24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs

M. A. Tryfonidou1, M. A. Oosterlaken-Dijksterhuis1, J. A. Mol1, T. S. G. A. M. van den Ingh2, W. E. van den Brom1, and H. A. W. Hazewinkel1

Departments of 1 Clinical Sciences of Companion Animals and 2 Pathology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A group of growing dogs supplemented with cholecalciferol (vitamin D3; HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D3/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of Pi and growth-regulating hormones between groups and no signs of vitamin D3 intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] was increased in HVitD vs. CVitD (demonstrated by [3H]1,25(OH)2D3 and increased renal 1alpha -hydroxylase gene expression), plasma 1,25(OH)2D3 levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)2D3 (demonstrated by [3H]1,25(OH)2D3 and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D3 toxicosis.

25-hydroxycholecalciferol; 1,25-dihydroxycholecalciferol; 24,25-dihydroxycholecalciferol; calcitonin; parathyroid hormone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED THAT the diet and the skin are sources of cholecalciferol (vitamin D3; see Ref. 27). Vitamin D3 is converted in the liver to 25-hydroxycholecalciferol [25(OH)D3] and with a sequential hydroxylation primarily in the kidney to 1,25-dihydroxycholecalciferol [1,25(OH)2D3] with aid of 1alpha -hydroxylase and to 24,25-dihydroxycholecalciferol [24,25(OH)2D3] with the aid of 24-hydroxylase. 1,25(OH)2D3 is the most biologically active vitamin D3 metabolite with regard to Ca metabolism and skeletal growth with a variety of target organs, including the skeleton, intestine, and kidney (7). 24,25(OH)2D3 is a biologically active metabolite mainly directed to the skeleton (6, 56) but also with putative actions at the intestine (39, 40, 59, 60).

The rate of renal synthesis of 1,25(OH)2D3 is directly responsive to plasma levels of Pi, growth hormone (GH), insulin-like growth factor I (IGF-I), parathyroid hormone (PTH), and calcitonin (CT; see Refs. 24, 36, and 41). Regulatory feedback on 1alpha -hydroxylase is provided by 1,25(OH)2D3 by induction of 24-hydroxylase activity and thus conversion of 1,25(OH)2D3 into less biologically active metabolites in its target tissues, including intestine, kidney, and bone (7, 52). In the kidney, 24-hydroxylase activity is enhanced by 1,25(OH)2D3 and downregulated by PTH (48, 62, 63), whereas in the intestine, 24-hydroxylase is enhanced by 1,25(OH)2D3 and downregulated by CT (3).

The period of rapid growth is a formidable challenge for vitamin D3 metabolism in preserving skeletal mineralization. There are few investigations in young intact animals that have studied the hormonal regulation of excessive vitamin D3 with respect to the activity of 1alpha -hydroxylase and 24-hydroxylase (4, 47, 57, 58). However, these studies confined their measurements to single-moment observations, possibly because of technical limitations. Therefore, there is insufficient knowledge concerning the time-dependent changes of vitamin D3 metabolism during elevated dietary vitamin D3 intake. Obtaining insight into the complexity of vitamin D3 homeostasis in relation to its regulating hormones and enzymes requires large research animals for long-term studies on the effect of dietary vitamin D3 supplementation. Dogs are of adequate size to allow for simultaneous and sequential sampling of blood and tissue material during the rapid growth period. Additional advantages are complete dependence on the dietary intake of vitamin D3 (29) and thus easy regulation of the vitamin D3 status without interpretation problems caused by seasonal variation of plasma vitamin D3 metabolite concentrations.

