Dietary 135-fold cholecalciferol supplementation severely disturbs the endochondral ossification in growing dogs

Domestic Animal Endocrinology 24 (2003) 265–285

Dietary 135-fold cholecalciferol supplementationseverely disturbs the endochondral

ossification in growing dogs

M.A. Tryfonidoua,∗, M.S. Holla, J.J. Stevenhagenb,C.J. Buurmanc, H.F. Delucad, M.A. Oosterlaken-Dijksterhuisa,

W.E. van den Broma, J.P.T.M. van Leeuwenc,H.A.W. Hazewinkela

a Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine,Utrecht University, Yalelaan 8, 3584 CM, Utrecht, The Netherlands

b TNO Nutrition and Food Research, Zeist, The Netherlandsc Department of Internal Medicine, Erasmus MC Rotterdam, Rotterdam, The Netherlands

d Department of Biochemistry, University of Wisconsin-Madison, Madison, WC, USA

Received 5 September 2002; accepted 13 January 2003

Abstract

The effects of excessive non-toxic dietary Vitamin D3 supplementation on Ca homeostasis withspecific effects on endochondral ossification and skeletal remodeling were investigated in a group ofgrowing Great Dane dogs supplemented with cholecalciferol (Vitamin D3; HVitD) versus a controlgroup (CVitD) (1350�g versus 11.4�g Vitamin D3 per kilogram diet) from 6 to 21 weeks of age.There were no differences between groups in plasma concentrations of total Ca, inorganic phos-phate, growth hormone, and insulin-like growth factor I and no signs of Vitamin D3 intoxicationin HVitD. For the duration of the study inHVitD compared toCVitD, plasma levels of parathyroidhormone (PTH) decreased, calcitonin (CT) increased, 25-hydroxycholecalciferol [25(OH)D3] in-creased 30- to 75-fold, 24,25-dihydroxycholecalciferol [24,25(OH)2D3] increased 12- to 16-fold,and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] decreased by approximately 40%. The latter wasattributed to the two-fold increased metabolic clearance rate in theHVitD versusCVitD accompa-nied by the absence of the anabolic effect of PTH on the production of 1,25(OH)2D3. FractionalCa absorption (α) did not differ between groups at 8 and 14 weeks of age, whereas at 20 weeksof ageα increased by only 16.4% inHVitD compared toCVitD. Excessive non-toxic Vitamin D3supplementation resulted in decreased bone remodeling and focal enlargement of the growth platewith morphology resembling those induced by administration of CT. Hypercalcitoninemia and the

∗ Corresponding author. Tel.:+31-30-2539-411; fax:+31-30-2518-126.E-mail address:[email protected] (M.A. Tryfonidou).

0739-7240/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0739-7240(03)00018-3

266 M.A. Tryfonidou et al. / Domestic Animal Endocrinology 24 (2003) 265–285

imbalanced relationship between 1,25(OH)2D3 and 24,25(OH)2D3 are potent candidates for thedisturbed endochondral ossification.© 2003 Elsevier Science Inc. All rights reserved.

Keywords:1,25-Dihydroxycholecalciferol; 24,25-Dihydroxycholecalciferol; Endochondral ossification; Caabsorption; Dog

1. Introduction

Nutrition plays an important role in the skeletal development of growing dogs[1]. Largebreed dogs are over-represented in juvenile skeletal disorders associated with disturbances ofendochondral ossification and/or skeletal remodeling, including for example osteochondro-sis, the radius curvus syndrome, enostosis, and the Wobbler syndrome. These disturbanceshave been related mainly to high intake of (a) food[2], (b) energy[3], (c) calcium (Ca)[4,5], (d) Ca and P[6], and (e) possibly cholecalciferol (Vitamin D3), although the role ofthe latter has not yet been elucidated.

Dogs are completely dependent on dietary sources for their Vitamin D3 requirements,due to restricted availability of 7-dihydroxycholesterol in the skin[7]. Vitamin D3 ishydroxylated in the liver to 25-hydroxycholecalciferol [25(OH)D3] and subsequently to1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] in the kidney. The latter two metabolites are reciprocally regulated. The rate of re-nal synthesis of 1,25(OH)2D3 is directly responsive to levels of plasma inorganic phosphate(Pi), growth hormone (GH), insulin-like growth factor I (IGF-I), and parathyroid hormone(PTH) [8–10], whereas regulatory feedback is provided by 1,25(OH)2D3 by inducing itsown catabolism[11].

The 1,25(OH)2D3 is the biologically most active metabolite of Vitamin D3 and facilitatesintestinal Ca absorption, renal Ca reabsorption, endochondral ossification, skeletal mineral-ization and remodeling[11,12]. The most abundantly present Vitamin D3 metabolites in theplasma of dogs [25(OH)D3 and 24,25(OH)2D3] do not have a well defined physiologicalrole. 25(OH)D3 may directly stimulate Ca absorption as known in man[13–15]althoughthis has been disputed by others[16]. 24,25(OH)2D3 has biological functions on bone byfacilitating skeletal mineralization[17], regulating chondrocyte differentiation[18], andpossibly affecting Ca absorption[19,20].

The period of fast growth has been associated in Great Danes with a juvenile excess inGH and IGF-I compared to Miniature Poodles[21]. Growth hormone and/or IGF-I excessis known to affect Vitamin D3 metabolism as characterized in rats[22], in pigs [23,24],and in man[25] by high plasma 1,25(OH)2D3 levels and low plasma 24,25(OH)2D3 levels.Similarly, growing Great Danes have higher plasma 1,25(OH)2D3 levels and a magnitudelower plasma 24,25(OH)2D3 levels than Miniature Poodles (unpublished data). Thus, GreatDanes seem to have a relative excess of 1,25(OH)2D3 and deficiency in 24,25(OH)2D3based (a) on the effects of the GH–IGF-I axis on Vitamin D3 metabolism and (b) on themaintenance of the plasma 25(OH)D3 levels at control levels, despite an eight-fold VitaminD3 supplementation[26]. This may give an indication for higher Vitamin D3 requirementsof growing large breed dogs than the NRC[28] recommendations that are based on studies

M.A. Tryfonidou et al. / Domestic Animal Endocrinology 24 (2003) 265–285 267

in growing Beagles experiencing a much shorter period of high GH release[27]. However,increased supplementation of Vitamin D3 can be detrimental as it may stimulate intestinalCa absorption and affect endochondral ossification and skeletal remodeling.

