The role of 1,25-dihydroxyvitamin D3 and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin D3-24-hydroxylase.

A single 325-pmol dose of 1,25-dihydroxyvitamin D₃ given to chicks fed a vitamin D-deficient diet containing 3% calcium and 0.6% phosphorus suppresses renal mitochondrial 25-hydroxyvitamin D₃-1α-hydroxylase and stimulates the 25-hydroxyvitamin D₃-24-hydroxylase as measured by in vitro assay. This alteration in the enzymatic activity takes place over a period of hours. The administration of parathyroid hormone rapidly suppresses the 25-hydroxyvitamin D₃-24-hydroxylase. The alterations in the hydroxylases by parathyroid hormone or 1,25-dihydroxyvitamin D₃ are not related to changes in serum clacium or phosphate but could be related to changes in intracellular levels of these ions. Actinomycin D or cycloheximide given in vivo reduces the 25-hydroxyvitamin D₃-24-hydroxylase activity rapidly which suggests that the turnover of the enzyme and its messenger RNA is rapid (1- and 5-h half-life, respectively). The half-lives of the hydroxylases are sufficiently short to permit a consideration that the regulation by 1,25-dihydroxyvitamin D₃ and parathyroid hormone may involve enzyme synthesis and degradation.     

Renal clearance of phosphate and calcium in vitamin D-deficient chicks: effect of calcium loading, parathyroidectomy, and parathyroid hormone administration.

Serum and renal clearance values of phosphate and calcium were measured and compared in 4 week-old vitamin D-deficient and vitamin D-replete chickens (Gallus gallus). D-deficient chicks had significantly lower body weights and serum calcium values; however, their renal functions were not different from D-replete controls. Serum calcium values in D-deficient birds did not change in response to parathyroid hormone (PTH) administration; however, they did drop significantly in response to parathyroidectomy (PTX). Serum phosphate values of D-deficient birds, but not D-replete birds, rose significantly after PTX. Clearance of phosphate is known to increase after administration of PTH. This conspicuous effect was absent in PTH-injected vitamin D-deficient chickens. PTX caused the excretion of phosphate to drop in both D-deficient and D-replete birds to near zero. Conversely, PTX of both D-deficient and D-replete chickens stimulated the excretion of more calcium than in controls. Calcium loading elevates the fractional excretion of calcium in both D-deficient and D-replete birds. It also causes a decrease in phosphate excretion in both groups, presumably by inhibiting the secretion of PTH. PTH administration to D-replete, calcium-loaded birds caused increased phosphate excretion (as it did in normal controls), an effect that was not seen in similarly treated D-deficient birds. Therefore, most renal functions studied after calcium loading, PTH administration, or PTX are not altered by vitamin D deficiency in the chicken. The major significant finding is that vitamin D-deficient chickens do not excrete increased amounts of phosphate in response to PTH stimulus.     

Chick kidney ferredoxin: Complementary DNA cloning and vitamin D effects on mRNA levels

Vertebrate ferredoxin is non-heme iron-sulfur protein found in steroideogenic tissues that serves as an electron shuttle in mitochondrial mixed function oxidase systems such as the 25-hydroxyvitamin D₃-1α-hydroxylase. A 2530-bp chick kidney ferredoxin cDNA was cloned, and the association between ferredoxin mRNA levels and the regulation of 1α-hydroxylase activity by vitamin D status was examined. The cDNA sequence indicates that the chick kidney mitochondrial mixed function oxidases use the same ferredoxin as do those in the chick testis and that the chick ferredoxin shares greater than 92% amino acid homology with mammalian ferredoxins. Southern blot analysis of genomic DNA indicates that there is a single copy of the ferredoxin gene present in the chick genome. Three species of mRNA, 1.8, 3.5 and 5.5 kb, were identified by Northern analysis. Slot blot analysis of poly A⁺ RNA from kidneys of vitamin D-deficient or -replete chicks indicates a 40% induction of ferredoxin message levels in the vitamin D-deficient chick kidney. This suggests that gene regulation of ferredoxin may be part of the mechanism of regulation for 25-hydroxyvitamin D₃-1α-hydroxylase activity in the chick kidney.     