To study the time-dependent changes and interactions of hormones implicated in Ca homeostasis during long-term dietary vitamin D3 supplementation and their effect on in vivo regulation of 1alpha -hydroxylase and 24-hydroxylase, a control group of dogs was investigated vs. an ample 100-fold vitamin D3-supplemented group for 18 wk immediately after partial weaning. Calciotropic and growth-regulating hormones were measured throughout the study. The influence of long-term vitamin D3 supplementation on parathyroid chief cell and thyroid C cell function was evaluated by dynamic tests. Furthermore, the gene expression of renal 1alpha -hydroxylase and 24-hydroxylase, and intestinal 24-hydroxylase, was determined, and the production and clearance rates of 1,25(OH)2D3 were investigated.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and diets. The Utrecht University Ethical Committee for Animal Care and Use approved all procedures. Sixteen Great Danes, originating from three different litters, were divided into the following two groups at 3 wk of age: a control group (n = 9, CVitD) and a dietary vitamin D3-supplemented group (n = 7, HVitD). Pups were raised on an extruded diet formulated to be comparable in energy, Ca, and phosphate content (450 kcal, 9.5 g, and 7.5 g/100 g dry matter diet, respectively). The control diet was formulated to contain the recommended amount of 500 IU vitamin D3/kg diet (1, 38), whereas the supplemented vitamin D3 diet was formulated to contain a total of 50,000 IU vitamin D3/kg diet. Diets were analyzed for their vitamin D3 content (55) by a reference laboratory (TNO Nutrition and Food Research, Zeist, The Netherlands), and the analyzed vitamin D3 content was 470 and 54,000 IU vitamin D3/kg diet for the CVitD and HVitD, respectively, with no detectable traces of ergocalciferol. From 3 until 6 wk of age, pups received their diet as a gruel in addition to the bitch milk and received dry diet exclusively later on. Body weight was measured biweekly, and food was provided at two times maintenance energy requirements of each dog (31) for the duration of the study.

Blood measurements. At 7, 10, 13, 16, and 19 wk of age, blood samples were collected after an overnight fast. Blood samples for the measurement of plasma total Ca and Pi levels were transferred to heparin tubes, centrifuged, and measured according to standard procedures (Beckman Industries).

Blood samples for hormone analysis were immediately transferred to EDTA-coated tubes and placed on ice until centrifuged. Plasma was stored at -20°C until analysis. Quantitative determination of 25(OH)D3 and 24,25(OH)2D3 was by a modified RIA (DiaSorin, Stillwater, MN). Before processing, both labeled standards {25-hydroxy[26,27-methyl-3H]cholecalciferol and 24R,25-dihydroxy[26,27-methyl-3H]cholecalciferol (sp act 16 and 15.4 GBq/mg, respectively; Amersham Pharmacia Biotech)} were added to plasma samples and to the standards of the RIA to determine individual sample recovery. Samples were extracted two times with ethylacetate-cyclohexane (1:1 vol/vol) and one time with methanol-ethylacetate-cyclohexane (4:5:5 vol/vol/vol; see Ref. 5), and 25(OH)D3 and 24,25(OH)2D3 were separated by solid-phase extraction using NH2 cartridges (Bakerbond spe Amino Disposable Extraction Columns, J. T. Baker) according to the method described by McGraw and Hug (34). The standard curves of both stable vitamin D3 metabolites showed good parallel dilution to the standard curve of the RIA. The intra- and interassay coefficients of variation (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. 1,25(OH)2D3 was extracted from plasma using acetonitrile followed by a two-step solid-phase extraction (C18 and silica gel cartridge; Waters Chromatography B. V. Etten, Leur, The Netherlands) and quantitatively determined by a radioreceptor assay based on the method described by Reinhardt et al. (44) and Hollis (28) with intra-assay and interassay CV of 5.7 and 6.6%, respectively. PTH was measured using an immunoradiometric assay for intact PTH (Nichols Institute, San Juan Capistrano, CA; see Ref. 54). The detection limit was 1 ng/l. The intra- and interassay CV were at 40 ng/l for 3.4 and 5.6%, respectively, and at 266 ng/l for 1.8 and 6.1%, respectively. CT was measured after extraction with ethanol by a homologous RIA as described before (25) with a detection limit of 25 ng/l. The intra- and interassay CV at 175 ng/l were 15.3 and 15.4%, respectively, and at 670 ng/l 4.5 and 9.2%, respectively. GH was measured by a homologous RIA, as described previously (20). 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 (37), with intra- and interassay CV of 4.7 and 15.6%, respectively.

Dynamic tests on C cell and chief cell function. Tests were performed after overnight food deprivation with a week between tests to avoid reciprocal influences. Ca infusion tests were performed at 6, 12, and 18 wk of age, whereas EDTA infusion tests were performed at 7, 13, and 19 wk of age in all dogs.

