24,25-dihydroxyvitamin D3 binds to catalase

There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) has endocrine actions. In the current work, we report that an endogenous binding protein for 24,25(OH)2D3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [3H]24,25(OH)2D3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)2D3 on 32P uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [3H]24,25(OH)2D3 specifically bound. Additional studies indicated that 25(OH)D3 was an effective competitor for binding, whereas 1,25(OH)2D3 only poorly displaced [3H]24,25(OH)2D3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (KD=5.6+/-2.7 nM) and a Bmax=209+/-34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [3H]24,25(OH)2D3 binding and catalase activity increased from 7- to 58-week-old birds, whereas in males, elevated levels of both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.     

Effect of 24,25-dihydroxyvitamin D3 in osteoclasts.

Previous results demonstrated that the administration of pharmacological doses of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to animals reduces bone resorption and increases bone volume with a decrease in osteoclast number. In order to clarify whether 24,25(OH)2D3 has an effect to inhibit osteoclastic bone resorption, the effect of 24,25(OH)2D3 on the formation and function of osteoclastic cells was examined in vitro. Treatment of hemopoietic blast cells, which are progenitors of osteoclasts, with parathyroid hormone (PTH) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulated the formation of osteoclast-like multinucleated cells in a dose-dependent manner. Although 24,25(OH)2D3 in itself had little effect on osteoclast-like multinucleated cells formation, it inhibited the stimulatory effect of PTH on the formation of osteoclastic cells. In addition, 24,25(OH)2D3 also inhibited the stimulation of resorption pit formation by osteoclasts under stimulation with PTH. In contrast, 1,25(OH)2D3 stimulated the formation and function of osteoclastic cells even at low concentrations, and the effect was additive to PTH. These results could not be explained by either an agonistic or antagonistic effect of 24,25(OH)2D3 on 1,25(OH)2D3, and are consistent with the assumption that 24,25(OH)2D3 has a unique inhibitory effect on the formation and function of osteoclasts. Because 24,25(OH)2D3 is shown to stimulate the degradation of 1,25(OH)2D3 and because the formation of 24,25(OH)2D3 is stimulated by 1,25(OH)2D3 not only in the kidney but also in many of its target tissues, including bone, the inhibitory effect of 24,25(OH)2D3 on osteoclastic bone resorption may play a role in the local modulation of the actions of osteotropic hormones in bone.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Bone turnover in rats treated with 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3

Female rats were given 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 0.25 g per 100 g body weight (bw), 25-hydroxyvitamin D₃ (25(OH)D₃), 1.7 g/100 g bw or 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) 1.7 g/100 g bw, subcutaneously three times a week for 12 weeks. Traditional variables pertaining to calcium homeostasis and growth, i.e. blood and urine calcium (Ca) and phosphate (P), serum levels of vitamin D₃ metabolites parathyroid hormone, (PTH), calcitonin (CT), prolactin (PRL) and growth hormone (GH) were measured every four weeks. This data pool was correlated with bone matrix turnover parameters, i.e. serum levels of alkaline phosphatase (ALP) and urinary hydroxyproline (u-HYP) excretion. After 12 weeks of treatment, 1,25(OH)₂D₃ significantly enhanced serum total and ionized Ca, urine Ca and urine P, and also diminished urine cAMP due to reduced renal function (creatinine clearance). However, 25(OH)D₃ administration had no such impact. 24,25(OH)₂D₃ opposed the effect of 1,25(OH)₂D₃ after 12 weeks by significantly augmenting serum P and diminishing serum levels of total Ca and ionized Ca. Cross sectional group analyses showed that criculating levels of ALP were directly related with serum 1,25(OH)₂D₃ and inversely related to serum 24,25(OH)₂D₃ and CT. Total u-HYP and per cent non-dialysable HYP (ndHYP) were reciprocally and positively correlated with serum PRL, respectively. However, no such relations were observed with serum GH.It appears that rats with elevated circulating levels of 1,25(OH)₂D₃ exhibit increased bone resorption, while augmented 24,25(OH)₂D₃ is associated with the opposite. Apparently, high bone turnover (i.e. reduced total urinary HYP and enhanced ndHYP) is associated with high serum PRL.     

A simple direct spectrophotometric assay for 24,25-dihydroxyvitamin D3

A simple reproducible assay to determine the concentration of 24,25-dihydroxyvitamin D₃ in blood plasma is described. This technique employs quantitation of the steroid by direct spectrophotometric analysis, rather than competitive protein binding assay after partition and high-pressure liquid chromatographic purification of plasma extracts. The concentration of metabolite observed in normal adult plasma is 2.4 ± 1.1 ng/ml.     

