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G. Satyanarayana Reddy - One of the best experts on this subject based on the ideXlab platform.
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Removal of C-ring from the CD-ring skeleton of 1α,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly
Archives of biochemistry and biophysics, 2006Co-Authors: G. Satyanarayana Reddy, Guochun Wang, Paul Vouros, G. Tayhas R. Palmore, Matthew Robinson, Lynn Gennaro, Pierre J. De Clercq, Maurits Vandewalle, Wu Young, Shi LingAbstract:Abstract It is now well established that 1α,25(OH) 2 D 3 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 1α,25(OH) 2 D 3 is metabolized into Calcitroic Acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1α,25(OH) 2 D 3 is metabolized into 1α,25(OH) 2 -3-epi-D 3 . 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 1α,25(OH) 2 D 3 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 1α,25(OH) 2 D 3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1α,25(OH) 2 D 3 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 1α,25(OH) 2 D 3 . SL 117 contains the same side chain as that of 1α,25(OH) 2 D 3 , 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 1α,25(OH) 2 D 3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1α,25(OH) 2 D 3 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 1α,25(OH) 2 D 3 , 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 1α,25(OH) 2 D 3 does not alter its target tissue metabolism significantly.
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23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic Acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic Acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway.
Archives of biochemistry and biophysics, 2006Co-Authors: G. Satyanarayana Reddy, Guochun Wang, G. Tayhas R. Palmore, John L. Omdahl, Kou-yi Tserng, Matthew Robinson, Domenick Vicchio, Alfred L. Yergey, Milan R. UskokovicAbstract:During the past two and half decades the elucidation of the metabolic pathways of 25OHD(3) and its active metabolite 1alpha,25(OH)(2)D(3) progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD(3) and 1alpha,25(OH)(2)D(3) are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1alpha,25(OH)(2)D(3) metabolism through C-24 oxidation pathway is 1alpha-OH-23- COOH-24,25,26,27-tetranor D(3) or Calcitroic Acid; the metabolism of 25OHD(3) into 23-COOH-24,25,26,27-tetranor D(3) has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D(3) as a water soluble metabolite of 24R,25(OH)(2)D(3) produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D(3) was also assumed as another end product of 25OHD(3) metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD(3) at low substrate concentration (3x10(-10)M) using [1,2-(3)H]25OHD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D(3) intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD(3) (5x10(-6)M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D(3) and 24-COOH-25,26,27-trinor D(3) in a ratio of 4:1. The same metabolite X containing both Acids in the same ratio of 4:1 was also produced when 24R,25(OH)(2)D(3) was used as the starting substrate. Previously, the trivial name of cholacalcioic Acid was assigned to 24-COOH-25,26,27-trinorvitamin D(3). Using the same guidelines, we now assign the trivial name of calcioic Acid to 23-COOH-24,25,26,27-tetranor D(3). In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic Acids as the end products of 25OHD(3) metabolism in rat kidney through C-24 oxidation pathway.
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Calcitroic Acid Is a Major Catabolic Metabolite in the Metabolism of 1α-Dihydroxyvitamin D2
Archives of biochemistry and biophysics, 2001Co-Authors: Duane R. Zimmerman, Timothy A. Reinhardt, Richard Kremer, Donald C. Beitz, G. Satyanarayana Reddy, R.l. HorstAbstract:Calcitroic Acid (1 alpha-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3)) is known to be the major water-soluble metabolite produced during the deactivation of 1 alpha,25-dihydroxyvitamin D(3). This deactivation process involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the Calcitroic Acid. Like 1 alpha,25-dihydroxyvitamin D(3), 1 alpha,25-dihydroxyvitamin D(2) is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1 alpha,25-dihydroxyvitamin D(2) undergoes side-chain cleavage. To investigate this possibility, we studied 1 alpha,25-dihydroxyvitamin D(2) metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic Calcitroic Acid on two reverse-phase HPLC columns of different chemistry. The putative Calcitroic Acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that Calcitroic Acid is a major terminal product for the deactivation of 1 alpha,25-dihydroxyvitamin D(2). Intermediates leading to the formation of the Calcitroic Acid in the 1 alpha,25-dihydroxyvitamin D(2) metabolism pathway are currently being studied.
