Identification of 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one as a product of metabolism of 25-hydroxycholecalciferol by liver microsomes

The ability of liver microsomes, sites of synthesis of 25-hydroxycholecalciferol, to further metabolize 25-hydroxycholecalciferol has been assessed. When liver microsomes were incubated with 25-hydroxycholecalciferol in the presence of cytosol, a metabolite was isolated that comigrated with 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3- one in three different chromatographic systems. The ultraviolet spectrum (220-350 nm) and mass spectrum of the purified metabolite were identical to that of synthetic 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. This study indicates that liver microsomes convert 25-hydroxycholecalciferol to 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. The significance of this metabolite, which has been shown previously by others to be produced by alveolar macrophages, has yet to be determined.     

Metabolism of cholecalciferol in land snails.

1. Radioactively labelled cholecalciferol was injected into the land snails Levantina hiersolyma and Theba pisana. Three metabolites (C, D and E), more polar than cholecalciferol, were found. 2. Metabolite C was found to be identical with 25-hydroxycholecalciferol. On injection of 25-hydroxy[26,27-3H]cholecalciferol, metabolite E was predominantly formed. Metabolite D was predominantly formed from cholecalciferol. Metabolites D and E differ from any known cholecalciferol metabolites. 3. The intestine was found to be the tissue capable of carrying out the transformation of 25-hydroxycholecalciferol into metabolite E. 4. 25-Hydroxycholecalciferol and metabolite E were localized in the digestive gland of the snail, the tissue responsible for the absorption of Ca2+ and its storage. Metabolite D was not localized in any specific tissue.     

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.     

Arachidonic acid metabolism in rabbit renal cortex. Formation of two novel dihydroxyeicosatrienoic acids

[1-14C]Eicosatetraenoic (arachidonic) acid was incubated with a low speed (17,000 X g) rabbit renal cortical supernatant or with a cortical microsomal suspension fortified with NADPH for 15 min at 37 degrees C. The products which were less polar than prostaglandins on reversed phase high performance liquid chromatography were identified by gas chromatography-mass spectrometry. Both the fortified microsomes and the low speed supernatant formed significant amounts of two novel metabolites, 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid. Other identified products were 19- and 20-hydroxyeicosatetraenoic acid, 19-oxoeicosatetraenoic acid, and in the low speed supernatant, eicosatetraen-1,20-dioic acid. The metabolites were not formed in significant amounts by high speed cortical supernatant or by nonfortified cortical microsomes. Carbon monoxide inhibited formation of these compounds, indicating that they may be formed by the cytochrome P-450-linked renal monooxygenase systems.     

Hepatic metabolism of ergot alkaloids in beef cattle by cytochrome P450

This study was conducted to investigate the involvement of cytochrome P450 3A (CYP3A) in the metabolism of ergotamine in beef liver microsomes. When incubated with liver microsomes, ergotamine was hydroxylated to metabolites M1 and M2. Similarly, its isomer was hydroxylated to M1-Iso and M2-Iso (8-hydroxy-derivatives). Further incubation resulted in a second hydroxylation of M1 and M2 to metabolites M3 and M4 (8,9-dihydroxy derivatives). Maximum formation of metabolites was reached after 20 min, and ergotamine and its isomer were almost totally metabolized after 60 min of incubation. The formation of these metabolites was completely dependent on the presence of NADPH or the NADPH generating system and was also dependent on microsome concentration. Ergotamine was converted at a rate of 2 nM/microgram microsome/min when incubated with bovine liver microsomes to produce a metabolite profile (M1, M2, M1-Iso and M2-Iso) similar to the metabolites produced (2.2 nM/microgram/min) when ergotamine was incubated with liver microsomes of dexamethasone treated rats. This work provides information on the modification of ergotamine in bovine liver microsomes by CYP3A, which is of importance in understanding the detoxification and the clearance of ergotamine and other ergot alkaloids by bovine.     

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

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

A comparison of the metabolism of 25-hydroxyvitamine D3 by chick renal tubules, homegenates and mitochondria

The metabolism of 25-hydroxycholecalciferol by isolated chick renal tubules has been investigated by the use of a new chromatographic method employing silica-treated paper as the supporting phase. This method has been found to be superior to Sephadex LH20 column chromatography in resolving the metabolites of 25-hydroxycholecalciferol, particularly in separating 1,25-dyhydroxycholecalciferol from other metabolites. With this method several heretofore undescribed metabolites of 25-hydroxycholecalciferol have been recognized and partially characterized.     

