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Hormonal regulation of calcium homeostasis in two breeds of dogs during growth at different rates¹

M. A. Tryfonidou^*,2, M. S. Holl^*, M. Vastenburg, M. A. Oosterlaken-Dijksterhuis^*, D. H. Birkenhäger-Frenkel, W. E. van den Brom^* and H. A. W. Hazewinkel^*

^* Department of Clinical Sciences of Companion Animals and and Division of Diagnostic Imaging,Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands; and and Department of Pathology, Erasmus University, Rotterdam, The Netherlands

² Correspondence:
Yalelaan 8 (phone: 31-30-2539411; fax: 31-30-2518126; E-mail:
M.A.Tryfonidou{at}vet.uu.nl).

	Abstract

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Growing giant-breed dogs are more susceptible to developing skeletal disorders than small-breed dogs when raised on diets with deficient or excessive Ca content. Differential hormonal regulation of Ca homeostasis in dogs with different growth rates was investigated in Great Danes (GD, n = 9) and Miniature Poodles (MP, n = 8). All animals were raised on the same balanced diet and under identical conditions. Calciotropic and growth-regulating hormones were measured. Production and clearance of 1,25-dihydroxycholecalciferol (1,25[OH]₂D₃) were investigated with the aid of [³H]-1,25(OH)₂D₃ and renal messenger RNA abundance of 1-hydroxylase and 24-hydroxylase. Intestinal, renal, and skeletal Ca handling were evaluated with the aid of ⁴⁵Ca balance studies. Skeletal development was evaluated by radiology and histomorphometry. Great Danes had greater (P < 0.001) growth rates than MP, as indicated by the 17-fold greater body weight gain, by increased longitudinal growth reflected in the increased (P < 0.05) gain in length of the radius and ulna, and by increased (P < 0.001) growth plate thickness. These findings were accompanied in GD by greater (P < 0.05) plasma GH and IGF-I concentrations. Effects were observed for vitamin D₃ metabolism, such as greater (P < 0.01) plasma 1,25(OH)₂D₃ concentrations due to decreased (P < 0.01) clearance rather than increased production of 1,25(OH)₂D₃, and decreased (P < 0.01) plasma 24,25-dihydroxycholecalciferol (24,25[OH]₂D₃) concentrations likely due to competitive inhibition of the production of 24,25(OH)₂D₃. These findings were accompanied in both breeds by a limited hormonal regulation of Ca and P absorption at the intestinal level, and in GD by increased (P < 0.05) renal reabsorption of inorganic P (P_i) compared with MP, resulting in greater (P < 0.01) P_i retention and greater (P < 0.01) plasma P_i concentrations. Bone turnover, resorption, and formation were greater (P < 0.01) in GD than in MP. In addition, GD had more irregular (P < 0.01) growth plates than MP, accompanied by disorders of endochondral ossification. It is suggested that in GD, increased calcitonin levels and/or a relative deficiency in 24,25(OH)₂D₃ at the growth-plate level may both be responsible for the retarded maturation of chondrocytes, resulting in retained cartilage cones and osteochondrosis, and this may be a pathophysiological factor for the increased susceptibility of large breed dogs to developing skeletal disorders.

Key Words: Absorption • Bones • Calcium • Cholecalciferol • Dogs • Somatotropin

	Introduction

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Dogs are completely dependent on dietary intake of cholecalciferol (vitamin D₃; How et al., 1994). Vitamin D₃ is hydroxylated in the liver to 25-hydroxycholecalciferol (25[OH]D₃) and subsequently in the kidney to 1,25-dihydroxycholecalciferol (1,25[OH]₂D₃) and 24,25-dihydroxycholecalciferol (24,25[OH]₂D₃). During growth, 1,25(OH)₂D₃ is directed to the skeleton, intestine, and kidney (Brown et al., 1999), whereas 24,25(OH)₂D₃ is mainly directed to the skeleton (Staal et al., 1997; Boyan et al., 2001). The rate of renal synthesis of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ is reciprocally regulated, with the former being directly responsive to plasma concentrations of inorganic P (P_i), GH, IGF-I, and parathyroid hormone (PTH) (Gray and Garthwaite, 1985; Nesbitt and Drezner, 1993; Murayama et al., 1999).

Giant-breed dogs are more susceptible to developing skeletal disorders compared with small-breed dogs when raised on diets with deficient (Hazewinkel et al., 1991; Nap et al., 1993b) or excessive (Goedegebuure and Hazewinkel, 1986) Ca content. It was observed that growing Great Danes (GD) and Miniature Poodles (MP) had similar plasma 25(OH)D₃ and 1,25(OH)₂D₃ concentrations, whereas plasma 24,25(OH)₂D₃ concentrations were a magnitude lower in GD than in MP (Hazewinkel and Tryfonidou, 2002). It was hypothesized that the susceptibility of developing skeletal disorders of giant-breed dogs may find part of its aetiopathogenesis in high plasma GH and IGF-I concentrations (Nap et al., 1992) and differences in vitamin D₃ metabolism (Hazewinkel and Tryfonidou, 2002). In order to evaluate this hypothesis, hormonal regulation of Ca homeostasis at the intestinal, renal, and skeletal level were investigated in growing giant- and small-breed dogs. Several parameters were evaluated, including calciotropic and growth-regulating hormones, production, and clearance of 1,25(OH)₂D₃, ⁴⁵Ca kinetics, and skeletal development.

