A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.     

1 alpha,25-Dihydroxycholecalciferol and a human myeloid leukaemia cell line (HL-60).

Human promyelocytic leukaemia cells (HL-60) can be induced to differentiate into mature granulocytes in vitro by 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], the active form of cholecalciferol. The differentiation-associated properties, such as phagocytosis and C3 rosette formation, were induced by as little as 0.12 nM-1 alpha,25(OH)2D3, and, at 12 nM, about half of the cells exhibited differentiation on day 3 of incubation. Concomitantly the viable cell number was decreased to less than half of the control. Among various derivatives of cholecalciferol examined, 1 alpha,25(OH)2D3 and 1 alpha,24R-dihydroxycholecalciferol were the most potent in inducing differentiation, followed successively by 1 alpha,24S-dihydroxycholecalciferol, 1 alpha-hydroxycholecalciferol, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol. A cytosol protein specifically bound to 1 alpha,25 (OH)2D3 was found in HL-60 cells. Its physical properties closely resembled those found in such target tissues as intestine and parathyroid glands. 1 alpha,25(OH)2D3 bound to the cytosol receptor was transferred quantitatively to the chromatin fraction. The specificity of various derivatives of cholecalciferol in inducing differentiation was well correlated with that of their association with the cytosol receptor. These results are compatible with the hypothesis that the active form of cholecalciferol induces differentiation of human myeloid leukaemia cells by a mechanism similar to that proposed for the classical concept of steroid hormone action.     

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.     

The effects of 24R,25-dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum.

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

Cholecalciferol metabolites binding in porcine parathyroid glands.

We studied the cytoplasmic and nuclear binding of 25-hydroxychole-calciferol and 1alpha,25-dihydroxycholecalciferol inside porcine parathyroid glands. Both sterols bind to cytoplasmic components, but a specific nuclear uptake was demonstrated only for 1alpha,25-dihydroxycholecalciferol. These findings support the hypothesis that mammalian parathyroid glands are a target organ for some cholecalciferol metabolites.     

The effect of 1 alpha, 25-dihydroxycholecalciferol on iron metabolism

Chronic administration of hypercalcemic doses of 1 alpha, 25-dihydroxycholecalciferol to intact, vitamin-D repleted rats for 4 weeks, enhanced net intestinal absorption of iron and liver iron stores. Daily net iron and calcium absorptions were found to be significantly correlated in both control and treated rats. In duodenal loop experiments, pretreatment with 1 alpha, 25-dihydroxycholecalciferol reversed the adverse effect of high Ca/Fe ratio on iron absorption. The increased intestinal absorption of iron did not result in a change of serum iron levels nor of total iron binding capacity due to the enhanced incorporation of absorbed iron into liver ferritin. The curve of uptake of 59Fe into circulating red cells of treated rats suggested retarded release of the isotope from stores. The hypothesis is advanced that the systemic metabolic defect (tissue hypoxia, raised erythropoietin levels) produced by 1 alpha, 25-dihydroxycholecalciferol is responsible for the disruption of the physiological coordination between iron stores and intestinal absorption.     

Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.     

The mechanism of end-organ resistance to 1 alpha,25-dihydroxycholecalciferol in the common marmoset.

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.     

A high-affinity cytosol binding protein for 1 alpha,25-dihydroxycholecalciferol in the uterus of Japanese quail.

Cytosol fractions prepared from the uterine mucosa of egg-laying Japanese Quail were analysed for binding of the metabolites of cholecalciferol. When the uterus was incubated at 37 degrees C with various radioactive metabolites of cholecalciferol, the nuclear fraction incorporated only 1 alpha,25-dihydroxy[3H]cholecalciferol. When the uterus was incubated at 0 degree C with 1 alpha,25-dihydroxy[3H]cholecalciferol, most of the radioactivity was found in the cytosol. Translocation of 1 alpha,25-dihydroxy[3H]cholecalciferol from the cytosol to the nucleus was temperature-dependent. The addition of 100-fold excess amounts of unlabelled 1 alpha-25-dihydroxycholecalciferol significantly diminished the nuclear binding of 1 alpha,25-dihydroxy[3H]cholecalciferol. The cytosol fraction contained a 3.5 S macromolecule that specifically binds 1 alpha,25-dihydroxy[3H]cholecalciferol. The dissociation constant was 0.39 nM and the maximal binding was 55 fmol/mg of protein. These results strongly suggest that the uterus in egg-laying birds is a target organ or 1 alpha,25-dihydroxycholecalciferol.     