The renal mitochondrial metabolism of 25-hydroxyvitamin D-3: a possible role for phospholipids

The effect of exogenous phospholipids on chick kidney mitochondrial 25-hydroxyvitamin D-3 metabolism was examined. Phosphatidylserine, phosphatidylcholine and phosphatidylinositol had no effect on either the 1- or 24-hydroxylation of 25-hydroxyvitamin D-3. Phosphatidylethanolamine and cardiolipin both brought about a dose-dependent decrease in the 1-hydroxylase activity in mitochondria from vitamin D-deficient chicks but not from vitamin D-replete chicks. There were no major differences in the phospholipid composition of mitochondria from vitamin D-deficient and -replete chicks nor in the fatty acid composition of these phospholipids. Preliminary kinetic studies suggest that cardiolipin acts as a noncompetitive inhibitor of the 1-hydroxylase in mitochondria isolated from vitamin D-deficient chicks. It does not appear to exert its effect by virtue of altering the distribution of substrate or products. Investigation of the effect of fatty acid methyl esters on the hydroxylase activities suggests that it may be the fatty acid moiety of the phospholipid, rather than the phosphate moiety in the polar head group, that is involved in the phospholipid effect on the hydroxylation of 25-hydroxyvitamin D-3.     

Inhibition of vitamin D metabolism by ethane-1-hydroxyl-1, 1-diphosphonate

The administration of disodium-ethane-1-hydroxy-1,1-diphosphonate (20 mg/kg body weight subcutaneously) to chicks given adequate amounts of vitamin D₃ causes a hypercalcemia, inhibits bone mineralization, and inhibits intestinal calcium transport. The administration of 1,25-dihydroxyvitamin D₃, a metabolically active form of vitamin D₃, restores intestinal calcium absorption to normal but does not restore bone mineralization in disodium-ethane-1-hydroxy-1,1-diphosphonate-treated chicks. In rachitic chicks, the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment does not further reduce the low intestinal calcium transport values while it nevertheless further reduces bone ash levels and increases serum calcium concentration.These observations prompted a more detailed study of the relationship between disodium-ethane-1-hydroxy-1,1-diphosphonate treatment and vitamin D metabolism. A study of the hydroxylation of 25-hydroxyvitamin D₃ in an in vitro system employing kidney mitochondria from chicks receiving disodium-ethane-1-hydroxy-1,1-diphosphonate treatment demonstrates a marked decrease in 1,25-dihydroxyvitamin D₃ production and a marked increase in the 24,25-dihydroxyvitamin D₃ production. In addition, the in vivo metabolism of 25-hydroxy-[26,27-³H]vitamin D₃ in disodium-ethane-1-hydroxy-1,1-diphosphonate treated chicks supports the in vitro observations. In rachitic chicks the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment markedly reduces the 25-hydroxyvitamin D₃-1-hydroxylase activity of kidney, but does not increase the 25-hydroxyvitamin D₃-24-hydroxylase.These results provide strong evidence that large doses of disodium-ethane-1-hydroxy-1,1-diphosphonate produce a marked effect on calcium metabolism via alterations in the metabolism of vitamin D as well as the expected direct effect on the bone.     

Opposing actions of methylxanthines and dibutyryl cyclic AMP on 1,25 dihydroxyvitamin D3 production and calcium fluxes in isolated chick renal tubules

In contrast to dibuturyl cyclic AMP, the methylxanthine phosphodiesterase inhibitors theophylline and caffeine were found to inhibit the conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃ in isolated renal tubules from vitamin D deficient chicks. This inhibition occurred at concentrations of methylxanthines which were shown to increase renal tubule cyclic AMP levels. No effect of theophylline or caffeine on 25 hydroxyvitamin D₃ metabolism in isolated chick renal mitochondria was detected. Because of a demonstrated inhibitory action of calcium (10 and 20 μmol/l) on renal mitochondrial conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃, the effect of theophylline and dibutyryl cyclic AMP on cellular calcium-45 efflux and total renal tubule calcium content was estimated. Theophylline 10 mmol/l was found to inhibit renal tubular calcium efflux and to increase total cellular calcium content, while dibutyryl cyclic AMP 1 mmol/l had the reverse effect on both parameters. Divergent actions of the methylxanthines and dibutyryl cyclic AMP on the formation of 1,25 dihydroxyvitamin D₃ and renal tubule calcium efflux and content support the hypothesis that intracellular calcium is an important regulator of renal vitamin D metabolism. The results indicate that observed actions of methylxanthines cannot always be ascribed to cyclic AMP accumulation.     