	Materials and Methods

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Animals and Diets

The Utrecht University Ethical Committee for Animal Care and Use approved all procedures. Nine purebred GD (GD group) and eight pure-bred MP (MP group), both originating from three different litters, were raised on an extruded diet (Table 1) formulated to meet the requirements for growing dogs (NRC, 1974). From 3 to 6 wk of age, pups received their diet as a gruel in addition to the bitch milk, and later on received dry diet exclusively. Body weight was measured biweekly, and food was provided at two times maintenance energy requirements of each dog (Lewis et al., 1987) for the duration of the study.

At 7, 10, 13, 16, 19, and 21 wk of age, blood samples were collected after overnight food deprivation. Blood samples for the measurement of plasma total Ca, P_i, alkaline phosphatase (AP), corticosteroid-induced AP (C-AP), and bile acid concentrations were transferred into tubes containing heparin, centrifuged, and then measured according to standard procedures (Beckman Industries Inc., Brea, CA); C-AP was determined as described previously (Teske et al., 1989).

Blood samples for the measurements of hormones were immediately transferred into EDTA-coated tubes and placed on ice until centrifuged. Plasma was stored at -20°C until analysis. Determination techniques of the plasma vitamin D₃ metabolites concentrations have been described earlier and validated for the dog (Tryfonidou et al., 2002a). In short, 25(OH)D₃ and 24,25(OH)₂D₃ were quantitatively determined by a modified RIA (DiaSorin, Stillwater, MN) after extraction (Bosch et al., 1983) and separation by solid-phase extraction (NH₂ cartridge; Bakerbond Spe Amino Disposable Extraction Columns, J.T. Baker, Philipsburg, NJ) (McGraw and Hug, 1990). The intra- and interassay CV for 25(OH)D₃ were 15.2 and 6.1%, respectively. The intra- and interassay CV for 24,25(OH)₂D₃ were 10.1 and 8.5%, respectively. The 1,25(OH)₂D₃ was quantitatively determined by a radioreceptor assay based on the method described by Reinhardt et al. (1984) and Hollis (1986) after extraction with acetonitrile followed by a two-step, solid-phase extraction (C18 and Silicagel cartridge; Waters Chromatography B.V., Etten Leur, The Netherlands) with an intra- and interassay CV of 5.7 and 6.6%, respectively. Plasma PTH concentrations were measured using an immunoradiometric assay for intact PTH (iPTH; Nichols Institute, San Juan Capistrano, CA) (Torrance and Nachreiner, 1989). The intra- and interassay CV were 3.4 and 5.6%, respectively. Plasma calcitonin (CT) concentrations were measured after extraction with ethanol by a homologous RIA, as described before (Hazewinkel et al., 1999), with a detection limit of 25 ng/L. The intra- and interassay CV were 15.3 and 15.4%, respectively. Basal plasma GH concentration was defined as the median of six consecutive time points (i.e., the initial one and five more at 30 min, and 1, 2, 3, and 4 h after the initial sample). Growth hormone was measured by a homologous RIA, as described previously (Eigenmann and Eigenmann, 1981). The intra- and interassay CV were 3.8 and 7.2%, respectively. Total IGF-I concentrations were measured by a heterologous RIA, as described previously (Nap et al., 1993c), with intra- and interassay CV of 4.7 and 15.6%, respectively.

Endogenous Metabolic Clearance Rate and Production Rate of 1,25(OH)₂D₃

At 19 wk of age, the metabolic clearance rate (MCR) of 1,25(OH)₂D₃ was determined in all dogs by techniques described previously (Dusso et al., 1989; Tryfonidou et al., 2002a). Briefly, after an i.v. administration of approximately 3.7 KBq of 1,25-dihydroxy[23,24(n)-³H]cholecalciferol (³H-1,25[OH]₂D₃, specific activity 10.5 GBq/mg, Amersham Pharmacia Biotech, Buckinghamshire, U.K.) the plasma disappearance curve of ³H-1,25(OH)₂D₃ was obtained, and a two-compartmental model was fitted to the plasma ³H-1,25(OH)₂D₃ concentration. The MCR of 1,25(OH)₂D₃ was calculated by the quotient of the injected dose (D) of ³H-1,25(OH)₂D₃ and the integral of plasma-specific activity of ³H-1,25(OH)₂D₃ as follows:

Metabolic clearance rate was expressed in L•kg of BW^-1•d^-1. The production rate (PR) of 1,25(OH)₂D₃, expressed in pmol•kg of BW^-1•d^-1, was derived from the following formula:

where endogenous circulating 1,25(OH)₂D₃ is the plasma 1,25(OH)₂D₃ concentration at 19 wk of age.