Vitamin D metabolism and its possible role in the developing chick embryo.

The relationship between bone formation and vitamin D metabolism was investigated in the developing chick embryo. Fertilized White Leghorn eggs were incubated at 38 degrees C in an incubator for 21 days. The fresh weight and calcium content of embryonic tibiae began to increase at day 12 and attained maximal values at day 19. Bone alkaline phosphatase and citrate decarboxylation activities, both of which represent osteoblastic activity, also began to increase at days 10-12, reached maximal values at day 19 and sharply declined thereafter. Both bone enzyme activities were highly correlated with CA2+-binding activity in the chorioallantoic membrane measured by the Chelex 100 assay. When mesonephric and metanephric homogenates were incubated with 25-hydroxy[3H]cholecalciferol, a marked and concomitant increase occurred in the metanephric 1 alpha- and 24-hydroxylase activity after day 14. The production of 1 alpha, 25-dihydroxycholecalciferol attained a maximal value at day 19 and decreased thereafter, whereas that of 24,25-dihydroxycholecalciferol continued to increase until hatching. The production rate of 1 alpha, 25-dihydroxycholecalciferol by the metanephros coincided with the changes in Ca2+-binding activity in the chorioallantoic membrane and osteoblastic activity. Since both intestinal calcium absorption and bone mineral mobilization do not occur in embryonic life, these results support the idea that 1 alpha, 25-dihydroxycholecalciferol may be involved directly in bone formation or induction of a calcium-binding protein in the chorioallantoic membrane.     

24R,25-dihydroxyvitamin D stimulates creatine kinase BB activity in chick cartilage cells in culture

In chick limb-bud cartilage cell cultures 24R,25-dihydroxycholecalciferol (24R,25(OH)2D3), but not 24S,25(OH)2D3, 1 alpha,25(OH)2D3 or 25(OH)D3, stimulates the activity of the brain type (BB) isozyme of creatine kinase (EC 2.7.3.2), the 'estrogen-induced protein' first identified in rat uterus. Cultures treated with bromodeoxyuridine, in which cartilage formation is inhibited, show no stimulation of creatine kinase BB by 24R,25(OH)2D3.     

Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.     

Structural analogs of 1alpha,25-dihydroxycholecalciferol: preparation and biological assay of 1alpha-hydroxypregnacalciferol.

The synthesis of 1alpha-hydroxypregnacalciferol, a side chain analog of 1alpha,25-dihydroxycholecalciferol (1alpha,25-dihydroxyvitamin D3), is described. Pregnenolone acetate was converted in five steps to 5-pregnen-1alpha,3beta-diol. Conversion of the diol to pregna-5,7-diene-1alpha,3beta diol diacetate followed by ultraviolet irradiation gave the corresponding previtamin derivative. Thermal isomerization, hydrolysis and chromatography then furnished the desired analog, 1alpha-hydroxypregnacalciferol. The compound was tested in vivo for its effect on intestinal calcium transport, serum calcium and phosphate levels and bone calcification, and in vitro for its effect on bone resorption. When given to intact rats, either as a single dose or in repeated daily doses, the analog even at high dose levels, exhibited no biological activity. The compound stimulated bone resorption in vitro, but only at high concentrations.     

Binding properties of serum vitamin D transport proteins in vertebrates for 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol in vitro.

1. The affinities of the specific vitamin D plasma transport proteins for 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol were studied in 34 vertebrate species. 2. Fish plasma proteins bound 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol with equal efficiency. 3. Vitamin D transport proteins in birds and a monotreme bound 25-hydroxycholecalciferol more efficiently than 24R, 25-dihydroxycholecalciferol; in one bird the two seco-steroids were bound with equal efficiency. 4. Transport proteins from marsupial and placental mammals bound 24R, 25-dihydroxycholecalciferol more efficiently than 24S, 25-dihydroxycholecalciferol. 5. Twelve mammal transport proteins bound 25-hydroxycholecalciferol and 24R, 25-dihydroxycholecalciferol with equal efficiency, however, in six mammals 25-hydroxycholecalciferol was more efficiently bound.     

Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide

Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha. Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit. The role of p110alpha in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect. Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.     

