![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands;
*
TNO Nutrition and Food Research, 3700 AJ Zeist, The Netherlands;
Department of Internal Medicine, Erasmus MC Rotterdam, 3000 DR Rotterdam, The Netherlands; and
**
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706
1To whom correspondence should be addressed. E-mail: M.A.Tryfonidou{at}vet.uu.nl.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-hydroxylase gene expression compared with cVitD dogs and compensated to a certain extent for the accelerated MCR of 1,25(OH)2D3. The moderately decreased plasma 1,25(OH)2D3 concentration can only partially explain the decreased Ca absorption in the hVitD dogs. Intestinal vitamin D receptor concentrations did not differ between groups and did not account for the decreased Ca absorption. We suggest that 24,25(OH)2D3 may downregulate Ca absorption.
KEY WORDS: • 1,25-dihydroxycholecalciferol • 24,25-dihydroxycholecalciferol • 45Ca balance • vitamin D receptor • dogs
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
), whereas after weaning, passive absorption decreases and active hormonally regulated absorption becomes more prominent (1
,2
). Cholecalciferol (vitamin D3) is essential for the hormonal regulation of intestinal Ca absorption. After its first hydroxylation in the liver into 25-hydroxycholecalciferol [25(OH)D3],2
it is hydroxylated mainly in the kidney into 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] with the aid of 1-hydroxylase and 24-hydroxylase, respectively. The upregulating effects of 1,25(OH)2D3 on active and passive Ca absorption have been well described (3
) although there is disagreement concerning the latter (4
). The biological activities of 1,25(OH)2D3 at the intestinal level are mediated mainly by binding to a high affinity receptor (vitamin D receptor; VDR) (5
). In addition, there is evidence that 1,25(OH)2D3 has rapid, nongenomic effects on Ca absorption (i.e., transcaltachia) that have been reported only in chicks and are transient, modulated locally and inconsistent with the plasma 1,25(OH)2D3 concentrations (4
). The endocrine effects of 24,25(OH)2D3 on Ca absorption have been demonstrated in several studies (6
–8
), including downregulation of VDR-related and of nongenomic effects of 1,25(OH)2D3 on Ca absorption (9
).
The in vivo effects of vitamin D3 toxicity are well established (10
–12
). The effects of vitamin D3 supplementation have been studied extensively in the elderly and children with vitamin D3 deficiency and/or negative Ca balance, including its effects on osteoporosis, renal failure and rickets (13
–15
). However, there is a lack of information concerning long-term effects of moderate vitamin D3 supplementation on the hormonal regulation of Ca metabolism in healthy, growing individuals in positive Ca balance.
Skeletal growth at high rates is a formidable challenge for both Ca and vitamin D3 metabolism, and any disturbance may have important consequences. Ca metabolism in relation to skeletal growth and development has been studied in different animals, including dogs (16
,17
). Because interlaboratory CV for mean plasma 25(OH)D3 concentrations may be 38% (18
), absolute plasma concentrations cannot be compared. However, pathophysiologic mechanisms studied in dogs may well serve for extrapolation to humans (19
,20
). Dogs are convenient models with which to study the influence of vitamin D3 on Ca metabolism because they are completely dependent on dietary intake of vitamin D3 (21
). This is attributed to the restricted availability of the vitamin D3 precursor, 7-dehydrocholesterol, in the skin. Because vitamin D3 status in dogs is easily controlled, seasonal variations of plasma vitamin D3 metabolite concentrations do not exist. The Great Dane breed provides a very attractive research model because it undergoes in a short time a period of gradual growth after weaning until 6 mo of age, simulating the growth curves of children. This dog breed has a body weight (BW) of 
6 kg at weaning and of 70 kg at growth-plate closure, a stage reached in a 12- to 15-mo period. In addition, its BW allows for sequential and simultaneous sampling of material for long-term studies.
To evaluate the long-term effects of moderate dietary vitamin D3 supplementation on the hormonal regulation of intestinal Ca absorption during growth, two groups of dogs were studied, i.e., a control group vs. an eightfold dietary-supplemented vitamin D3 group. Dogs were studied from weaning at 6 wk until 4 mo of age and Ca absorption, and calciotropic and growth-regulating hormones were measured. In addition, the production and metabolic clearance of 1,25(OH)2D3 were investigated.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Male (n = 6) and female (n = 8) pure-bred Great Dane dogs (Utrecht University, The Netherlands), originating from three different litters, were divided into two groups at 3 wk of age: a control group (n = 7, cVitD) and a dietary vitamin D3-supplemented group (n = 7, hVitD). Pups were fed one of two dry pellet diets (Table 1
) formulated to be comparable in energy, Ca and phosphate (P) concentrations as confirmed by analysis of (Table 2
). The control diet contained the recommended amount of 12.5 µg vitamin D3/kg diet (22
,23
), whereas the supplemented vitamin D3 diet contained a total of 100 µg vitamin D3/kg diet. Diets were analyzed for their vitamin D3 content (24
) in a reference laboratory (TNO Nutrition and Food Research, Zeist, The Netherlands). At 3 wk of age, partial weaning began and the pups received their diet as a gruel in addition to the bitch milk. Pups received their dry diet exclusively by 6 wk of age. Food intake was adapted biweekly to actual BW and provided at two times maintenance energy requirements of each dog (25
) for the duration of the study. The Utrecht University Ethical Committee for Animal Care and Use approved all procedures.
