Plasma Calcitriol Concentrations Research Articles

Caloric Intake in Renal Patients: Repercussions on Mineral Metabolism.

The aim of this paper is to review current knowledge about how calorie intake influences mineral metabolism focussing on four aspects of major interest for the renal patient: (a) phosphate (P) handling, (b) fibroblast growth factor 23 (FGF23) and calcitriol synthesis and secretion, (c) metabolic bone disease, and (d) vascular calcification (VC). Caloric intake has been shown to modulate P balance in experimental models: high caloric intake promotes P retention, while caloric restriction decreases plasma P concentrations. Synthesis and secretion of the phosphaturic hormone FGF23 is directly influenced by energy intake; a direct correlation between caloric intake and FGF23 plasma concentrations has been shown in animals and humans. Moreover, in vitro, energy availability has been demonstrated to regulate FGF23 synthesis through mechanisms in which the molecular target of rapamycin (mTOR) signalling pathway is involved. Plasma calcitriol concentrations are inversely proportional to caloric intake due to modulation by FGF23 of the enzymes implicated in vitamin D metabolism. The effect of caloric intake on bone is controversial. High caloric intake has been reported to increase bone mass, but the associated changes in adipokines and cytokines may as well be deleterious for bone. Low caloric intake tends to reduce bone mass but also may provide indirect (through modulation of inflammation and insulin regulation) beneficial effects on bone. Finally, while VC has been shown to be exacerbated by diets with high caloric content, the opposite has not been demonstrated with low calorie intake. In conclusion, although prospective studies in humans are needed, when planning caloric intake for a renal patient, it is important to take into consideration the associated changes in mineral metabolism.

Noteworthy Idiosyncrasies of 1α,25‐Dihydroxyvitamin D3 Kinetics for Extrapolation from Mouse to Man

Calcitriol or 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] is the active ligand of the vitamin D receptor (VDR) that plays a vital role in health and disease. Vitamin D is converted to the relatively inactive metabolite, 25‐hydroxyvitamin D3 [25(OH)D3] by CYP27A1 and CYP2R1 in the liver, then to 1,25(OH)2D3 by a specific, mitochondrial enzyme, CYP27B1 (1α‐hydroxylase) that is present primarily in the kidney. Degradation of both metabolites is mostly carried out by the more ubiquitous mitochondrial enzyme, CYP24A1. Despite that calcitriol inhibits its formation and degradation, allometric scaling revealed strong interspecies (mouse, rat, dog and human) correlations of the net calcitriol clearance (CL from dose/AUC∞), production rate (PR), and basal, plasma calcitriol concentration (CBL) with body weight (BW). A closer scrutiny of the collated literature data revealed that the basal plasma concentration of calcitriol was not taken into consideration in the estimation of AUC∞ (with subtraction of CBL) and CL after exogenous calcitriol dosing in both animals and humans. This led to an overestimation of AUC∞ and underestimation of CL. After correction, the allometric equations [plots of log(CL) or log(PR) versus log(body weight) or log(BW)] were: CL=0.654xBW0.807 and PR=36.6xBW0.718. The CL of calcitriol was further influenced by diseased states, and was found to be reduced in chronic kidney disease but increased in cancer when compared to healthy humans. These changes, however, were not discerned with allometric scaling since all clinical data appeared as a cluster of closely similar values on the allometric plot. PBPK‐PD (physiologically‐based pharmacokinetic‐pharmacodynamic) modeling (Ramakrishnan et al 2016), however, confirmed the dynamic interactions between calcitriol and Cyp27b1/Cyp24a1, showing the increase in CL as well as decrease in PR in a dose‐dependent fashion for the intravenous, murine data of Quach et al (2015). The PBPK model was successful in predicting data from the escalating oral and intravenous doses of calcitriol given to cancer patients upon scale‐up.

Noteworthy idiosyncrasies of 1α,25-dihydroxyvitamin D3 kinetics for extrapolation from mouse to man: Commentary.

