calcium:creatinine or phosphorus:creatinine, nor did it significantly affect any measure of urinary excretion of the minerals.

Table 4

Effect of 25-hydroxyvitamin D3 (25[OH]D₃) supplementation on urine mineral excretions in five castrated male adult cats

25(OH)D₃ concentration (µg/kg)	Calcium			Phosphorus			Magnesium
25(OH)D₃ concentration (µg/kg)	Ca:Cr	mg/kg BW/day*	Excreted (mg)/consumed (mg) × 100^†	P:Cr	mg/kg BW/day	Excreted (mg)/consumed (mg) × 100	Mg:Cr^†	mg/kg BW/day	Excreted (mg)/ consumed (mg) × 100*
0 (pretrial)	–	–	–	0.95 (0.74–1.51)	29.8 (21.7–38.9)	26.1 (20.5–29.0)	0.02 (0.01–0.04)^a	0.63 (0.18–1.45)	4.23 (1.03–10.2)
4.9	0.039 (0.018–0.078)	0.80 (0.42–1.48)	0.69 (0.34–1.49)	0.99 (0.81–1.13)	27.0 (22.4–38.1)	24.1 (20.1–33.5)	0.03 (0.01–0.05)^a	0.80 (0.19–1.54)	7.43 (0.87–13.6)
8.4	0.035 (0.013–0.057)	1.0 (0.42–1.52)	0.85 (0.37–1.32)	0.99 (0.78–1.02)	28.1 (18.5–34.8)	24.0 (15.8–30.4)	0.03 (0.01–0.06)^b	0.88 (0.19–2.08)	4.96 (1.63–15.7)
11.8	0.033 (0.015–0.058)	0.71 (0.43–1.48)	0.60 (0.43–1.28)	0.91 (0.79–1.00)	31.3 (24.6–41.0)	28.3 (22.7–35.2)	0.02 (0.02–0.05)^a	0.77 (0.15–1.57)	3.24 (1.31–15.0)
P value	0.522	0.247	0.247	0.534	0.683	0.299	0.018	0.175	0.06

Non-normal observations are expressed as median (range); all other observations are expressed as mean (range)

†

Where letter annotations are not the same, differences between supplementation amount were significant (P <0.05, or P ⩽0.0625 when Friedman analysis was used)

Ca:Cr = urine calcium (mg/dl):creatinine (mg/dl) ratio; P:Cr = urine phosphorus (mg/dl):creatinine (mg/dl) ratio; Mg:Cr = urine magnesium (mg/dl):creatinine (mg/dl) ratio; BW = body weight

Discussion

At the lowest dietary 25(OH)D₃ supplementation (4.9 µg/kg), plasma concentrations of 25(OH)D₃ in the study cats were similar to concentrations we found in the cats when given the pretrial diet. A consensus on circulating 25(OH)D₃ concentrations indicating vitamin D sufficiency in cats has not been reached and multiple variables such as assay technique, signalment, disease and physiological variation affect the RI and therapeutic target range.³⁰ In our study, vitamin D status, as seen by plasma concentrations of 25(OH)D₃, were compared with pretrial concentrations and with recent publications. A study by Titmarsh et al reported vitamin D status in healthy cats and compared with those with feline immunodeficiency virus.⁵ The median serum 25(OH)D₃ in the healthy cats in this study was 44.7 ng/ml (range 14.9–61.0), which is similar to our cats pretrial and lowest supplement (4.9 µg/kg) concentrations. In humans, serum values between 25 and 50 ng/ml are considered safe and sufficient for skeletal health.³¹

These results may indicate that dietary 25(OH)D₃ is greater than 10 times more potent than D₃ for affecting plasma 25(OH)D₃ concentration of cats, and therefore vitamin D status of cats. However, this conclusion may be conditional with presenting vitamin D status. Heaney et al describe for human subjects a very steep, linear increase in serum 25(OH)D₃ concentration with increasing D₃ oral dose, until an inflection occurs when high D₃ dosing causes only a modest rise in serum 25(OH)D₃ concentration.³² These investigators suggest that production of 25(OH)D₃ is a saturable process and that limited available 25-hydroxylase accounts for a curvilinear relationship between D₃ dose and serum 25(OH)D₃ concentration. Hence, in the cats, a dietary D₃ supplementation much less than that measured in the pretrial diet (<91 µg/kg) may be adequate for sustaining an apparently sufficient range in plasma concentrations of 25(OH)D₃ indicating vitamin D sufficiency (eg, ⩾20 ng/ml).³³ The recommended allowance for D₃ in diets of cats is only 7.0 µg/kg of diet, which is <10% of the D₃ content of the pretrial diet.^19,33 A worthy subject of future research will be the description of plasma 25(OH)D₃ response of adult cats to increasing dietary D₃ concentration when they were previously depleted of vitamin D.

