ABSTRACT
BACKGROUND: Bites constitute a public health problem worldwide. AIM: To characterize epidemiologically bites by animals happened in the province of Los Andes (2005-2007). METHOD: Descriptive, retrospective epidemiological study. Studied variables: Bitten person (BP), accident by bite, biting animal and bite. It was not feasible to obtain more updated information by law of patient protection. RESULTS: 2,360 BP were assisted in the emergency unit of San Juan de Dios Hospital and Rio Blanco clinic. An annual average rate of 729 BP/100,000 inhabitants and 1.99 daily average was recorded. The male gender was most affected (53.5%) and the age group from 6 to 10 years old. (Rate: .521/100,000). Most frequent topographic location was the lower limb, except in children under 5 year olds in whom predominated head and neck. The biting animal according to frequency was: the dog (67.1%) spider (7.1%) and cat (3.9%). The animal property was 35.6% known and 30.7% own. The most frequent problems were: nonspecific allergy; toxic effect by spider bites and among the infections, the disease made by cats scratch stands out. Regarding the record system, the biting animal complaint was applied to 47.6% of the BP and the 92.8% of the recorded information was incomplete. CONCLUSIONS: Bites reported higher rates in the province of Los Andes than the average of the country (729 versus 188/100,000), standing out the higher magnitude in 6 to 10 year-old-children. It is noticed that the record is low and incomplete. In this province, no bite control programs or updated studies have been carried out.
Subject(s)
Bites and Stings/epidemiology , Adolescent , Adult , Age Distribution , Animals , Bites and Stings/classification , Cats , Child , Child, Preschool , Chile/epidemiology , Dogs , Emergency Medical Services , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Rabbits , Rats , Retrospective Studies , Seasons , Spider Bites/epidemiology , Young AdultABSTRACT
Background: Bites constitute a public health problem worldwide. Aim: To characterize epidemiologically bites by animals happened in the province of Los Andes (2005-2007). Method: Descriptive, retrospective epidemiological study. Studied variables: Bitten person (BP), accident by bite, biting animal and bite. It was not feasible to obtain more updated information by law of patient protection. Results: 2,360 BP were assisted in the emergency unit of San Juan de Dios Hospital and Rio Blanco clinic. An annual average rate of 729 BP/100,000 inhabitants and 1.99 daily average was recorded. The male gender was most affected (53.5%) and the age group from 6 to 10 years old. (Rate: .521/100,000). Most frequent topographic location was the lower limb, except in children under 5 year olds in whom predominated head and neck. The biting animal according to frequency was: the dog (67.1%) spider (7.1%) and cat (3.9%). The animal property was 35.6% known and 30.7% own. The most frequent problems were: nonspecific allergy; toxic effect by spider bites and among the infections, the disease made by cats scratch stands out. Regarding the record system, the biting animal complaint was applied to 47.6% of the BP and the 92.8% of the recorded information was incomplete. Conclusions: Bites reported higher rates in the province of Los Andes than the average of the country (729 versus 188/100,000), standing out the higher magnitude in 6 to 10 year-old-children. It is noticed that the record is low and incomplete. In this province, no bite control programs or updated studies have been carried out.
Introducción: Las mordeduras constituyen un problema de salud pública mundial. Objetivo: Caracterizar epidemiológicamente las mordeduras por animales ocurridos en la provincia de Los Andes (2005-2007). Método: Estudio epidemiológico descriptivo retrospectivo. Variables estudiadas: Persona mordida (PM), accidente por mordedura, animal mordedor y mordedura. No fue factible obtener información más actualizada por ley de protección del paciente. Resultados: Se atendieron 2.360 PM en los Servicios de Urgencia del Hospital San Juan de Dios y Clínica Río Blanco. Se registró una tasa anual promedio de 729 PM/100.000 hbts. y promedio diario 1,99. El género más afectado fue el masculino (53,5%), y el grupo etario de 6 a 10 años (tasa:1.521/100.000). La ubicación topográfica más frecuente fue extremidad inferior, excepto en niños bajo 5 años de edad en quienes predominó cabeza-cuello. El animal mordedor según frecuencia fue: perro (67,1%) araña (7,1%) y gato (3,9%). La propiedad del animal fue 35,6% conocida y 30,7% propios. Las complicaciones más frecuentes fueron alergia no específica, efecto tóxico por mordedura de arañas y entre las infecciones destaca la enfermedad por rasguño de gato. La notificación de animal mordedor se aplicó a 47,6% de las PM; 92,8% de la información contenida estaba incompleta. Conclusiones: Las mordeduras en la Provincia de Los Andes presentaron tasas mayores que el promedio país (729 versus 188/100.000), destacando la mayor magnitud en niños de 6 a10 años. En la provincia no se han realizado programas de control de mordeduras o estudios actualizados.