Calcitriol or 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] is the active ligand of the vitamin D receptor (VDR) that plays a vital role in health and disease. Vitamin D is converted to the relatively inactive metabolite, 25-hydroxyvitamin D3 [25(OH)D3 ], by CYP27A1 and CYP2R1 in the liver, then to 1,25(OH)2 D3 by a specific, mitochondrial enzyme, CYP27B1 (1α-hydroxylase) that is present primarily in the kidney. The degradation of both metabolites is mostly carried out by the more ubiquitous mitochondrial enzyme, CYP24A1. Despite the fact that calcitriol inhibits its formation and degradation, allometric scaling revealed strong interspecies correlation of the net calcitriol clearance (CL estimated from dose/AUC∞ ), production rate (PR), and basal, plasma calcitriol concentration with body weight (BW). PBPK-PD (physiologically based pharmacokinetic-pharmacodynamic) modeling confirmed the dynamic interactions between calcitriol and Cyp27b1/Cyp24a1 on the decrease in the PR and increase in CL in mice. Close scrutiny of the literature revealed that basal levels of calcitriol had not been taken into consideration for estimating the correct AUC∞ and CL after exogenous calcitriol dosing in both animals and humans, leading to an overestimation of AUC∞ and underestimation of the plasma CL. In humans, CL was decreased in chronic kidney disease but increased in cancer. Collectively, careful pharmacokinetic data analysis and improved definition are achieved with PBPK-PD modeling, which embellishes the complexity of dose, enzyme regulation, and disease conditions. Allometric scaling and PBPK-PD modeling were applied successfully to extend the PBPK model to predict calcitriol kinetics in cancer patients.

Phosphorus restriction does not prevent the increase in fibroblast growth factor 23 elicited by high fat diet.

This study was designed to evaluate the influence of phosphorus (P) restriction on the deleterious effects of high fat diets on mineral metabolism. Twenty-four rats were allotted to 3 groups (n = 8 each) that were fed different diets for 7 months. Rats in group 1 were fed normal fat-normal P (0.6%) diet (NF-NP), rats in group 2 were fed high fat- normal P diet (HF-NP) and rats in group 3 were fed high fat-low P (0.2%) diet (HF-LP). Blood, urine and tissues were collected at the end of the experiments. When compared with the control group (NF-NP), rats fed HF diets showed increases in body weight, and in plasma concentrations of triglycerides and leptin, and decreased plasma calcitriol concentrations. In rats fed HF-NP plasma fibroblast growth factor 23 (FGF23) was higher (279.6±39.4 pg/ml vs 160.6±25.0 pg/ml, p = 0.018) and renal klotho (ratio klotho/GAPDH) was lower (0.75±0.06 vs 1.06±0.08, p<0.01) than in rats fed NF-NP. Phosphorus restriction did not normalize plasma FGF23 or renal klotho; in fact, rats fed HF-LP, that only ingested an average of 22.9 mg/day of P, had higher FGF23 (214.7±32.4 pg/ml) concentrations than rats fed NF-NP (160.6±25.0 pg/ml), that ingested and average of 74.4 mg/day of P over a 7 month period. In conclusion, our results demonstrate that severe P restriction over a prolonged period of time (7 months) does not normalize the increase in circulating FGF23 induced by HF diets. These data indicate that the deleterious effects of high fat diet on the FGF23/klotho axis are not eliminated by reduced P intake.