Plasma 25(OH)D₃ concentrations of the cats increased with increasing dietary 25(OH)D₃ concentration and appeared linear over the trialed range of supplementations, which is similar to that described in humans.^1,34 Plasma 25(OH)D₃ concentration increased by a mean of about 14 ng/ml for every 0.1 µg/kg^0.67 of 25(OH)D₃ ingested. The results of a recent study of 91 elderly men and women indicate a similar degree of responsiveness to oral 25(OH)D₃.⁷ Highly efficient gut absorption of 25(OH)D₃ and lack of need for metabolism by liver are attributes of 25(OH)D₃ suggested to account for the linear response in plasma 25(OH)D₃ to ingested 25(OH)D₃.

Our findings for the α-epimers in this trial support the postulation that C-3 epimerization occurs before or simultaneously with 25-hydroxylation of D vitamers.³⁵ The findings also indicate that C-3 epimerization in cats does not substantially contribute to the removal of ingested 25(OH)D₃, whereas the epimerization may importantly impact metabolic elimination of ingested D₃. The enzymatic reactions responsible for C-3 epimerization have not yet been identified, but they are reported to occur in cells of several tissues, including the liver.³⁶ The physiological importance of α-epimers is unclear, although in vitro and a few in vivo studies indicate that the α-epimers have calciotropic activities, albeit less than those of β-epimers.³⁷ If the functionality of C-3 epimerization is identified in the future, it may be especially important to cats, a species for which α-epimers appear uniquely abundant in plasma.

Plasma 24,25(OH)₂D₃ concentrations among the cats increased with increasing dietary 25(OH)D₃ supplementation. This trend is consistent with 24-hydroxylation being a metabolic means for removal of excess 25(OH)D₃, irrespective of whether cats ingest the 25(OH)D₃ or produced it through 25-hydroxylation of D₃.³⁸

The highest dietary 25(OH)D₃ supplementation (11.8 µg/kg), or an equivalence in daily intake of 25(OH)D₃ of 0.22 µg/kg^{0. 67}, may have been excessive. In one of the five cats, the β-epimer concentration of 25(OH)D₃ wasin excess of the cited laboratory RI upper limit (>68 ng/ml).²⁰ Plasma concentrations of magnesium and total and iCa were also increased, albeit slightly. As plasma 1,25(OH)₂D₃ concentration was not affected by 25(OH)D₃ dietary supplementation, ligand activity of 25(OH)D₃ toward intestinal vitamin D receptors may have mediated the increased plasma calcium and magnesium concentrations. While estimates of vitamin D receptor affinity for 25(OH)D₃ are 50- to 150-fold less than the affinity for 1,25(OH)₂D₃, plasma concentrations of 25(OH)D₃ observed in the cats were more than 150-fold greater than of those of 1,25(OH)₂D₃.⁹ The highest supplementation with 25(OH)D₃ did not significantly affect urine mineral excretion. However, with the medium dietary supplementation, which was estimated to be 8.4 µg/kg, a slight but significant increase in the urinary magnesium:creatinine ratio was seen. As calcium and magnesium concentrations in plasma are regulated through renal excretions of the minerals, daily ingestion of ⩾0.22 µg/kg^0.67 of 25(OH)D₃ may theoretically raise the risk for ammonium-magnesium-phosphate (struvite) or calcium oxalate urolithiasis. In future studies, urine pH and mineral supersaturation indexes should be evaluated for assessment of urolithiasis risk.

Results of several studies that indirectly determined urinary mineral excretion rates can be compared with our findings. Dobenecker et al quantified renal excretion of phosphorus in healthy adult cats fed high phosphorus diets.³⁹ When the cats were fed the two control diets containing 0.5–0.6% dietary phosphorus, renal phosphorus excretion was 23 and 14 mg/kg BW/day, respectively. In our pretrial phase, when dietary phosphorus was 1.0%, average phosphorus excretion was higher, a mean of 30 mg/kg BW/day. However, our value was low compared with the urinary excretion of phosphorus by cats given a high sodium phosphate diet (1.7% phosphorus), which Dobenecker et al reported to be 83 mg/kg BW/day.³⁹ Paßlack et al found that urinary excretion of calcium in healthy cats was on average 0.69 mg/kg BW/day when fed 0.65% sodium and 1.0% calcium on a dry matter basis.²⁴ When our diets were supplemented with 25(OH)D₃ urinary calcium excretions by our cats were slightly higher than these, although the sodium and calcium content of our diets were similar to those of Paßlack et al.²⁴ Calcium excretions when our cats were given the D₃ supplemented diet were not determined as pretrial urine samples were frozen before analysis. We found that freezing variably decreased urine concentrations of calcium, whereas concentrations of phosphorus and magnesium were unaffected.