The present study was designed to elucidate the effects of excessive non-toxic dietaryVitamin D3 supplementation on Ca homeostasis with specific effects on endochondralossification and skeletal remodeling. In adult dogs, chronic daily intake of 500�g VitaminD3 per kilogram body weight (BW) exceeding by∼1000 times the NRC[28] VitaminD3 recommendations has been reported to cause Vitamin D3 toxicity with impaired kidneyfunction and calcifications[29]. Therefore,∼100-fold NRC-recommended[28] Vitamin D3supplementation was chosen in order to recognize the possible implications of excessivenon-toxic Vitamin D3 intake. Animals were monitored for signs of Vitamin D3 intoxicationby standard biochemical measurements and assessment of the kidney function. Skeletalgrowth and development were determined with the aid of biochemical measurements,45Cabalance studies, radiology, and histomorphometry and interpreted in light of the plasmalevels of the calciotropic hormones and intestinal Ca absorption.

2. Materials and methods

2.1. Animals and diets

The Utrecht University Ethical Committee for Animal Care and Use approved all pro-cedures. Sixteen (eight males and eight females) Great Dane dogs, originating from threedifferent litters (Utrecht University, The Netherlands), were divided into two groups at 3weeks of age: a control group (n = 9,CVitD) and a dietary Vitamin D3 supplemented group(n = 7, HVitD). Pups were raised on a dry pellet diet (Table 1) formulated to be compa-rable for energy, Ca, and P content. The supplemented Vitamin D3 diet was aimed to have∼100-fold greater Vitamin D3 per kilogram diet content than the recommended amountof 12.5�g Vitamin D3 per kilogram diet for growing dogs[30]. Diets were analyzed fortheir Vitamin D content[31] in a reference laboratory (TNO Nutrition and Food Research,Zeist, The Netherlands) (Table 2). The puppies received their diet as gruel in addition to thebitch milk by 3 weeks of age and exclusively fed dry diet by 6 weeks of age. Food intakewas adjusted biweekly to the actual BW and provided at two times maintenance energyrequirements of each dog[32] for the duration of the study.

2.2. Ca absorption measurements

Ca absorption was measured with the aid of one tracer45Ca, by techniques as describedpreviously and validated for the dog[33]. In short, the actual food intake was measured ineach dog and Ca intake (VI ) was calculated for each investigation period. The endogenousfecal excretion of45Ca (Vf ) was determined by collecting the feces for 3 consecutive daysafter an intravenous (i.v.) dose of 0.1, 0.25, and 0.4 MBq45Ca as45CaCl2 water (specificactivity 271.88 MBq/mg, NEN Life Science Products, Boston, USA) at 8, 14, and 20 weeksof age, respectively.Vf was calculated from the quotient of the part of the injected dose (Rf3)that was excreted in the feces during 3 days and the integral of plasma specific activityC(t)


Table 1Composition of the experimental diets

Ingredient Control cholecalciferol diet (g/kg) High cholecalciferol diet (g/kg)

Corn 528 528Chicken 200 200Poultry fat 75.5 75.5Brewers rice 74.2 74.2Mineral mixturea 35.4 35.4Beet pulp 34.0 34.0Egg 25.0 25.0Brewers yeast 10.0 10.0Menhaden oil 8.0 8.0Vitamin mixture Cb 6.7 0Vitamin mixture Hc 0 6.7Ground flax 2.8 2.8DL-methionine 0.4 0.4

a Composition of the mineral mixture (amount in 35.4 g): calcium carbonate (Ca= 4.41 g); monosodiumphosphate (P= 2.61 g); ferrous sulfate (Fe= 2.55 g); zinc oxide (Zn= 2.12 g); cupric sulfate (Cu= 159 mg);manganese oxide (Mn= 283 mg); manganese sulfate (Mn= 205 mg); potassium iodide (I= 32 mg); cobaltcarbonate (Co= 5.3 mg); with potassium chloride as the remainder.

b Composition of the vitamin mixture (amount in 6.7 g): Vitamin A (13.2 mg); Vitamin C (367 mg); chole-calciferol (22.1�g); Vitamin E (226 mg); thiamin (27 mg); riboflavin (168 mg); nicotinic acid (28 mg); calciumpantothenate (52 mg); pyridoxine-RCl (13 mg); biotin (0.76 mg); folic acid (2.5 mg); inositol (17 mg); cyanocobal-amin (0.30 mg); choline chloride (228 mg); with rice hulls as the remainder.

c Composition of the vitamin mixture (amount in 6.7 g): Vitamin A (13.2 mg); Vitamin C (367 mg); chole-calciferol (2204�g); Vitamin E (226 mg); thiamin (27 mg); riboflavin (168 mg); nicotinic acid (28 mg); calciumpantothenate (52 mg); pyridoxine-HC1 (13 mg); biotin (0.76 mg); folic acid (2.5 mg); inositol (17 mg); cyanocobal-amin (0.30 mg); choline chloride (228 mg); with rice hulls as the remainder.

according to the formula:Vf = Rf 3/∫ 3

0 C(t) dt. Although, in a similar manner, the urine lossof 45Ca (Vu) could be calculated asVu = Ru3/

∫ 30 C(t) dt with Ru3 the part of the injected

dose that was excreted in the urine during 3 days, the specific activity of45Ca in urine wastoo low for reliable counting and thusVu was determined by analysis of the total urinary

Table 2Analysis of the experimental diets

Item Control cholecalciferol dieta High cholecalciferol dietb

g/100 g diet dry matterCrude protein 26.90 26.83Crude lipid 14.65 15.44Ash 5.10 5.20Calcium 0.94 0.95Phosphate 0.80 0.85

�g/kg dietCholecalciferol 11.4c 1350c

a Moisture 7.1 g/100 g diet.b Moisture 10.1 g/100 g diet.c No detectable traces of ergocalciferol.