Vitamin E protects against extraskeletal calcification in uremic rats fed high fat diets

BackgroundHigh fat diets are implicated in the pathogenesis of metabolic syndrome, obesity and renal disease. Previous studies have revealed that high fat diets promote vascular calcification in uremic rats. Moreover, vitamin E has been shown to prevent uremic calcifications in genetically obese Zucker rats fed standard diet. The objective of this study was to investigate the influence of vitamin E supplementation on the development of extraskeletal calcifications in non-obese (wild type) uremic rats fed high fat diets.MethodsWistar rats (n = 32) were preconditioned by feeding either a normal (NF) or high fat (HF) diet for 45 days and subsequently were subjected to 5/6 nephrectomy (Nx). Just before performing the first Nx step, a blood sample (Pre-Nx) was obtained. After Nx rats were switched to a diet with 0.9% phosphorus and supplemented with calcitriol. Also, after Nx, half of the rats from each group (NF and HF) were treated with vitamin E (VitE) in the diet (30,000 mg/kg) and the other half were maintained on basic VitE requirements (27 mg/kg). Thus, rats were allotted to four experimental groups: Nx-NF (n = 8), Nx-NF-VitE (n = 8), Nx-HF (n = 8) and Nx-HF-VitE (n = 8). At the time of sacrifice (day 66), blood and tissue samples were obtained.ResultsFeeding a HF diet for 45 days did not increase body weight but elicited hyperglycemia, hypertriglyceridemia, an increase in plasma fibroblast growth factor 23 and a reduction in plasma calcitriol concentrations. After Nx, rats fed HF diet showed substantial extraskeletal calcification with aortic calcium content that was higher than in rats fed NF diet. Supplementation with VitE significantly (p < 0.05) reduced aortic (from 38.4 ± 8.8 to 16.5 ± 1.4 mg/g), gastric (from 5.6 ± 2.7 to 1.2 ± 0.4 mg/g) and pulmonary (from 1.8 ± 0.3 to 0.3 ± 0.2 mg/g) calcium content in rats on HF diets.ConclusionsUremic rats fed HF diets developed more severe extraosseous calcifications than their normocaloric-fed counterparts and dietary VitE supplementation protected against uremic calcifications in rats fed HF diets. Thus, eating energy-rich foods should be discouraged in patients with renal disease and their deleterious effect may be ameliorated with adequate antioxidant supply.

Stimulating effects of a diet negative in dietary cation-anion difference on calcium absorption from the rumen in sheep.

The concept of feeding anionic salts in late gestation is widely used to prevent milk fever in dairy cows. While the effects of these diets on renal Ca excretion and tissue responsiveness towards parathyroid hormone have clearly been demonstrated, data on a potential impact on gastrointestinal Ca absorption are conflicting. Therefore, the aim of this study was to investigate the influence of feeding a diet negative in dietary cation-anion difference (DCAD) on ruminal mineral concentrations, fermentation products, electrophysiological properties of rumen epithelia and Ca flux rates. For this purpose, sheep were kept for 3 weeks on diets that were either positive or negative in DCAD. The induction of a compensated hyperchloremic metabolic acidosis could be demonstrated by increased plasma Cl and enhanced concentrations of ionised Ca, while plasma concentrations of HCO3- and base excess were decreased with the low DCAD diet. Neither transmural potential differences nor fermentation products were affected, but ruminal concentrations of Cl and Mg as well as the relation of ionised to total Ca were increased. Ussing chamber experiments revealed alterations of electrophysiological parameters and an increase in the electroneutral component of Ca flux rates from the mucosal to the serosal side of rumen epithelium. As plasma calcitriol concentrations were not affected, it can be concluded that the administration of anionic salts results in a vitamin D-independent stimulation of ruminal Ca transport.

Species-specific responses of N homeostasis and electrolyte handling to low N intake: a comparative physiological approach in a monogastric and a ruminant species