|
|
At 14 and 20 wk of age, true Ca absorption (Va) was measured with the aid of one tracer, i.e., 45Ca, by techniques described previously and validated for dogs (26
,27
). The use of only one tracer is justified by the following observations: 1) after intravenous (IV) administration of 45Ca, the endogenous fecal excretion (Vf) of 45Ca is measurable for a maximum of 3 d; 2) after oral administration of 45Ca, the nonabsorbed tracer (VF) is measurable for at most 4 d in the feces; and 3) the contribution of Vf by the tracer to VF is negligible. Therefore, only one tracer can be used in the balance studies provided that the oral 45Ca dose is given at least 3 d after the IV administration and with the assumption that the Ca balance is in a steady state during the 7-d investigation period.
In short, dogs were individually housed in metabolic cages during the 45Ca balance studies. The actual food intake was measured and Ca intake (VI) was calculated for each investigation period. The endogenous fecal excretion of 45Ca (Vf) was determined by collection of the feces for 3 consecutive days after an IV dose of 0.25 and 0.4 MBq 45Ca as 45CaCl2 in water (specific activity 271.88 MBq/mg, NEN Life Science Products, Boston, MA) at 14 and 20 wk of age, respectively. Vf was calculated from the quotient of the part of the injected dose (R3) that was excreted in the feces over 3 d and the integral of plasma specific activity C(t) according to the following formula:
 |
On d 4 of each metabolic study, a dose of 45Ca equivalent to the IV dose was administrated orally and feces were collected for 4 consecutive days. The fecal content of 45Ca was determined to calculate the total fecal content of Ca (VF). Va was calculated by the formula Va = VI - (VF - Vf). All parameters were expressed in mmol Ca/(kg body · d). The fractional absorption of Ca () was defined as = Va/VI.
Feces were collected and pooled for each part of the 3- and 4-d investigation periods, weighed and homogenized with a known quantity of distilled water with a blender. Triplicate samples were dried for 24 h at 60°C, weighed, ashed for 6 h at 550°C, resuspended in 10 mL of hydrochloric acid (37% v/v) and then distilled water was added to a total volume of 100 mL. The activity of 45Ca was determined in 0.4 mL of the supernatant mixed thoroughly with 4 mL scintillation fluid (Ultima Gold, Packard Bioscience BV, Groningen, The Netherlands) in a liquid scintillation counter (1212 Rackbeta, LKB Wallac, Turku, Finland).
Blood measurements.
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 and inorganic phosphate (Pi) concentrations were transferred into heparin tubes, centrifuged at 3000 x g for 10 min at 4°C and measured according to standard procedures (Beckman Industries, Brea, CA).
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.
25(OH)D3 and 24,25(OH)2D3 were determined quantitatively 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 (specific activity 16 GBq/mg and 15.4 GBq/mg, respectively, Amersham Pharmacia Biotech, UK), were added to plasma samples and to the standards of the RIA to determine individual sample recovery. Samples were extracted twice with ethylacetate:cyclohexane (1:1, v/v) and once with methanol:ethylacetate:cyclohexane (4:5:5, v/v/v) (28
), 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, Phillipsburg, NJ) according to the method of McGraw and Hug (29
). The standard curves of both stable vitamin D3 metabolites paralleled the standard dilution curve of the RIA. 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.
1,25(OH)2D3 was extracted from plasma using acetonitrile followed by a two-step solid-phase extraction (C18 and Silicagel 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. (30
) and Hollis (31
) with an intra- and interassay CV of 5.7 and 6.6%, respectively.
Parathyroid hormone (PTH) was measured using an immunoradiometric assay for intact PTH (iPTH, Nichols Institute, San Juan Capistrano, CA) (32
). The intra- and interassay CV were 3.4 and 5.6%, respectively. Calcitonin (CT) was measured after extraction with ethanol by a homologous RIA (33
). The intra- and interassay CV were 15.3 and 15.4%, respectively.
Growth hormone (GH) was measured by a homologous RIA (34
). The intra- and interassay CV were 3.8 and 7.2%, respectively. Insulin-like growth factor I (IGF-I) was measured by a heterologous RIA as described previously (35
) with intra- and interassay CV of 4.7 and 15.6%, respectively. At 21 wk of age, GH and IGF-I were not determined.
Endogenous metabolic clearance rate (MCR) and production rate (PR) of 1,25(OH)2D3.