Ca content over the period of 3 days. On day 4 of each metabolic study, a dose of45Caequivalent to the i.v. dose was orally administered and feces were collected for 4 consecutivedays. The fecal content of45Ca was determined to calculate the total fecal content of Ca(VF). True Ca absorption (Va) was calculated by the formula:Va = VI − (VF − Vf ). Thefractional absorption of Ca (α) was defined as:α = Va/VI × 100%. Assuming a steadystate condition during each investigation period of 7 days, then turnover= input = outputand thusT = Va + V−

0 = Vu + Vf + V+0 whereV−

0 andV+0 are the calcium resorption

and accretion, respectively. Consequently,V−0 = T − Va andV+

0 = T − (Vu + Vf ). Allparameters were expressed in mmol per kg body per day.

2.3. Blood measurements

At 7, 13, and 19 weeks of age blood samples were collected after an overnight fast. Bloodsamples for the measurement of Ca, Pi , alkaline phosphatase (AP), corticosteroid-inducedAP (C-AP), and bile acid levels were transferred into heparine tubes, centrifuged and mea-sured according to standard procedures (Beckman Industries Inc., Brea, USA). C-AP wasdetermined as described previously[34]. Blood samples for the analysis of Ca2+ werecollected anaerobically in heparinized syringes (PICO 50, Radiometer Copenhagen, Den-mark), were placed on melting ice and analyzed within 2 h after collection with the aid ofthe ionized Ca analyzer (ABL 605, Radiometer Copenhagen).

Determination techniques of the plasma Vitamin D3 metabolites levels have been de-scribed earlier and validated for the dog[26]. In short, 25(OH)D3 and 24,25(OH)2D3 werequantitatively determined by a modified radioimmunoassay (RIA) (DiaSorin, Stillwater,Minnesota, USA) after extraction[35] and separation by solid phase extraction (NH2 car-tridge; Bakerbond spe Amino Disposable Extraction Columns, J.T. Baker, Philipsburg,USA) [36]. 1,25(OH)2D3 was quantitatively determined by a radioreceptor assay based onthe method described by Reinhardt et al.[37] and Hollis [38] after extraction with ace-tonitrile followed by a two-step solid phase extraction (C18 and Silicagel cartridge; WatersChromatography B.V. Etten Leur, The Netherlands). PTH was measured using an immuno-radiometric assay for intact PTH (iPTH, Nichols Institute, San Juan Capistrano, CA, USA)[39]. Calcitonin (CT) was measured after extraction with ethanol by a homologous RIA[40]. GH was measured by a homologous RIA[41]. Total IGF-I levels were measured bya heterologous RIA[42].

2.4. Kidney function

At 7, 13, and 19 weeks of age plasma creatinin and urea levels were determined bystandard procedures (Beckman Industries Inc., Brea, USA). After completion of every45Ca balance study, the glomerular filtration rate (GFR) was determined at 8, 14, and 20weeks of age with the aid of 99Tcm-Diethylenetriaminepentaacetic acid (99Tcm-DTPA) asdescribed before[43].

In addition to theVu of Ca, mean daily urine excretion of Pi (VuP, mmol per kg bodyper day) was measured during the45Ca balance studies over a period of 3 days, when dogswere housed in metabolic cages. Tubular Reabsorption of Pi (TRP) was determined by the


formula: TRP = 1 − (CRPi /CRCreatinin), where TRP is expressed in percentage of thefiltrated Pi and CR is the clearance rate expressed in ml kg per body per minute determinedfor either factorsX (i.e. P or creatinin). CR was calculated from CR= (Xu × Vu)/Xpland corrected for BW, whereXu is the concentration of the factor in the daily urine,Vu thevolume of urine in a day,Xpl the plasma concentration of the factorX.

2.5. Endogenous metabolic clearance rate (MCR) of 1,25(OH)2D3

At 19 weeks of age the MCR of 1,25(OH)2D3 was determined in eightCVitD and sevenHVitD dogs by techniques as described previously[26,44]. In short, after an i.v. admin-istration of approximately 3.7 kBq 1�,25-dihydroxy[23,24(n)-3H]cholecalciferol (3H-1,25(OH)2D3, specific activity 10.5 GBq/mg, Amersham Pharmacia Biotech, Buckinghamshire,UK) the plasma disappearance curve of3H-1,25(OH)2D3 was obtained and a two-compart-mental model was fitted to the plasma3H-1,25(OH)2D3 concentration. The MCR of 1,25(OH)2D3 was calculated by the quotient of the injected doseD of 3H-1,25(OH)2D3 and theintegral of plasma specific activity of3H-1,25(OH)2D3 as follows: MCR= D/

∫ ∞0 C(t) dt,

where MCR is given in l per kg body per day.

2.6. Vitamin D receptor (VDR) analysis

At the end of the study (at 21 weeks of age), animals were euthanized with an overdoseof Na-pentobarbital and after immediate laparotomy, the mucosal layer was sampled fromthe following defined intestinal segments: proximal and distal duodenum, beginning, mid-dle and end of the jejunum, ileum, and colon. Nuclear extracts were prepared as describedby Sandgren and DeLuca[45]. Briefly, segments were rinsed with ice-cold TED saline(50 mmol/l Tris–HCl, pH 7.4, 1.5 mmol/l EDTA, 5 mmol/l DTT, 10�g/ml TPCK, 1�g/mlleupeptin, 1�g/ml pepstatin) and the mucosal layer was scraped and washed three timesin TED saline by centrifugating for 10 min at 3000× g and 4◦C. Samples were homog-enized in 4 vol. (w/v) KTED-300 (TED, 300 mmol/l KCl) in a glass Teflon homogenizer.Homogenates were centrifuged at, 100,000× g for 1 h at 4◦C and supernatants (nuclearextracts) were stored at−70◦C until assayed. Concentration of VDR was measured by aVDR specific enzyme-linked immunoassay[46] and expressed as femtomole per milligrammucosal protein.