In our former studies low crude protein (LCP) intake influenced N homeostasis and electrolyte handling in goats. We hypothesised that due to rumino-hepatic nitrogen (N) recycling adaptation of N homeostasis and adjustment of electrolyte handling to LCP intake differs between goats and monogastric animals. Therefore, an experiment similar to that with goats was conducted with rats. Two feeding groups received a diet either containing 20 or 8 % crude protein (as fed basis) for 5 weeks and intake and excretion of N, calcium (Ca) and phosphorus (P) were determined. To detect systemic and endocrine adaptation to LCP intake plasma concentrations of urea, Ca, phosphate (Pi), insulin-like growth factor 1 (IGF-1), 1,25-dihydroxyvitamin D3 (calcitriol), parathyroid hormone (PTH) and cross-linked telopeptide of type I collagen (CTX) were measured. Adjustment of renal electrolyte transport was assessed by detecting protein expression of key proteins of renal Pi transport. All data were compared with the data of the goat experiment. LCP intake decreased plasma urea concentration stronger in goats than in rats. In both species urinary N excretion declined, but faecal N excretion decreased in goats only. Furthermore, in goats urinary Ca excretion decreased, but in rats urinary Ca concentration increased. Decreased plasma IGF-1 and calcitriol concentrations were found in goats only. Thus, renal Ca excretion appears to be a common target in adaptation of electrolyte homeostasis in both species, but is regulated differently.

Investigation of the pathophysiological mechanism for altered calcium homeostasis in hyperthyroid cats

To investigate possible pathophysiological mechanisms (reduced plasma calcitriol concentrations and/or presence of concurrent or masked chronic kidney disease) for hypocalcaemiain hyperthyroid cats. Prospective cohort study. Routine plasma biochemical parameters, plasma parathyroid hormone and calcitriol concentrations, ionized calcium concentrations, and venous pH, were measured at diagnosis and following treatment of hyperthyroidism. Linear regression analysis was used to determine predictors of ionized calcium concentration. Hyperthyroid cats (n=45) had lower ionized calcium concentrations than healthy geriatric cats (n=52), however, ionized calcium concentrations were higher in hyperthyroid cats with concurrent or masked chronic kidney disease than non-azotaemic hyperthyroid cats. Plasma calcitriol concentrations were higher in hyperthyroid cats than control cats. Plasma total thyroxine concentration and venous pH were independent predictors of ionized calcium concentration. Plasma total thyroxine concentration was also a predictor of ionized calcium concentration after adjustment for plasma parathyroid hormone and calcitriol concentrations. Hypocalcaemia in hyperthyroid cats is not associated with the presence of concurrent or masked chronic kidney disease or reduced plasma calcitriol concentrations. Increased thyroid hormone concentrations might influence ionized calcium concentrations through a mechanism, yet to be determined, that is independent of control by parathyroid hormone and calcitriol.

Parathyroid hormone concentration in geriatric cats with various degrees of renal function

To determine whether cats in the nonazotemic stages of chronic kidney disease have increased plasma parathyroid hormone (PTH) concentrations as a compensatory physiologic mechanism to maintain plasma phosphate concentration within the reference interval. Prospective longitudinal study. 118 client-owned geriatric cats with various degrees of renal function. For each cat, a blood sample was obtained for plasma biochemical analysis and determination of plasma PTH concentration, and a urine sample was obtained for determination of urine specific gravity at study entry (baseline) and after 12 months. For a subset of 30 cats, plasma calcitriol concentration was determined at baseline. Cats were categorized into 1 of 3 groups on the basis of kidney function at the end of 12 months. At baseline and after 12 months, plasma concentrations of variables associated with calcium homeostasis were compared between the 3 groups and also within groups over time. Multivariable linear regression was used to identify variables associated with plasma PTH concentration. Plasma PTH concentration was significantly increased in cats that developed azotemia, compared with PTH concentration in cats that remained nonazotemic, and PTH concentration increased before changes in plasma calcium and phosphate concentrations were detected. A moderate positive association between plasma calcitriol and PTH concentrations was identified. Plasma PTH concentration was associated with age and plasma urea, creatinine, and total calcium concentrations in the final multivariable model. Results suggested that renal secondary hyperparathyroidism can develop prior to azotemia in cats, even in the absence of hyperphosphatemia and hypocalcemia.