Echanges d'acides gras in vitro entre mitochondries, microsomes et surnageants cytoplasmiques de cellules de pommes de terre et de chou-fleur

When potato tuber or cauliflower buds mitochondria, containing fatty acids labelled from [1-¹⁴C]acetate, are incubated in a small volume of cytoplasmic supernatant with unlabelled microsomes isolated from the same species, the specific radioactivity of the mitochondrial fatty acids decreases and the fatty acids of the microsomes and supernatant become radioactive. The same result is obtained when labelled microsomes are mixed with unlabelled mitochondria. These data suggest an exchange of fatty acids from microsomes to mitochondria and in the opposite way. All types of fatty acids are transferred, the major ones being the most actively exchanged. There is no breakdown of fatty acids and no subsequent synthesis during the transfer since the distribution of the total radioactivity remains constant among the various fatty acids. The same transfers are observed when potato mitochondria are incubated with cauliflower microsomes or reciprocally.

The cytoplasmic supernatant plays an important role in these phenomena; the labelled fatty acids of the supernatant can be transferred to unlabelled microsomes or mitochondria from the same tissue. The transfer is less active from a supernatant of one species to organelles of another species.     

Benzanthrone: a new substrate for hepatic microsomal cytochrome P-450

The metabolism of benzanthrone, a commonly used dy intermediate, by rat hepatic microsomes was investigated using thin layer chromatography (TLC) analysis. Incubation of benzanthrone with hepatic microsomes in the presence of NADPH generating system produced at least seven fluorescent metabolites on TLC plates. TLC spots numbered II, III, IV, V and VI were the major metabolites obtained from hepatic microsomes with the Rf values of 0.53, 0.45, 0.38, 0.33 and 0.26, respectively. Metabolites VII and VIII were faint bands with Rf values of 0.08 and 0.04, respectively. Preincubation of hepatic microsomes with either 1-benzyl-imidazole (10(-4)M) or SKF-525 A (10(-4)M) or metyrapone (10(-3)M) or flushing with carbon monoxide substantially inhibited the benzanthrone metabolism. alpha-Naphtho-flavone (10(-4)M) did not cause any change in hepatic microsomal metabolism of benzanthrone. Oral administration of benzanthrone to animals yielded at least six urinary metabolites. TLC spots numbered II, III, IV, V and VI in the urine were same as those of hepatic microsomal metabolites. However, one of the urinary metabolite numbered IX which stays at the origin of TLC plate with the Rf value of 0.05 may be a conjugate. Our results suggest that benzanthrone acts as a substrate for hepatic heme protein, cytochrome P-450 and that some of the metabolites are excreted in urine.     

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Microsomal monooxygenation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Synthesis and identification of cis and trans monohydroxylated products.

Liver microsomal hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur on the cyclohexyl ring at positions 3 and 4. Four metabolites were isolated by selective solvent extraction and purifed by high-pressure liquid chromatography. cis-4-, trans-4-, cis-3-, and trans-3-OH derivatives of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were synthesized and their chromatographic, mass spectral, and nuclear magnetic resonance characteristics matched those of the metabolites. The position of ring hydroxylation and the identity of each geometric isomer were established by nuclear magnetic resonance using a shift reagent in conjunction with spin decoupling techniques. Microsomes from rats pretreated with phenobarbital showed a sixfold increase in hydroxylation rate (19.5 vs. 3.3 nmol per mg per min). The induction was quite selective for cis-4 hydroxylation (19-fold); however, induction of trans-4 (threefold), cis-3 (threefold), and trans-3 (twofold) hydroxylation did occur. Quantitatively the cis-4-hydroxy metabolite was 67of the total product by phenobarbital-induced microsomes and 21% for normal microsomes. Microsomes from animals pretreated wit- 3-methyl-cholanthrene gave about the same rate and product distribution that normal microsomes gave. A mixture of 80% carbon monoxide-20% oxygen inhibited formation of all four hydroxy metabolites with the inhibition ranging from 55 to 78%.     