1-Hydroxylase and 24-Hydroxylase Messenger Ribonucleic Acid Abundance

The abundance of messenger RNA (mRNA) for 1-hydroxylase and 24-hydroxylase was determined by techniques described previously (Tryfonidou et al., 2002a) at the end of the study in seven GD and seven MP (i.e., at 21 wk of age). Animals were euthanized with an overdose of Na-pentobarbital and biopsies from the kidney were sampled, immediately frozen in liquid nitrogen, and stored at -70°C until required for RNA isolation. Real-time PCR, based on the high-affinity, double-stranded DNA-binding dye SYBR green I (BMA, Rockland, ME), was performed in triplicate in a spectrofluorimetric thermal cycler (iCycler, BioRad, Hercules, CA). Cycling conditions were optimized for the reaction of each target gene. Primer pairs (Table 2) were designed using PrimerSelect software (DNASTAR, Inc., Madison, WI). For each experimental sample, the mRNA abundance of target (1-hydroxylase and 24-hydroxylase) and ß-actin as endogenous reference were determined from the appropriate standard curve in an autologous experiment. The amount of target was divided by the amount of endogenous reference to obtain a normalized target value. Each of the normalized target values was divided by the normalized target value of the calibrator (MP group) to generate n-fold relative abundance levels.

At 8, 14, and 20 wk of age, Ca kinetics were determined with the aid of one tracer (⁴⁵Ca) by techniques validated for the dog and previously described (Hazewinkel et al., 1991; Schoenmakers et al., 1999; Tryfonidou et al., 2002a). Radioactive ⁴⁵Ca is equally available and absorbed in the same preference as stable Ca and represents less than 1/1,000 of total daily dietary Ca intake. During each 7-d investigation period, animals were kept individually in metabolic cages allowing for individual daily collection of feces and urines. For each dog, the actual food intake was measured and Ca intake (V_ICa) was calculated for each investigation period. The plasma disappearance curve of ⁴⁵Ca was obtained after an i.v. dose of 15 KBq of ⁴⁵Ca/kg of BW as ⁴⁵CaCl₂ water (specific activity = 271.88 MBq/mg, NEN Life Science Products, Boston, MA). By means of a computerized nonlinear, least squares fitting procedure, a bi-exponential function

was fitted to the plasma ⁴⁵Ca concentrations. Calcium turnover (T_Ca) of the plasma equivalent pool was calculated by the quotient of the injected dose (D) of ⁴⁵Ca and the integral of plasma specific activity of ⁴⁵Ca as follows:

with T_Ca expressed in mmol of Ca•kg BW^-1•d^-1. The endogenous fecal excretion of ⁴⁵Ca (V_fCa) was determined by collecting the feces for three consecutive days after the i.v. dose of ⁴⁵Ca and was calculated from the quotient of the part of the injected dose (R_f3) that was excreted in the feces during 3 d and the integral of plasma specific activity, C(t), according to the following formula:

Although, in a similar manner, the urinary loss of ⁴⁵Ca (V_uCa) could be calculated as follows:

However, with R_u3, the part of the injected dose that was excreted in the urine during 3 d, the specific activity of ⁴⁵Ca in urine was too low for reliable counting. Therefore, V_uCa was determined by analysis of the total urinary Ca content over the period of 3 d. On the d 4 of each metabolic study, a dose of ⁴⁵Ca equivalent to the i.v. dose was orally administrated, and feces were collected for four consecutive days. The fecal content of ⁴⁵Ca was determined to calculate the total fecal content of Ca (V_FCa). True Ca absorption (V_aCa) was calculated by the formula:

The fractional absorption of Ca (_Ca) was defined as follows:

Assuming a steady-state condition during each 7-d investigation period, turnover = input = output, and thus T_Ca = V_aCa + V_{0 Ca}^- = V_uCa + V_fCa + V_{0 Ca}⁺, where V_{0 Ca}^- and V_{0 Ca}⁺ are Ca resorption and accretion, respectively. Consequently, V_{0 Ca}^- = T_Ca - V_aCa and V_{0 Ca}⁺ = T_Ca - (V_uCa + V_fCa), and Ca retention (_Ca) is given by _Ca = V_{0 Ca}⁺ - V_{0 Ca}^- = V_aCa - (V_uCa + V_fCa). All parameters were expressed in mmol •kg of BW^-1•d^-1.

P_i Balance Studies

Simultaneously with the ⁴⁵Ca balance studies, intestinal apparent P_i absorption (V_AP, mmol •kg of BW^-1•d^-1) was determined with the aid of the classic balance. Phosphate intake (V_IP) was calculated for each 7-d investigation period. The daily fecal content of P_i (V_FP, mmol •kg of BW^-1•d^-1) was determined, and the fraction of the P_i absorption ('_P, %) was defined as follows:

where V_IP - V_FP represents V_AP, and thus '_P was not corrected for the endogenous fecal losses of P_i, it represented the apparent fractional P_i absorption. Mean daily urine excretion of P_i (V_uP, mmol •kg BW^-1•d^-1) was measured during the ⁴⁵Ca balance studies over a period of 3 d, when dogs were housed in metabolic cages. The apparent retention of P_i was defined as follows:

Tubular reabsorption of P_i (TRP) was determined by the formula:

where TRP is expressed as a percentage of the filtrated P_i, and CR is the clearance rate expressed in mL•kg of BW^-1•min^-1 determined for either factors X (i.e., P or creatinin). The CR was calculated from CR = (X_u•V_u)/X_pl and corrected for BW, where X_u is the concentration of the factor in the daily urine, V_u the volume of urine in a day, and X_pl the plasma concentration of the factor X.