Subject(s)
Humans , Animals , Male , Female , Infant, Newborn , Infant , Child, Preschool , Adolescent , Adult , Middle Aged , Cats , Dogs , Rabbits , Rats , Young Adult , Bites and Stings/epidemiology , Spider Bites/epidemiology , Seasons , Bites and Stings/classification , Chile/epidemiology , Retrospective Studies , Age Distribution , Emergency Medical ServicesABSTRACT
Recently, it was reported that stimulation of the infralimbic cortex produces a feedforward inhibition of central amygdala neurons. The interest of this observation comes from the fact that the central nucleus is the main output station of the amygdala for conditioned fear responses and evidence that the infralimbic cortex plays a critical role in the extinction of conditioned fear. However, the identity of the neurons mediating this infralimbic-evoked inhibition of the central nucleus remains unknown. Likely candidates are intercalated amygdala neurons. Indeed, these cells receive glutamatergic afferents from the infralimbic cortex, use GABA as a transmitter, and project to the central amygdala. Thus, the present study was undertaken to test whether, in adult rats, the infralimbic cortex can affect the activity of intercalated neurons. To this end, disinhibition of the infralimbic cortex was induced by local infusion of the non-competitive GABA-A receptor antagonist picrotoxin. Subsequently, neuronal activation was determined bilaterally within the amygdala using induction of the immediate early gene Fos. Infralimbic disinhibition produced a significant increase in the number of Fos-immunoreactive intercalated cells bilaterally whereas no change was detected in the central nucleus. In the basolateral amygdaloid complex, increases in the number of Fos-immunoreactive cells only reached significance in the contralateral lateral nucleus. These results suggest that glutamatergic inputs from the infralimbic cortex directly activate intercalated neurons. Thus, our findings raise the possibility that the infralimbic cortex inhibits conditioned fear via the excitation of intercalated cells and the consequent inhibition of central amygdala neurons.
Subject(s)
Amygdala/metabolism , Interneurons/metabolism , Limbic System/metabolism , Neural Pathways/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Amygdala/drug effects , Animals , Conditioning, Classical , Fear/physiology , GABA Antagonists/administration & dosage , Injections, Intraventricular , Interneurons/drug effects , Limbic System/drug effects , Male , Neural Pathways/drug effects , Picrotoxin/administration & dosage , Proto-Oncogene Proteins c-fos/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
In a recent study, we investigated the metabolism of 1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), a potent synthetic vitamin D3 analog in the isolated perfused rat kidney and proposed that the enhanced biological activity of 1alpha,25(OH)2-20-epi-D3 is in part due to its metabolism into stable bioactive intermediary metabolites derived via the C-24 oxidation pathway (Siu-Caldera et al. [1999] J. Steroid. Biochem. Mol. Biol. 71:111-121). It is now well established that 1alpha,25(OH)2D3 and its analogs are metabolized in target tissues not only via the C-24 oxidation pathway but also via the C-3 epimerization pathway. As the perfused rat kidney does not express the C-3 epimerization pathway, we could not identify other possible bioactive metabolites of 1alpha,25(OH)2-20-epi-D3 such as 1alpha,25(OH)2-20-epi-3-epi-D3, derived via the C-3 epimerization pathway. Therefore, we studied the metabolism of 1alpha,25(OH)2-20-epi-D3 in rat osteosarcoma cells (UMR 106) which express both the C-24 oxidation and the C-3 epimerization pathways. Our results indicate that 1alpha,25(OH)2-20-epi-D3 is metabolized in UMR 106 cells into several metabolites which included not only the previously known metabolites of the C-24 oxidation pathway but also three new metabolites which were labeled as metabolites X, Y1, and Y2. Metabolite X was unequivocally identified as 1alpha,25(OH)2-20-epi-3-epi-D3. Even though definite structure identification of the metabolites, Y1 and Y2 was not achieved in our present study, we determined