Enzyme studies on the esterification of vitamin D in rat tissues

1. The mechanism of vitamin D esterification in the rat was studied with liver, small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and [1-(3)H]cholecalciferol, [U-(14)C]ergocalciferol and [4-(14)C]cholesterol as substrates. 2. No esterification of vitamin D could be detected with liver preparations nor with homogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreatic juice esterified [1-(3)H]cholecalciferol with oleic acid, and specificity studies indicated that a cholesterol-esterifying enzyme was using vitamin D as substrate. 4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The specificity of the esterification reaction is discussed in relation to (a) the molecular structure of the substrates and (b) their availability, in a micellar solution, to the enzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin D in vivo during absorption from the small intestine and while it is transported in blood.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Investigations on vitamin D esters synthesized in rats. Turnover and sites of synthesis

1. The tissue contents of vitamin D alcohol and ester were estimated in rats 5, 10, 24, 48 and 72hr. after peroral administration of [1-(3)H]cholecalciferol. 2. The total vitamin D in liver decreased in an exponential fashion from 19% of the dose at 5hr. to 0.6% at 72hr., but the ester content remained at a relatively constant low value from 5hr., so that by 72hr. it represented 67% of the total vitamin D. Vitamin D ester in kidney increased slowly to 48hr., but by 72hr. it was only 10% of the total vitamin D. 3. The small intestine, unlike liver and kidney, contained a higher content of vitamin D ester 10hr. after administration than at later times, and it is postulated that some vitamin D was esterified during absorption from the alimentary tract. 4. Plasma contained vitamin D ester at all time intervals, and it is suggested that ester found in liver and kidney could have been transported to these sites in the blood. 5. Thoracic-duct lymph was found to transport 43% of a peroral dose of vitamin D in 12hr., of which 1.4% was esterified. The fatty acid components of the lymph vitamin D ester, determined by two-dimensional thinlayer chromatography, were mainly palmitate (31%), stearate (25%), oleate (16%) and linoleate (16%). This pattern was similar to that previously found in liver.     

Metabolism of 1alpha-hydroxyvitamin D3 in the chick.

Chicks convert both orally and intravenously administered 1alpha-hydroxy[6-3H]vitamin D3 rapidly to 1alpha,25-dihydroxy[6-3H]vitamin D3. The maximal accumulation of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine precedes the intestinal absorption response to 1alpha-hydroxyvitamin D3 by at least 2 hours. Oral administration results in the highest concentrations of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine, giving a level about 1.5 times that achieved with an intravenous dose. On the other hand, an oral dose of 1alpha-hydroxy[6-3H]vitaminD3 gives much lower amounts of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 in bone and blood than an intravenous dose, which suggests that the 1alpha-hydroxy[6-3H]vitamin D3 may not be utilized as well by the oral route as by an intravenous route. Liver homogenates from both rat and chick convert 1alpha-hydroxy[6-3H]vitamin D3 to 1alpha,25-dihydroxy[6-3H]vitamin D3. However, intestinal homogenates from chick, but not rat, can also cary out this conversion, which may account for the higher concentration of 1alpha,25-dihydroxy[6-3H]vitamin D3 found in the intestine of chicks given an oral dose of 1alpha-hydroxy[6-3H]vitamin D3.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

The photobiogenesis and metabolism of vitamin D.

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. P450-dependent mixed function oxidase system is 1alpha,25(OH)2D. When calcium and phosphate homeostasis prevails the renal 25-OH-D-1alpha-hydroxylase activity is limited and instead a non-cytochrome P450 mixed function oxidase metabolizes 25-OH-D to 24R,25(OH)2D. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitary hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined. The regulation of the photometabolism of provitamin D3 to vitamin D3, the role and significance of the side-chain metabolism of 1,25(OH)2D by the small intestine, and the metabolism of 25-OH-D to 24R,25(OH)2D by chondrocytes and its stimulation of protein synthesis in these cells are just a few issues that will require further investigation.     

Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.     