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Metabolism of 1α,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells: In vitro biological activities of the natural metabolites of 1α,25-dihydroxyvitamin D3 produced in HL-60 cells
Steroids, 2001Co-Authors: D.sunita Rao, Milan R. Uskokovic, Moray J Campbell, H P Koeffler, Seiichi Ishizuka, P Spagnuolo, G. Satyanarayana ReddyAbstract:Abstract The secosteroid hormone, 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1α,25(OH) 2 D 3 and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1α,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, 1α,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 1α,25-dihydroxy-3-epi-vitamin-D 3 . We performed the present study first to examine in detail the metabolism of 1α,25(OH) 2 D 3 in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1α,25(OH) 2 D 3 in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1β- 3 H] 1α,25(OH) 2 D 3 and demonstrated that these cells metabolize 1α,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 1α,25(OH) 2 D 3 derived via the C-24 oxidation pathway namely, 1α,24(R),25-trihydroxyvitamin D 3 , 1α,25-dihydroxy-24-oxovitamin D 3 and 1α,23(S),25-trihydroxy-24-oxovitamin D 3 [1α,23(S),25(OH) 3 -24-oxo-D 3 ] were almost as potent as 1α,25(OH) 2 D 3 in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1α,23(S),25(OH) 3 -24-oxo-D 3 which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1α,25(OH) 2 D 3 to examine further its effects on these cells. Our results indicated that 1α,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 1α,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 1α,25(OH) 2 D 3 in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1α,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 1α,25(OH) 2 D 3 may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1α,25(OH) 2 D 3 .
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Double bond in the side chain of 1α,25-dihydroxy-22-ene-vitamin D3 is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney
The Journal of steroid biochemistry and molecular biology, 2001Co-Authors: D.sunita Rao, R.l. Horst, Kou-yi Tserng, D Balkundi, Milan R. Uskokovic, Jeffrey W. Clark, G. Satyanarayana ReddyAbstract:Abstract 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] 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 1α,25-dihydroxyvitamin D2 [1α,25(OH)2D2] like 1α,25(OH)2D3, is also converted into the same final product, Calcitroic Acid. This finding indicated that 1α,25(OH)2D2 also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1α,25(OH)2D2 when compared to the side chain of 1α,25(OH)2D3, 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 1α,25(OH)2D2. We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D2 related compounds. We compared the metabolism of 1α,25-dihydroxy-22-ene-vitamin D3 [1α,25(OH)2-22-ene-D3], a synthetic vitamin D analog whose side chain differs from that of 1α,25(OH)2D3 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 1α,25(OH)2-22-ene-D3 and 1α,25(OH)2D3 are converted into common metabolites namely, 1α,24(R),25-trihydroxyvitamin D3 [1α,24(R),25(OH)3D3], 1α,25-dihydroxy-24-oxovitamin D3 [1α,25(OH)2-24-oxo-D3], 1α,23(S),25-trihydroxy-24-oxovitamin D3 and 1α,23-dihydroxy-24,25,26,27-tetranorvitamin D3. This finding indicates that the double bond in the side chain of 1α,25(OH)2-22-ene-D3 is reduced during its metabolism. Along with the aforementioned metabolites, 1α,25(OH)2-22-ene-D3 is also converted into two additional metabolites namely, 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3. Furthermore, we did not observe direct conversion of 1α,25(OH)2-22-ene-D3 into 1α,25(OH)2D3. These findings indicate that 1α,25(OH)2-22-ene-D3 is first converted into 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3. Then the double bonds in the side chains of 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3 undergo reduction to form 1α,24(R),25(OH)3D3 and 1α,25(OH)2-24-oxo-D3, respectively. Thus, our study indicates that the double bond in 1α,25(OH)2-22-ene-D3 is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1α,25(OH)2-22-ene-D3 metabolism indicating that prior C-24 hydroxylation of 1α,25(OH)2-22-ene-D3 is required for the double bond reduction to occur.