In short, during the Ca stimulation tests, dogs were kept in a sitting position, and calcium gluconate (13.75 mg calcium gluconate/ml; Sandoz Pharma, Basel, Switzerland) was administered for 3-5 s through an indwelling catheter in the cephalic vein. The total dose was 0.28 ml calcium gluconate/kg body wt, equivalent to a dose of 2.52 mg Ca2+/kg body wt. Blood samples were taken by jugular venipuncture at -5, 0, 1, 2, 4, 8, and 16 min after the initiation of the Ca infusion.

During the EDTA infusion tests, dogs were kept in right lateral recumbency, and 30 mg Na3EDTA/kg body wt in 1 ml of 0.9% NaCl were administered at a constant infusion rate of 0.25 ml · kg body wt-1 · min-1 through an indwelling catheter in the cephalic vein. Blood samples were obtained through an indwelling catheter in the jugular vein at -5, 0, 1, 2, 4, 8, and 16 min after the initiation of the EDTA infusion. Dogs were monitored closely during the infusion for the occurrence of clinical and electrocardiographic signs of hypocalcemia.

Plasma PTH, CT, and Ca2+ levels were determined at all time points. Blood samples for the analysis of Ca2+ were collected anaerobically in heparinized syringes (PICO 50; Radiometer, Copenhagen, Denmark), placed in melting ice, and analyzed within 2 h after collection with the aid of an ionized calcium analyzer (ABL 605; Radiometer).

Preinfusion plasma levels of Ca2+, PTH, and CT measured at -5 and 0 min served as the baseline and were defined as Ca<UP><SUB>Baseline</SUB><SUP>2+</SUP></UP>, PTHBaseline, and CTBaseline, respectively. Postinfusion maximal-response plasma levels were defined in the Ca infusion tests as Ca<UP><SUB>max</SUB><SUP>2+</SUP></UP>, PTHmin, and CTmax and in the EDTA infusion tests as Ca<UP><SUB>min</SUB><SUP>2+</SUP></UP>, PTHmax, and CTmin, respectively. In each group, the areas under the curve above zero and above baseline were calculated from the stimulated values. The mean response levels above zero (RL0) and above baseline (RLBaseline) were derived from the corresponding AUC corrected for the duration of the response (t, min), i.e., AUC/t.

The Ca set point for PTH release (CaSfor PTH), i.e., the plasma Ca2+ level that inhibits PTH secretion to 50% of its maximal stimulated value, was calculated according to the model described by Brown (9). The parathyroid gland function was analyzed with a logistic model containing the four parameters equation: y = [(A - D)/1 + (x/C)a] + D, in which y is the plasma PTH level, A is the maximal plasma PTH level during hypocalcemic stimulation (i.e., PTHmax), D is the nonsuppressible plasma PTH level during hypercalcemic inhibition (i.e., PTHmin), x is the plasma Ca2+ level, C is the CaSfor PTH, and a is the slope of the logarithmically conversed mathematical function described below. The equation can be rewritten as z = (y - D)/(A - D) = 1/[1 + (x/C)a] and thus z* = (1/z- 1 = (x/C)a. Using the PTHmax and PTHmin values from the data, z and thus z* can be calculated and by applying linear regression on the logarithmically transformed variables z* and x, the values of C and a can be determined. Plasma levels obtained after the completion of the EDTA infusion were excluded from the analysis, since this would cause hysteresis (10). Because of the time delay in Ca2+-driven PTH secretion, values also obtained at t = 1 min were not used.

After euthanasia, at 21 wk of age, both left and right thyroid and parathyroid glands were removed in total and fixed in 10% buffered formalin. After fixation, a longitudinal section was made, and the material was embedded routinely in paraffin. Sections were stained with hematoxylin and eosin for routine histological investigation and were examined blindly. Activity of the parathyroid glands was estimated from the cellular and nuclear size, aspect of the cytoplasma, and number of mitotic figures. The activity of the C cells was estimated histologically from the number of C cells, the cellular and nuclear size, and the aspect of cytoplasma.

1alpha -Hydroxylase and 24-hydroxylase gene expression. Gene expression of 1alpha -hydroxylase and 24-hydroxylase was determined at the middle and the end of the study in all dogs. At 10 wk of age, five duodenal forceps biopsies were taken under endoscopic guidance with the dog under general anesthesia. In addition, at 11 wk of age, two kidney biopsies were obtained with the aid of fine needle biopsy under guidance of echography with the dog under general anesthesia. At the end of the study, i.e., at 21 wk of age, the animals were killed with an overdose of pentobarbital sodium, and biopsies from the kidney and mucosa of the proximal duodenum were sampled. Kidney and intestinal biopsies were frozen immediately in liquid nitrogen and stored at -70°C until required for RNA isolation.