2.7. Radiology

At 9, 15, and 21 weeks of age radiographs of the right radius and ulna were made.Skeletal growth of the dogs was assessed radiographically by measuring the length of theradius (RL) and ulna (UL) along the axis of the bone using a curved ruler and correcting forgeometric magnification. Skeletal development was assessed by evaluating the developmentof the secondary ossification centers of the elbow joint, i.e. the anconeal process, medialhumeral epicondyl, and olecranon apophysis, and of the distal ulna the styloid process andarchitecture of the distal ulnar metaphysis (DUM) according to techniques described earlier[47].


2.8. Bone histomorphometry

At 10 and 21 weeks of age, bone biopsies were obtained from the 9th left and right rib,respectively. In addition to routine histological evaluation, histomorphometry was carriedout in longitudinal slices containing the growth plate and 2.5 cm metaphysis of the rib withthe aid of semi-automated image analyzing system, using the KS 400 software package (CarlZeiss Vision, Germany), a program running language for image analysis applications (Dr.M. Terlou, Faculty of Biology, Utrecht University, NL). The mean growth plate thickness(GPl.Th in millimeters) of the growth plate was determined from the mean of the completearea of the growth plate. The standard deviation of the mean thickness of the growth plate(GPl.ThS.D. in millimeters) was determined from the mean of 100 measurements at fixedspaced sites in samples fixated in alcohol and stained with Goldner’s Trichrome. In theentire area of the metaphyseal bone, which was parallel to and at 1 mm fixed distance fromthe growth plate, the percentage of mineralized trabecular bone (Md.Tb.V in percentage)was determined in samples fixated in 80% alcohol, undecalcified, stained with Von Kossa.Absolute osteoclast number (N.Oc) was determined from the mean number of osteoclastsof at least 15 microscopic fields (25× objective) in slices stained with Goldner’s Trichrome.

2.9. Statistical analysis

Statistical analyses were performed using the SPSS for Windows 10.1 (SPSS Inc, Chicago,USA). Differences between groups were analyzed by the two-tailed Studentst-test after test-ing for homogeneity of variance with Levene’s test, while differences between ages withingroups were analyzed by multiple comparisons (Tukey test) in an univariate ANOVA forrepeated measures. The significance of differences in VDR concentration between groupswas tested by the non-parametric Wilcoxon–Mann–WhitneyU test because the standarddeviation of the mean was in the majority greater than 50% of the mean. The differ-ence in growth plate regularity within each group was analyzed by comparing the ratioof GPl.ThS.D./GPl.Th due to a decrease in GPl.Th with increasing age. Values were con-sidered to be significant whenP < 0.05. Results are presented as mean± standard error ofthe mean (S.E.M.).

3. Results

Dogs from both groups had good general health with an undisturbed growth curve (Fig. 1)and complete uptake of the amount of food that was administered daily. Consequently,energy and food intake per kilogram metabolic BW did not differ between groups. Thesupplemented Vitamin D3 group (HVitD) consumed approximately 135-fold more VitaminD3 per kilogram body in comparison to the control group (CVitD) (Table 2).

3.1. Ca absorption

VI was similar in both groups at all ages (Fig. 2). Vf was significantly lower in theHVitDgroup in comparison to theCVitD group at 8 and 14 weeks of age, whereas it was not


Fig. 1. Undisturbed growth curve from weaning (6 weeks) to 21 weeks of age during 135-fold cholecalciferolsupplementation in Great Dane dogs.

significantly different between groups at 20 weeks of age inHVitD versusCVitD (Fig. 2).Vu accounted for less than 0.3% of theVI and ranged from 0.01±0.002 to 0.03±0.002 mmolper kg body per day and did not differ between groups.α decreased significantly with agefor both groups and was significantly lower at 20 weeks of age in comparison to 8 and 14weeks of age for both groups.Va andα did not differ significantly between both groups at8 and 14 weeks of age, whereas they were significantly higher in theHVitD versusCVitDgroup at 20 weeks of age (Fig. 2). V+

0 andV−0 were significantly lower only at 20 weeks of

age in theHVitD versus theCVitD group (Fig. 2).

3.2. Biochemical profiles

The biochemical profiles for both groups of dogs at 7, 13, and 19 weeks of age areshown inTable 3. There were no relevant differences between groups in the plasma lev-els of Ca, Pi , and AP, whereas plasma Ca2+ levels were higher in theHVitD versus theCVitD group only at 19 weeks of age. In both groups, C-AP was undetectable and bileacids ranged within the reference limits for dogs, i.e. 1–10�mol/l, for the duration ofthe study. Plasma 25(OH)D3 and 24,25(OH)2D3 levels increased significantly in bothgroups with age. Plasma 25(OH)D3 levels were significantly increased by 29-, 75-, and56-fold in theHVitD versusCVitD group at 7, 13, and 19 weeks of age, respectively.Plasma 24,25(OH)2D3 levels were significantly increased by 12- to 16-fold in theHVitDversusCVitD group with increasing age. At 7 weeks of age plasma 1,25(OH)2D3 levels


Fig. 2. Effects of cholecalciferol supplementation from partial weaning (3 weeks) to 21 weeks of age on the45Cabalance parameters: Ca intake (VI ), intestinal true Ca absorption (Va), fractional Ca absorption (α), endogenousfecal excretion (Vf ), urine Ca losses (Vu), Ca accretion (V+

0 ) and Ca resorption (V−0 ). Data are presented as

means± S.E.M. (∗) P ≤ 0.05 and (∗∗) P ≤ 0.01 between groups at the same age, (A) P ≤ 0.05 as compared tothe fractional Ca absorption at 8 weeks of age within the group, (B) P ≤ 0.05 as compared to the fractional Caabsorption at 14 weeks of age within the group.

were not different between groups, whereas they were significantly decreased by 40 and36% at 13 and 19 weeks of age in theHVitD versusCVitD group. At all ages, plasmaPTH levels were significantly lower in theHVitD versusCVitD group. Plasma CT levelswere higher in theHVitD versusCVitD group with significance at 7 and 13 weeks of age.There were no differences in the plasma GH and IGF-I levels between groups during thestudy.