Milan R. Uskokovic - One of the best experts on this subject based on the ideXlab platform.
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23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic Acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic Acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway.
Archives of biochemistry and biophysics, 2006Co-Authors: G. Satyanarayana Reddy, Guochun Wang, G. Tayhas R. Palmore, John L. Omdahl, Kou-yi Tserng, Matthew Robinson, Domenick Vicchio, Alfred L. Yergey, Milan R. UskokovicAbstract:During the past two and half decades the elucidation of the metabolic pathways of 25OHD(3) and its active metabolite 1alpha,25(OH)(2)D(3) progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD(3) and 1alpha,25(OH)(2)D(3) are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1alpha,25(OH)(2)D(3) metabolism through C-24 oxidation pathway is 1alpha-OH-23- COOH-24,25,26,27-tetranor D(3) or Calcitroic Acid; the metabolism of 25OHD(3) into 23-COOH-24,25,26,27-tetranor D(3) has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D(3) as a water soluble metabolite of 24R,25(OH)(2)D(3) produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D(3) was also assumed as another end product of 25OHD(3) metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD(3) at low substrate concentration (3x10(-10)M) using [1,2-(3)H]25OHD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D(3) intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD(3) (5x10(-6)M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D(3) and 24-COOH-25,26,27-trinor D(3) in a ratio of 4:1. The same metabolite X containing both Acids in the same ratio of 4:1 was also produced when 24R,25(OH)(2)D(3) was used as the starting substrate. Previously, the trivial name of cholacalcioic Acid was assigned to 24-COOH-25,26,27-trinorvitamin D(3). Using the same guidelines, we now assign the trivial name of calcioic Acid to 23-COOH-24,25,26,27-tetranor D(3). In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic Acids as the end products of 25OHD(3) metabolism in rat kidney through C-24 oxidation pathway.
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highly active analogs of 1α 25 dihydroxyvitamin d3 that resist metabolism through c 24 oxidation and c 3 epimerization pathways
Steroids, 2001Co-Authors: Milan R. Uskokovic, Anthony W. Norman, Percy S Manchand, George P Studzinski, Moray J Campbell, Phillip H Koeffler, Atsuko Takeuchi, Mei Ling Siucaldera, Sunita D Rao, Satyanarayana G ReddyAbstract:Abstract The secosteroid hormone 1α,25-dihydroxyvitamin D3 [1α,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 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 1α,25(OH)2-3-epi-D3. A rational design for the synthesis of potent analogs of 1α,25(OH)2D3 is developed based on the knowledge of the various metabolic pathways of 1α,25(OH)2D3. 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 D3 analogs 1α,25(OH)2-16,23-E-diene-26,27-hexafluoro-19-nor-D3 (Ro 25–9022), 1α,25(OH)2-16,23-Z-diene-26,27-hexafluoro-19-nor-D3 (Ro 26–2198), and 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-D3 (Ro 25–6760), as indicated by their antiproliferative activities.
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Metabolism of 1α,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells: In vitro biological activities of the natural metabolites of 1α,25-dihydroxyvitamin D3 produced in HL-60 cells
Steroids, 2001Co-Authors: D.sunita Rao, Milan R. Uskokovic, Moray J Campbell, H P Koeffler, Seiichi Ishizuka, P Spagnuolo, G. Satyanarayana ReddyAbstract:Abstract The secosteroid hormone, 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1α,25(OH) 2 D 3 and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1α,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, 1α,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 1α,25-dihydroxy-3-epi-vitamin-D 3 . We performed the present study first to examine in detail the metabolism of 1α,25(OH) 2 D 3 in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1α,25(OH) 2 D 3 in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1β- 3 H] 1α,25(OH) 2 D 3 and demonstrated that these cells metabolize 1α,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 1α,25(OH) 2 D 3 derived via the C-24 oxidation pathway namely, 1α,24(R),25-trihydroxyvitamin D 3 , 1α,25-dihydroxy-24-oxovitamin D 3 and 1α,23(S),25-trihydroxy-24-oxovitamin D 3 [1α,23(S),25(OH) 3 -24-oxo-D 3 ] were almost as potent as 1α,25(OH) 2 D 3 in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1α,23(S),25(OH) 3 -24-oxo-D 3 which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1α,25(OH) 2 D 3 to examine further its effects on these cells. Our results indicated that 1α,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 1α,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 1α,25(OH) 2 D 3 in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1α,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 1α,25(OH) 2 D 3 may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1α,25(OH) 2 D 3 .