Frozen intestinal and kidney tissue sampled at the middle of the study was resuspended in Qiagen lysis buffer, homogenized using a Polytron, and centrifuged for 3 min at 5,000 g at room temperature. Frozen tissue sampled at 21 wk of age was ground in liquid nitrogen prefrozen cups of a microdismembrator (Micro-Dismembrator U; B. Braun Biotech International, Melsungen, Germany) using two cycles of 45 s at 2,200 rpm. Thirty milligrams of milled tissue (kidney or intestine) were resuspended in Qiagen lysis buffer and centrifuged for 3 min at 5,000 g at room temperature. The supernatant was applied to a Qiagen minicolumn (Qiagen, Hilden, Germany), and total RNA was isolated according to the manufacturer's protocol.

RNA was ethanol precipitated and resuspended in RNAsecure (1×; Ambion, Austin, TX) that was activated at 60°C for 10 min. After DNase I treatment (DNAfree kit; Ambion), RNA was ethanol precipitated again and resuspended in 20 µl RNase-free water. Total RNA (2 and 3 µg) was used in a cDNA-synthesis reaction with 40 and 60 µl final volume (Reverse Transcription System; Promega) according to the manufacturer's instructions for the material sampled at the middle and the end of the study, respectively.

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; Bio-Rad, Hercules, CA). Data were collected and analyzed with the provided application software. For each real-time PCR reaction, 1.67 µl (of the 60-µl stock) of cDNA were used in a reaction volume of 50 µl containing 1× PCR buffer, 2 mM MgCl2, 1:100,000 dilution of SYBR green I, 10 nM fluorescein calibration dye (Bio-Rad), 200 µM dNTPs, 20 pmol forward primer, 20 pmol reverse primer, and 1.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Roche, Branchburg, NJ). Cycling conditions were optimized for the reaction of each target gene. Primer pairs (Table 1) were designed using PrimerSelect software (DNASTAR, Madison, WI).

                              
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Table 1.   Sense and antisense primer pairs used in real-time PCR

Melt curves (iCycler; Bio-Rad) and agarose gel electrophoresis were used to examine each sample for purity, and standard sequencing procedures (ABI PRISM 310 Genetic Analyzer; Applied Biosystems) were used to verify the analytical specificity of the PCR products. Standard curves constructed by plotting the log of the starting amount vs. the threshold cycle were generated using serial 10-fold dilutions of known amounts of PCR products (from a conventional PCR). The amplification efficiency, E (%) = [10(1/-s) - 1] × 100 (s = slope), of each standard curve was determined and appeared to be >90% over a large dynamic range (6-8 orders of magnitude). For each experimental sample, the amounts of target (1alpha -hydroxylase and 24-hydroxylase) and beta -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 (i.e., CVitD) to generate n-fold relative expression levels.

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 eight CVitD and seven HVitD dogs with the aid of a bolus injection with 1alpha ,25-dihydroxy[23,24(n)-3H]cholecalciferol {[3H]1,25(OH)2D3, sp act 10.5 GBq/mg; Amersham Pharmacia Biotech, UK} by techniques described previously (19, 23). In short, after an intravenous administration of ~3.7 KBq [3H]1,25(OH)2D3, blood samples were drawn at 4, 6, 8, 10, 15, 20, 30, 45, 60, and 90 min and at 2, 3, 4, 5, 6, 10, 12, and 24 h after the injection; transferred immediately to EDTA-coated tubes; and placed on melting ice until centrifuged and processed further. The plasma disappearance curve of [3H]1,25(OH)2D3 was obtained by counting plasma samples (0.5 ml) with 4 ml scintillation fluid (Ultima Gold; Packard Bioscience, Groningen, The Netherlands) in a liquid scintillation counter (1212 Rackbeta; LKB Wallac, Turku, Finland) for 30 min/sample. By means of a computerized nonlinear least-squares fitting procedure, a biexponential function C(t) = Ae-at + Be-bt was fitted to the plasma [3H]1,25(OH)2D3 concentrations. 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: MCR = Dint <UP><SUB>0</SUB><SUP>∞</SUP></UP>C(t)dt = D[(A/a) + (B/b)], where MCR is given in liters per kilogram body weight per day. The production rate (PR) of 1,25(OH)2D3 (expressed in pmol · kg body wt-1 · day-1) was derived from the formula PR = MCR × endogenous circulating 1,25(OH)2D3, where the endogenous circulating 1,25(OH)2D3 is the plasma 1,25(OH)2D3 level at 19 wk of age.