3.3. Kidney function

Plasma creatinin and urea levels and GFR did not differ between groups and were withinthe reference range for dogs (Table 3) [43]. VuP did not differ between groups and was1.34±0.08, 1.05±0.05, and 0.99±0.04 mmol per kg body per day at 8, 14, and 20 weeksof age, respectively.

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Table 3Plasma bioehemical profiles during 135-fold cholecalciferol supplementation in Great Dane dogs from partial weaning (3 weeks) to 21 weeks of agea,b

CVitD (n = 9) 11.4�g cholecalciferol/kg diet HVitD (n = 7) 1350�g cholecalciferol/kg diet

7 weeks 13 weeks 19 weeks 7 weeks 13 weeks 19 weeks

Cholecatciferol intake(�g kg per body per day)

0.37± 0.01 0.35± 0.01 0.30± 0.01 50.6± 1.2∗∗ 46.2± 0.4∗∗ 40.3± 0.6∗∗

Ca (mmol/l) 2.86± 0.03 2.95± 0.02 2.90± 0.02 2.99± 0.03† 3.01± 0.03 2.91± 0.03Ca2+ (mmol/l) 1.51± 0.05 1.49± 0.01 1.48± 0.04 1.49± 0.03 1.51± 0.01 1.51± 0.01∗∗Pi (mmol/l) 2.81± 0.04 2.74± 0.02 2.69± 0.03 2.87± 0.06 2.85± 0.06 2.79± 0.05AP (IU/l) 170.1± 15.8 196.6± 15.5 189.7± 12.7 176.1± 14.0 201.1± 14.2 175.7± 13.9Creatinin (�mol/l) 37.3± 2.0 51.4± 2.4 61.8± 1.3 28.9± 1.1 45.4± 0.7 56.7± 1.4Urea (mmol/l) 1.8± 0.2 2.9± 0.1 4.2± 0.2 2.5± 0.1 3.4± 0.1 4.8± 0.2GFR (ml kg per body per minute) 5.2± 0.3 5.9± 0.2 4.0± 0.1 4.9± 0.1 6.1± 0.4 3.9± 0.125(OH)D3 (nmol/l) 17.5± 3 14.7± 2.8 22.4± 2.7A 517± 89∗∗ 1101± 164∗∗A 1255± 188∗∗A

24,25(OH)2D3 (nmol/l) 25.1± 3.5 39.9± 3.2A 48.5± 4.8A 302± 43∗∗ 647± 113∗∗A 769± 58∗∗A

1,25(OH)2D3 (pmol/l) 286± 17 255± 9 244± 12 289± 16 152± 7∗∗A 156± 16∗A

iPTH (ng/l) 89.3± 32.1 61.8± 6.5 41.2± 6.3 27.9± 6.2∗ 22.9± 3.7∗∗ 16.8± 2.3∗∗CT (ng/l) 91.7± 14.7 160.0± 29.4 140.6± 40.3 158.6± 28.7∗ 266.2± 36.0∗ 240.3± 69.3GH (�g/l) 19.7± 2.4 9.7± 1.5 6.6± 1.1 13.7± 1.7 8.6± 1.0 5.4± 0.9IGF-I (�g/l) 216± 24 273± 24 413± 27 213± 17 275± 22 335± 25MCR of l,25(OH)2D3

(l per kg body per day)– – 0.26± 0.04 – – 0.52± 0.03∗∗

a Data is presented as means± S.E.M. (∗) P ≤ 0.05 and (∗∗) P ≤ 0.01 between groups at the same age, (†) P < 0.05 between groups at the same age but consideredto be a relevant difference, (A) P < 0.05 as compared to 7 weeks of age.

b Ca, calcium; Ca2+, ionized Ca; Pi , inorganic phosphate; AP, alkaline phosphatase; GFR, glomerular flitration rate; 25(OH)D3, 25-hydroxycholecalciferol;24,25(OH)2D3, 24,25-dihydroxycholecalciferol; 1,25(OH)2D3, 1,25-dihydroxycholeca1ciferol; iPTH, intact parathyroid hormone; CT, calcitonin; GH, growth hormone;IGF-I, insulin-like growth factor 1; MCR, metabolic clearance rate.


Table 4Parameters of skeletal growth (mean±S.E.M.) and development in growing Great Dane dogs raised on a balanceddiet differing only in Vitamin D3 content

Age (week) Length of radius (cm) Length of ulna (cm)

CVitD HVitD CVitD HVitD

9 8.1± 0.2 9.0± 0.3 9.9± 0.2 10.9± 0.315 12.6± 0.1 13.2± 0.2 14.9± 0.1 15.7± 0.321 15.8± 0.2 16.5± 0.3 18.8± 0.2 19.7± 0.3

Increase in length (%)

9–15 55.5± 2.2 47.2± 2.5∗ 50.0± 2.1 44.9± 2.215–21 25.7± 1.3 24.9± 1.3 26.7± 0.7 25.0± 1.4

Development of the secondary ossification centers of the elbow joint and of the distal ulna

9 weeks of age 15 weeks of age 21 weeks of age

CVitD HVitD CVitD HVitD CVitD HVitD

AnconeusNo ossification 9 7 1 – – –Some ossification, no fusion – – 3 – – –Complete ossification, no fusion – – 3 5 1 –Complete ossification, fusion – – 2 2 8 7

Olecranal apophysisIrregular shape 6 4 – – – –Round – 3 – – – –Rounded edges 3 – 4 1 – –Complete ossification – – 5 3 9 7

Medial humeral epicondylRound 1 3 – – – –Rounded edges 8 4 – – – –Compete ossification – – 9 7 9 7

Ulnar styloid processRectangular 6 2 – – – –Square 3 5 – – – –Young apex – – 2 1 – –Apex visible, no fusion – – 5 2 – –Complete ossification – – 2 4 9 7

Distal ulnar metaphysisFlattened or rounded 2 3 4 3 3 3Cartilage cone – – 5 3 3 2Irregular structure – – 2 4 6 4Normal shape 7 4 1 1 2 –

Two groups of Great Dane dogs were raised from partial weaning (3 weeks) until 21 weeks of age on a balancediet differing only in its Vitamin D3 content:CVitD group (n = 9) with 11.4�g andHVitD (n = 7) with 1350�gVitamin D3/kg diet.