that the metabolite Y1 is produced from 1alpha,25(OH)2-20-epi-D3 and the metabolite Y2 is produced from 1alpha,25(OH)2-20-epi-3-epi-D3. We also noted the production of both 1alpha,25(OH)2-20-epi-3-epi-D3 and the two metabolites Y1 and Y2 in different rat osteosarcoma cells (ROS 17/2.8) which express only the C-3 epimerization pathway but not the C-24 oxidation pathway. Furthermore, we investigated the metabolism of 1alpha,25(OH)2-20-epi-D3 in the isolated perfused rat kidney in an earlier study. The results of this study indicated that the rat kidney unlike rat osteosarcoma cells did not produce either 1alpha,25(OH)2-20-epi-3-epi-D3 or the metabolites Y1 and Y2. Thus, it appears that the metabolites Y1 and Y2, like 1alpha,25(OH)2-20-epi-3-epi-D3, are produced only in specific tissues. Preliminary biological activity of each new metabolite is assessed by measuring its ability to generate VDR-mediated gene transcription. 1alpha,25(OH)2-20-epi-3-epi-D3 was found to be almost equipotent to 1alpha,25(OH)2-20-epi-D3 while the metabolites, Y1 and Y2 were found to be less active. The metabolite Y1 when compared to the metabolite Y2 has higher biological activity and its potency is almost equal to 1alpha,25(OH)2D3. In summary, we report for the first time tissue specific metabolism of 1alpha,25(OH)2-20-epi-D3 into several bioactive metabolites which are derived not only via the previously established C-24 oxidation and C-3 epimerization pathways but also via a new pathway. (c) 2001 Wiley-Liss, Inc.
Subject(s)
Calcitriol/metabolism , Animals , Calcitriol/pharmacology , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Genes, Reporter , Models, Chemical , Osteosarcoma , Oxidation-Reduction , Rats , Receptors, Calcitriol/metabolism , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
To clarify physiological role of the carbon 3 (C-3) epimerization of 1alpha,25(OH)(2)D(3) and biologic significance of a 3-epi metabolite of 1alpha,25(OH)(2)D(3), we examined biologic activities of the 3-epimers of 1alpha,25(OH)(2)D(3) and 1alpha,25(OH)(2)-16-ene-D(3) analogs in terms of modulation of cell cycle phase distribution and cell-surface CD11b antigen expression of HL-60 cells, transactivation of vitamin D target genes in transfected cells, stimulation of VDR/RXRalpha heterodimer formation in a rabbit reticulocyte lysates transcription/translation system, stimulation of VDR/RXRalpha/VDRE complex formation, and induction of HL-60 cell apoptosis. The analogs tested here were 1) 1alpha,25(OH)(2)D(3), 2) 1alpha,25(OH)(2)-3-epi-D(3), 3) 1alpha,25(OH)(2)-16-ene-D(3), 4) 1alpha,25(OH)(2)-16-ene-3-epi-D(3), 5) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-D(3), 6) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-3-epi-D(3), 7) 1alpha,25-(OH)(2)-16-ene-20-epi-23-yne-D(3), and 8) 1alpha,25(OH)(2)-16-ene-20-epi-23-yne-3-epi-D(3). When compared to the 3-natural (beta) analogs, the 3-epi (alpha) analogs were biologically significantly less active. The findings support the hypothesis that the C-3 epimerization is an inactivation pathway of 1alpha,25(OH)(2)D(3) and its analogs in vitamin D target tissues. We also found that the 3-epi analogs, but not the 3-natural (beta) analogs, were the potent inducers of apoptosis of HL-60 cells. These results suggest that the analogs could be divided into two groups, in which the 3-epi analogs were the potent inducers of apoptosis of HL-60 cells, and the 3-natural analogs were the potent modulators of HL-60 cell growth and differentiation. This is the first report demonstrating that the 3-epimerization of the hydroxyl group at C-3 of the A-ring of 1alpha,25(OH)(2)D(3) plays an important role to modulate HL-60 cell differentiation and apoptosis.
Subject(s)
Calcitriol/pharmacology , Leukemia, Promyelocytic, Acute/pathology , Animals , Apoptosis/drug effects , Calcitriol/analogs & derivatives , Cattle , Cell Cycle/drug effects , Cell Differentiation/drug effects , Dimerization , HL-60 Cells/drug effects , Humans , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Stereoisomerism , Transcription Factors/metabolism , Transcriptional Activation/drug effectsABSTRACT
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.