24,24-Difluoro-25-hydroxyvitamin D3-enhanced bone mineralization in rats. Comparison with 25-hydroxyvitamin3 and vitamin D3

The biological activity of 24,24-difluoro-25-hydroxyvitamin D₃ was assessed using elevation of serum phosphorus and healing of rickets of vitamin D-deficient rats. Various levels of 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃ were administered daily for 2 weeks in the dose range of 6.5 to 3250 pmol after feeding rats a low phosphorus, vitamin D-deficient diet for 3 weeks. Vitamin D₃ was concurrently tested at dose levels of 650 and 3250 pmol. 24,24-Difluoro-25-hydroxyvitamin D₃ is approximately equipotent with 25-hydroxyvitamin D₃ in stimulation of growth, mineralization of rachitic bone, and elevation of serum inorganic phosphorus. Radiological manifestations of rickets were also equally improved by 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃. Compared with vitamin D₃, these compounds were approximately 5 to 10 times more active in mineralization using rats on a low phosphorus, vitamin D-deficient diet. The functional role, if any, for 24-hydroxylated vitamin D compounds, such as 24,25-dihydroxyvitamin D₃, therefore remains obscure. It appears that vitamin D compounds that cannot be 24-hydroxylated evoke no disorder in bone mineralization.     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

Kidney microsomal 25- and 1α-hydroxylase in vitamin D metabolism: catalytic properties,molecular cloning,cellular localization and expression during development

Both a 25-hydroxylation and a 1α-hydroxylation are necessary for the conversion of vitamin D₃ into the calcium-regulating hormone 1α,25-dihydroxyvitamin D₃. According to current knowledge, the hepatic mitochondrial cytochrome P450 (CYP) 27A and microsomal CYP2D25 are able to catalyze the former bioactivation step. Substantial 25-hydroxylase activity has also been demonstrated in kidney. This paper describes the molecular cloning and characterization of a microsomal vitamin D₃ 25- and 1α-hydroxylase in kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D₃ and 1α-hydroxyvitamin D₃ and, in addition, 1α-hydroxylation of 25-hydroxyvitamin D₃. The cDNA encodes a protein of 500 amino acids. Both the DNA sequence and the deduced peptide sequence of the renal enzyme are homologous with those of the hepatic vitamin D₃ 25-hydroxylase CYP2D25. Genomic Southern blot analysis suggested the presence of a single gene for CYP2D25 in the pig. Immunohistochemistry experiments indicated that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. These findings indicate a tissue-specific developmental regulation of CYP2D25. The results from the current and previous studies on renal vitamin D hydroxylations imply that CYP2D25 has a biological role in kidney.     

Cold resistance of the brain during hibernation. Temperature sensitivity of the partial reactions of the Na+, K+-ATPase.

The regulatory effect of calcium added in vitro on 25-hydroxycholecalciferol metabolism was studied in kidney mitochondria and in renal tubules from vitamin D-deficient chicks. The addition of calcium (0.05 – 0.2 mm) to mitochondrial suspensions prepared with calcium-chelating agents caused a marked and dose-related stimulation of 1-hydroxylation. A sharp decline in the activity was induced by higher concentrations of calcium (0.3 – 0.7 mm). A similar but less striking biphasic effect of calcium on 1-hydroxylation was observed in mitochondria prepared in the absence of calcium chelating agents. The effect of calcium was not a consequence of accelerated mitochondrial translocation of either exogenous NADP or Mg²⁺ but was related to mitochondrial calcium content. The addition of inhibitors of the calcium uptake, i.e., LaCl₃ or ruthenium red, or a calcium ionophore (A 23187) significantly inhibited the calcium-induced stimulation of the 1-hydroxylation reaction. Similar calcium effects were also observed in renal tubules isolated from intact, but not from parathyroidectomized, vitamin D-deficient chicks. These data strongly suggest that mitochondrial calcium plays an important role in the regulation of 1-hydroxylase activity in kidney.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Isolation,identification, and biological activity of 23,25,26-trihydroxyvitamin D3, an in vitro and in vivo metabolite of vitamin D3

Kidney homogenates from vitamin D₃-supplemented chicks incubated with 25-hydroxyvitamin D₃ [25(OH)D₃] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D₃ [23,25,26(OH)₃D₃] by loss of radioactivity from 25-hydroxy[23,24-³H]vitamin D₃ and 25-hydroxy-[26,27-methyl-³H]vitamin D₃, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D₃, and structurally characterized as 23,25,26-trihydroxyvitamin D₃. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH)₃D₃ or 25(OH)D₃ over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.     

Regulation of vitamin D metabolism.