3.4. Endogenous metabolic clearance of 1,25(OH)2D3

The MCR of 1,25(OH)2D3 at 19 weeks of age was increased two-fold in theHVitD versusCVitD group (Table 3).

3.5. Intestinal VDR content

VDR levels at the end of the study did not differ between groups and decreased graduallyin the distal segments of the intestine ranging from 350±33 fmol/mg protein in the proximalduodenum to 86± 19 fmol/mg protein in the colon.

3.6. Radiology

Length of the radius and ulna was greater in theCVitDversusHVitD group but the increasein length did not differ between groups (Table 4). The development of the secondary centersof ossification was variable in both groups, however at 21 weeks of age all dogs of bothgroups were at the same stage of development (Table 4). There were no obvious differencesbetween groups in the structure of the DUM except for that none of theHVitD dogs hadnormal structure of the DUM at the end of the study (Table 4).

3.7. Histomorphometry

Results of the histomorphometric analysis of the growth plate and metaphyseal area ofthe rib biopsies are presented inTable 5. At 10 weeks of age GPl.Th and GPl.ThS.D./GPl.Thwere significantly higher in theHVitD versusCVitD group. With increasing age, GPl.Thdecreased significantly only in theCVitD group. GPl.ThS.D./GPl.Th did not change withage in theCVitD group, whereas it increased significantly with age in theHVitD group. At10 weeks of age Md.Tb.V did not differ between groups and increased with age in both

Table 5Histomorphometric parameters of the growth plate and metaphyseal area of the rib of growing Great Dane dogsraised on diet differing only in Vitamin D3 content

10 weeks of age 21 weeks of age

CVitD HVitD CVitD HVitD

GP1.Th (mm) 1.14± 0.03 1.27± 0.05∗ 0.98± 0.03B l.26 ± 0.07∗∗GP1.ThS.D. (mm) 0.18± 0.02 0.26± 0 02 0.27± 0.09 0.49± 0.07GPl.ThS.D./GPl.Th (%) 15.7± 1.3 20.3± 0 7∗∗ 17.6± 1.3 38.7± 3.4∗∗A

Md.Tb.V (%) 15.6± 1.4 17.7± 0 7 22.4± 1.3B 22.4± 1.6A

N.Oc (n) 10.0± 0.3 9.4± 0.7 7.6± 0.4B 6.6± 0.3B

Two groups of dogs were raised from partial weaning (i.e. at 3 weeks) until 21 weeks of age on a balance dietdiffering only in its Vitamin D3 content:CVitD group (n = 9) with 11.4�g andHVitD (n = 7) with 1350�gVitamin D3/kg diet. GPl.Th, mean growth plate thickness; GPl.ThS.D., standard deviation of the GPl.Th; Md.Tb.V,mineralized trabecular bone percentage of the total bone area; N.Oc, Osteoclast number. Data is expressed asmean± S.E.M.(∗∗) P < 0.01 compared to theCVitD group; (A) P < 0.05 and (B) P < 0.01 compared to 10weeks of age.


groups, with no differences between groups at 21 weeks. At both measured points N.Ocdid not differ between groups and decreased significantly with age.

Routine histological evaluation revealed in both groups irregularities of the costal growthplate that were variable in presentation and severity. There were focal disturbances ofendochondral ossification with lack of mineralization of the intercellular substance atthe general line of mineralization. Consequently hypertrophic chondrocytes protruded inthe metaphyseal area (adjacent primary spongiosa), i.e. retained cartilage. These focaldisturbances were restricted in theCVitD group, whereas were more expanded in theHVitD group occupying most often one-third to half of the area of the growth plate,protruding into the metaphyseal area (primary and secondary spongiosa) for several mil-limeters with necrosis at the bordering cancellous bone in selected most severe cases(Fig. 3).

Fig. 3. Physeal growth plate of the 9th rib of Great Danes at 21 weeks of age raised on balanced food with 11.4�gVitamin D3/kg according to the recommendations of the NRC[28] (CVitD group; A) and 1350�g Vitamin D3/kg(HVitD group; B–D). Dogs of theHVitD group had irregular growth plates with variable severity due to focaldisorders of endochondral ossification characterized by arrest of chondrocyte apoptosis, retarded formation ofprimary spongiosa and protrusion of the growing cartilage in the metaphyseal area with necrosis in the mostsevere cases (arrow). 1, cartilage; 2, growth plate; 3, metaphyseal area.


4. Discussion

The 135-fold Vitamin D3 supplementation in growing dogs compared to age matchedcontrols induced major changes in the plasma levels of Vitamin D3 metabolites for theduration of the study. Vitamin D3 supplementation was accompanied by an increment infractional Ca absorption of only 16.4% solely at the end of the study and severe disturbancesof endochondral ossification. Both groups of dogs were raised on a diet differing only inits Vitamin D3 content and food consumption was restricted to the energy requirementsof growing dogs and thus Ca and P intake did not differ between groups. Dogs grew ac-cording to the curves described for Great Danes in earlier studies[26,42]. In addition, theHVitD group had plasma Ca, Pi, creatinin, and urea levels comparable to theCVitD group,plasma Ca2+ increased only at the end of the study, GFR in the optimal reference range forall dogs in both groups[43], and renal excretion of Ca and P at control levels. Thus, thechronic dietary 135-fold supplementation with Vitamin D3 was not accompanied by clini-cal signs of Vitamin D3 intoxication, such as severe hypercalcemia, hyperphosphatemia, orkidney failure. The effects of excessive non-toxic dietary Vitamin D3 supplementation dur-ing growth on skeletal development are here discussed in light of the calciotropic hormonesand influences on Ca homeostasis.