Dose-Response Effects of an Aqueous Formulation of Calcitriol and Evaluation of Renal Function in Nonpregnant Cows

Dose responses of plasma calcitriol, calcium (Ca), bone metabolic markers and glomerular filtration rate (GFR) were evaluated in four nonpregnant Holstein cows treated subcutaneously with an aqueous formulation of calcitriol at four doses in a 4 × 4 Latin-square design. Calcitriol, Ca, and markers of bone metabolism were analyzed in plasma samples. GFR was measured in predose and day 5 samples. Plasma calcitriol and Ca concentrations increased dose-dependently. The calcitriol dose was positively correlated with the area under the concentration-time curve of plasma calcitriol. Bone formation markers tended to increase from day 3 onward for all doses. No significant changes in GFR were noted. Thus, exogenous calcitriol administered between 0.0625 and 0.5 µg/kg body weight elicited dose-dependent increases in both plasma calcitriol and Ca and elevated bone formation markers without affecting renal function in nonpregnant cows.

Effects of an Oleaginous Calcitriol on Changes in Plasma Calcitriol, Calcium and Bone Metabolic Markers in Dairy Cows

The present study evaluated the effects of calcitriol dissolved in an oleaginous vehicle (calcitriol-OLE) on changes in plasma calcitriol, calcium and bone metabolic markers in nonpregnant, nonlactating Holstein cows. Five cows were treated intramuscularly or subcutaneously with calcitriol-OLE and oleaginous vehicle alone using a 5 × 5 Latin square design. Additionally, cows were also treated intravenously with calcitriol dissolved in an aqueous vehicle (calcitriol-AQU) for comparison. The plasma calcitriol concentrations after intramuscular and subcutaneous calcitriol-OLE administrations peaked at 24 and 12 hr, respectively, remained significantly elevated until day 3, returned to the respective control levels on day 5 and decreased significantly on day 7. In cows given intravenous calcitriol-AQU, the calcitriol levels decreased with linearity on day 1. The plasma calcium levels rose from 12 hr post-dose and peaked on day 2 for both preparations and in all three administration routes. Significantly increased calcium levels continued until day 5 in the intramuscular and intravenous routes and day 7 in the subcutaneous route. The plasma osteocalcin concentrations significantly increased from day 3 for calcitriol-OLE and from day 5 for calcitriol-AQU, whereas the bone resorption markers, tartrate-resistant acid phosphatase isoform 5b and hydroxyproline, decreased during this time. These results suggest that either intramuscular or subcutaneous injection of calcitriol-OLE extends and maintains supraphysiological calcitriol levels in the plasma and prolongs hypercalcemia. Moreover, exogenous calcitriol in normocalcemic cows increases the plasma osteocalcin concentration and decreases the plasma levels of bone resorption markers probably due to hypercalcemia.

Response of plasma bone markers to a single intramuscular administration of calcitriol in dairy cows

To elucidate the effects of an exogenous calcitriol (1,25-dihydroxyvitamin D 3) on plasma bone markers, the formation item osteocalcin (OC), undercarboxylated OC (ucOC) and bone-specific alkaline phosphatase (BALP), and the resorption parameter tartrate-resistant acid phosphatase isoform 5b (TRAP5b) and hydroxyproline (HYP) were measured in conjunction with plasma calcitriol and calcium (Ca) concentrations in dairy cows receiving calcitriol or its vehicle according to a 2 × 2 crossover design. Calcitriol (0.5 μg/kg, i.m.) increased significantly its plasma level during 6 h to day 2 and plasma Ca concentration during 12 h to day 7 compared to the vehicle. Also, plasma OC and ucOC started to rise from day 3 and 1, respectively, and remained elevated until day 7. No change in plasma BALP, TRAP5b or HYP associated with calcitriol treatment was noted. These results demonstrate that exogenous calcitriol stimulates osteoblasts to biosynthesise OC, a determinant of the bone formation in cows.