Metabolism and pharmacokinetics of 24,25-dihydroxyvitamin D3 in the vitamin D3-replete rat

The time course of in vivo metabolism of 24,25-dihydroxyvitamin D3 in rats has been examined. Several tissues were surveyed in an effort to discover new metabolites of 24,25-dihydroxyvitamin D3 and to estimate the concentrations of previously identified metabolites. Rapidly growing male rats were dosed with 24,25-dihydroxyvitamin D3 orally until plasma concentrations of 24,25-dihydroxyvitamin D3 were at steady state. 24,25-Dihydroxyvitamin [3-3H]D3 was then administered. At 10 min and 1, 6, 15, 24, 96, and 192 h after dosing, the animals were killed, and plasma, liver, intestine, and bones were analyzed with a newly developed gradient straight-phase high performance liquid chromatography system. The high performance liquid chromatography system is capable of base-line resolution of most of the major vitamin D metabolites. 24,25-Dihydroxyvitamin D3 clearance from plasma, liver, and kidney but not intestine followed a two-compartment model. 24,25-Dihydroxyvitamin D3 disappeared from plasma with a half-life of 0.55 h (fast phase) and 73.8 h (slow phase). Only two lipid-soluble metabolites of 24,25-dihydroxyvitamin D3 were detected: 24-oxo-25-hydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3. These compounds circulate at very low concentrations in the plasma (50 pg/ml of plasma).     

In vitro and in vivo glucuronidation of 24,25-dihydroxyvitamin D3.

Glucuronidation of 24,25-dihydroxyvitamin D3 has been investigated in in vitro and in vivo experiments. Three positional isomers of 24,25-dihydroxyvitamin D3 monoglucuronide were synthesized from 24,25-dihydroxyprovitamin D3 derivatives with Koenigs-Knorr reaction and used as standard samples. In the presence of the rat liver microsomal fraction and uridine-5'-diphosphoglucuronic acid, 24,25-dihydroxyvitamin D3 gave 3- and 24-glucuronides as the main products in almost equal amounts, but only a small amount of the corresponding 25-glucuronide was obtained. 24,25-Dihydroxyvitamin D3 monoglucuronide was deconjugated with rat intestine homogenate, which indicated the entero-hepatic circulation of 24,25-dihydroxyvitamin D3. After the administration of 24,25-dihydroxyvitamin D3 to rats, its 3- and 24-glucuronides were identified from the bile as inferred from the in vitro experiment. However, the in vivo glucuronidation occurred at the 24-position in preference to the 3-position, and the corresponding 25-glucuronide was not detected. These glucuronides were identified in comparison with standard samples based on their chromatographic behavior during high-performance liquid chromatography and data obtained from liquid chromatography-electrospray ionization-mass spectrometry, which was helpful in identifying these compounds.     

Synthesis in vitro of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by interferon-gamma-stimulated normal human bone marrow and alveolar macrophages

Cultured human macrophages from normal donors were examined for their capability to metabolize 25-hydroxyvitamin D3 (25-(OH)D3). Upon exposure to recombinant human interferon-gamma (IFN-gamma) both bone marrow-derived macrophages (BMM) and pulmonary alveolar macrophages (PAM) produced a polar 25-(OH)D3 metabolite which was purified from conditioned media and unequivocally identified as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by UV-absorbance spectrophotometry and mass spectrometry. The BMM and PAM also synthesized a second 25-(OH)D3 metabolite which was structurally identified as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). The time course of 25-(OH)D3 metabolism by macrophages suggested that the production of 24,25-(OH)2D3 was stimulated by high intracellular levels of 1,25-(OH)2D3 and not by IFN-gamma. The 1,25-(OH)2D3 obtained from BMM and PAM promoted macrophage-like differentiation of promyelocytic HL-60 leukemia cells and inhibited IFN-gamma production by normal human lymphocytes. Our data suggest that locally high levels of 1,25-(OH)2D3 in the microenvironment of IFN-gamma-stimulated BMM and PAM may modulate the function of hormone-responsive cells.     

Assay of 24,25-dihydroxyvitamin D by competitive protein binding

A competitive protein binding radioassay for 24,25-dihydroxyvitamin D in human serum has been developed, which is relatively simple and rapid. Acetonitrile is used for sample extraction and protein precipitation. column chromatography is then performed in a Sep-pak cartridge. High pressure liquid chromatography follows. The dried eluate is assayed using rat serum as the source of binding protein. Since 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 are equipotent in their competitive displacement of tritiated 25-hydroxyvitamin D3 from at serum, 25-hydroxyvitamin D3 can be used as the assay standard.     