At 19 wk of age, the MCR of 1,25(OH)2D3 was determined with the aid of a bolus injection of 1, 25-dihydroxy[23,24(n)-3H]cholecalciferol [3H-1,25(OH)2D3, specific activity 10.5 GBq/mg, Amersham Pharmacia Biotech, UK] by techniques described previously (36
,37
). In short, after an IV 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 and were immediately transferred into EDTA-coated tubes and put on ice until centrifuged and further processed. 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 BV) in a liquid scintillation counter (1212 Rackbeta, LKB Wallac) for 30 min per sample. Using 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 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:
 |
where MCR is expressed in L/(kg body · d). The PR 1,25(OH)2D3 was derived from the formula: PR [pmol/(kg body · d)] = MCR [L/(kg body · d)] · endogenous circulating 1,25(OH)2D3 (pmol/L), where the endogenous circulating 1,25(OH)2D3 is the plasma 1,25(OH)2D3 concentration at 19 wk of age.
1-hydroxylase gene expression.
Gene expression levels of 1-hydroxylase were determined at the middle and the end of the study in all dogs. At 11 wk of age, two kidney biopsies were obtained by 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 dogs were killed with an overdose of Na-pentobarbital and biopsies from the kidney were sampled. Kidney biopsies were immediately frozen in liquid nitrogen and stored at -70°C until required for RNA isolation.
RNA isolation and cDNA synthesis.
Frozen kidney tissue sampled at 11 wk of age was resuspended in Qiagen lysis buffer, homogenized using a Polytron, and centrifuged for 3 min at 5000 x g at room temperature. Frozen kidney tissue sampled at 21 wk of age was ground in liquid nitrogen prefrozen cups of a micro-dismembrator (Micro-Dismembrator U, B. Braun Biotech International GmbH, Melsungen, Germany) using 2 cycles of 45 s at 2200 rpm. Milled tissue (30 mg) was resuspended in Qiagen lysis buffer and centrifuged for 3 min at 5000 x g at room temperature. The supernatant was applied to a Qiagen mini-column (Qiagen GmbH, Hilden, Germany) and total RNA was isolated according to the manufacturer’s protocol.
RNA was ethanol precipitated and resuspended in RNAsecure (1X; Ambion, Austin, TX), which was activated at 60°C for 10 min. After DNase I treatment (DNAfreeTM kit, Ambion), RNA was again ethanol precipitated and resuspended in 20 µL of RNase-free water. Total RNA (2 and 3 µg) was used in a cDNA-synthesis reaction with a final volume of 40 and 60 µL (Reverse Transcription System, Promega, Madison, WI) according to the manufacturer’s instructions, for the material sampled at the middle and the end of the study, respectively.
Real-time polymerase chain reaction (PCR).
Real-time PCR, based on the high affinity double-stranded DNA-binding dye SYBR green I (SYBR green I, BMA, Rockland, ME), was performed in triplicate in a spectrofluorometric thermal cycler (iCycler, BioRad). Data were collected and analyzed with the application software provided. For each real-time PCR reaction, 1.67 µL (from the 40- or 60-µL stock) of cDNA was used in a reaction volume of 50 µL containing 1X PCR buffer, 2 mmol/L MgCl2, 1:100,000 dilution of SYBR green I, 10 nmol/L fluorescein calibration dye (Bio-Rad, Hercules, CA), 200 µmol/L dNTP, 20 pmol forward primer, 20 pmol reverse primer, and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Roche, Branchburg, NJ). Cycling conditions were optimized for the reaction of each target gene. Primer pairs were designed using PrimerSelect software (DNASTAR, Madison, WI). The primer pair of ß-actin based on the human sequence (M10277) distinguishes cDNA from pseudogene DNA and the sense (F) and antisense (R) primers for its amplification were 5'-GATATCGCCGCGCTCGTCGTC-' (exon 2; 1102–1122) and 5'-GGCTGGGGTGTTGAAGGTCTC-3' (exon 4; 2038–2058), respectively. Based on the human sequence of 1-hydroxylase (AB005989) the F and R primers 5'-CCGGAGCTTGGCAGACATC-3' (exon 1; 103–121) and 5'-GAGGAGGAGCGGGGCCAGGAGACT-3' (exon 3;417–440), respectively, were used for amplification of a 337-bp fragment. Based on the homologous canine sequence of this PCR product, a 134-bp fragment was designed with the F and R primers 5'- GGCGGCCCCTGCCCTCGTC -3' (exon 2) and 5'-CCTCGCCTTCCGCGGTGAGCA-3' (exon 2,3), respectively.
Melt curves (iCycler, BioRad) 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] x 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 1-hydroxylase and ß-actin as endogenous references 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 (cVitD group) to generate relative expression levels.
VDR analysis.
