Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite that ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1.Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet.Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 5/25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected.Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol.Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites

The comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites were investigated to evaluate: (1) optimization of sensitivity (2) crossreactivity of these compounds in their respective assays and (3) side chain steric requirements of the vitamin D molecule for optimum binding to the calciferol binding protein or bovine thymus receptor. Disequilibrium assay conditions improved assay sensitivity 30-fold for the calciferol assay and approx 3-fold for metabolites in the 25-hydroxycalciferol and 1,25-dihydroxycalciferol assays. Ergocalciferol compounds were uniformly less efficient in their association with the proteins tested than were their cholecalciferol counterparts, with one exception. In the calciferol assay, cholecalciferol had greater affinity for the the calciferol binding protein than did ergocalciferol. In the 25-hydroxycalciferol assay affinity for the calciferol binding protein was 25-hydroxycholecalciferol = 24,25-dihydroxycholecalciferol greater than 25-hydroxyergocalciferol greater than 25S,26-dihydroxycholecalciferol greater than 24,25-dihydroxyergocalciferol greater than 25,26-dihydroxyergocalciferol. In the assay for 1,25-dihydroxycalciferol, bovine thymus receptor recognized 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol equally. From the forthcoming data it appears that hydroxyl and/or methyl groups on the calciferol side chain alter the ability of these physiological compounds to associate with the calciferol binding protein.     

The metabolism of cholecalciferol in the liver of Japanese quail (Coturnix coturnix japonica) with particular reference to the effects of oestrogen.

1. Studies were carried out in vitro with the livers of Japanese quail that had been fed from hatching on diets supplying their full requirements for vitamin D. 2. 25-Hydroxycholecalciferol was the major metabolite when liver homogenates of egg-laying female and oestrogen-treated quail of both sexes were incubated with [3H]cholecalciferol. 3. Very little 25-hydroxycholecalciferol was generated from liver homogenates of adult male and immature quail. Instead the cholecalciferol was converted into one or more compounds less polar than 25-hydroxycholecalciferol and into a number of highly polar metabolites, some of which were water-soluble. 4. Oestrogen not only stimulated the 25-hydroxylation of cholecalciferol but also protected both cholecalciferol and 25-hydroxycholecalciferol from degradation by the enzymic pathways active in immature and male birds. 5. These actions of oestrogen may be of physiological significance in relation to the high requirements of laying birds for 1,25-dihydroxycholecalciferol to support the intense metabolism of calcium associated with egg-shell calcification.     

1,25-dihydroxycholecalciferol: the metabolite of vitamin D responsible for increased intestinal calcium transport

1,25-Dihydroxycholecalciferol was prepared from [26,27-³H]-25-hydroxycholecalciferol and from [1,2-³H]-25-hydroxycholecalciferol enzymatically and purified chromatographically. Injection of 62.5 pmoles of 1,25-dihydroxycholecalciferol intravenously into vitamin D-deficient chicks resulted in the accumulation of a maximum of 5.9% of the dose in the intestine. During the 12 hr period following injection, this radioactivity was found almost entirely as 1,25-dihydroxycholecalciferol. It has previously been shown that intestinal calcium absorption is initiated by 1,25-dihydroxycholecalciferol during this period. These results provide strong evidence that the 1,25-dihydroxycholecalciferol is not metabolized further before it initiates intestinal calcium absorption.     

24-Hydroxylation of 1,25-dihydroxyergocalciferol. An unambiguous deactivation process