Subject(s)
Calcitriol/metabolism , Calcitriol/pharmacology , Animals , Calcitriol/analogs & derivatives , Calcitriol/chemistry , Humans , Molecular Structure , Oxidation-Reduction , Structure-Activity Relationship , Vitamin D/analogs & derivatives , Vitamin D/chemistry , Vitamin D/metabolism , Vitamin D/pharmacologyABSTRACT
New C,D-ring side-chain-modified sulfone 4a, with natural 1alpha, 3beta-hydroxyl groups but lacking the 25-hydroxyl group characteristic of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1), has been prepared and characterized. Novel synthetic features include: (1) chemoselective oxidation of only a primary silyl ether in a primary-secondary bis-silyl ether intermediate and (2) smooth reductive etherification without interference by a neighboring sulfonyl group. Sulfone 4a, but not its 1beta, 3alpha-diastereomer 4b, is powerfully antiproliferative and transcriptionally active in vitro but desirably noncalcemic in vivo. Although sulfone 4a, designed to resemble Leo Pharmaceutical Co.'s KH-1060 (3), is recognized by catabolic enzymes, the selective biological profile of sulfone 4a is likely not due to its metabolites that are formed in only minor amounts.
Subject(s)
Calcitriol/analogs & derivatives , Calcitriol/chemical synthesis , Sulfones/chemical synthesis , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding, Competitive , Calcitriol/chemistry , Calcitriol/pharmacology , Cell Division/drug effects , Graft Rejection , Humans , Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Islets of Langerhans Transplantation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Receptors, Calcitriol/metabolism , Stereoisomerism , Structure-Activity Relationship , Sulfones/chemistry , Sulfones/pharmacology , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Calcitriol/analogs & derivatives , Calcitriol/chemistry , Calcitriol/metabolism , Oxygen/metabolism , Animals , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Osteosarcoma/metabolism , Rats , Time Factors , Tumor Cells, CulturedABSTRACT
1alpha,24(R)-Dihydroxyvitamin D3 [1alpha,24(R)(OH)2D3], a synthetic vitamin D3 analog, has been developed as a drug for topical use in the treatment of psoriasis. At present, the target tissue metabolism of 1alpha,24(R)(OH)2D3 is not understood completely. In our present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in the isolated perfused rat kidney. The results indicated that 1alpha,24(R)(OH)2D3 is metabolized in rat kidney into several metabolites, of which 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxovitamin D3, 1alpha,23(S),25-trihydroxy-24-oxovitamin D3, and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D3 are similar to the previously known metabolites of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In addition to these aforementioned metabolites, we also identified two new metabolites, namely 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3. The two new metabolites do not possess the C-25 hydroxyl group. Thus, the metabolism of 1alpha,24(R)(OH)2D3 into both 25-hydroxylated and non-25-hydroxylated metabolites suggests that 1alpha,24(R)(OH)2D3 is metabolized in the rat kidney through two pathways. The first pathway is initiated by C-25 hydroxylation and proceeds further via the C-24 oxidation pathway. The second pathway directly proceeds via the C-24 oxidation pathway without prior hydroxylation at the C-25 position. Furthermore, we demonstrated that rat kidney did not convert 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] into 1alpha,25(OH)2D3. This finding indicates that the rat kidney does not possess the classical vitamin D3-25-hydroxylase (CYP27) activity. However, from our present study it is apparent that prior hydroxylation of 1alpha(OH)D3 at the C-24 position in the 'R' orientation allows 25-hydroxylation to occur. At present, the enzyme responsible for the C-25 hydroxylation of 1alpha,24(R)(OH)2D3 is unknown. Our observation that the side chain of 1alpha,24(R)(OH)2D3 underwent 24-ketonization and 23-hydroxylation even in the absence of the C-25 hydroxyl group suggests that 1alpha,25(OH)2D3-24-hydroxylase (CYP24) can perform some steps of the C-24 oxidation pathway without prior C-25 hydroxylation. Thus, we speculate that CYP24 may be playing a dual role in the metabolism of 1alpha,24(R)(OH)2D3.