Nuclear extracts were prepared as described by Sandgren and DeLuca (38
). Briefly, at the end of the study (i.e., at 21 wk of age) after killing and immediate laparotomy, the mucosal layer was sampled from the following defined intestinal segments: proximal and distal duodenum, beginning, middle and end of the jejunum, ileum and colon. Segments were rinsed with ice-cold TED saline (50 mmol/L Tris-HCl, pH 7.4, 1.5 mmol/L EDTA, 5 mmol/L dithiothreitol, 10 mg/L TPCK, 1 mg/L leupeptin, 1 mg/L pepstatin) and the mucosal layer was scraped and washed three times in TED saline by centrifugation for 10 min at 3000 x g and 4°C. Samples were homogenized in 4 volumes (wt/v) KTED-300 (TED, 300 mmol/L KCl) in a glass Teflon homogenizer. Homogenates were centrifuged at 100,000 x g for 1 h at 4°C and supernatants (nuclear extracts) were stored at -70°C until further assayed. The concentration of VDR was measured by a VDR-specific ELISA (39
) and expressed as fmol/mg mucosal protein.
Statistical analysis.
Statistical analyses were performed using the SPSS for Windows 10.1 (SPSS Inc, Chicago, IL). Differences within groups were analyzed in a repeated measures ANOVA. Differences between groups were analyzed by the unpaired two-tailed Student’s t test, after testing for homogeneity of variance with Levene’s test. The significance of differences in VDR concentration between groups was tested by the nonparametric Wilcoxon-Mann-Whitney U test because of the considerably high SD of the mean (i.e., in the majority >50% of the mean). Differences were considered significant when P < 0.05. Results are presented as means ± SEM.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Ca absorption.
At all ages, VI did not differ between groups (Table 3
). The Vf did not differ between groups at 14 wk of age, whereas at 20 wk of age, it was significantly higher in the hVitD group than in the cVitD group. At both ages, Va and were significantly lower in the hVitD group than in the cVitD group.
|
The hVitD group consumed approximately 8 times as much vitamin D3/kg BW as the cVitD group (Table 3)
. Plasma Ca concentrations did not differ between groups throughout the study, ranging from 2.80 ± 0.05 to 3.00 ± 0.02 mmol/L. Similarly, plasma P concentrations did not differ and ranged from 2.71 ± 0.05 to 2.96 ± 0.03 mmol/L. Plasma 25(OH)D3 concentrations did not differ significantly between groups for the duration of the study (Fig. 1
). In the hVitD group, plasma 24,25(OH)2D3 concentrations were significantly greater than the cVitD group beginning at the age of 13 wk (Fig. 1)
. In the hVitD group, plasma 1,25(OH)2D3 concentrations were significantly lower at 10, 13 and 21 wk of age than in the cVitD group (Fig. 1)
. Plasma PTH, CT, GH and IGF-I concentrations did not differ between groups throughout the study and stayed within limits of the reference values in growing Great Dane dogs (35
,40
).
|
At 19 wk of age, the MCR and PR of 1,25(OH)2D3 were significantly greater in the hVitD group than in the cVitD group (Fig. 2
).
|
-hydroxylase.
At 11 wk of age, renal gene expression levels of 1-hydroxylase did not differ between groups (data not presented), whereas at 21 wk of age, they were 12.9-fold greater (with P < 0.01) in the hVitD group compared with the cVitD group.
Intestinal VDR concentration.
At the end of the study, VDR concentrations did not differ between groups and decreased gradually and progressively in the distal segments of the intestine in both groups, ranging from 349 ± 35 fmol/mg protein in the proximal duodenum to 92 ± 20 fmol/mg protein in the colon.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) and had growth curves, radiological and histological skeletal development (not shown here), and Ca metabolism consistent with previous studies in healthy growing Great Danes (35
). The hVitD group also grew well with growth rates similar to those of the cVitD group (41
). Chronic intake of food fortified with 8 times the amount of vitamin D3 had major influences on the formation of the biologically important vitamin D3 metabolites and resulted in downregulation of intestinal Ca absorption. The mechanisms implicated in the depression of intestinal Ca absorption are discussed.
Fractional Ca absorption () was significantly lower in the hVitD compared with the cVitD group during the study and was reflected in the slightly but significantly increased Vf at the end of the study (42
). Despite the lower , the hVitD group retained its Ca homeostasis as indicated by maintenance of plasma PTH concentrations at control levels (43
). Vitamin D3 metabolism in relation to Ca absorption is influenced by a wide variety of minerals and hormones of which plasma Ca, Pi, PTH, CT, GH, IGF-I and vitamin D3 metabolites concentrations are the most important. Because these concentrations did not differ between groups during the study, we assume that they did not differently affect vitamin D3 metabolism in the two groups. The high growth rate and corresponding high GH and IGF-I levels may contribute to the higher plasma 1,25(OH)2D3 concentrations in Great Dane dogs (44
) when compared with healthy children (i.e., 101 ± 31 pmol/L) (45
). It remains to elucidated whether vitamin D3 metabolism differs during growth at high and slow growth rates.