Kidney Function

After completion of every ⁴⁵Ca balance study, at 8 (GD group only), 14, and 20 wk of age, kidney function was analyzed by measurement of plasma creatinin and urea concentration with the aid of standard procedures (Beckman Industries Inc.) and by determination of the glomerular filtration rate (GFR) with the aid of 99T^cm-diethylenetriaminepentaacetic acid as described before (van den Brom and Biewenga, 1981).

Radiology

At 9, 15, and 21 wk of age, radiographs of the right radius and ulna were made. Skeletal growth of the dogs was assessed radiographically by measuring the length of the radius (Rd_L) and ulna (U_L) along the axis of the bone using a curved ruler and correcting for geometric magnification. Skeletal development was assessed by evaluating the development of the secondary ossification centers of the elbow joint (i.e., the anconeal process, medial humeral epicondyl, and olecranon apophysis), and of the distal ulna, the styloid process, and architecture of the distal ulnar metaphysis [DUM]) according to techniques described earlier (Voorhout et al., 1994; Schoenmakers et al., 2000a).

Bone Histomorphometry

At 10 and 21 wk of age, bone biopsies were obtained from the 9th left and right ribs, respectively. Histomorphometry was carried out in longitudinal slices containing the growth plate and 2.5-cm metaphysis of the rib with the aid of a semiautomated image analyzing system using the KS 400 software package (Carl Zeiss Vision, Germany), a program running language for image analysis applications (M. Terlou, Utrecht University, The Netherlands). The mean growth plate thickness (GPl.Th, in mm) was determined from the mean of the complete area of the growth plate. The standard deviation of the GPl.Th (GPl.Th_SD, in mm) was determined from the mean of 100 measurements at fixed space sites in samples fixated in alcohol and stained with Goldner’s Trichrome. In the area of the metaphyseal bone, which was parallel to and at 1 mm fixed distance from the growth plate, the percentage of mineralized trabecular bone (Md.Tb.V) was determined in samples fixated in 80% alcohol, undecalcified, and stained with Von Kossa. Absolute osteoclast number (N.Oc) was determined from the mean number of osteoclast of at least 15 microscopic fields (25x objective) in slices stained with Goldner’s Trichrome.

	Statistical Analysis

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Statistical analyses were performed using SPSS for Windows 10.1 (SPSS Inc, Chicago, IL). Differences in growth curves between groups were analyzed with a covariance analysis. Most differences between groups at each time point were analyzed by ANOVA for repeated measures, except for the mRNA abundance and histomorphometric data that were analyzed by the two-tailed Students’ t-test after testing for homogeneity of variance with Levene’s test. The two-tailed Pearson’s correlation coefficient was used to analyze the correlation between the plasma concentrations of GH and IGF-I and their relationship with age within each breed. Values were considered to be significant when P < 0.05. The difference in growth plate regularity between breeds of dogs with considerable differences in size was analyzed by comparing the ratio of GPl.Th_SD:GPl.Th between groups. Results are presented as mean ± SEM.

	Results

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Food intake was restricted according to energy requirements and did not differ between groups. Animals grew well (Figure 1); BW increased by 1.55 kg/wk on average in the GD group and by 0.09 kg/wk in the MP group, and growth rates were greater (P < 0.001) in the GD vs. MP group.

View larger version (13K): [in this window] [in a new window]   Figure 1. Growth curves of two groups of dogs with considerable difference in BW raised on the same balanced food from partial weaning at 3 wk to 21 wk of age. Data are expressed as means ± SEM.

Results are presented in Figures 2 and 3. Plasma Ca concentrations did not differ between groups for the duration of the study, whereas most plasma P_i concentrations were greater (P < 0.01) in the GD vs. MP group. Plasma AP concentrations were greater (P < 0.01) in the GD vs. MP group at all ages, whereas C-AP was undetectable and bile acids in both groups were within the reference limits for dogs (Kirk, 2002) (i.e., 1 to 10 µmol/L for the duration of the study). Plasma 25(OH)D₃ and 24,25(OH)₂D₃ concentrations were lower (P < 0.01), whereas plasma 1,25(OH)₂D₃ concentrations were greater (P < 0.01) in the GD vs. MP group for the duration of the study. Plasma PTH concentrations did not differ between groups. Plasma CT concentrations were similar between groups at the start of the study and greater (P < 0.05) for the remainder of the study in the GD vs. MP group. Basal plasma GH and IGF-I concentrations were significantly greater for the duration of the study in the GD vs. MP group. Basal plasma GH concentrations decreased with age in the GD group (r = -0.631, P < 0.01), whereas they did not change with age in the MP group. The correlation of plasma IGF-I concentrations with age was strongly positive (r = 0.700, P < 0.01) in the GD group and weakly positive in the MP group (r = 0.328, P = 0.034).