The difference of biological activity among four diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-lactone

All four possible diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-lactone (1 alpha,25-(OH)2D3-26,23-lactone) were chemically synthesized and were compared to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3) in terms of their stimulation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in vitamin D-deficient rats (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal cytosol receptor for 1 alpha,25-(OH)2D3. The receptor binding affinity results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1 alpha,25(OH)2D3. The RCI obtained for 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 7.90, for 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 2.27, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 0.17, for 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone 0.22 and for the in vivo produced 1 alpha,25(OH)2D3-26,23-lactone the RCI was only 0.17. Also the four diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone all stimulated intestinal calcium transport, reaching a maximum 8 h after administration. Compared with the stimulation of intestinal calcium transport by 1 alpha,25(OH)2D3, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/4 as effective, 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/20 as effective, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/74 as effective and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/53 as effective. Similarly, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone were estimated to be 3 and 20 times less active than 1 alpha,25-(OH)2D3 in elevation of serum calcium. However, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone decreased the serum calcium levels 24 h after administration. 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone reduced serum calcium concentrations to a greater extent than 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone. These results indicate that the biological activities of the diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone were quite different among four stereochemical configurations.     

Effects of 1 alpha,25-dihydroxycholecalciferol on sodium-ion translocation across chick intestinal brush-border membrane.

By utilizing isolated brush-border vesicles, Na+ transport across the luminal membrane of chick small intestine was found to be a composite of (i) a saturable (Km 10mM-Na+) amiloride-sensitive Na+/H+ antiport and (ii) a potential-sensitive conductive pathway. No evidence was obtained for the existence of a Na+/Cl- symport system. With the exception of the duodenum, luminal Na+ transfer in the entire small intestine was subject to regulation by vitamin D. Repletion of vitamin D-deficient chicks with 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] significantly decreased net Na+ uptake by isolated membrane vesicles (by approximately 30%). The sterol suppresses the conductive pathway (25-45% inhibition) as well as the Na+/H+ antiport system. Kinetic analysis of the latter revealed that 1 alpha,25(OH)2D3 altered Vmax (from 12.9 to 4.8 nmol of Na+/20s per mg of protein), but did not change Km. Diminution of Na+ transfer, entailing an increase in the electrochemical transmembrane Na+ gradient, provides an explanation of the simultaneously observed stimulatory action of 1 alpha,25(OH)2D3 on Na+-gradient-driven solute transport in chick small intestine. Indirect evidence was obtained that the luminal plasma membrane of chick small intestine displays a definite H+ permeability that is positively affected by 1 alpha,25(OH)2D3.     

Comparative studies on the 25-hydroxylations of cholecalciferol and 1α-hydroxycholecalciferol in perfused rat liver

The 25-hydroxylations of [(3)H]cholecalciferol and 1alpha-hydroxy[(3)H]cholecalciferol in perfused rat liver were compared. Results showed that about twice as much 1alpha(OH)D(3) (1alpha-hydroxycholecalciferol) was incorporated into the liver as cholecalciferol. 25-Hydroxy[(3)H]cholecalciferol and 1alpha-25-dihydroxy[(3)H]cholecalciferol were not incorporated significantly. Livers isolated from vitamin D-deficient rats formed the 25-hydroxy derivatives of cholecalciferol and 1alpha(OH)D(3) respectively linearly with time for at least 120min. The rate of 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxycholecalciferol) production increased exactly 10-fold on successive 10-fold increases in the dose of 1alpha(OH)D(3), suggesting that hepatic 25-hydroxylation of 1alpha(OH)D(3) is not under metabolic control. On the other hand, the rate of conversion of cholecalciferol into 25(OH)D(3) (25-hydroxycholecalciferol) did not increase linearly with increase in the amount of cholecalciferol in the perfusate. The 25-hydroxylation of cholecalciferol seemed to proceed at a similar rate to that of 1alpha(OH)D(3) at doses of less than 1nmol, but with doses of more than 2.5nmol, the conversion of cholecalciferol into 25(OH)D(3) became much less efficient, though the linear relation between the amounts of substrate and product was maintained. A reciprocal plot of data on the 25-hydroxylation of cholecalciferol gave two K(m) values of about 5.6nm and 1.0mum, whereas that for the 25-hydroxylation of 1alpha(OH)D(3) gave a single K(m) value of about 2.0mum. These results suggest that there are two modes of 25-hydroxylation of cholecalciferol in the liver, which seem to be closely related to the mechanism of control of 25(OH)D(3) production by the liver.