The decreased Ca absorption in the hVitD group is most likely explained by the only difference between the groups, i.e., the vitamin D3 intake and consequently, the different plasma concentrations of the vitamin D3 metabolites. Surprisingly, the hVitD group had plasma 25(OH)D3 concentrations not differing from the control group, plasma 1,25(OH)2D3 concentrations lower than the cVitD group at 10, 13 and 21 wk of age, and plasma 24,25(OH)2D3 concentrations significantly higher than the cVitD group starting at 13 wk of age. Plasma 25(OH)D3 concentrations are influenced by a variety of factors, including vitamin D3 intake (46
), successive hydroxylation into 1,25(OH)2D3 and 24,25(OH)2D3 (47
), and negative feedback from 1,25(OH)2D3 on 25-hydroxylation (48
). None of the factors regulating plasma 1,25(OH)2D3 and 24,25(OH)2D3 concentrations differed between groups except for vitamin D3 intake (49
). Thus, during positive Ca balance, high vitamin D3 intake (as in the hVitD group) enhances 24-hydroxylase activity and results in enhanced production of 24,25(OH)2D3 (11
,12
) and probably competitive inhibition of the synthesis of 1,25(OH)2D3 from 25(OH)D3 as has been suggested in Ca-deficient rats (50
). Accordingly, between 10 and 13 wk of age in the hVitD group, plasma 24,25(OH)2D3 concentrations started to increase, whereas plasma 1,25(OH)2D3 concentrations were lower than in the cVitD group. The MCR of 1,25(OH)2D3 at 19 wk of age was significantly higher in the hVitD group than in the cVitD group, reflecting the upregulated 24-oxidation pathway of 1,25(OH)2D3 and resulting in downregulation of plasma 1,25(OH)2D3 concentrations. The latter is consistent with earlier reports of in vivo studies in rats (11
,51
). To counterbalance the increased MCR, the PR of 1,25(OH)2D3 increased in the hVitD group; this was demonstrated at 19 wk of age by using [3H]-1,25(OH)2D3 and by the 12.9-fold increase in the renal gene expression levels of 1-hydroxylase at 21 wk of age compared with the cVitD group. Consequently, the combination of enhanced production of 24,25(OH)2D3 and 1,25(OH)2D3 resulted in successive hydroxylation of the substrate 25(OH)D3 and the maintenance of the plasma 25(OH)D3 concentrations in the hVitD group at the same levels as those in the cVitD group.
The attenuated in the hVitD group during the in vivo 45Ca balance studies can be attributed in part to a decreased biologic effect of the moderately decreased plasma 1,25(OH)2D3 concentrations. Biological activity of nuclear hormones, including 1,25(OH)2D3, is mediated by regulation of their receptor levels in target tissues. Intestinal VDR concentrations in the hVitD group were maintained at the same levels as in the cVitD group and thus did not account for the observed attenuation of in the hVitD group. The most important determinant of has been reported to be the plasma 1,25(OH)2D3 concentration in healthy women (52
). Regression analysis reported by Morris et al. (52
) predicted an 18.7% difference in to be related to a 60% decrease in plasma 1,25(OH)2D3 concentrations. Consequently, at 13 wk of age in the hVitD group, it is questionable whether the 18% decreased plasma 1,25(OH)2D3 concentration decreased to the same magnitude, i.e., 18.7%. Accordingly, at 20 wk of age in the hVitD group, plasma 1,25(OH)2D3 concentrations similar to the cVitD group were accompanied by a 15% lower . Thus, in the hVitD group, the decrease in is not adequately explained by the moderate decrease in plasma 1,25(OH)2D3 concentrations. The remainder of the difference in between the cVitD and hVitD group may be due to a counterregulating effect of the increased plasma 24,25(OH)2D3 concentrations on intestinal Ca absorption. Although disputed by others (53
), 24,25(OH)2D3 has been suggested to have endocrine effects on the intestine, in addition to the described actions on bone, cartilage and fracture healing (54
). The effects of 24,25(OH)2D3 on intestinal Ca absorption can be executed by 1) an allosteric effect of 24,25(OH)2D3 on 1,25(OH)2D3 binding to the VDR (9
), 2) a binding domain/receptor for 24,25(OH)2D3 independent of the VDR (6
), and 3) a suppressive effect of 24,25(OH)2D3 on the rapid nongenomic actions of 1,25(OH)2D3 on P absorption (7
) and transcaltachia (8
) in the intestine. Transcaltachia has so far been reported only in chicks and whether it is applicable in mammalian species requires further study. Therefore, in the hVitD group a putative inhibitory effect of the elevated plasma 24,25(OH)2D3 concentrations on intestinal Ca transport can be expected.