View larger version (9K): [in this window] [in a new window]   Figure 2. Plasma concentration of total calcium (Ca), inorganic phosphorus (Pi), and alkaline phosphatase (AP) in two groups of dogs, raised on the same balanced food. Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 between groups at the same age.

View larger version (23K): [in this window] [in a new window]   Figure 3. Plasma concentration of 25-hydroxycholecalciferol (25[OH]D3), 24,25-dihydroxycholecalciferol (24,25[OH]2D3), 1,25-dihydroxycholecalciferol (1,25[OH]2D3), GH, and IGF-I, parathyroid hormone (PTH), and calcitonin (CT) in two groups of dogs, raised on the same balanced food. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01 between groups at the same age.

At 19 wk of age, the MCR of 1,25(OH)₂D₃ was less (P < 0.01) in the GD vs. MP group, whereas the PR of 1,25(OH)₂D₃ did not differ between groups (Figure 4).

View larger version (21K): [in this window] [in a new window]   Figure 4. Production rate (PR) and metabolic clearance rate (MCR) of 1,25-dihydroxycholecalciferol (1,25[OH]2D3) at 19 wk of age in two groups of dogs with considerable difference in growth rate raised on the same balanced food from partial weaning at 3 wk to 21 wk of age. Data are presented as mean ± SEM, **P < 0.01 between groups.

Messenger Ribonucleic Acid Abundance of 1-Hydroxylase and 24-Hydroxylase

At 21 wk of age, mRNA abundance of renal 1-hydroxylase and 24-hydroxylase did not differ between groups.

⁴⁵Ca Balance Studies

Results are presented in Table 3. The V_ICa was similar in both groups and decreased with age. The T_Ca was 1.5-fold greater in the GD than in the MP group at all ages. The V_fCa was less in the GD vs. MP group at 8 (P < 0.01) and 20 wk of age (P < 0.05), whereas it was not significantly different between groups at 14 wk of age. The V_uCa did not differ between groups during the study. The V_aCa did not differ significantly between groups for the duration of the study, whereas at 8 and 14 wk of age, _Ca was significantly greater in the GD vs. MP group, and at 20 wk of age, it did not differ between groups. However, the mean difference in between groups at 8 and 14 wk of age was 6.7 and 7.4%, respectively, less than the reported 10 to 15% difference that is considered to be of biological significance (Heaney et al., 1988). Both V_{0 Ca}⁺ and V_{0 Ca}^- were significantly greater in the GD vs. MP group for the duration of the study (Figure 5). The _Ca was greater (P < 0.05) in the GD vs. MP group at 8 wk of age, whereas it was not significantly different between group for the remainder of the study (Figure 5).

View larger version (14K): [in this window] [in a new window]   Figure 5. Calcium accretion (V0 Ca+), resorption (V0 Ca-), and retention (Ca) in two groups of dogs with a considerable difference in growth rate raised on the same balanced food from partial weaning at 3 wk to 21 wk of age. Data are presented as mean ± SEM, **P < 0.01 between groups.

The V_IP, V_AP, and '_P did not differ between groups at all ages (Table 4). At all measure points, V_uP was less (P < 0.05), whereas '_P and TRP were greater (P < 0.01 and 0.05, respectively) in the GD vs. MP group (Table 4).

Plasma urea and creatinin concentrations were within the reference range for dogs (Kirk, 2002). The GFR did not differ between groups and was within the reference range for dogs (Table 4) (Biewenga and van den Brom, 1981).

Radiology

The actual R_L and U_L at all ages and the increase in length between 9 and 21 wk of age were greater in the GD vs. MP group (Table 5). The development was less uniform in the GD than in the MP group. Regardless of the course of the development of the different secondary ossification centra, both breeds had reached the same stage of development by 21 wk of age. The majority of the GD group had normal-shaped DUM at 9 wk of age, abnormally flattened DUM with increasing incidence of cartilage cones at 15 wk of age, and an irregular structure of the DUM at 21 wk of age. Four dogs of the MP group had flattened or rounded DUM at 9 wk of age that normalized with age; there were no cartilage cones or irregularities of the structure of DUM at all ages.

The GPl.Th and GPl.Th_SD:GPl.Th were greater in the GD compared to the MP group both at 10 and 21 wk of age (Table 6). The Md.Tb.V increased with age and did not differ between groups. The N.Oc decreased with age and was greater (P < 0.01) in the GD vs. MP group.

	Discussion

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Both GD and MP grew according to the growth curves described in earlier studies (Nap et al., 1993a,c). The accelerated growth rate in the GD group, with respect to the MP group, was reflected in the 17-fold greater BW gain/wk and the accelerated increase in length of the radius and ulna during the study. High growth rates in the GD group were accompanied by significantly greater basal plasma GH concentrations that decreased with age, as reported by others (Nap et al., 1992; Favier et al., 2001), and were contrary to the findings in the MP group. Growth hormone is a recognized indicator of growth acting directly and indirectly by stimulating the production of IGF-I in the liver and in target cells (Ohlsson et al., 1998). Accordingly, plasma IGF-I concentrations were significantly greater in the GD vs. MP group. However, there was no correlation between the plasma concentrations of GH and IGF-I. In adult dogs, plasma IGF-I concentrations are reportedly related to body size (Eigenmann et al., 1984a,b). It is questionable whether IGF-I concentration in the plasma is an indicator of growth rate since it is not correlated with growth rate in numerous other species (Zangger et al., 1987). Also, IGF-I originates predominantly from the liver, and elimination of the IGF-I production by the liver does not influence growth (Sjogren et al., 1999).