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Double bond in the side chain of 1α,25-dihydroxy-22-ene-vitamin D3 is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney
The Journal of steroid biochemistry and molecular biology, 2001Co-Authors: D.sunita Rao, R.l. Horst, Kou-yi Tserng, D Balkundi, Milan R. Uskokovic, Jeffrey W. Clark, G. Satyanarayana ReddyAbstract:Abstract 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] 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 1α,25-dihydroxyvitamin D2 [1α,25(OH)2D2] like 1α,25(OH)2D3, is also converted into the same final product, Calcitroic Acid. This finding indicated that 1α,25(OH)2D2 also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1α,25(OH)2D2 when compared to the side chain of 1α,25(OH)2D3, 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 1α,25(OH)2D2. We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D2 related compounds. We compared the metabolism of 1α,25-dihydroxy-22-ene-vitamin D3 [1α,25(OH)2-22-ene-D3], a synthetic vitamin D analog whose side chain differs from that of 1α,25(OH)2D3 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 1α,25(OH)2-22-ene-D3 and 1α,25(OH)2D3 are converted into common metabolites namely, 1α,24(R),25-trihydroxyvitamin D3 [1α,24(R),25(OH)3D3], 1α,25-dihydroxy-24-oxovitamin D3 [1α,25(OH)2-24-oxo-D3], 1α,23(S),25-trihydroxy-24-oxovitamin D3 and 1α,23-dihydroxy-24,25,26,27-tetranorvitamin D3. This finding indicates that the double bond in the side chain of 1α,25(OH)2-22-ene-D3 is reduced during its metabolism. Along with the aforementioned metabolites, 1α,25(OH)2-22-ene-D3 is also converted into two additional metabolites namely, 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3. Furthermore, we did not observe direct conversion of 1α,25(OH)2-22-ene-D3 into 1α,25(OH)2D3. These findings indicate that 1α,25(OH)2-22-ene-D3 is first converted into 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3. Then the double bonds in the side chains of 1α,24,25(OH)3-22-ene-D3 and 1α,25(OH)2-24-oxo-22-ene-D3 undergo reduction to form 1α,24(R),25(OH)3D3 and 1α,25(OH)2-24-oxo-D3, respectively. Thus, our study indicates that the double bond in 1α,25(OH)2-22-ene-D3 is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1α,25(OH)2-22-ene-D3 metabolism indicating that prior C-24 hydroxylation of 1α,25(OH)2-22-ene-D3 is required for the double bond reduction to occur.
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1α 25 dihydroxy 16 ene 23 yne vitamin d3 and 1α 25 dihydroxy 16 ene 23 yne 20 epi vitamind3 analogs of 1α 25 dihydroxyvitamin d3 that resist metabolism through the c 24 oxidation pathway are metabolized through the c 3 epimerization pathway
Archives of Biochemistry and Biophysics, 2000Co-Authors: Satyanarayana G Reddy, Paul Vouros, A. Weiskopf, Percy S Manchand, Mei Ling Siucaldera, Sunita D Rao, Norbert Astecker, Gino J Sasso, Milan R. UskokovicAbstract: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.
G S Reddy - One of the best experts on this subject based on the ideXlab platform.
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Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways.
Steroids, 2001Co-Authors: M R Uskokovic, M L Siu-caldera, A W Norman, Percy S Manchand, George P Studzinski, Moray J Campbell, Atsuko Takeuchi, H P Koeffler, D S Rao, G S ReddyAbstract: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.
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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.
Steroids, 2001Co-Authors: D S Rao, S. Ishizuka, Moray J Campbell, M R Uskokovic, H P Koeffler, P Spagnuolo, G S ReddyAbstract: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).