Metabolism of arachidonic acid by isolated rat hepatocytes,renal cells and by some rabbit tissues: Detection of vicinal diols by mass fragmentography

Purified cytochromes P-450 (LM₂ and PB-B₂) in a reconstituted system and epoxide hydrolase were recently found to metabolize arachidonic (eicosatetraenoic) acid to four vicinal dihydroxyeicosatrienoic acids. These metabolites were chemically synthetized from octadeuterated arachidonic acid and employed as internal standards for mass fragmentography. Isolated rat hepatocytes and renal cells were incubated with arachidonic acid (0.1 mM; 37°C, 15 min) and, following extractive isolation and reversed-phase HPLC, formation of 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid was demonstrated by mass fragmentography using a capillary GC column. Furthermore, these diols were also detected in rabbit liver and renal cortex and they therefore appear to be formed endogenously. Formation of vicinal diols was also studied in cell free systems. Rabbit liver and renal cortical microsomes were incubated with NADPH (1 mM) and arachidonic acid (0.15 mM) for 15 min at 37°C and, besides 11,12-dihydroxy- and 14,15-dihydroxyeicosatrienoic acid, small amounts of 8,9-dihydroxy- and 5,6-dihydroxyeicosatrienoic acid could be detected by mass fragmentography. Renal as well as hepatic monooxygenases can thus epoxidize each of the four double bonds of arachidonic acid. In contrast, rabbit lung microsomes and NADPH metabolize arachidonic acid mainly to prostaglandins and 19-hydroxy- and 20-hydroxyarachidonic acid, while only small amounts of 11,12-dihydroxyeicosatrienoic acid could be found. Monooxygenase metabolism of arachidonic acid by epoxidation might therefore be a significant pathway for the metabolism of this essential fatty acid in isolated rat renal cells and hepatocytes but presumably not in the lung.     

Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6 beta-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver, 6 beta-Hydroxylation was the most important pathway of biotransformation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2 beta-, 15 beta- and 15 alpha-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6 beta-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6 beta-alcohol was the only metabolite formed from AD at a rate greater than 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti-P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2 beta-, 6 beta-, 15 alpha- and 15 beta-positions (by 31-56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6 beta-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6 beta-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6 beta-hydroxylation.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.     

Inhibition of rat liver calciferol 25-hydroxylase activity with anticonvulsant drugs.

Diphenylhydantoin or phenobarbital adminstered for 25 days to Vitamin D-deficient rats inhibited liver calciferol 25-hydroxylase activity. This inhibition was observed with either total homogenate or the microsomal fraction. Eight days following cessation of phenobarbital treatment, liver calciferol 25-hydroxylase activity had returned to control value. Addition of diphenylhydantoin or phenobarbital in vitro to liver homogenate or microsomes isolated from rachitic untreated animals also inhibits the enzymic activity. These data suggest that the impaired conversion of Vitamin D₃ to its 25-hydroxylated metabolite may be the cause of low plasma 25-hydroxycholecalciferol levels in anticonvulsant treated patients.     

Metabolism of benzo[a]pyrene in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 38 h with ¹⁴C-labeled benzo[a]pyrene; autoclaved cultures were used as controls. Metabolites were isolated by a sequential extraction procedure and further studied by chromatography or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The soluble metabolites amounted to 1–2.2% in the case of parsley cells, and 19–28% in the case of soybean cells. These metabolites varied in polarity, some being soluble in organic solvent or aqueous buffer while other metabolite fractions were soluble only in hot aqueous sodium dodecylsulphate. In addition, a significant amount of an insoluble metabolite fraction was isolated from the culture fluid as well as the cellular material of soybean suspension cultures.Abbreviations BP benzo[a]pyrene - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Biotransformation of tamoxifen in a human endometrial explant culture model

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.     

Acetate and arachidonate incorporations into lipids of ovine chorioallantoic tissue at day 24 of pregnancy

Incorporation of acetate and arachidonic acid into lipid classes was examined in chorioallantoic membranes obtained from sheep at Day 24 of pregnancy. Conceptus tissues were incubated in vitro with 5 mM acetate, 0.042 mM arachidonate, 0.45 muCi [1-14C]acetate, and 5.0 muCi [5,6,8,9,11,12,14,15-3H]arachidonate for 3 and 6 h. After incubation, tissue lipid fractions were extracted, isolated, and examined for radiolabel incorporations. Medium was extracted and analyzed for radiolabeled metabolites. Metabolic pathways commonly associated with fatty acid metabolism were confirmed to be present. Acetate was utilized for de novo synthesis of free cholesterol and free fatty acid. Fatty acids containing radiolabel from both acetate and arachidonate were mainly esterified in phospholipid and triglyceride, major lipid classes found in chorioallantoic tissue. Labeled metabolites of acetate were not sufficient for analytical measurement in medium. Metabolites of arachidonic acid from lipoxygenase and cyclooxygenase pathways were determined in medium after incubation. Results suggest that, within Day 24 ovine chorioallantoic tissue, utilization of exogenous arachidonate and de novo lipogenesis from acetate function in a parallel and anabolic mode appropriate for membrane expansion.     

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

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