Norris et al evaluated the effect of magnesium-deficient diets on serum and urine magnesium concentrations in adult healthy cats.⁴⁰ Cats studied by these investigators had a urinary magnesium:creatinine ratio of 0.008 ± 0.004 when fed a magnesium replete diet that contained half the concentrations of magnesium in our diets.⁴⁰ Compared with these ratios, our urine magnesium:creatine ratios were about 2.5–3.75-fold greater. Though greater, our dietary 25(OH)D₃ supplementations did not significantly affect the ratios, except for when 8.4 µg/kg of 25(OH)D₃ was fed. The magnesium concentration in our diet is 2.5-fold higher than the National Research Council recommended allowance for adult cats (400 mg/kg dry matter) but within the range of over-the-counter adult feline diets.¹⁹

The main limitation of our study was its small sampling of middle-aged, castrated male cats. While significant effects of dietary 25(OH)D₃ content on vitamin D metabolites were found, evaluation of a larger study population may have indicated a significant increase in urine magnesium:creatinine ratio with increasing 25(OH)D₃ ingestion. Initially, six cats were enrolled in the study. One cat was excluded due to development of struvite urocystoliths. Of the remaining five, one cat had a prior treated hyperthyroidism while another cat had biochemical evidence of mild chronic kidney disease. We do not believe that these conditions biased our findings on effects of dietary 25(OH)D₃ supplementation. Our findings indicate that a future study of a larger population of cats is warranted.

Conclusions

A dietary 25(OH)D₃ supplementation of approximately 5.0 µg/kg on an as-fed basis for 9 weeks was well tolerated by adult cats. The supplementation was the equivalence of daily ingestion of approximately 0.09 µg of 25(OH)D₃ per metabolic BW (kg^0.67). It supported circulating 25(OH)D₃ concentrations in adult cats that were consistent with vitamin D sufficiency and did not cause extraordinarily high urinary concentrations or excretions of calcium, phosphorus or magnesium. However, greater 25(OH)D₃ supplementations that were trialed increased plasma calcium and magnesium concentrations, but only slightly. The 25(OH)D₃ supplementation did not appear to substantially change plasma abundances of calcitriol and C-3 α-epimeric forms of plasma 25(OH)D₃ and 24,25(OH)₂D₃. This finding lessens concern about quantifying circulating α-epimeric forms when dietary 25(OH)D₃ supplementation might be used.

Supplemental Material

Table 1:

Guaranteed and proximate analysis of nutrients and ingredients for custom, extruded, dry-expanded feline diet as reported by the manufacturer and measured

Supplemental Material

Table 2:

Clinical hematology observations for five healthy adult male castrated cats after feeding with a diet supplemented with vitamin D₃ (81 μg/kg) for 13 weeks or diet supplemented with 25(OH)D₃ for nine weeks at measured concentrations of either 4.9, 8.4 or 11.8 μg/kg

Footnotes

Acknowledgements

The authors thank DSM Nutritional Products, Parsippany, NJ, USA, for the provision of the diets for the study.

Accepted: 22 February 2022

Supplementary material

The following files are available online:

Table 1: Guaranteed and proximate analysis of nutrients and ingredients for custom, extruded, dry-expanded feline diet as reported by the manufacturer and measured

Table 2: Clinical hematology observations for five healthy adult male castrated cats after feeding with a diet supplemented with vitamin D₃ (81 μg/kg) for 13 weeks or diet supplemented with 25(OH)D₃ for nine weeks at measured concentrations of either 4.9, 8.4 or 11.8 μg/kg

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was funded by grants from the Phi Zeta Veterinary Honor Society at the University of Missouri, DSM Nutritional Products, Parsippany, NJ, USA and the Nestlé Purina Endowed Program for Small Animal Nutrition at the University of Missouri.

Ethical approval

The work described in this manuscript involved the use of experimental animals and the study therefore had prior ethical approval from an established (or ad hoc) committee as stated in the manuscript.

Informed consent

Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (either experimental or nonexperimental animals) for the procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Janelle C Corugedo

Robert C Backus

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