Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains.

To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3.     

Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3

A cytochrome P-450 that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3 (P-450cc24: P-450cholecalciferol24) was purified to electrophoretic homogeneity from the kidney mitochondria of female rats treated with vitamin D3 (Ohyama, Y., Hayashi, S., and Okuda, K. (1989) FEBS Lett. 255, 405-408). The molecular weight was 53,000, and its absorption spectrum showed peaks characteristic of cytochrome P-450. The turnover number was 22 min-1 and the specific content was 2.8 nmol/mg protein. The N-terminal amino acid sequence, Arg-Ala-Pro-Lys-Glu-Val-Pro-Leu-, is different from the N-terminal sequence of any other cytochrome P-450s so far reported. Upon reconstitution with the electron-transferring system of the adrenal mitochondria, the enzyme showed a high activity in hydroxylating 25-hydroxyvitamin D3 as well as 1 alpha,25-dihydroxyvitamin D3 at position 24. However, the purified enzyme hydroxylated neither vitamin D3 nor 1 alpha-hydroxyvitamin D3. The enzyme was also inactive toward xenobiotics. The enzyme hydroxylated 25-hydroxyvitamin D3 at position 24 but not at 1 alpha, indicating that the enzyme is distinct from that catalyzing 1 alpha-hydroxylation. The reaction followed Michaelis-Menten kinetics, and the Km value for 25-hydroxyvitamin D3 was 2.8 microM. Both vitamin D3 and 1 alpha-hydroxyvitamin D3 inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 in a competitive, concentration-dependent manner. 25-Hydroxyvitamin D3 24-hydroxylase activity was significantly inhibited by 7,8-benzoflavone, ketoconazole, and CO, whereas it was only slightly inhibited by aminoglutethimide, metyrapone, and SKF-525A. Mouse antibodies raised against the cytochrome P-450 inhibited the reaction about 70% and reacted with the P-450cc24 in immunoblotting but did not react with other kinds of cytochrome P-450 in rat liver microsomes and mitochondria.     

Soluble 25-hydroxyvitamin D3-1 alpha-hydroxylase from kidney mitochondria of rachitic pigs.

1. Mitochondria isolated from the kidneys of rachitic pigs have been shown to contain an active 25-hydroxyvitamin D3-1 alpha-hydroxylase. From these mitochondria a cytochrome P-450 has been solubilized with a specific content of 0.02-0.04 nmol/mg protein. 2. In the presence of a bovine adrenal NADPH-ferredoxin reductase, bovine adrenal ferredoxin and NADPH, the cytochrome P-450 supported the formation of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. 3. The hydroxylation reaction was linear with time up to 40 min, and with the amount of enzyme up to 0.03 nmol cytochrome P-450. The pH optimum for the reaction was 7.4, and the apparent Km was 3 x 10(-10) mol/mg protein. 4. The results show that 25-hydroxyvitamin D3 is metabolized in mammals by the same enzyme system as has been demonstrated in birds.     

Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1): a novel reaction by CYP27A1

The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Increased intestinal chromatin template activity. Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes.

1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase. The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol. This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney. The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours. Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo. To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes. After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone. This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol. The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3. It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

Separation of the cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3

The cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 have been separated. The cytochrome P-450 fractions required NADPH, mitochondrial ferredoxin and ferredoxin reductase for catalytic activity. The present report demonstrates that different forms of cytochrome P-450 are involved in 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 and provides a basis for further purification and characterization of these enzymes.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Studies on the mechanism of action of 1 alpha, 24-dihydroxyvitamin D3. II. Specific binding of alpha, 24-dihydroxyvitamin D3 to chick intestinal receptor

The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.     

Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17alpha,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17alpha-hydroxyvitamin D3 and 17alpha,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 --> 20-hydroxyvitamin D3 --> 20,23-dihydroxyvitamin D3 --> 17alpha,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.     

Isolation and identification of vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid.

It has been shown that Solanum malacoxylon contains 1 alpha,25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1 alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them comigrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1 alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1 alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.     

Isolation and identification of 2alpha,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3

Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.     

Properties of guinea-pig kidney 25-hydroxyvitamin D3 1 alpha-hydroxylase assayed by isotope dilution-mass spectrometry.

1. A highly specific and accurate method based on isotope dilution-mass spectrometry was used for characterization of the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase in untreated guinea pigs with a normal vitamin D status. In previous work, the properties of the enzyme had been determined in rachitic animals only. 2. With intact mitochondria, the reaction required the presence of citric acid-cycle intermediates. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had an inhibitory effect on the isocitrate-supported reaction, indicating that energy-dependent transhydrogenation is of importance. Mitochondrial respiratory-chain inhibitors (cyanide, rotenone, antimycin A) had no effect on the hydroxylation. CO had an inhibitory effect, suggesting participation of a species of cytochrome P-450 in the reaction. A fraction solubilized from mitochondria by cholate became catalytically active in 1 alpha-hydroxylation of 25-hydroxyvitamin D3 after addition of ferredoxin and ferredoxin reductase. The isocitrate-supported reaction catalysed by crude mitochondria had an apparent Km of about 1 microM. 3. An atmosphere containing 50% O2 was found to be necessary for optimal activity. It is thus possible that O2 may be a limiting factor under normal conditions in vivo. 4. The results demonstrate that the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is a cytochrome P-450-dependent mixed-function oxidase with properties similar to those previously reported for the same enzyme system in chicken. The present assay and animal system seem to be suitable for further studies on the mechanism of regulation of the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase under conditions when the vitamin D status is normal.