Application of a Reservoir-Type Calcitriol Transdermal Patch in Dairy Cattle

Our objective was to evaluate the effects of a reservoir-type calcitriol (5 mg/animal) transdermal patch on plasma calcitriol and calcium (Ca) concentrations in dairy cattle. A group of six heifers received three different types of patches: an encapsulated reservoir solution containing calcitriol alone (CAL), calcitriol with dodecylamine (CAL+D), or vehicle (CONT). The patches were applied to the skin of the tail for duration of 2 days at intervals of at least 3 weeks. Cattle that received CAL or CAL+D showed significant increases in plasma calcitriol and Ca concentrations on day 2 and days 2 and 3, respectively. The areas under the plasma concentration-time curve (AUC) values for plasma calcitriol and Ca in the CAL and CAL+D groups increased significantly compared to the CONT group. The data of this study describe evidence of measurable transdermal absorption of exogenous calcitriol from a patch and its sufficient biological action to elevate plasma Ca concentrations.

The vitamin D receptor and its ligand 1α,25-dihydroxyvitamin D3 in Atlantic salmon (Salmo salar)

Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr-smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (<1 mM Ca(2+)) to the plasma (~1.25 mM Ca(2+)) and to strongly hypercalcic (8-12 mM Ca(2+)). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D(3) and 24,25(OH)(2)D(3) did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr-smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

Phosphate Transport in Pig Proximal Small Intestines during Postnatal Development: Lack of Modulation by Calcitriol

The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [<1 week postpartum (pp)], suckling (3–4 weeks pp), and weaned (>6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16–21 pg/ml) and differed significantly from those in respective con animals (60–69 pg/ml) and vitamin D3-treated def piglets (50–56 pg/ml). However, newborn and suckling def piglets had normal Ca (∼3.0 mmol/liter) and Pi (∼2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4–6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170–224 nmol/(cm2·h)] and decreased by approximately 80% during the first week of life before remaining constant [30–50 nmol/(cm2·h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9–2.2 nmol/(mg protein·10 sec); suckling piglets, 0.4–0.6 nmol/(mg protein·10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein·10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5–0.7 mmol/liter) were observed. In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.

Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol.

The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [<1 week postpartum (pp)], suckling (3-4 weeks pp), and weaned (>6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16-21 pg/ml) and differed significantly from those in respective con animals (60-69 pg/ml) and vitamin D3-treated def piglets (50-56 pg/ml). However, newborn and suckling def piglets had normal Ca (approximately 3.0 mmol/liter) and Pi (approximately 2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4-6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170-224 nmol/(cm2 x h)] and decreased by approximately 80% during the first week of life before remaining constant [30-50 nmol/(cm2 x h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9-2.2 nmol/(mg protein 10 sec); suckling piglets, 0.4-0.6 nmol/(mg protein x 10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein x 10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5-0.7 mmol/liter) were observed. In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum.

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca- and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly direct Na+ gradient. The Na+-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol.1(-1) sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na+ concentrations in the order of 5 mmol.1(-1). Inorganic phosphate accumulation was not affected by a K+ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4 Na+-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with Vmax = 0.42 +/- 0.08 nmol.mg-1 protein per 15S-1 and Km = 0.03 +/- 0.01 mmol.1(-1) (n = 4, mean +/- SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of Vmax by 35-80%, irrespective of the level of P intake. In the presence of an inwardly directed Na+ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pHout/pHin = 5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na+. In summary, in goats Na+ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.

Mechanisms of intestinal phosphate transport in small ruminants.

In order to study the localization and mechanisms of intestinal phosphate transport in sheep and goats, unidirectional inorganic phosphate (Pi) flux rates across isolated stripped epithelial tissues were measured in vitro by applying the Ussing-chamber technique. In the first experiment the tissues were obtained from animals which had been kept on an adequate dietary P supply. In the second experiment the animals had either been kept on an adequate Ca and P supply or were Ca- and/or P-depleted. Significant net Pi absorption was measured in all segments of the small intestine and in the proximal colon of sheep and in the duodenum and jejunum of goats. Since the experiments were carried out in the absence of any electrochemical gradient, this clearly indicates the presence of active mechanisms for Pi transport in the intestinal tract of small ruminants. In sheep jejunum, reduction of mucosal Na concentration to 1.8 mM or serosal application of ouabain (0.1 mM) resulted in significant decreases of net Pi absorption of the same order of magnitude, indicating that about 65% of active Pi transport in sheep jejunum is mediated by a Na-dependent active transport mechanism. The mechanism for the remaining Na(+)-independent active Pi transport has not yet been identified. Dietary P depletion caused hypophosphataemia and induced a significant stimulation of net Pi absorption in goat duodenum and jejunum. This increase was independent of dietary Ca supply and was not associated with increased plasma calcitriol concentrations. This suggests substantial differences in hormonal regulation of Pi transport in small ruminants in comparison with single-stomached species.