Statistical analysis. Statistical analyses were performed using SPSS for Windows 10.1 (SPSS). Homogeneity of variance was tested according to Levene. Differences between groups were analyzed by the two-sided Student's t-test. The AUC of the basal plasma PTH and CT levels were calculated for the duration of the study for both groups. Values were considered to be significant at P < 0.05. Results are presented as means ± SE. For the analysis of the pre- and postinfusion plasma levels within the group, baseline values were compared with the corresponding maximal response values by a one-sided Student's t-test for paired data. Differences in RL0, RLBaseline, and CaSfor PTH between groups were analyzed by the two-sided Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Dogs had good general health, grew well, and consumed the total amount of food that was offered daily. Consequently, energy and food intake per kilogram metabolic body weight (kg0.75) did not differ between groups. HVitD consumed ~135 times more vitamin D3/kg body wt compared with CVitD. The mean growth rate per week for the entire study period was 1.5 ± 0.4 and 1.6 ± 0.3 kg body wt/wk of age for CVitD and HVitD, respectively, and was not significantly different between groups.

Blood measurements. Plasma Ca levels did not differ between groups for the duration of the study (Fig. 1). Plasma Pi levels did not differ between groups and ranged from 2.68 ± 0.03 to 2.95 ± 0.03 mmol/l for the duration of the study. Plasma levels of 25(OH)D3 were 30- to 70-fold increased, and levels of 24,25(OH)2D3 were 12- to 16-fold increased in HVitD vs. CVitD for the duration of the study (Fig. 1). Plasma 1,25(OH)2D3 levels did not differ between groups at 7 wk of age, whereas for the remainder of the study they were decreased significantly in HVitD vs. CVitD (Fig. 1). Most basal plasma PTH levels and the PTH AUC were significantly lower in HVitD vs. CVitD for the duration of the study (Fig. 1). Basal plasma CT levels were highly variable, being mainly increased in the beginning period of the study in HVitD vs. CVitD (Fig. 1). The CT AUC for the duration of the study was increased significantly in HVitD vs. CVitD. Plasma GH and IGF-I levels did not differ between groups and increased and decreased with age from 19.7 ± 2.4 to 6.6 ± 1.1 µg/l and from 216 ± 24 to 360 ± 27 µg/l, respectively.


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Fig. 1.   Plasma levels of total Ca (A), parathyroid hormone (PTH; B), calcitonin (CT; C), 25-hydroxycholecalciferol [25(OH)D3; D], 24,25-dihyroxycholecalciferol [24,25(OH)2D3; E], and 1,25-dihydroxycholecalciferol [1,25(OH)2D3; F] in two groups of dogs raised on a diet with different vitamin D3 content (CVitD: 470 IU vitamin D3/kg diet, n = 9 and HVitD: 54,000 IU vitamin D3/kg diet, n = 7) from 3 to 21 wk of age. Data are presented as means ± SE. * P < 0.05 and dagger P < 0.01 from CVitD, as analyzed by the 2-sided Student's t-test.

Ca and EDTA infusion tests. Plasma Ca<UP><SUB>Baseline</SUB><SUP>2+</SUP></UP> levels did not differ between groups in the beginning (6 and 7 wk) and middle (12 and 13 wk) of the study (on average 1.52 ± 0.03 mmol/l), whereas they were significantly increased at the end of the study (18 and 19 wk) in HVitD vs. CVitD (i.e., 1.54 ± 0.01 vs. 1.48 ± 0.04 mmol/l, respectively). Plasma PTHBaseline levels were significantly lower in all tests and at all ages in HVitD vs. CVitD (Fig. 1). Plasma CTBaseline levels were increased significantly at 6 and 7 wk of age and were variably higher later on in the study in HVitD vs. CVitD (Fig. 1).