1,24,25-Trihydroxyergocalciferol was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyergocalciferol and from chick kidney homogenates incubated with 24,25-dihydroxyergocalciferol. The identity was established by ultraviolet absorbance, sensitivity to periodate, nuclear magnetic resonance, and mass spectrometry. The new metabolite had an affinity equal to 1,24,25-trihydroxycholecalciferol for the bovine-thymus and chick-intestinal 1,25-dihydroxyvitamin D receptor and had an affinity twice that of 1,24,25-trihydroxycholecalciferol for the rat-intestinal receptor. It was 3- and 6-fold less competitive than either 1,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol, respectively, for the rat plasma vitamin D transport protein. 1,24,25-Trihydroxyergocalciferol was at least 10-fold less active than 1,25-dihydroxycholecalciferol, 1,25-dihydroxyergocalciferol, and 1,24,25-trihydroxycholecalciferol at stimulating intestinal-calcium transport and was also relatively ineffective at stimulating bone-calcium resorption in rats. Moreover, in rats, [3H]1,24,25-trihydroxyergocalciferol was cleared from plasma approximately 40% faster than [3H]1,24,25-trihydroxycholecalciferol. These data suggest that C-24 hydroxylation of 1,25-dihydroxyergocalciferol represents a significant in vivo deactivation step, whereas equivalent deactivation of 1,25-dihydroxycholecalciferol seems to involve metabolic steps subsequent to C-24 hydroxylation (C-24 ketonization). C-24 ketonization of 1,25-trihydroxyergocalciferol would not be anticipated due to the presence of the 24(S)-methyl group. These results reveal further dissimilarities between ergocalciferol and cholecalciferol metabolism in mammals and suggest a mechanism for the lesser tendency of ergocalciferol to cause hypercalcemia relative to cholecalciferol.     

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).     

Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin D3

It was originally postulated, primarily on the basis of experiments employing actinomycin D, that calciferol (vitamin D) mediated its characteristic physiological responses in the intestine via the activation of information stored in the intestinal genome. A more recent alternative hypothesis suggested that actinomycin D blocked the biological response to calciferol by inhibiting the mandatory metabolism of cholecalciferol to 1,25-dihydroxycholecalciferol. Presented in this paper are the results of recent experiments studying the effects of both actinomycin D and cycloheximide on the metabolism, subcellular localization, and action of cholecalciferol or its metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Actinomycin D was found to inhibit calcium transport stimulated by cholecalciferol or its metabolites without inhibiting their metabolism or localization in the target tissue, the intestinal mucosa. However, actinomycin D had to be administered in four doses at 2-hr intervals to block the stimulation of calcium transport by 1,25-dihydroxycholecalciferol. Actinomycin D was also found not to lower the renal levels of 25-hydroxycholecalciferol-1-hydroxylase, which were measured in vitro. In contrast, cycloheximide was found to inhibit the localization of the sterols in the intestine. Also cycloheximide lowered the renal enzyme levels which were measured in vitro following administration of the antibiotic in vivo. From these data it can be calculated that the 25-hydroxycholecalciferol-1-hydroxylase appears to have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approximately 3 hr. Thus, the inhibition of intestinal calcium transport by these two antibiotics may in fact occur at two different target organs; cycloheximide by a lowering of the kidney levels of 25-hydroxycholecalciferol-1-hydroxylase and actinomycin D by blocking the action of 1,25-dihydroxycholecalciferol in the intestine.     

Vitamin D3 metabolites in rat epididymis: high 24,25-dihydroxy vitamin D3 levels in the cauda region

Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D3 ([3H]25(OH)D3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [3H]-25(OH)D3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D3 and the 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH)2D3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH)2D3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in rat reproductive tissues.     

Effect of oestrogen and 1,25-dihydroxycholecalciferol on 25-hydroxycholecalciferol metabolism in primary chick kidney-cell cultures.

Primary cultures of chick kidney cells convert 25-hydroxycholecalciferol into more-polar metabolites. Cells from vitamin D-deficient chicks have high 25-hydroxycholecalciferol 1 alpha-hydroxylase (1 alpha-hydroxylase) activity, but no 25-hydroxycholecalciferol 24-hydroxylase (24-hydroxylase) activity. Physiological concentrations of 1,25-dihydroxycholeclaciferol suppress 1 alpha-hydroxylase and induce 24-hydroxylase activity. The inhibition of 1 alpha-hydroxylase preceded the induction of 24-hydroxylase. In contrast, oestradiol-17 beta had no effect on the activity of either hydroxylase under a variety of experimental conditions. These results clearly demonstrate that 1,25-dihydroxycholecalciferol, but not oestrogen, acts directly on the kidney cells to regulate the metabolism of 25-hydroxycholecalciferol.     

Seasonal fluctuations in serum concentrations of vitamin D metabolites in normal subjects.