In conclusion, 24-hydroxylase activity is the key to explain the changes in vitamin D3 metabolites and in Ca absorption during chronic moderate vitamin D3 supplementation. Further studies of 24-hydroxylase activity are necessary before all questions are answered. The results of this study indicate that chronic dietary vitamin D3 supplementation of growing dogs in positive Ca balance has a downregulating effect on Ca absorption accompanied by moderately decreased plasma 1,25(OH)2D3 and increased plasma 24,25(OH)2D3 concentrations. The vitamin D3–supplemented group retained its Ca homeostasis, as indicated by the maintenance of the plasma PTH concentrations within control levels. The attenuated Ca absorption is only partially due to the low plasma 1,25(OH)2D3, a consequence of accelerated degradation mediated by 24-hydroxylase. In addition, 24,25(OH)2D3 may have a downregulating effect on intestinal Ca absorption in growing dogs mediated either by its own receptor or an allosteric effect on the VDR.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
, fractional Ca absorption; BW, body weight; CT, calcitonin; cVitD, control vitamin D3; GH, growth hormone; hVitD, high vitamin D3; IGF-I, insulin like growth factor I; IV, intravenous; MCR, endogenous metabolic clearance; 25(OH)D3, 25-hydroxycholecalciferol; 1,25(OH)2D3, 1,25-dihydroxycholecalciferol; 24,25(OH)2D3, 24,25-dihydroxycholecalciferol; PCR, polymerase chain reaction; Pi, inorganic phosphate in plasma; PR, production rate; PTH, parathyroid hormone; Va, true calcium absorption; VDR, vitamin D receptor; Vf, endogenous fecal calcium excretion; VF, total fecal content of 45Ca after oral administration; VI, calcium intake. 
Manuscript received 11 April 2002. Initial review completed 8 May 2002. Revision accepted 23 May 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Pansu, D., Bellaton, C. & Bronner, F. (1983) Developmental changes in the mechanisms of duodenal calcium transport in the rat. Am. J. Physiol. 244:G20-G26.
2. Ghishan, F. K., Parker, P., Nichols, S. & Hoyumpa, A. (1984) Kinetics of intestinal calcium transport during maturation in rats. Pediatr. Res. 18:235-239.[Medline]
3. Wasserman, R. H. & Fullmer, C. S. (1995) Vitamin D and intestinal calcium transport: facts, speculations and hypotheses. J. Nutr. 125:1971S-1979S.
4. Norman, A. W. (1990) Intestinal calcium absorption: a vitamin D-hormone-mediated adaptive response. Am. J. Clin. Nutr. 51:290-300.
5. Brown, A. J., Dusso, A. & Slatopolsky, E. (1999) Vitamin D. Am. J. Physiol. 277:F157-F175.
6. Wilhelm, F., Ross, F. P. & Norman, A. W. (1986) Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25-dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding—24R,25-dihydroxyvitamin D3 regulates 1,25-dihydroxyvitamin D3 binding to its chick intestinal receptor. Arch. Biochem. Biophys. 249:88-94.[Medline]
7. Nemere, I. (1996) Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3. Endocrinology 137:2254-2261.[Abstract]
8. Nemere, I. (1999) 24,25-Dihydroxyvitamin D3 suppresses the rapid actions of 1,25-dihydroxyvitamin D3 and parathyroid hormone on calcium transport in chick intestine. J. Bone Miner. Res. 14:1543-1549.[Medline]
9. Wilhelm, F. & Norman, A. W. (1985) 24R,25-Dihydroxyvitamin D3 regulates 1,25-dihydroxyvitamin D3 binding to its chick intestinal receptor. Biochem. Biophys. Res. Commun. 126:496-501.[Medline]
10. Jacobus, C. H., Holick, M. F., Shao, Q., Chen, T. C., Holm, I. A., Kolodny, J. M., Fuleihan, G. E. & Seely, E. W. (1992) Hypervitaminosis D associated with drinking milk. N. Engl. J. Med. 326:1173-1177.[Abstract]
11. Beckman, M. J., Johnson, J. A., Goff, J. P., Reinhardt, T. A., Beitz, D. C. & Horst, R. L. (1995) The role of dietary calcium in the physiology of vitamin D toxicity: excess dietary vitamin D3 blunts parathyroid hormone induction of kidney 1-hydroxylase. Arch. Biochem. Biophys. 319:535-539.[Medline]
12. Shepard, R. M. & DeLuca, H. F. (1980) Plasma concentrations of vitamin D3 and its metabolites in the rat as influenced by vitamin D3 or 25-hydroxyvitamin D3 intakes. Arch. Biochem. Biophys. 202:43-53.[Medline]
13. Lips, P. (2001) Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr. Rev. 22:477-501.
14. Martin, K. J. & Gonzalez, E. A. (2001) Strategies to minimize bone disease in renal failure. Am. J. Kidney Dis. 38:1430-1436.[Medline]
15. Chesney, R. W. (2001) Vitamin D deficiency and rickets. Rev. Endocr. Metab. Disord. 2:145-151.[Medline]
16. Carpenter, K. J. (1991) Contribution of the dog to the science of nutrition. J. Nutr. 121:S1-S7.
17. Geddes, A. D. (1996) Animal Models of Bone Disease. Bilezikian, J. P. Raisz, L. G. Rodan, G. A. eds. Principles of Bone Biology 1996:1343-1354 Academic Press San Diego, CA. .