Bone resorbing activities of 24-hydroxy stereoisomers of 24-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

R and S isomers of 24-OH-D₃ and 24,25-(OH)₂D₃ were tested for their effects on bone resorption $\text{in vitro}$. 24(R), 25-(OH)₂D₃ was more active than 24(S),25-(OH)₂D₃. Likewise, 24(R)-OH-D₃ was more active than 24(S)-OH-D₃. The bone resorbing activity of 24(R)-OH-D₃ was equivalent to that of 25-OH-D₃; 24(R),25-(OH)₂D₃ was somewhat less potent. The results indicate that there is discrimination between the isomers of these compounds at the level of the responding tissue.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Isolation and identification of 24-hydroxyvitamin D2 and 24,25-dihydroxyvitamin D2

The isolation and identification of two metabolites of vitamin D₂ found in mammalian and avian species are reported. They are 24-hydroxyvitamin D₂ and 24,25-dihydroxyvitamin D₂. Their existence suggests that 24-hydroxylation occurs in a sterospecific manner in the 24R position and adds further support to the theory that vitamin D₂ metabolism qualitatively parallels that of vitamin D₃.     

Identification of 24,25,26,27-tetranor-23-hydroxyvitamin D3 as a product of the renal metabolism of 24,25-dihydroxyvitamin D3

By cochromatography, mass spectrometry, and chemical derivatization, we have shown that a metabolite isolated from the perfused rat kidney incubated with 24-(R),25-dihydroxyvitamin D3 is indistinguishable from chemically synthesized 24,25,26,27-tetranor-23-hydroxyvitamin D3. The new metabolite is also produced from 24-oxo-25-hydroxyvitamin D3 but not from 23(S),25-dihydroxyvitamin D3. Enzymes required for the synthesis of the new metabolite are absent in the vitamin D deplete animal but are induced along with the 25-hydroxyvitamin-D3 24-hydroxylase by vitamin D repletion. The pathway of 24,25-dihydroxyvitamin D3 metabolism in the perfused kidney is stimulated by pre-treatment of the rat with large doses of vitamin D3, suggesting that the pathway is a degradative one.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Increased placental concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in pregnant rats treated with human chorionic gonadotropin

Pregnant rats were injected intrajugularly with 2500 i.u. human chorionic gonadotropin (HCG) toward the end of gestation (days 18-19) and 7.0 pmoles of tritiated 25-hydroxyvitamin D3 [( 3H]25(OH)D3) the following day. They were sacrificed ten to 24 hours later. [3H]25(OH)D3 and the in vivo produced [3H]24,25-dihydroxyvitamin D3 [( 3H]24,25(OH)2D3) in lipid extracts from maternal serum, kidneys, placenta and fetal tissues were separated by Sephadex LH-20 chromatography, and high performance liquid chromatography (HPLC). HCG treatment of pregnant rats increased significantly 25(OH)D3 levels in the placenta and kidneys and 24,25(OH)2D3 level in the placenta. Fetal metabolites levels were unaffected by HCG treatment. Serum and kidney levels of 25(OH)D3 and 24,25(OH)2D3 in pregnant rats were significantly lower than in non-pregnant rats. Serum and kidney levels of both metabolites in non-pregnant female rats treated with HCG did not differ from the untreated controls. HCG may, therefore, be involved in regulation of fetoplacental vitamin D metabolism.     

Structural study on the effect of 24,25-dihydroxyvitamin D3 on epiphyseal growth plate in suckling mice

The in vivo effects of 24,25(OH)2D3 on cellular structure and organization, matrix metachromasia and mineralization were studied in epiphyseal growth plate of normal neonatal mice. A relatively low dose of the metabolite, 40 ng/kg body weight, significantly increased the overall size of humeral growth plate and the zone of cellular proliferation. By and large, the tissue's response to the metabolite did not change with the increase in dose administered except for a decrease in the number of chondroblasts. 24,25(OH)2D3 led to significant increases in the metachromatic reaction of the cartilaginous matrix, but appeared to depress the mineralization process. Qualitative structural changes were noted in chondroblasts and hypertrophic chondrocytes. 24,25(OH)2D3 affected the osteoblastic and osteocytic populations of cells in the metaphysis and diaphysis of the humerus. High doses of 24,25(OH)2D3 brought about distinct atrophic changes in the above cells. These findings indicate that excessive doses of 24,25(OH)2D3 in an intact animal may lead to retardative effects upon bone growth.