The response of the small intestine to vitamin D. Correlation between calcium-binding-protein production and increased calcium absorption

1. The synthesis of calcium-binding protein, a protein produced in the small intestine in response to vitamin D, was investigated with a view to determining whether calcium-binding-protein production could be correlated with the stimulation of calcium absorption by vitamin D. 2. A radioimmunological assay, which can quantitatively estimate calcium-binding-protein concentrations as low as 1μg/g wet wt., was used to detect the synthesis of soluble calcium-binding protein. 3. When used on intestinal supernatants from chicks dosed with vitamin D, calcium-binding protein was not detectable at 8h but was present after 12h at a concentration of 8.6μg/g wet wt.; in agreement with this an increase in calcium absorption due to vitamin D was detected at 12h but not at 8h. 4. The synthesis of calcium-binding protein was also monitored directly by making use of the ability of the iodinated antiserum to bind specifically to nascent calcium-binding protein chains on intestinal polyribosomes; in this way calcium-binding-protein synthesis could be detected 8h after dosage with vitamin D. Further, the binding reaction indicated a near linear increase in the calcium-binding-protein-synthesizing capacity over a 16h period. 5. From the amount of calcium-binding protein present 12 and 24h after vitamin D administration it is calculated that calcium-binding-protein mRNA is produced at approx. 1mol/min per intestinal cell. 6. It is concluded that the high correlation between the initiation of calcium-binding-protein synthesis and the stimulation of calcium absorption by vitamin D strengthens the proposal that calcium-binding protein plays an important role in calcium transport.     

Pancreatic vitamin D-dependent calcium binding protein: biochemical properties and response to vitamin D

The biochemical properties of a chick pancreatic calcium binding protein (CaBP) and its response to vitamin D status and dietary calcium and phosphorus levels were studied and compared with the known vitamin D-dependent CaBPs present in the chick intestine and kidney. Pancreatic CaBP is homologous to the intestinal CaBP on the basis of immunological cross-reactivity, molecular size (28,200 Da), and charge properties (chromatographic mobility on DEAE-Sephadex in the presence of either EDTA or Ca2+). Pancreatic levels of CaBP respond to changes in vitamin D status and dietary Ca and P level in a fashion similar to the intestinal CaBP. Thus, in the absence of dietary vitamin D, both pancreatic and intestinal CaBPs were essentially undetectable, while in the presence of dietary vitamin D, a low dietary P (0.05%) elevated the pancreatic and intestinal CaBP 1.5X and 1.6X, respectively, compared to the CaBP levels present with normal dietary Ca and P (1.0%, 1.0%). The tissue levels of pancreatic CaBP (6-10 ng/mg protein) are about 0.2% of the intestine (5000 ng/mg protein) and 1% of the kidney CaBP (700 ng/mg protein). However, when corrections are made for the CaBP distribution in the tissues and expressed as CaBP concentration per CaBP-containing cells, the pancreatic CaBP level was 30% of the intestine and 10% of the kidney. Collectively, these results suggest that the chick pancreatic vitamin D-dependent CaBP is a homologous protein to the intestinal CaBP, both with regards to its relative cellular concentration as well as in its response to changing dietary levels of Ca and P.     

25-Azavitamin D3, an inhibitor of vitamin D metabolism and action.

25-Azavitamin D3 inhibited both the bone calcium mobilization and intestinal calcium transport responses of rats to vitamin D3 but not to 25-hydroxyvitamin D3. Although 25-azavitamin D3 had no effect on the response of bone to 1alpha,25-dihydroxyvitamin D3, it did diminish the response of the intestine to that metabolite. 25-Azavitamin D3 increased liver vitamin D content and reduced the concentration of 25-hydroxyvitamin D3 required to inhibit the metabolism of vitamin D3 (75 and 200 microgram) were similar to the doses of 25-azavitamin D3 required to inhibit the action of vitamin D3 in vivo (50 and 150 microgram). 25-Azavitamin D3 is thus a vitamin D antagonist, acting for the most part via inhibition of the liver 25-hydroxylation of vitamin D3.     

Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat.

1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

The skeleton as an intracrine organ for vitamin D metabolism

The endocrine hormone, 1alpha,25-dihydroxyvitamin D(3) (1,25D) is an important regulator of calcium and phosphorus homeostasis. In this context, 1,25D is generally recognized as necessary for the maintenance of a healthy skeleton through its actions on the small intestine. In this review, we highlight the direct effects of 1,25D on the constituent cells of the bone, actions that are independent of effects on the intestine and kidney. We also consider the evidence that 25D levels, not 1,25D levels, correlate best with parameters of bone health, and that the bone itself is a site of metabolic conversion of 25D into 1,25D, by virtue of its expression of the 25-hydroxyvitamin D 1alpha-hydroxylase, CYP27B1. We review the evidence that at least osteoblasts and chondrocytes, and possibly also bone resorbing osteoclasts, are capable of such metabolic conversion, and therefore that these cells likely participate in autocrine and paracrine loops of vitamin D metabolism. We conclude that the skeleton is an intracrine organ for vitamin D metabolism, challenging the long-held notion that 1,25D is solely an endocrine hormone.     

Stimulation of spd-glucose transport A novel effect of vitamin D on intestinal membrane transport

Vitamin D stimulates absorption of spd-glucose in chick jejunum and ileum by a specific action on the maximal velocity of Na⁺-gradient driven spd-glucose transport across the brush-border membrane of intestinal cells. Induction of spd-glucose transport by either vitamin D-3 or 1,25-dihydroxyvitamin D-3 in embryonic intestine can be blocked by inhibitors of RNA and protein synthesis.     

Effects of different metabolites of vitamin D3 and of calcium concentration on the intestinal absorption of strontium]

The effect of calcium concentration and vitamin B3 25 OHD3 and 1.25 diOHD3 upon intestinal strontium transport was studied in vitamin D deficient rats with duodenal perfusion in situ. When the calcium concentration was increased, the strontium passive absorption was decreased. The vitamin D3 and its derivatives increased intestinal strontium transport significantly and this increment was not modified by calcium. In our experiment, calcium is competitive with strontium only in passive absorption.     

Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.     

Calbindin D9k is not required for 1,25-dihydroxyvitamin D3-mediated Ca2+ absorption in small intestine

The exact role of calbindin D9k in vitamin D-mediated calcium absorption has been debated but remains unsettled. In 129/OlaHsd mice, calbindin D9k was found highest in duodenum (36-50%) and kidney (24-34%) followed by stomach, lung and uterus. Age does not affect the relative distribution of calbindin D9k but it does decline with age in duodenum of both male and female 129/Ola mice. Recently, we produced a null calbindin D9k mutant 129/OlaHsd mouse; this mouse proved to be indistinguishable from the wild-type in phenotype and in a serum calcium level regardless of age or gender. We have now examined directly whether the mutant mouse can absorb calcium from the intestine in response to the active form of vitamin D. The calbindin D9k null mutant mouse is fully able to absorb calcium from the intestine in response to 1,25-dihydroxyvitamin D3. It is, therefore, clear that calbindin D9k is not required for vitamin D-induced intestinal calcium absorption.     

Methylation of the vitamin D-dependent CaBP gene (calbindin 9 Kd) does not mediate tissue or vitamin D regulation

The vitamin D-dependent intestinal calcium binding protein gene is predominantly expressed in the intestine. In this report we have examined the possibility that methylation of the gene might play a role in its tissue-specific expression employing genomic Southern analysis. None of the Hpa II and Hha I sites examined by the indicated probes in and around the gene were found to be methylated in the intestine, kidney and liver. No change in the methylation of these sites was detected in response to 1,25-dihydroxy-vitamin D3 administration to vitamin D-deficient rats under conditions which stimulate the expression of the gene. These results indicate that the rat intestinal calcium binding protein gene is not methylated in these tissues, at the indicated sites and, therefore, methylation seems not to be involved in the regulation of this gene's expression.