During the Ca infusion tests at all ages and in both groups, plasma levels of Ca<UP><SUB>max</SUB><SUP>2+</SUP></UP>, PTHmin, and CTmax were significantly different with respect to the preinfusion levels. The RL0 of PTH was at all ages significantly lower in HVitD vs. CVitD, whereas the RLBaseline of PTH did not differ between groups (Table 2). The RL0 and RLBaseline of CT were significantly higher in HVitD vs. CVitD only at 6 and 12 wk of age (Table 2).

                              
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Table 2.   Hypervitaminosis D3 in growing dogs: characteristics of the plasma PTH and CT levels during provocation with the aid of dynamic tests

During the EDTA infusion tests at all ages and in both groups, plasma levels of Ca<UP><SUB>min</SUB><SUP>2+</SUP></UP>, PTHmax, and CTmin were significantly different with respect to the preinfusion levels. The RL0 of PTH was lower in HVitD vs. CVitD only at 7 and 19 wk of age, whereas the RLBaseline of PTH was significantly lower in HVitD vs. CVitD only at 19 wk of age (Table 2). Both RL0 and RLBaseline of CT were higher in HVitD vs. CVitD only at 7 wk of age (Table 2).

The CaSfor PTH was significantly lower in HVitD vs. CVitD at the beginning and middle of the study, whereas is did not differ between groups at the end of the study (Fig. 2).


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Fig. 2.   The Ca set point for PTH release (CaSfor PTH in mmol/l) presented as mean ± SE and the sigmoid relationship between fractional PTH release and plasma Ca2+ levels (in mmol/l) during hypervitaminosis D3 in growing dogs from partial weaning at 3 wk until 21 wk of age. *P < 0.05 and dagger P < 0.01 from CVitD, as analyzed by 2-sided Student's t-test.

There were no obvious histological differences with respect to the parathyroid glands and the C cell compartment of the thyroid between groups.

Gene expression levels of 1alpha -hydroxylase and 24-hydroxylase. At all ages, gene expression levels of renal 1alpha -hydroxylase, 24-hydroxylase, and intestinal 24-hydroxylase were increased significantly in HVitD vs. CVitD, respectively (Fig. 3). There were no age-dependent differences within each group.


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Fig. 3.   Gene expression levels of renal 1alpha -hydroxylase (A), 24-hydroxylase (B), and intestinal 24-hydroxylase (C) during hypervitaminosis D3 in growing dogs from 3 to 21 wk of age. Data are expressed as mean n-fold relative expression levels + SE; *P < 0.05 and dagger P < 0.01 vs. CVitD at the same age, as analyzed by 2-sided Student's t-test. There were no differences within groups, as analyzed with the paired Student's t-test.

MCR and PR of 1,25(OH)2D3. At 19 wk of age, MCR and PR of 1,25(OH)2D3 were significantly higher in HVitD vs. CVitD (Fig. 4).


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Fig. 4.   Metabolic clearance rate (MCR) and production rate (PR) of 1,25(OH)2D3 at 19 wk of age during hypervitaminosis D3 in growing dogs from partial weaning at 3 wk until 21 wk of age. Data are presented as means ± SE; n = 7. *P < 0.05 and dagger P < 0.01 from CVitD, as analyzed by 2-sided Student's t-test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary 135-fold vitamin D3 supplementation during growth had striking influences on the main vitamin D3 metabolites in plasma and was accompanied by hypoparathyroidism, lowered CaSfor PTH, and hypercalcitoninemia. Plasma Pi, GH, and IGF-I levels could be excluded as differentially regulating factors, since they did not differ between groups, leaving Ca2+, PTH, CT, vitamin D3 intake, and vitamin D3 metabolites as the main regulating factors in vitamin D3 metabolism for the total duration of the study.