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Natural metabolites of 1α,25‐dihydroxyvitamin D3 retain biologic activity mediated through the vitamin D receptor
Journal of cellular biochemistry, 2000Co-Authors: Hanna Harant, G S Reddy, D. Spinner, Ivan J. D. LindleyAbstract:1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(OH)-24,25,26,27-tetranor-23-COOH-D(3); Calcitroic Acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1alpha,25(OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(OH)(2)D(3) or the metabolites 1alpha,24R,25(OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,23S,25(OH)(3)D(3) resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed even for Calcitroic Acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(OH)(2)D(3) from VDR than the parent hormone. Thus, we report that several natural metabolites of 1alpha,25(OH)(2)D(3) retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.
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Human osteoblasts in culture metabolize both 1 alpha, 25-dihydroxyvitamin D3 and its precursor 25-hydroxyvitamin D3 into their respective lactones
Endocrinology, 1995Co-Authors: M L Siu-caldera, L. Zou, M. Ehrlich, E R Schwartz, S. Ishizuka, G S ReddyAbstract:1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], the hormonal form of vitamin D3, is further metabolized in the kidney and intestine through the carbon 24 (C-24) oxidation pathway initiated by C-24 hydroxylation, and the carbon 23 (C-23) oxidation pathway initiated by C-23 hydroxylation. The C-24 oxidation pathway leading to the formation of Calcitroic Acid has been previously reported to be present in bone cells, but the C-23 oxidation pathway leading to the formation of 1 alpha, 25-(OH)2D3-26,23-lactone has not been described in bone cells, even though 1 alpha, 25-(OH)2D3-26,23-lactone is noted to have a significant effect on bone formation. Therefore, in the present study, we investigated the production of 1 alpha, 25-(OH)2D3-26,23-lactone in normal human osteoblasts, and our studies revealed that human osteoblasts possess the activity of both 24- and 23-hydroxylases constitutively. Thus, 1 alpha, 24(R),25-(OH)3D3, 1 alpha, 25-(OH)2-24-oxo-D3, 1 alpha, 23(S), 25-(OH)3-24-oxo-D3, 1 alpha, 23-(OH)2...
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1,25-Dihydroxy-24-OXO-16ene-vitamin D3, a renal metabolite of the vitamin D analog 1,25-dihydroxy-16ene-vitamin D3, exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.
Endocrinology, 1994Co-Authors: J M Lemire, D C Archer, G S ReddyAbstract:The hormone, 1,25-(OH)2D3, is metabolized into 1,25-(OH)2-24-OXO-D3, in kidney prior to conversion to its final inactive product, Calcitroic Acid. Similarly, 1,25-(OH)2-24OXO-16eneD3, is produced in the kidney from the Vitamin D analog, 1,25-(OH)2-16eneD3, but resists further hydroxylation. The analog's metabolite was synthesized and its biologic activity compared to the parent compound. Naive SJL/J mice, 4 weeks old, were immunized with neuroantigen in adjuvant to induce experimental autoimmune encephalomyelitis [EAE]. Treatment with 1,25-(OH)2-24OXO-16eneD3 was given at 0.05, 0.15 and 0.3 microgram I.P., on alternate days, starting 3 days prior and for up to 5 days post immunization and compared to a similar treatment with 0.1 microgram 1,25-(OH)2D3 or 1,25-(OH)2-16eneD3. Suppression of EAE was observed with 0.15 microgram 1,25-(OH)2-24OXO-16eneD3, comparable to the suppression induced with the parent compound and more potent than 1,25-(OH)2D3. However, no hypercalcemia was seen in mice treated with 0.1...
Glenville Jones - One of the best experts on this subject based on the ideXlab platform.
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Calcioic Acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D3 and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D3.