Oral Vitamin D3 supplementation in theHVitD group induced striking changes in theplasma levels of the calciotropic hormones, i.e. Vitamin D3 metabolites, PTH, and CTlevels. Plasma total Ca, Pi , GH, and IGF-I levels did not differ between groups and thuswere assumed not to play a differential regulatory role on Vitamin D3 metabolism be-tween groups. The 135-fold Vitamin D3 supplementation resulted in a major increase of theplasma 25(OH)D3 levels accompanied by a significant increase in plasma 24,25(OH)2D3levels compared to theCVitD group, which is a well established phenomenon[48,49]. Inhypervitaminosis D3, plasma 25(OH)D3 and 24,25(OH)2D3 levels may rise far above thebinding capacity of the Vitamin D binding protein (DBP) and displace 1,25(OH)2D3 fromDBP resulting in increase of the free plasma 1,25(OH)2D3 levels[50]. The latter may haveattributed to increased biologic activity of 1,25(OH)2D3 without concomitant increase inthe total plasma 1,25(OH)2D3 levels inHVitD. In the absence of hypercalcemia (in thebeginning and middle of the study) and of increased total plasma 1,25(OH)2D3 levels,either increased free plasma 1,25(OH)2D3 levels or high plasma 25(OH)D3 levels in theHVitD group mimicking the biologic actions of 1,25(OH)2D3 on parathyroid level[51]may result in down regulation of plasma PTH levels. Decreased plasma PTH levels indicateeither a decrease in production of PTH[52], an increase in sensitivity of the chief-cellsto stimuli [53], or a modification of the secretory profile of the parathyroid gland[54].Hypercalcitoninemia has been related in dogs to C-cell hyperplasia due to chronic hyper-calcemia[4,55,56]. However, hypercalcemia, the main mediator of hypercalcitoninemia[57], was only present at 19 weeks of age in theHVitD group. Taking into considerationthe inverse relationship of the plasma PTH and CT levels, it seems conceivable to sug-gest a direct influence of one of the Vitamin D3 metabolites either on CT production andsecretion, or on the responsiveness of the C-cells to stimuli. The relation of Vitamin D3metabolism to C-cells remains to be elucidated but is supported (a) by the presence of aCa sensing receptor (CaR) in chief- and C-cells which may influence the secretion of therespective hormones PTH and CT[53] and (b) by the up regulating effect of 1,25(OH)2D3


on CaR gene expression of the parathyroid gland in Vitamin D-deplete and -replete rats[58,59].

Plasma 1,25(OH)2D3 levels did not change with age in theCVitD group. On the con-trary, in theHVitD plasma 1,25(OH)2D3 levels decreased with age and were significantlydecreased by 40 and 36% at 13 and 19 weeks of age, respectively, compared to theCVitDgroup. This decrease in plasma 1,25(OH)2D3 levels is in accordance with earlier studies onVitamin D3 excess in rats[60] and is more pronounced than that encountered during moder-ate (i.e. eight-fold) Vitamin D3 supplementation in growing dogs over the same life period[26]. In theHVitD group, low plasma PTH levels indicated an attenuation of the anabolicaction of PTH on the production of 1,25(OH)2D3 [61]. In addition, MCR of 1,25(OH)2D3was two-fold higher in theHVitD than in theCVitD group indicating up regulation of24-oxidation pathway and thus increased conversion of 1,25(OH)2D3 into less biologicallyactive products in the target organs[49,62]. Summarizing, the absence of the anabolic ef-fect of PTH on the production of 1,25(OH)2D3 [49] accompanied by the enhanced MCR of1,25(OH)2D3 resulted in a modifying down regulating effect on the plasma 1,25(OH)2D3levels.

45Ca balance studies demonstrated that, despite the prominently lower plasma1,25(OH)2D3 levels in the middle and end of the study in theHVitD versusCVitD group,α was similar between groups at 14 weeks of age, whereas it was increased in theHVitDcompared to theCVitD group at 20 weeks of age. The up regulation ofα in theHVitD groupcan be attributed either to an increase of the free plasma 1,25(OH)2D3 levels or to the highplasma 25(OH)D3 levels. Excessive 25(OH)D3 may compete for binding at VDR sites andproduce similar effects to 1,25(OH)2D3 in the intestine, i.e. stimulate Ca absorption[63].In support of the biologic effect of 25(OH)D3 are the in vivo studies in man giving evidencethat 25(OH)D3 has a direct effect on Ca absorption even at physiological concentration[13,14]. Heaney et al.[15] reported in man an increment of the fractional Ca absorptionof 0.000299 for every rise of 1 nmol/l in plasma 25(OH)D3 levels. By extrapolating thisrelationship to the data of the present study and supposing that 25(OH)D3 stimulates Caabsorption also in the dog, the striking rise of plasma 25(OH)D3 levels in theHVitD group,would have been expected to evoke an increase inα of 14.9% at 8 weeks up to 35.8% at20 weeks of age compared to theCVitD group. However, the increase inα was significantonly at 20 weeks of age, being lower than the expected rise, i.e. just 16.4% higher in theHVitD group thanCVitD group. The blunted increase in Ca absorption at all ages in theHVitD group cannot be anticipated by an alteration in the abundance of intestinal VDR asthey were maintained in theHVitD group at the same levels as in theCVitD group. A mech-anism counter regulating Ca absorption may be provided either by relatively limited tissueresponsiveness to 25(OH)D3 by maintenance of the VDR at control level and/or by theincreased plasma 24,25(OH)2D3 levels in theHVitD group as has been described in chickintestine[19,64]. Two-fold increased plasma 24,25(OH)2D3 levels have been associatedwith the depression of intestinal Ca absorption in growing dogs receiving moderate dietaryVitamin D3 supplementation[26]. However, this consideration is rather tentative with con-troversy in the literature[19,20,64]and the counter regulating effect of 24,25(OH)2D3 onCa absorption in dogs needs further investigation.