Despite the same vitamin D₃ intake in both groups, plasma concentrations of 25(OH)D₃, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ were lower, greater, and a magnitude lower, respectively, in the GD vs. MP group. Plasma concentrations of all vitamin D₃ metabolites are a function of production and clearance rates. The production of 25(OH)D₃ is loosely regulated, depending mainly on the amount of substrate (Stravitz et al., 1996) and negative feedback from 1,25(OH)₂D₃ (Reinholz and DeLuca, 1998). Thus, production of 25(OH)D₃ in the GD group was presumably less than that of the MP group due to negative feedback from the increased plasma 1,25(OH)₂D₃ concentrations. Clearance of 25(OH)D₃ depends on hydroxylation and production of 24,25(OH)₂D₃ and 1,25(OH)₂D₃ (Horst and Reinhardt, 1997), and apparently it was not increased in the GD vs. MP group. Accordingly, competitive inhibition of 24,25(OH)₂D₃ production induced by increased plasma concentrations of GH and IGF-I (Wongsurawat et al., 1984; Zoidis et al., 2002) was indicated by the decreased plasma 24,25(OH)₂D₃ concentrations in the GD vs. MP group, in accordance with earlier observations (Hazewinkel and Tryfonidou, 2002) and supported by the similar renal mRNA abundance of 24-hydroxylase in both groups. In addition, the production of 1,25(OH)₂D₃ did not differ between groups, as indicated by the PR of 1,25(OH)₂D₃ and mRNA abundance of renal 1-hydroxylase.

It was demonstrated that the greater plasma 1,25(OH)₂D₃ concentrations in the GD group, which undergoes a period of GH excess during growth, were the result of a decreased MCR of 1,25(OH)₂D₃ rather than an increase in PR of 1,25(OH)₂D₃ with respect to the MP group. High plasma 1,25(OH)₂D₃ concentrations have been reported in acromegaly (Lund et al., 1981) and after GH administration (Goff et al., 1990; Denis et al., 1994). So far, IGF-I has been proposed to be the mediator of the actions of GH increasing renal production of 1,25(OH)₂D₃ (Nesbitt and Drezner, 1993), whereby maximal stimulation is achieved under hypophosphatemic conditions (Gray and Garthwaite, 1985; Nesbitt and Drezner, 1993). However, the above mechanism was elucidated by administration of supraphysiological amounts of GH and/or IGF-I and was not confirmed under physiological conditions. Catabolism of 1,25(OH)₂D₃ is mainly dependent on 24-hydroxylase, which is regulated by 1,25(OH)₂D₃ itself (Brown et al., 1999), and by P_i (Wu et al., 1996), 24,25(OH)₂D₃ (Matsumoto et al., 1988; Yamato et al., 1989), PTH (Shinki et al., 1992), GH, and IGF-I (Wu et al., 1997). High plasma GH and IGF-I concentrations have been related to low plasma 24,25(OH)₂D₃ concentrations (Lund et al., 1981), and GH and IGF-I have been reported to induce a decrease in 24-hydroxylase activity (Wu et al., 1997). It is reasonable to suggest that GH and IGF-I have two fronts of action in order to favor increased plasma 1,25(OH)₂D₃ concentrations: decreasing 1,25(OH)₂D₃ clearance under physiological conditions at fast growth and increasing 1,25(OH)₂D₃ production under conditions of fast GH excess.

There were no differences in intestinal Ca and P_i absorption between groups despite the distinct differences in plasma concentration of growth-regulating factors and vitamin D₃ metabolites. Growth hormone may influence Ca absorption by increasing plasma 1,25(OH)₂D₃ concentrations by stimulating the production of 1,25(OH)₂D₃ (Goff et al., 1990) or by decreasing the clearance of 1,25(OH)₂D₃, as demonstrated here. The 1,25(OH)₂D₃ is a well known up-regulator of active Ca and P absorption (Wasserman and Fullmer, 1995), with disputed effects on passive Ca absorption (Lee et al., 1990; Norman, 1990). Consequently, greater _Ca and '_P would have been expected in the GD vs. MP group. However, model analysis discerning passive and active Ca absorption in growing dogs revealed that at the V_ICa reported in this study, passive Ca absorption would account for approximately 70% of the V_aCa (Tryfonidou et al., 2002b), indicating limited hormonal regulation of Ca absorption during this life stage at sufficient V_ICa (Lee et al., 1990). States of insufficient V_ICa are evoked more in giant-breed dogs with foods containing a much higher Ca content than in small-breed dogs. For example, osteoporosis is induced in GD when raised on a diet with 0.55 g/100 g of dietary DM, and only in MP when raised on a diet with 0.05 g Ca/100 g of dietary DM (Hazewinkel et al., 1991; Nap et al., 1993a). It is reasonable to suggest that large-breed dogs with high Ca needs and rapid growth rates are armed with additional protective mechanisms for the production of 1,25(OH)₂D₃ in order to be able to facilitate _Ca at low V_ICa and to keep up with the needs of the rapidly growing skeleton.