Vitamin D can be regarded as a prohormone and its most potent metabolite, 1, 25-dihydroxyvitamin D₃, a hormone which mobilizes calcium and phosphate from bone and intestine. In true hormonal fashion, the biosynthesis of 1, 25-dihydroxyvitamin D₃ by kidney mitochondria is feed-back regulated by serum calcium and serum phosphorus levels. The lack of calcium brings about a secretion of parathyroid hormone which stimulates 1, 25-dihydroxyvitamin D₃ synthesis while low blood phosphorus stimulates 1, 25-dihydroxyvitamin D₃ synthesis even in the absence of the parathyroid glands. For such regulation to occur, vitamin D must be present probably because 1, 25-dihydroxyvitamin D₃ itself is needed for the regulation. The molecular and cellular mechanisms whereby 1, 25-dihydroxyvitamin D₃ synthesis is regulated are unknown despite many recent reports. Likely the elucidation of these mechanisms must await a detailed investigation of the enzymology of the renal 25-hydroxyvitamin D₃-1α-hydroxylase. In addition to the regulation at the 25-hydroxyvitamin D₃-1α-hydroxylase step, vitamin D metabolism is regulated at the hepatic vitamin D-25-hydroxylase level. This regulation is a suppression of the hydroxylase by the hepatic level of 25-hydroxyvitamin D₃ itself by an unknown mechanism. Much remains to be learned concerning the regulation of this newly discovered endocrine system but already the findings are not only relevant to calcium homeostasis but also to an understanding of a variety of metabolic bone diseases.     

Evidence for aqueous soluble vitamin D-like substances in the calcinogenic plant, Tristetum flavescens

A crude aqueous extract of the leaves of $\text{T. flavescens}$ when administered orally to vitamin D-deficient chicks produced significant increases in plasma phosphate but had little effect on plasma calcium. When chicks, fed a high strontium diet to inhibit endogenous 1,25(OH)₂ vitamin D₃ production and intestinal calcium transport, were dosed with the extract or synthetic 1,25(OH)₂D₃⁴⁵Ca absorption from the duodenum $\text{in vivo}$ was stimulated, whereas vitamin D₃ was ineffective. Partial purification of the crude extract on a Sephadex GH25 column yielded two factors, one of which mimicked 1,25 (OH)₂D₃ activity in chicks fed the high strontium diet while the other produced a significant increase in plasma phosphate. The presence of these substances, together with previously demonstrated organic solvent soluble vitamin D-type activity, may account for the calcinogenic nature of the plant.     

1 alpha, 25-difluorovitamin D3: an inert vitamin D analog

1α,25-Difluorovitamin D₃ has been synthesized by reacting 1,25-dihydroxyvitamin D₃-3-acetate with diethylaminosulfurtrifluoride followed by hydrolysis. Retention of configuration of the fluoro group in this reaction was demonstrated by physical studies using 1α-fluoro and 1β-fluorovitamin D₃ models. The 1,25-difluorovitamin D₃ compound possessed no vitamin D-like activity demonstrating the importance of 1α- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D₃ had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D₃ has anti-25-hydroxylase activity, it appears the introduction of a fluoro group on the 1 position diminishes interaction of the vitamin D molecule with the 25-hydroxylase system.     

Circadian rhythm of 1 alpha,25-dihydroxyvitamin D3 production in egg-laying hens.

The activity of renal 25-hydroxyvitamin D₃(25-OH-D₃)-1α- and 24-hydroxylase and the plasma concentrations of vitamin D metabolites were investigated in relation to the ovulatory cycle in egg-laying hens. The time after ovulation was estimated from the position of the egg in the oviduct and the dry weight of the egg-shell. The $\text{in}\text{vitro}$ renal 25-OH-D₃-1α-hydroxylase activity was significantly enhanced 14–16 hr after ovulation, whereas 25-OH-D₃-24-hydroxylase activity remained unchanged. The plasma level of 1α,25-dihydroxyvitamin D [1α,25-(OH)₂-D] was also increased 14–16 hr after ovulation in accord with the enhancement of the renal 1α-hydroxylase activity. The plasma level of 24,25-dihydroxyvitamin D did not change during the ovulatory cycle. These results strongly suggest that 1α,25-(OH)₂-D₃ production in the kidney varies in a circadian rhythm during the ovulatory cycle in egg-laying hens.     

Regulation of vitamin D metabolism by calcium and phosphate ions in isolated renal tubules.