Vitamin D3 supplementation had only moderate effects on skeletal mineralization andremodeling.V+

0 was higher thanV−0 at all measured points and regardless of the group


(Fig. 2) as may be expected in animals with a growing skeleton. This was reflected in theincreasing Md.Tb.V of the costal metaphyseal area with age in both groups (Table 5). Boneturnover decreased with age in both groups and at the end of the study it was significantlylower in theHVitD than in theCVitD group as indicated by the lowerV+

0 andV−0 , although

this was not reflected in the plasma AP levels and the N.Oc. However, plasma AP levelshave been reported not to correlate with the bone-isoform originating from active osteoblast[65], and the N.Oc is not necessarily related to the level of resorption activity. In addition tothat, histomorphometry is restricted to the costal metaphyseal area, whereas45Ca balancestudies give a whole body estimation of theV+

0 andV−0 1,25(OH)2D3 stimulates skeletal

mineralization as well as resorption[66], whereas 24,25(OH)2D3 stimulates mineralizationwithout affecting resorption[67], and CT attenuates bone resorption[57]. Increased plasma24,25(OH)2D3 levels in combination with lowered plasma 1,25(OH)2D3 levels explain thelower bone turnover/remodelling in theHVitD versusCVitD group[68], whereas increasedCT levels indicate retraction of the ruffle border of the osteoclasts resulting in decreasedbone resorption without affecting the N.Oc.

There is evidence that excessive non-toxic dietary Vitamin D3 intake in large breed dogsdirectly influences endochondral ossification. Radiological evaluation of the right forelimbrevealed that the development of the secondary ossification centers was not influenced byVitamin D3 supplementation. Evaluation of the structure of the distal ulna revealed irregularstructure and the presence of cartilage cones in both groups indicating that even in the controldogs endochondral ossification did not proceed undisturbed. However, at the end of thestudy allHVitD dogs revealed abnormal structure. Histomorphometric evaluation of costalgrowth plate at 10 and 21 weeks of age revealed that GPl.Th decreased significantly withage in theCVitD group, which is in agreement with other reports on growing Great Danes[4,69]. In addition, the regularity of the frontline between growth plate and metaphysealbone remained constant with age in theCVitD group. To the contrary, in theHVitD groupgrowth plates were irregular at 10 weeks and even more so at 21 weeks of age. The increasein irregularity of theHVitD group reflected the increasing severity of disturbances of theendochondral ossification with age (Fig. 3).

Chondrocyte proliferation, hypertrophy, and mineralization at growth plate level are sub-ject to complex autocrine, paracrine, and endocrine regulation. Disturbed endochondralossification seen in theHVitD group was related to increased plasma 24,25(OH)2D3 levelsand the decreased plasma 1,25(OH)2D3 levels, as well as to the increased plasma CT levels.High Ca intake resulting in high Ca retention has been related to osteochondrosis in growinglarge breed dogs[4,6] and may be a contributing factor in theHVitD group. At 20 weeksof age Ca retention was 5.3 mmol per kg body per day in theHVitD group,∼8.5 mmol perkg body per day in dogs raised on a diet with 3.3 g Ca/100 g diet (DM)[70], whereas itwas 4.1 mmol per kg body per day in theCVitD group. 24,25(OH)2D3 has been reportedto cause resting zone cells to mature in the endochondral lineage and become responsive to1,25(OH)2D3 [71] which is further responsible for the terminal chondrocyte differentiationand mineralization of the matrix[72]. We hypothesize that the 16- to 18-fold increasedplasma 24,25(OH)2D3 concentration in combination with the 30–40% decrease in plasma1,25(OH)2D3 levels may be implicated in the abnormal development of endochondral os-sification. The morphological findings in this study with thickening of the growth platesresemble those of other studies in turtles[73], rabbits[74], and rats[75,76] treated with


salmon CT. The described mechanisms of focal thickening of the growth plate due to CTexcess resulting in disturbances of endochondral ossification are not univocal and have beenattributed either to a direct effect of CT on the maturation of the hypertrophic cells[75] or adown regulating effect of CT in skeletal metaphyseal remodelling[76]. The causative roleof hypercalcitoninism in osteochondrosis of dogs as suggested by others[4,55] is supportedby the hypercalcitoninism with concomitant manifestation of osteochondrosis of the costalgrowth plates in theHVitD group.

In conclusion, the 135-fold Vitamin D3 supplementation in growing dogs from 3 to 21weeks of age resulted in decreased plasma PTH levels and increased plasma CT levelsand major changes in the plasma levels of the Vitamin D3 metabolites, i.e. increase inthe plasma 25(OH)D3 and 24,25(OH)2D3 levels and decrease in the plasma 1,25(OH)2D3levels. The decrease in plasma 1,25(OH)2D3 levels was attributed to a combination of en-hanced metabolic clearance and probably attenuation of the anabolic action of the loweredplasma PTH levels on the synthesis of 1,25(OH)2D3. Despite the 135-fold Vitamin D3 sup-plementation and on average 50-fold increase in plasma 25(OH)2D3 levels, Ca absorptionremained at control levels and increased significantly only at the end of the study. The at-tenuated increase in Ca absorption may be attributed to maintenance of the intestinal VDRlevels at control levels, a counter regulating effect of 24,25(OH)2D3 on Ca absorption, ora combination of the above. Excessive, non-toxic dietary Vitamin D3 supplementation re-sulted at the end of the study in decreased skeletal remodeling and severe disturbances ofendochondral ossification attributed to direct effects on the maturation process of hyper-trophic chondrocytes, as well as indirect effects on the remodelling of the metaphyseal area.Important candidates that may disturb endochondral ossification were either (a) increasedplasma 24,25(OH)2D3 levels in combination with decreased plasma 1,25(OH)2D3 levels,(b) increased plasma CT levels, or (c) a combination of the above. Dietary intake of VitaminD3 135-fold higher than the NRC recommendations during the period of fast growth in largebreed dogs does not cause clinical signs of Vitamin D3 intoxication, except for disturbancesof endochondral ossification.

Acknowledgements

Stable metabolites of 25(OH)D3 and 24,25(OH)2D3 were kindly provided by Dr. J.P.van de Velden (Solvay Pharmaceuticals, Weesp, The Netherlands). The authors would liketo acknowledge the clinic attendants for good caring of the pups and their assistance inperforming the experiments, as well as the assistance of the Biochemical Laboratory. Wethank Yvonne Pollak for the technical assistance in the GFR measurements.

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Dietary 135-fold cholecalciferol supplementation severely disturbs the endochondral ossification in growing dogs