At a similar _Ca for both groups, the GD group had significantly decreased V_fCa at 8 and 20 wk of age compared with the MP group, resulting in greater _Ca at 8 wk of age in the GD vs. MP group. The V_fCa is a function of both secretion of endogenous Ca into the intestinal lumen and of its reabsorption in the more distal part of the intestine. Thus, the differences in V_fCa between groups can be anticipated by a difference in Ca secretion into, rather than reabsorption from, the intestinal lumen. The endogenous secretion of Ca has been reported to be independent of V_ICa and plasma Ca concentrations (Heaney and Skillman, 1964) but dependent on P_i intake (Heaney and Recker, 1994); this was true for both groups. Consequently, the difference in endogenous secretion of Ca, and thus V_fCa, can be related to a change in the permeability of the intestinal wall (i.e., the tight junctional permeability). Vitamin D sterols have been reported to have a genomic effect on assembly and permeability of the tight-junctional complexes (Chirayath et al., 1998), although further studies are necessary to elucidate and define the potential effect of vitamin D sterols on the secretion of Ca into the intestinal lumen.

Kidney function was optimal in both groups and there were no differences in V_uCa between groups, whereas V_uP was decreased in the GD vs. MP group. Differences in V_uCa were not detectable since it accounted for a very small fraction of the V_ICa. Because both groups had similar V_IP and _P, the greater TRP in the GD group resulted in greater '_P, which was reflected in the greater plasma P_i concentrations compared with the MP group. Plasma PTH concentrations did not differ between groups and thus can be excluded as a differentially regulating factor of renal P_i handling (Hammerman et al., 1980). The significantly greater plasma concentrations of GH, IGF-I, and 1,25(OH)₂D₃ in the GD group indicated a well-established positive effect on P_i reabsorption (Corvilain and Abramow, 1964; Denis et al., 1994; Brown et al., 1999). Maintenance of a positive P_i balance is required at rapid growth rates for the growth of soft tissues, energy metabolism, and bone mineralization.

Bone turnover was parallel to growth rate, resulting in striking differences on the skeletal level between the two groups. More than 99% of the total body Ca is located in the skeleton, and thus T_Ca, determined with the aid of ⁴⁵Ca kinetics, reflects mainly bone turnover of Ca. Regardless of breed, V_{0 Ca}⁺ was greater than V_{0 Ca}^- at all ages and, together with the positive _Ca, served for the mineralization of the growing skeleton. This was reflected in the increasing Md.Tb.V with age in the metaphysis of the ninth rib without differences between groups at corresponding ages. Both groups thus had a positive Ca balance and maintained Ca homeostasis, as indicated by plasma PTH concentrations not differing between groups and staying at reference levels (Schoenmakers et al., 2000b). Calcium turnover was significantly increased in the GD, with greater V_{0 Ca}^- and V_{0 Ca}⁺ with respect to the MP group. Accordingly, in the GD group, increased V_{0 Ca}^- was reflected in the greater N.O_C, whereas increased V_{0 Ca}⁺ was reflected in the increased plasma AP concentrations at all measured points with respect to the MP group. This rise was presumed to be of osteoblast origin since hepatic origin could be excluded by normal bile acids and C-AP levels were undetectable. The 1,25(OH)₂D₃ is a mediator of bone formation as well as bone resorption by supporting osteoclastogenesis and resulting in the high N.O_C seen in the GD group (Suda et al., 1995). To the contrary, 24,25(OH)₂D₃ is a mediator of bone formation without promoting bone resorption (Ono et al., 1996). Thus, 24,25(OH)₂D₃ is a counterbalancing factor for bone turnover, and correspondingly high plasma 24,25(OH)₂D₃ concentrations have been associated with low bone turnover (Mortensen et al., 1993). Accordingly, in the GD group, high bone turnover was accompanied by relatively high plasma 1,25(OH)₂D₃ concentrations and low plasma 24,25(OH)₂D₃ concentrations (Mortensen et al., 1993) in combination with high plasma GH and IGF-I concentrations (Denis et al., 1994; Ohlsson et al., 1998), contrary to the findings in the MP group.