The Journal of steroid biochemistry and molecular biology, 2018Co-Authors: Martin Kaufmann, Corine Martineau, Alice Arabian, Mary Traynor, René St-arnaud, Glenville JonesAbstract:Abstract Calcitroic Acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3) proceeds via analogous steps culminating in calcioic Acid; however this C23-truncated Acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2D3 was found to restore normal fracture repair in Cyp24a1−/− mice devoid of 24,25-(OH)2D3. We set out to study the multi-step catabolism of D3 metabolites in vivo using LC–MS/MS methods in vehicle or 24,25-(OH)2D3-treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2D3 (7 ng/mL) and 25−OH-D3-26,23-lactone (4 ng/mL). We also detected 24-oxo-25−OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1−/− mice. In 24,25-(OH)2D3-treated Cyp24a1+/- mice, serum 24,25-(OH)2D3 rose to 200 ng/mL while 25-OH-D3-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2D3 Calcioic Acid was increased to 0.030 ng/mL for 24,25-(OH)2D3-treated Cyp24a1+/- mice. In 24,25-(OH)2D3-treated Cyp24a1−/− mice, serum 24,25-(OH)2D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2D3 dose. Serum 1,25-(OH)2D3 levels were suppressed in 24,25-(OH)2D3-treated Cyp24a1+/- and Cyp24a1−/− mice. Circulating 1,24,25-(OH)3D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2D3. While undetectable in vehicle-treated Cyp24a1−/− mice, 1,24,25-(OH)3D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2D3-treated nulls suggesting conversion of 24,25-(OH)2D3 to 1,24,25-(OH)3D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic Acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.
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Bioengineering Anabolic Vitamin D-25-Hydroxylase Activity into the Human Vitamin D Catabolic Enzyme, Cytochrome P450 CYP24A1, by a V391L Mutation
The Journal of biological chemistry, 2011Co-Authors: Martin Kaufmann, David E. Prosser, Glenville JonesAbstract:CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) to different products: Calcitroic Acid or 1α,25-(OH)2D3-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D3, the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D3 (1α-OH-D3), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino Acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)2D3-24-hydroxylase into an anabolic 1α-OH-D3-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)2D3. Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)2D3 via C24 hydroxylation, it can also make Calcitroic Acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)2D3 from 1α-OH-D3, but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D3 is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D3 above the heme for hydroxylation.
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Metabolism and Catabolism of Vitamin D, Its Metabolites, and Clinically Relevant Analogs
Vitamin D, 2010Co-Authors: Glenville JonesAbstract:This chapter discusses the current state of knowledge of vitamin D metabolism and the specific enzymes involved. Vitamin D3 undergoes a two-step metabolic activation involving sequential hydroxylations at 25- and 1α-carbons by cytochrome P450-based hydroxylases (CYP2R1 and CYP27B1) to give first the main circulating form 25-hydroxyvitamin D3 and then a hormonally active form 1α,25-dihydroxyvitamin D3. The plant-derived vitamin D2 undergoes the same activation steps. This review highlights the recent finding of extra-renal sites of CYP27B1 expression and the physiological implications of this discovery. 1α,25-Dihydroxyvitamin D3 is inactivated by another cytochrome P450 enzyme (CYP24A1) which produces a series of metabolic products culminating in either a side chain-truncated biliary excretory form, Calcitroic Acid, or a 26,23-lactone derivative. This chapter also discusses the current knowledge of the metabolism of the clinically relevant analogs of vitamin D, ranging from prodrug forms (e.g. 1(OH)D) that require a step or more of activation to produce a biologically active form to the calcitriol analogs, which are active as administered. Differences between metabolism-sensitive and metabolism-resistant vitamin D analogs are discussed in the context of evaluating the relative importance of analog metabolism in their mechanism of action. The review ends by attempting to predict future directions in the field, focussing on determination of CYP structure, the knowledge gained from mouse CYP knockouts and future vitamin D drug design.
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Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH)2D3-26,23-lactone
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: David E. Prosser, Martin Kaufmann, Brendan M. O'leary, Valarie Byford, Glenville JonesAbstract:Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1α,25-(OH)2D3, leading to the excretory form, Calcitroic Acid, and 23-hydroxylation, culminating in 1α,25-(OH)2D3-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino Acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design.