Binding properties of goat intestinal vitamin D receptors as affected by dietary calcium and/or phosphorus depletion.

The binding capacity (Bmax) and the affinity (Kd) of the intestinal vitamin D receptor (VDR) have been studied using mucosa preparations from the duodenum, jejunum and proximal colon of male growing goats which had been kept in a two-factorial (2 x 2) trial on Ca and/or P deficient diets for 9 weeks. This treatment resulted in significant changes of different parameters of Ca and P homeostasis. Irrespective from the level of Ca intake, P depletion caused significant hypophosphatemia with corresponding hypercalcemia. In both Ca depleted groups the calcitriol concentrations in plasma significantly increased by more than 100% in comparison with normal Ca supply. No changes were recorded for plasma calcitriol concentrations in response to P depletion with an adequate Ca supply. Plasma PTH levels were only increased significantly in Ca depletion with adequate P supply. Irrespective of different feeding regimens, the highest Bmax values were found in the jejunum. In all intestinal segments tested, the Bmax values were significantly decreased by P depletion as compared with an adequate P supply. No effects on the Bmax of VDR were observed in response to changes of Ca supply. The Kd values of the VDR were neither affected by different intestinal localizations nor by Ca and/or P depletion. From the present results, it has to be concluded that the physiological relevance of VDR down-regulation may not be related to P homeostasis rather than to Ca homeostasis by minimizing the hypercalcemia induced by P depletion.

Vitamin D, parathormone, and calcitonin profiles in persons with long-standing spinal cord injury

Rapid immobilization after acute spinal cord injury (SCI) leads to increased bone resorption, net calcium efflux from the bone, hypercalciuria, depressed parathormone (PTH) and increased calcitonin release. However, the effects, if any, of long-standing SCI on calcium regulatory system is not well understood. We measured plasma concentrations of 25 hydroxy (OH) vitamin D, 1,25(OH)2 vitamin D (calcitriol), intact PTH molecule, calcitonin, ionized calcium [Ca++] and phosphorus in 40 clinically stable men with long-standing SCI of 3-year to 50-year duration (22 persons with paraplegia and 18 persons with quadriplegia). The results were compared with those obtained in 14 able-bodied control men. Plasma PTH concentration in the SCI group was significantly lower than that found in the able-bodied controls despite virtually identical concentrations of ionized calcium. Likewise, plasma calcitriol concentration in the SCI group was significantly lower than the value found in the able-bodied control group and lower in persons with quadriplegia than in those with paraplegia. In contrast, plasma calcitonin concentration in the quadriplegic group was significantly higher than that in persons with paraplegia and insignificantly higher than that in the control group. No significant difference was noted in serum ionized calcium between the study groups. PTH and calcitriol levels were positively related to one another (r = 0.35, p < .01) and negatively related to the level of injury (r = −0.43, p < .002 and r = −0.54, p < .001, respectively). In conclusion, long-standing SCI is associated with significant depression of calcitriol and PTH concentrations despite normal ionized calcium concentration. These findings can be explained by an increased calcium release leading to suppression of PTH, which in turn down regulates calcitriol biosynthesis. Negative correlations between the level of injury and both PTH and calcitriol levels suggest bones in the paralyzed structures as the primary source of calcium release in the SCI patients.