Striking differences were encountered with respect to the regularity of the growth plate and skeletal development. The GPl.Th decreased significantly with age in both groups in accordance with earlier reports (Goedegebuure and Hazewinkel, 1986). It was significantly thicker in the GD vs. MP group, reflecting growth velocity (Vollmerhaus et al., 1981). However, growth plates were more irregular in the GD vs. MP group as indicated by their relative greater GPl.Th_SD/GPl.Th at both measured points. Accordingly, at 9 wk of age, the majority of dogs in the GD group had normal development of the DUM, whereas at 15 wk of age, increased incidence of structural irregularities were encountered in combination with retained cartilage cones in the metaphysis. Disturbance of chondrocyte maturation and subsequent of matrix calcification may occur focally and will lead to arrest of chondrocyte apoptosis, retarded formation of primary spongiosa and thus result in protrusion of the growing cartilage in the metaphysis with formation of retained cartilage cones (i.e., osteochondrosis). Osteochondrosis was observed in growing GD dogs raised on excessive Ca intake (Goedegebuure and Hazewinkel, 1986; Schoenmakers et al., 2000a). Similar lesions to osteochondrosis were also observed in turtles and rabbits treated with CT (Belanger et al., 1973; Tarsoly and Bucher, 1973). Chondrocyte proliferation, hypertrophy, and mineralization at the growth plate level are subject to complex autocrine, paracrine, and endocrine regulation. In the GD group, stimulation of prechondrocytes by GH and clonal expansion of chondrocytes by GH-induced IGF-I endocrine and paracrine actions support the high rates of cartilage growth (Ohlsson et al., 1998). Rapid growth rates may demand analogous rates in vitamin D₃ metabolism. Both 1,25(OH)₂D₃ and 24,25(OH)₂D₃ are produced and regulated at the growth plate level (Schwartz et al., 1992; 2001) and influence endochondral bone formation by distinct mechanisms (Boyan et al., 2001). It has been reported that 24,25(OH)₂D₃ causes resting zone cells to mature in the endochondral lineage and become responsive to 1,25(OH)₂D₃, which is further responsible for the terminal chondrocyte differentiation and mineralization of the matrix (Boyan et al., 2001). Since the GD group had relatively low plasma 24,25(OH)₂D₃ concentrations, it is reasonable to suggest that there may have been relative deficiency in 24,25(OH)₂D₃ at the growth plate level. As a consequence, this relative deficiency in 24,25(OH)₂D₃ may be responsible for the retarded maturation of chondrocytes resulting in retained cartilage cones and osteochondrosis. However, there also may be factors implicated in the pathogenesis of osteochondrosis—for example, CT that circulates in plasma at greater concentrations in the GD than in the MP group. Future studies are needed to resolve these issues.

Collectively, growing GD were characterized by greater growth rates than MP, as reflected in the 17-fold greater BW gain and the increased longitudinal skeletal growth. The differences in the plasma GH and IGF-I concentrations in the two breeds were accompanied by striking differences in vitamin D₃ metabolism. In the GD group, the high plasma GH and IGF-I concentrations may have resulted in greater plasma 1,25(OH)₂D₃ concentrations than in the MP group due to decreased metabolic clearance, rather than increased production of 1,25(OH)₂D₃. In addition, they may have inhibited production of 24,25(OH)₂D₃, resulting in plasma 24,25(OH)₂D₃ concentrations a magnitude lower than in the MP group. Differences in plasma vitamin D₃ metabolite concentrations did not influence intestinal Ca and P_i absorption. However, TRP was upregulated in the GD compared with the MP group, thereby facilitating the more positive P_i balance required for the rapid growth rates. Regardless of the growth rate, Ca balance was positive throughout the study and mineralization of the skeleton increased with age in both groups. However, in the GD group, bone turnover was significantly greater than in the MP group, with significantly increased bone resorption and formation. Disturbances in endochondral ossification due to retarded chondrocyte maturation were only prevalent in the GD group. High plasma CT concentrations and/or decreased plasma concentrations of 24,25(OH)₂D₃, the vitamin D₃ metabolite known to stimulate chondrocyte differentiation, may be responsible for this pathology that is frequently encountered in fast-growing dogs.

	Implications

Top Abstract Introduction Materials and Methods Statistical Analysis Results Discussion Implications Literature Cited

 
Growing giant-breed dogs are more susceptible to developing skeletal disorders than small-breed dogs when raised on a diet with deficient or excessive Ca content. Even at an optimal calcium intake, giant- breed dogs have more irregular growth plates with manifestation of moderate disorders of endochondral ossification than small-breed dogs. This phenomenon may be related to differential regulation of Ca homeostasis and skeletal growth between breeds. Giant-breed dogs grow rapidly and have high plasma concentrations of growth-regulating factors, which in turn may influence vitamin D₃ metabolism, whereby 1,25-dihydroxyvitamin D₃ is favored over 24,25-dihydroxyvitamin D₃. Consequently, a relative deficiency of 24,25-dihydroxyvitamin D₃ may be a pathophysiological factor responsible for the increased incidence of skeletal disorders, in addition to the potential growth factors and calcitonin. It remains to be elucidated whether vitamin D₃ supplementation would facilitate optimal growth in giant-breed dogs.

	Footnotes

 
¹ Stable metabolites of 25(OH)D₃ and 24,25(OH)₂D₃ were kindly provided by J. P. van de Velden (Solvay Pharmaceuticals, Weesp, The Netherlands). The authors would like to acknowledge the clinic attendants for care of the pups and their assistance in performing the experiments, as well as the assistance of the Biochemical Laboratory. We thank Y. Pollak for technical assistance with the GFR measurements. The critical reading of G. Voorhout is highly appreciated.

Received for publication August 15, 2002. Accepted for publication February 3, 2003.

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