Serum concentrations of 25-hydroxycholecalciferol (25-OHD), 24,25-dihydroxycholecalciferol (24,25-(OH)2D), and 1,25-dihydroxycholecalciferol (1,25-(OH)2D) were measured at monthly intervals throughout the year in eight normal subjects. 25-OHD was measured by competitive protein-binding assay after Sephadex LH 20 chromatography, 24,25-(OH)2D by competitive protein-binding assay after Sephadex LH 20 and high-pressure chromatography, and 1,25-(OH)2D by radioimmunoassay after the same separation procedure as for 24,25-(OH)2D. A seasonal variation, apparently dependent on exposure to ultraviolet light, was found for all three metabolites. A study in six other normal subjects showed that there was no diurnal rhythm in any of the metabolites. Oral administration of 2 microgram 1,25-(OH)2D caused a sharp rise in serum concentrations of 1,25-(OH)2D and no change in the concentrations of the two other metabolites, but by 12 hours the 1,25-(OH)2D concentration had returned to the basal value. The concentrations of all three metabolites studied vary according to the season. Thus to interpret these concentrations in any subject the normal range for the particular season must be referred to.     

Competitive protein binding assay for 24,25-dihydroxycholecalciferol

A competitive protein binding assay which measures 24,25-dihydroxycholecalciferol in human serum has been developed using the binding protein from vitamin D-deficient rat kidney. As 25-hydroxycholecalciferol and 25,26-dihydroxycholecalciferol also interact with the binding protein, possible interference by these compounds in the assay has been overcome by preparative chromatography of serum extracts on Sephadex LH 20 prior to assay. The mean serum level of 24,25-dihydroxycholecalciferol in seven normal volunteers was 1.68 ± 0.82 ng/ml whereas patients receiving large therapeutic doses of vitamin D were found to have higher levels. None was detectable in the serum of a vitamin D-deficient patient.     

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

A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) 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)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) 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 D(3) toxicosis.     

Effect of experimental diabetes on levels of 25-hydroxycholecalciferol in pregnant rats and fetuses

Induction of diabetes in female rats by streptozotocin administration 7 days before mating led to an increase in maternal and fetal calcemia at day 21 of gestation. The plasma levels of 25-hydroxycholecalciferol (25 OH D3) were increased in the diabetic mother whereas the 25 OHD3 contents in entire fetuses were greatly decreased in comparison with control values obtained in both normal pregnant rat and normal fetuses. Our results obtained in pregnant rat were different from those found in the literature concerning non pregnant animals in which only 1,25-dihydroxycholecalciferol was affected by diabetes.     

Stimulation of (3H)uridine incorporation into nuclear RNA of rat kidney by vitamin D metabolites

Within 30–60 min after administration of 25-hydroxycholecalciferol or 30 min after 1,25-dihydroxycholecalciferol, the incorporation of [³H]uridine into the nuclear RNA of kidney is stimulated 1.6-fold or 3-fold, respectively. The results suggest that 1,25-dihydroxycholecalciferol is the active form responsible for the stimulation of RNA synthesis. It is suggested that specific RNA and protein synthesis may be involved in the renal reabsorption of ions initiated by vitamin D or its metabolites.     

Production of C-24- and C-23-oxidized metabolites of 1,25-dihydroxycholecalciferol by cultured kidney cells (LLC PK1) and their presence in kidney in vivo.

1,25-Dihydroxy[3H]cholecalciferol was converted into several more-polar metabolites by a cultured pig kidney cell line (LLC PK1). The production of metabolites was stimulated by pretreating the cells with unlabelled 1,25-dihydroxycholecalciferol. A similar profile of metabolites was observed on high-pressure-liquid-chromatographic analysis of an extract from the kidneys of rats dosed intravenously with 1,25-dihydroxy[3H]cholecalciferol. Among the metabolites detected were 1,24,25-trihydroxycholecalciferol, 1,25-dihydroxy-24-oxocholecalciferol, 1,23,25-trihydroxy-24-oxocholecalciferol and 1,25-dihydroxycholecalciferol-26,23-lactone. The results are in accord with data reported for intestinal 1,25-dihydroxycholecalciferol metabolism [Napoli, Pramanik, Royal, Reinhardt & Horst (1983) J. Biol. Chem. 258, 9100-9107]. These data indicate that C-23- and C-24-oxidation of 1,25-dihydroxycholecalciferol are phenomena common to calciferol target tissues, and that regulation of 1,25-dihydroxycholecalciferol homoeostasis is dependent on the rate of its metabolism in addition to the rate of its synthesis.     