18. Lips, P., Chapuy, M. C., Dawson-Hughes, B., Pols, H. A. & Holick, M. F. (1999) An international comparison of serum 25-hydroxyvitamin D measurements. Osteoporos. Int. 9:394-397.[Medline]
19. Cloutier, M., Gagnon, Y., Gascon-Barre, M., Brossard, J. H. & D’Amour, P. (1997) Adaptation of parathyroid function to intravenous 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs. J. Endocrinol. 155:133-141.[Abstract]
20. Monier-Faugere, M. C., Geng, Z., Friedler, R. M., Qi, Q., Kubodera, N., Slatopolsky, E. & Malluche, H. H. (1999) 22-Oxacalcitriol suppresses secondary hyperparathyroidism without inducing low bone turnover in dogs with renal failure. Kidney Int. 55:821-832.[Medline]
21. How, K. L., Hazewinkel, H.A.W. & Mol, J. A. (1994) Dietary vitamin D dependence of cat and dog due to inadequate cutaneous synthesis of vitamin D. Gen. Comp. Endocrinol. 96:12-18.[Medline]
22. National Research Council (1974) Nutrient Requirements of Dogs 1974 National Academy Press Washington DC. .
23. AAFCO (2000) 2000 Association of American Feed Control Officials, Inc. AAFCO MN. http:\\www.aafco.org.
24. van den Berg, H., Boshuis, P. G. & Schreurs, W.H.P. (1986) Determination of vitamin D in fortified and nonfortified milk powder and infant formula using a specific radioassay after purification by HPLC. J. Agric. Food Chem. 34:264-268.
25. Lewis, L. D., Morris, M. L. & Hand, M. S. (1987) Nutrients. Lewis, L. D. Morris, M. L. Hand, M. S. eds. Small Animal Nutrition III 1987:1-25 Mark Morris Associates Topeka, KS. .
26. Hazewinkel, H.A.W., van den Brom, W. E., van ’t Klooster, A. Th., Voorhout, G. & Van Wees, A. (1991) Calcium metabolism in Great Dane dogs fed diets with various calcium and phosphorus levels. J. Nutr. 121:S99-S106.
27. Schoenmakers, I., Hazewinkel, H.A.W. & van den Brom, W. E. (1999) Excessive Ca and P intake during early maturation in dogs alters Ca and P balance without long-term effects after dietary normalization. J. Nutr. 129:1068-1074.
28. Bosch, R., Visser, W. J., Thijssen, J. H. & Duursma, S. A. (1983) Synthesis of [10S(19)-3H]-dihydrotachysterol2 from ergocalciferol and preliminary investigations into its metabolic fate in rats. J. Steroid Biochem. 18:441-447.[Medline]
29. McGraw, C. A. & Hug, G. (1990) Simultaneous measurement of 25-hydroxy, 24,25-dihydroxy-, and 1,25-dihydroxyvitamin D without use of HPLC. Med. Lab. Sci. 47:17-25.[Medline]
30. Reinhardt, T. A., Horst, R. L., Orf, J. W. & Hollis, B. W. (1984) A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J. Clin. Endocrinol. Metab. 58:91-98.[Abstract]
31. Hollis, B. W. (1986) Assay of circulating 1,25-dihydroxyvitamin D involving a novel single-cartridge extraction and purification procedure. Clin. Chem. 32:2060-2063.
32. Torrance, A. G. & Nachreiner, R. (1989) Human-parathormone assay for use in dogs: validation, sample handling studies, and parathyroid function testing. Am. J. Vet. Res. 50:1123-1127.[Medline]
33. Hazewinkel, H.A.W., Schoenmakers, I., Pelling, D., Snijdelaar, M., Wolfswinkel, J. & Mol, J. A. (1999) Biological potency and radioimmunoassay of canine calcitonin. Domest. Anim. Endocrinol. 17:333-344.[Medline]
34. Eigenmann, J. E. & Eigenmann, R. Y. (1981) Radioimmunoassay of canine growth hormone. Acta Endocrinol. 98:514-520.
35. Nap, R. C., Mol, J. A. & Hazewinkel, H.A.W. (1993) Age-related plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-I) in Great Dane pups fed different dietary levels of protein. Domest. Anim. Endocrinol. 10:237-247.[Medline]
36. Gray, R. W., Caldas, A. E., Wilz, D. R., Lemann, J., Jr., Smith, G. A. & DeLuca, H. F. (1978) Metabolism and excretion of 3H-1,25-(OH)2-vitamin D3 in healthy adults. J. Clin. Endocrinol. Metab. 46:756-765.[Abstract]
37. Dusso, A., Lopez-Hilker, S., Lewis-Finch, J., Grooms, P., Brown, A., Martin, K. & Slatopolsky, E. (1989) Metabolic clearance rate and production rate of calcitriol in uremia. Kidney Int. 35:860-864.[Medline]
38. Sandgren, M. E. & DeLuca, H. F. (1989) An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor. Anal. Biochem. 183:57-63.[Medline]
39. Uhland-Smith, A., Prahl, J. M. & DeLuca, H. F. (1996) An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. J. Bone Miner. Res. 11:1921-1925.[Medline]
40. Schoenmakers, I., Mol, J. A. & Hazewinkel, H.A.W. (2000) Hormonal Ca regulation and calcium setpoint in offsprings of bitches with different calcium intakes during pregnancy. J. Anim. Physiol. Anim. Nutr. 83:1-14.