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Enzymes involved in the activation and inactivation of vitamin D
Trends in biochemical sciences, 2004Co-Authors: David E. Prosser, Glenville JonesAbstract:Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1α-hydroxylase contains the component CYP27B1, which completes the activation pathway to the hormonal form 1α,25-dihydroxyvitamin D 3 . A five-step inactivation pathway from 1α,25-(OH) 2 D 3 to Calcitroic Acid is attributed to a single multifunctional CYP, CYP24A1, which is transcriptionally induced in vitamin D target cells by the action of 1α,25-(OH) 2 D 3 . On the basis of alignments and crystal structures of other CYPs, homology models of vitamin-D-related CYPs have been generated. Two human forms of rickets caused by mutations of CYP2R1 and CYP27B1, as well as mouse knockout models of CYP27A1, CYP27B1 and CYP24A1, are helping us to establish the full in vivo physiological roles of the vitamin-D-related hydroxylases.
Moray J Campbell - One of the best experts on this subject based on the ideXlab platform.
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highly active analogs of 1α 25 dihydroxyvitamin d3 that resist metabolism through c 24 oxidation and c 3 epimerization pathways
Steroids, 2001Co-Authors: Milan R. Uskokovic, Anthony W. Norman, Percy S Manchand, George P Studzinski, Moray J Campbell, Phillip H Koeffler, Atsuko Takeuchi, Mei Ling Siucaldera, Sunita D Rao, Satyanarayana G ReddyAbstract:Abstract The secosteroid hormone 1α,25-dihydroxyvitamin D3 [1α,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 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 1α,25(OH)2-3-epi-D3. A rational design for the synthesis of potent analogs of 1α,25(OH)2D3 is developed based on the knowledge of the various metabolic pathways of 1α,25(OH)2D3. 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 D3 analogs 1α,25(OH)2-16,23-E-diene-26,27-hexafluoro-19-nor-D3 (Ro 25–9022), 1α,25(OH)2-16,23-Z-diene-26,27-hexafluoro-19-nor-D3 (Ro 26–2198), and 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-D3 (Ro 25–6760), as indicated by their antiproliferative activities.
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Metabolism of 1α,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells: In vitro biological activities of the natural metabolites of 1α,25-dihydroxyvitamin D3 produced in HL-60 cells
Steroids, 2001Co-Authors: D.sunita Rao, Milan R. Uskokovic, Moray J Campbell, H P Koeffler, Seiichi Ishizuka, P Spagnuolo, G. Satyanarayana ReddyAbstract:Abstract The secosteroid hormone, 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1α,25(OH) 2 D 3 and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1α,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, 1α,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 1α,25-dihydroxy-3-epi-vitamin-D 3 . We performed the present study first to examine in detail the metabolism of 1α,25(OH) 2 D 3 in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1α,25(OH) 2 D 3 in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1β- 3 H] 1α,25(OH) 2 D 3 and demonstrated that these cells metabolize 1α,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 1α,25(OH) 2 D 3 derived via the C-24 oxidation pathway namely, 1α,24(R),25-trihydroxyvitamin D 3 , 1α,25-dihydroxy-24-oxovitamin D 3 and 1α,23(S),25-trihydroxy-24-oxovitamin D 3 [1α,23(S),25(OH) 3 -24-oxo-D 3 ] were almost as potent as 1α,25(OH) 2 D 3 in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1α,23(S),25(OH) 3 -24-oxo-D 3 which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1α,25(OH) 2 D 3 to examine further its effects on these cells. Our results indicated that 1α,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 1α,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 1α,25(OH) 2 D 3 in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1α,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 1α,25(OH) 2 D 3 may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1α,25(OH) 2 D 3 .
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Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways.
Steroids, 2001Co-Authors: M R Uskokovic, M L Siu-caldera, A W Norman, Percy S Manchand, George P Studzinski, Moray J Campbell, Atsuko Takeuchi, H P Koeffler, D S Rao, G S ReddyAbstract: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.
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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.
Steroids, 2001Co-Authors: D S Rao, S. Ishizuka, Moray J Campbell, M R Uskokovic, H P Koeffler, P Spagnuolo, G S ReddyAbstract: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).