Separation of nanogram quantities of hydroxy metabolites of vitamin D3 in plasma by thin-layer chromatography on silica gel

Thin-layer chromatography (TLC) on silica gel coated HPTLC plates, using chloroform—ethanol—water as mobile phase, is highly effective in the quantitative separation of biologically active metabolites of vitamin D. The combination of TLC and competitive protein-binding assay results in a rapid, sensitive and reproducible method for the analysis of nanogram quantities of metabolites of vitamin D₃ (25-hydroxycholecalciferol, 24,25-dihydroxycholecalciferol and 25,26-dihydroxycholecalciferol) in plasma samples.     

Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats.

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.     

The localization of 1,25-dihydroxycholecalciferol in bone cell nuclei of rachitic chicks

1. A simple technique has been developed to obtain subcellular fractions of chick bone. The method yielded 60-70% of total DNA in the nuclear debris fraction and 80-90% of total (14)C recovered in bone after a dose of radioactive vitamin D. 2. After a dose of [4-(14)C,1,2-(3)H(2)]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the time-course of total (14)C radioactivity in the epiphysis, metaphysis and diaphysis of proximal tibiae was measured. The maximum concentrations were reached at 6h, corresponding to a similar peak of radioactivity in blood, decreasing until 24h and indicating the dependence on the circulating (14)C and on the blood supply of the three bone components. 3. The (14)C radioactivity of cholecalciferol and 25-hydroxycholecalciferol (expressed per mg of DNA) followed the pattern of incorporation of total (14)C radioactivity in all three bone components. The more polar metabolite fraction reached a peak of radioactivity at 6-9h and maintained its concentration over the 24h period studied in all anatomical bone components. 4. After a dose of [4-(14)C,1-(3)H]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the subcellular distribution was studied. At 24h after dosing, the nuclear fraction contained 27% and the supernatant fraction had 67% of total (14)C recovered in the bone filtrate. When the (14)C in the residual bone fragments was included, the nuclear fraction contained up to 35% of the total radioactivity in the bone. 5. The subcellular distribution pattern of individual vitamin D metabolites indicated that the purified nuclear fraction concentrated the polar metabolite, which lost (3)H at C-1, so that 77% of the radioactivity could be accounted for by 1,25-dihydroxycholecalciferol. The supernatant fraction contained smaller amounts of 1,25-dihydroxycholecalciferol (9%), with 66% of 25-hydroxycholecalciferol forming the major metabolite, corresponding to its concentration found in blood at 24h. 6. The preferential accumulation of 1,25-dihydroxycholecalciferol in the nuclear fraction and the overall pattern of other metabolites, found previously in intestinal tissue, suggests a similar mechanism of action in bone to that postulated for the intestinal cell in calcium translocation.     

Cholecalciferol and placental calcium transport

Transplacental movement of calcium from mother to fetus is essential for normal fetal development. In most species, fetal plasma calcium levels are higher than maternal levels at term. The role of cholecalciferol metabolites, with specific emphasis on 1,25-dihydroxycholecalciferol (1,25(OH)2D), in placental calcium transport and maintenance of the fetomaternal gradient has been extensively investigated. In rats, there is not an absolute demand for 1,25(OH)2D for maintenance of fetal calcium homeostasis in utero, even though it is essential for maintenance of maternal plasma calcium levels. However, in sheep, the absence of 1,25(OH)2D results in disruption of both maternal and fetal calcium homeostasis. It is known that rat and human placentas contain specific cytosolic binding proteins for 1,25(OH)2D that are similar to the well-characterized intestinal receptor. Two calcium-binding proteins (CaBP) have been detected in rat and human placentas: a protein immunologically identical to the vitamin D-dependent CaBP and a calcium-dependent ATPase. The levels of CaBP in rat placenta have been shown to increase in response to exogenously administered 1,25(OH)2D but cannot be obliterated with maternal vitamin D deficiency. No relationship has been shown between 1,25(OH)2D and placental Ca-ATPase in any species. Thus, the mechanism of action of 1,25(OH)2D in maintenance of the transplacental calcium gradient in sheep is unknown. In the pregnant rat (and perhaps human), 1,25(OH)2D is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate.     

Vitamin D metabolite-binding proteins in human tissue

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD₃) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD₃-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport α-globulin. A cathodal, 1,25-(OH)₂D₃-binding protein, which sedimented at 3–4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.