The acute and long-term effects of Ca2+ and Pi on vitamin D metabolism were studied in vitro with isolated renal tubules from vitamin D-deficient and vitamin D-supplemented chicks. Ca2+ depletion, achieved by isolating renal tubules in Ca2+-free buffers, led to suppression of 1 alpha-hydroxylase activity. Re-introduction of Ca2+ during incubation caused an acute stimulation of this enzyme. This stimulatory effect of Ca2+ was prevented by prior treatment of Ca2+-depleted renal tubules for 6 h with 1,25-dihydroxycholecalciferol. Ca2+ and Pi produced marked acute affects on 1 alpha-hydroxylase activity, which persisted for the whole 8 h experimental period, in Ca2+-depleted renal tubules from vitamin D-deficient chicks. The effects of either ion were influenced by the concentration of the other. However, the effects of these ions could not be reproduced in either Ca2+-depleted renal tubules from vitamin D-supplemented chicks or in renal tubules from vitamin D-deficient chicks, isolated in Ca2+-containing buffers. Isolation of renal tubules from vitamin D-supplemented chicks in Ca2+-containing buffers and subsequent incubation for 8 h in the presence of increased [Ca2+] led to a modest but statistically significant suppression of 1 alpha-hydroxylase and stimulation of 24-hydroxylase activity. It is concluded that the acute effects of Ca2+ and Pi on 1 alpha-hydroxylase activity of Ca2+-depleted renal tubules from vitamin D-deficient chicks are not regulatory but the results of the experimental conditions. In contrast the long-term effects of Ca2+ on both hydroxylases of renal tubules from vitamin D-supplemented chicks may be of physiological significance.     

Vitamin D in adipose tissue and serum 25-hydroxyvitamin D after roux-en-Y gastric bypass

Vitamin D is stored in body fat. The purpose of this study was to determine vitamin D concentration in abdominal fat of obese patients who underwent roux‐en‐Y gastric bypass (RYGB), and to describe changes in serum 25‐hydroxyvitamin D (25(OH)D) levels in relation to loss of body fat. Subjects from a single clinic who were scheduled for RYGB were invited into the study. Abdominal subcutaneous, omental, and mesenteric fat were obtained at time of surgery. Adipose vitamin D₂ and vitamin D₃ concentrations were measured by high‐performance liquid chromatography (HPLC). Weight and serum 25(OH)D were assessed at baseline and every 3 months up to 1 year. Seventeen subjects were included, and fat samples were available from eleven. Total vitamin D content in subcutaneous abdominal fat was 297.2 ± 727.7 ng/g tissue, and a wide range was observed (4–2,470 ng/g). Both vitamin D₂ and vitamin D₃ were detected in some of the fat samples. At baseline, 25(OH)D was 23.1 ± 12.6 ng/ml. Average weight loss was 54.8 kg at 12 months, of which ～40 kg was fat mass. Despite daily vitamin D intake of ≥2,500 IU throughout the study, no significant increase in serum 25(OH)D was observed, with mean serum concentration of 25(OH)D at 1 year of 26.2 ± 5.36 ng/ml (P = 0.58). We conclude that vitamin D in adipose tissue does not significantly contribute to serum 25(OH)D despite dramatic loss of fat mass after RYGB.     

25,26-dihydroxyvitamin D3 is not a major intermediate in 25-hydroxyvitamin D3-26,23-lactone formation

Structural similarities between 25S,26-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃-26,23-lactone and their concomitant multifold increase in the plasma of animals treated with pharmacological doses of vitamin D₃ suggest a precursor-product relationship. However, a single dose of 25S,26-[³H]dihydroxyvitamin D₃ given to rats treated chronically with pharmacological amounts of vitamin D₃ did not result in detectable plasma 25-[³H]hydroxyvitamin D₃-26,23-lactone. Multiple doses of synthetic 25S,26-dihydroxyvitamin D₃ given to vitamin D₃-deficient rats treated chronically with pharmacological amounts of vitamin D₂ also did not result in detectable plasma 25-hydroxyvitamin D₃-26,23-lactone. Furthermore, homogenates prepared from vitamin d-deficient chickens, dosed with 1,25-dihydroxyvitamin D₃, converted 25-[³H]hydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. But these same homogenates did not convert 25S,26-[³H]dihydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. These data indicate that 25,26-dihydroxyvitamin D₃ is not an intermediate in 25-hydroxyvitamin D₃26, 23-lactone formation.