41. Schoenmakers, I., Hazewinkel, H.A.W., Voorhout, G., Carlson, C. S. & Richardson, D. (2000) Effects of diets with different calcium and phosphorus contents on the skeletal development and blood chemistry of growing great danes. Vet. Rec. 147:652-660.
42. Heaney, R. P. & Recker, R. R. (1994) Determinants of endogenous fecal calcium in healthy women. J. Bone Miner. Res. 9:1621-1627.[Medline]
43. Feldman, D. (1999) Vitamin D, parathyroid hormone, and calcium: a complex regulatory network. Am. J. Med. 107:637-639.[Medline]
44. Favier, R. P., Mol, J. A., Kooistra, H. S. & Rijnberk, A. (2001) Large body size in the dog is associated with transient GH excess at a young age. J. Endocrinol. 170:479-484.[Abstract]
45. Taylor, A. F. & Norman, M. E. (1984) Vitamin D metabolite levels in normal children. Pediatr. Res. 18:886-890.[Medline]
46. Schmidt-Gayk, H., Bouillon, R. & Roth, H. J. (1997) Measurement of vitamin D and its metabolites (calcidiol and calcitriol) and their clinical significance. Scand. J. Clin. Lab. Invest. Suppl. 227:35-45.[Medline]
47. Horst, R. L. & Reinhardt, T. A. (1997) Vitamin D Metabolism. Feldman, D. Glorieux, F. H. Wesley Pike, J. eds. Vitamin D 1997:13-32 Academic Press San Diego, CA. .
48. Reinholz, G. G. & DeLuca, H. F. (1998) Inhibition of 25-hydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in rats. Arch. Biochem. Biophys. 355:77-83.[Medline]
49. Henry, H. L. (1997) The 25-Hydroxyvitamin D 1a-Hydroxylase. Feldman, D. Glorieux, F. H. Wesley Pike, J. eds. Vitamin D 1997:57-68 Academic Press San Diego, CA. .
50. Matsumoto, T., Ikeda, K., Yamato, H., Morita, K., Ezawa, I., Fukushima, M., Nishii, Y. & Ogata, E. (1988) Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats. Biochem. J. 250:671-677.[Medline]
51. Vieth, R., Milojevic, S. & Peltekova, V. (2000) Improved cholecalciferol nutrition in rats is noncalcemic, suppresses parathyroid hormone and increases responsiveness to 1, 25-dihydroxycholecalciferol. J. Nutr. 130:578-584.
52. Morris, H. A., Need, A. G., Horowitz, M., O’Loughlin, P. D. & Nordin, B. E. (1991) Calcium absorption in normal and osteoporotic postmenopausal women. Calcif. Tissue Int. 49:240-243.[Medline]
53. Fleet, J. C. & Wood, R. J. (1999) Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in caco-2 cells. Am. J. Physiol. 276:G958-G964.
54. Boyan, B. D., Sylvia, V. L., Dean, D. D. & Schwartz, Z. (2001) 24,25-(OH)2D3 regulates cartilage and bone via autocrine and endocrine mechanisms. Steroids 66:363-374.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. de Fornel-Thibaud, G. Blanchard, L. Escoffier-Chateau, S. Segond, F. Guetta, D. Begon, F. Delisle, and D. Rosenberg Unusual Case of Osteopenia Associated With Nutritional Calcium and Vitamin D Deficiency in an Adult Dog J. Am. Anim. Hosp. Assoc., January 1, 2007; 43(1): 52 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Narayanan, V. A. T. Sepulveda, M. Falzon, and N. L. Weigel The Functional Consequences of Cross-talk between the Vitamin D Receptor and ERK Signaling Pathways Are Cell-specific J. Biol. Chem., November 5, 2004; 279(45): 47298 - 47310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Tryfonidou, M. S. Holl, M. Vastenburg, M. A. Oosterlaken-Dijksterhuis, D. H. Birkenhager-Frenkel, W. E. van den Brom, and H. A. W. Hazewinkel Hormonal regulation of calcium homeostasis in two breeds of dogs during growth at different rates J Anim Sci, June 1, 2003; 81(6): 1568 - 1580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Tryfonidou, J. van den Broek, W. E. van den Brom, and H.A.W. Hazewinkel Intestinal Calcium Absorption in Growing Dogs Is Influenced by Calcium Intake and Age but Not by Growth Rate J. Nutr., November 1, 2002; 132(11): 3363 - 3368. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