Effects of hypervitaminosis D3 on the main vitamin D3 metabolites. At states of excessive vitamin D3 as in HVitD, the abundant substrate is metabolized by the loosely regulated 25-hydroxylase (53), and plasma 25(OH)D3 levels increase (47). In HVitD, the 12- to 16-fold increased plasma 24,25(OH)2D3 levels were attributed to the increased renal 24-hydroxylase activity indicated by the striking 10.5- and 6-fold increase of the renal 24-hydroxylase gene expression levels at both measure points compared with CVitD. Plasma 25(OH)D3 and 24,25(OH)2D3 levels may rise far above the binding capacity of the vitamin D-binding protein (DBP) and displace 1,25(OH)2D3 from DBP, resulting in an increase of the free plasma 1,25(OH)2D3 levels (43). The latter may have resulted in increased biological activity of 1,25(OH)2D3 without a concomitant increase in the total plasma 1,25(OH)2D3 levels. The lowered total plasma 1,25(OH)2D3 levels in HVitD were mainly a consequence of increased MCR of 1,25(OH)2D3 in HVitD vs. CVitD with conversion of 1,25(OH)2D3 into less biologically active products (33), including 1,24,25-trihydroxycholecalciferol (14). In support, the significantly increased renal and intestinal 24-hydroxylase gene expression indicated upregulation of the 24-oxidation pathway. At the same time, in HVitD, the PR of 1,25(OH)2D3 was increased significantly, being in accordance with the 9.3- and 8-fold increases in the renal 1alpha -hydroxylase gene expression at both measure points compared with CVitD. Renal synthesis of 1,25(OH)2D3 is regulated tightly by Ca, 1,25(OH)2D3, PTH, and CT (7, 35, 36). Increased plasma Ca2+ levels at the end of the study did not seem to affect the production of 1,25(OH)2D3 in HVitD, whereas the decreased plasma 1,25(OH)2D3 levels indicated withdrawal of homologous negative feedback on the production of 1,25(OH)2D3. Plasma PTH levels were decreased significantly for the duration of the study in HVitD and thus can be excluded as a relevant upregulating factor of the production of 1,25(OH)2D3 (49). Plasma CT levels were, however, at high levels in HVitD, being a potential upregulator of the production of 1,25(OH)2D3 (49). In accordance, Beckman et al. (4) hold increasing plasma CT levels during hypercalcemia responsible for stimulating the production of 1,25(OH)2D3. It seems conceivable to suggest that the stimulatory effect of CT on 1,25(OH)2D3 production is an additional defensive mechanism in growing animals to maintain positive Ca balance required for optimal skeletal mineralization.

24-Hydroxylase is a potent key regulator. In hypervitaminosis, D3 24-hydroxylase seems to play a counterregulating key role (3, 4). 24-Hydroxylase has been reported to be upregulated by 1,25(OH)2D3 at the transcriptional level (61) and downregulated by PTH (62, 63), more so at the kidney level than at the intestinal level (48) and by CT at the intestinal level (3). Relevant upregulators of 24-hydroxylase may have been the increased free plasma 1,25(OH)2D3 levels, as well as 25(OH)D3, which has been reported to act directly on target organs of 1,25(OH)2D3 both at supraphysiological (12) and physiological concentrations (16, 26). Hypoparathyroidism in HVitD and thus reduction of the downregulatory effect of PTH on kidney 24-hydroxylase assured an active 24-hydroxylase pathway for the regulation of the circulating 25(OH)D3 and 1,25(OH)2D3. Hypercalcitoninism resulted accordingly in HVitD in an increment of 24-hydroxylase gene expression of a smaller magnitude on an intestinal than on a renal level. In summary, the increased renal synthesis of 1,25(OH)2D3 was provided with an effective means of its deactivation by upregulation of the 24-oxidation pathway in target organs (intestine and kidney), and consequently plasma 1,25(OH)2D3 levels were lower in HVitD than in CVitD, as has been reported to occur in rats by Beckman et al. (4).

