Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Removal of C-ring from the CD-ring skeleton of 1alpha,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly

It is now well established that 1alpha,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1alpha,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1alpha,25(OH)2D3 is metabolized into 1alpha,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1alpha,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1alpha,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1alpha,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1alpha,25(OH)2D3. SL 117 contains the same side chain as that of 1alpha,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1alpha,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1alpha,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 does not alter its target tissue metabolism significantly.     

1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1alpha,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the previously well established main side chain modification pathway, is initiated by hydroxylation at C-24 of the side chain. The C-3 epimerization pathway, the newly discovered A-ring modification pathway, is initiated by epimerization of the hydroxyl group at C-3 of the A-ring. The end products of the metabolism of 1alpha,25(OH)2D3 through the C-24 oxidation and the C-3 epimerization pathways are calcitroic acid and 1alpha,25-dihydroxy-3-epi-vitamin-D3 respectively. During the past two decades, numerous noncalcemic analogs of 1alpha,25(OH)2D3 were synthesized. Several of the analogs have altered side chain structures and as a result some of these analogs have been shown to resist their metabolism through side chain modifications. For example, two of the analogs, namely, 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-D3] and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-D3], have been shown to resist their metabolism through the C-24 oxidation pathway. However, the possibility of the metabolism of these two analogs through the C-3 epimerization pathway has not been studied. Therefore, in our present study, we investigated the metabolism of these two analogs in rat osteosarcoma cells (UMR 106) which are known to express the C-3 epimerization pathway. The results of our study indicate that both analogs [1alpha,25(OH)2-16-ene-23-yne-D3 and 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3] are metabolized through the C-3 epimerization pathway in UMR 106 cells. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-D3 [1alpha,25(OH)2-16-ene-23-yne-3-epi-D3] was confirmed by GC/MS analysis and its comigration with synthetic 1alpha,25(OH)2-16-ene-23-yne-3-epi-D3 on both straight and reverse-phase HPLC systems. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-3-epi-D3] was confirmed by GC/MS and 1H NMR analysis. Thus, we indicate that vitamin D analogs which resist their metabolism through the C-24 oxidation pathway, have the potential to be metabolized through the C-3 epimerization pathway. In our present study, we also noted that the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 is about 10 times greater than the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-D3. Thus, we indicate for the first time that certain structural modifications of the side chain such as 20-epi modification can alter significantly the rate of C-3 epimerization of vitamin D compounds.     

Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS)

The development of novel gene expression systems for cytochrome P450s (CYPs) together with a revolution in analytical mass spectrometry with the emergence of liquid chromatography/mass spectrometry (LC/MS) has opened the door to answering some long-standing questions in Vitamin D metabolism. Our studies focused on: (1) elucidating the role of CYP24 in 25-OH-D3 and 1alpha,25-(OH)2D3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D3 metabolism in CYP24-knockout (CYP24-XO) cells and; (4) comparing 1alpha-OH-D2 metabolism in the CYP24-XO mouse in vivo and in vitro. Methodology employed CYP24 over-expression and knockout systems in conjunction with state-of-the-art analytical LC/MS, diode array, and radioisotopic detection methods. We found that CYP24 metabolizes 25-OH-D3 and 1alpha,25-(OH)2D3 at similar rates in vitro, but that for 25-OH-D3 but not 1alpha,25-(OH)2D3, this rate is strongly influenced by the concentration of DBP. Unlike their wild type littermates, the administration of 25-OH-D3 to CYP24-XO mice results in no measurable 24,25-(OH)2D3 production. When neonatal murine keratinocytes are prepared from wild type and CYP24-XO mice there was no measurable production of 24,25-(OH)2D3 or 1alpha,24,25-(OH)2D3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [3H] 1alpha-OH-D2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.     

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.     

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).     

Double bond in the side chain of 1alpha,25-dihydroxy-22-ene-vitamin D(3) is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1alpha,25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] like 1alpha,25(OH)(2)D(3), is also converted into the same final product, calcitroic acid. This finding indicated that 1alpha,25(OH)(2)D(2) also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1alpha,25(OH)(2)D(2) when compared to the side chain of 1alpha,25(OH)(2)D(3), has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1alpha,25(OH)(2)D(2). We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D(2) related compounds. We compared the metabolism of 1alpha,25-dihydroxy-22-ene-vitamin D(3) [1alpha,25(OH)(2)-22-ene-D(3)], a synthetic vitamin D analog whose side chain differs from that of 1alpha,25(OH)(2)D(3) only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1alpha,25(OH)(2)-22-ene-D(3) and 1alpha,25(OH)(2)D(3) are converted into common metabolites namely, 1alpha,24(R),25-trihydroxyvitamin D(3) [1alpha,24(R),25(OH)(3)D(3)], 1alpha,25-dihydroxy-24-oxovitamin D(3) [1alpha,25(OH)(2)-24-oxo-D(3)], 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D(3). This finding indicates that the double bond in the side chain of 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Along with the aforementioned metabolites, 1alpha,25(OH)(2)-22-ene-D(3) is also converted into two additional metabolites namely, 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Furthermore, we did not observe direct conversion of 1alpha,25(OH)(2)-22-ene-D(3) into 1alpha,25(OH)(2)D(3). These findings indicate that 1alpha,25(OH)(2)-22-ene-D(3) is first converted into 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Then the double bonds in the side chains of 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3) undergo reduction to form 1alpha,24(R),25(OH)(3)D(3) and 1alpha,25(OH)(2)-24-oxo-D(3), respectively. Thus, our study indicates that the double bond in 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1alpha,25(OH)(2)-22-ene-D(3) metabolism indicating that prior C-24 hydroxylation of 1alpha,25(OH)(2)-22-ene-D(3) is required for the double bond reduction to occur.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Crystal structure of CYP105A1 (P450SU-1) in complex with 1alpha,25-dihydroxyvitamin D3

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.     

Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.     

Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).