Effects of hypervitaminosis D3 on PTH secretion and production. Although basal plasma PTH levels and RL0 of PTH induced by an elevation or decrease of Ca2+ were lower in HVitD vs. CVitD, the RLBaseline of PTH induced by elevation of Ca2+ did not differ between groups. This indicated that, in HVitD, regardless of the lower basal PTH secretion levels, the chief cells retained their responsiveness to an elevation or decrease of Ca2+. Exceptionally, at the end of the study, RLBaseline of PTH induced by a decrease of Ca2+ was significantly lower in HVitD vs. CVitD, indicating a decrease in the maximal secretion rate of PTH and a probable initiation of hypoparathyroidism. The decreased PTH secretion rate resulting in decreased basal plasma PTH levels may be attributed either to increased sensitivity of the parathyroid gland to any given plasma Ca2+ level, to a decrease in PTH production, to an adjustment of the degradation of the newly synthesized PTH at the chief cell level, or any combination. This consideration is rather tentative, since PTH gene expression levels and the secretory profile of the parathyroid gland are not available (i.e., intact PTH in relation to carbon-terminal PTH fragments). Increased sensitivity of the parathyroid gland in the beginning and middle of the study is indicated by the significantly lower CaSfor PTH in HVitD vs. CVitD. The shift of the CaSfor PTH to the left can be attributed to induction of the chief cell Ca-sensing receptor (CaR; see Ref. 11). Accordingly, 1,25(OH)2D3 administration has been reported to result in a shift of the CaSfor PTH to the left (17, 18, 32) and in increased CaR gene expression of the parathyroid gland in vitamin D-deplete and -replete rats (8, 13). At a lower CaSfor PTH, the production of PTH is not necessarily decreased, although increased free plasma 1,25(OH)2D3, the abundantly circulating 25(OH)D3, and increased plasma Ca2+ levels have been reported to have an inhibiting effect on the production of PTH (50, 51). Histological evaluation of the activity of the parathyroid glands at the end of the study did not show any obvious differences between HVitD and CVitD dogs. A modified demand for intact biologically active PTH may also have been achieved by adjustment of the parathyroid secretion profile toward an increased secretion of carbon-terminal fragments of PTH, as reported in hypercalcemic states or treatment with 1,25(OH)2D3 (15) without necessarily a decrease in production of PTH.

Effects of hypervitaminosis D3 on CT secretion. The principal regulator of CT secretion is the increase in plasma Ca2+ levels (2). Only in the beginning and middle of the study were the increased CT secretion and increased responsiveness of C cells to stimuli indicated by the significantly higher RL0 and RLBaseline of CT in HVitD vs. CVitD during stimulation or depression of CT secretion. These findings were independent of the basal plasma Ca2+ levels and indicated increased CT production with or without C cell hyperplasia (22, 30, 42). However, C cell hyperresponsiveness seemed to be elapsing with the duration of the study, since the differences in CT secretion and responsiveness between groups were diminished at the end of the study. The latter was also verified by histological evaluation at the same time point. Responsiveness of the C cells to changes of the plasma Ca2+ levels and the production of CT may be mediated by the extracellular CaR of C cells (11, 21). It remains to be elucidated which positive regulator or mediator may have resulted in increased secretion of CT and responsiveness of the C cells in HVitD, including endocrine systems directly or indirectly connected to bone metabolism (45). The biological significance of an increase in CT during states of positive Ca balance, as during growth, is mainly directed at avoiding hypercalcemia by decreasing osteoclastic resorption and enhancing deposition of Ca in bone (46).

Conclusively, during 135-fold vitamin D3 supplementation in growing dogs despite the increase in 1,25(OH)2D3 production, plasma 1,25(OH)2D3 levels were decreased as a result of an apparently even greater catabolism of 1,25(OH)2D3 by 24-hydroxylase. Downregulation of the PTH secretion and hypercalcitoninism provided extra protection against hypercalcemia by cessation of bone resorption and thus reduction of the liberation of Ca. There was only a slight increase in plasma Ca2+ levels at the end of the study, suggesting that the total burden of vitamin D3 might have reached a critical stage. However, there was no clear shift toward a vitamin D3 toxic state with the typical clinical signs of impaired growth, hypercalcemia and hyperphosphatemia, and kidney failure. In spite of the 135-fold vitamin D3 supplementation, HVitD dogs grew well and retained normophosphatemia and normal plasma total Ca levels for most of the study. Efficient hormonal counteraction with a key role for 24-hydroxylase prevented the development of vitamin D3 toxicosis during the course of the study.


    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). We acknowledge the clinic attendants for good care of the pups and their assistance in performing the experiments, and the assistance of the Biochemical Laboratory and the Department of Anaesthesiology. Drs. E. den Hertog, M. Diaz, A. van Dongen, and G. Voorhout are acknowledged for assistance in sampling the intestinal and kidney biopsies. We thank Dr. J. E. van Dijk for excellent assistance in histological evaluation.


    FOOTNOTES

Address for reprint requests and other correspondence: M. A. Tryfonidou, Dept. of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht Univ., Yalelaan 8, 3584 CM Utrecht, The Netherlands (E-mail: M.A.Tryfonidou{at}vet.uu.nl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 19, 2002;10.1152/ajpendo.00236.2002

Received 6 May 2002; accepted in final form 24 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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