Subject(s)
Cholecalciferol/analogs & derivatives , Dermatologic Agents/metabolism , Dihydroxycholecalciferols/metabolism , Kidney/metabolism , Animals , Calcitriol/metabolism , Calcitriol/pharmacology , Cholecalciferol/isolation & purification , Dermatologic Agents/pharmacology , Dihydroxycholecalciferols/pharmacology , In Vitro Techniques , Kidney/drug effects , Male , Mass Spectrometry , Perfusion , Rats , Rats, Sprague-Dawley , Spectrophotometry, UltravioletABSTRACT
In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1alpha, 25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] by identifying 1alpha,24(S),25,28-tetrahydroxyvitamin D(2) [1alpha,24(S),25, 28(OH)(4)D(2)] as a major renal metabolite of 1alpha,25(OH)(2)D(2) [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1alpha, 25(OH)(2)D(2). We perfused rat kidneys in vitro with 1alpha, 25(OH)(2)[26,27-(3)H]D(2) (5 x 10(-10)M) and demonstrated that 1alpha,24(R),25-trihydroxyvitamin D(2) [1alpha,24(R),25(OH)(3)D(2)] and 1alpha,24(S),25,28(OH)(4)D(2) are the only two major physiological metabolites of 1alpha,25(OH)(2)D(2). In the same perfusion experiments, we also noted that there is no conversion of 1alpha,25(OH)(2)D(2) into 1alpha,25,28-trihydroxyvitamin D(2 )[1alpha,25,28(OH)(3)D(2)]. Moreover, 1alpha,24(S),25,28(OH)(4)D(2) is not formed in the perfused rat kidney when synthetic 1alpha,25, 28(OH)(3)D(2) is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1alpha,25(OH)(2)D(2) occurs only after 1alpha,25(OH)(2)D(2) is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1alpha, 24(R),25(OH)(3)D(2) in rat kidney is not known. Recently, it was found that 1alpha,25(OH)(2)D(3)-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D(3) compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1alpha,24(R),25(OH)(3)D(2) to form 1alpha, 24(S),25,28(OH)(4)D(2). The biological activity of 1alpha,24(S),25, 28(OH)(4)D(2), determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1alpha,24(S),25,28(OH)(4)D(2) exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1alpha,25(OH)(2)D(2) in kidney under physiological conditions.
Subject(s)
Calcium/metabolism , Ergocalciferols/metabolism , Kidney/metabolism , Animals , Cattle , Ergocalciferols/chemistry , Hydroxylation , Ion Transport , Male , Rats , Rats, Sprague-DawleyABSTRACT
Induction of growth arrest and monocyte differentiation of HL-60 leukemia cells by 1alpha,25 dihydroxyvitamin D3 (1alpha,25(OH)2D3) is well established. By contrast, we have observed, that 1alpha,25(OH)2D3 and its metabolites play separate roles in clonal expansion and survival of differentiating HL-60 cells. Cells that had differentiated by 48 h (CD14 positive) grew slower than control cells, whereas CD14 negative cells were growing faster at this time point. Inhibiting 1alpha,25(OH)2D3 or 1alpha,25(OH)2-20-epi-D3 metabolism, by the 25(OH)D3-24-hydroxylase inhibitor ketoconazole, abolished hyperproliferation of CD14 negative cells. Instead, both the onset of differentiation and subsequent apoptosis were enhanced. These events were associated with immediate up-regulation of the cyclin-dependent kinase inhibitor p21(waf1) and a lack of sustained expression, respectively. Stimulation and inhibition of growth by vitamin D3-related compounds was observed to be concentration and metabolite specific. Low amounts of 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated HL-60 cell growth. At higher concentrations, 1alpha,25(OH)2-20-epi-D3 was a more potent inducer than 1alpha,24,25(OH)3-20-epi-D3 of HL-60 differentiation; 1alpha,25(OH)2-20-epi-24-oxo-D3 was exclusively pro-differentiative at all concentrations. 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated proliferation of KG-1a leukemia cells, but neither of these compounds nor 1alpha,25(OH)3-20-epi-24-oxo-D3 exerted pro-differentiative effects on these cells. These findings shed new light on the pro- and anti-proliferative effects of 1alpha,25(OH)2D3 and lead to the postulate that metabolism of 1alpha,25(OH)2D3 and its 20-epi analog regulates different subsets of genes so as to co-ordinate population expansion and the differentiation process. Furthermore, 1alpha,25(OH)2D3 metabolism and/or sensitivity to the effects of metabolites may be altered in transformed cells to derive a clonal advantage.
Subject(s)
Apoptosis/physiology , Calcitriol/analogs & derivatives , Calcitriol/metabolism , Cell Differentiation/physiology , Cytochrome P-450 Enzyme System , Apoptosis/drug effects , Calcitriol/pharmacology , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Division/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Enzyme Inhibitors/pharmacology , HL-60 Cells , Humans , Ketoconazole/pharmacology , Models, Biological , Steroid Hydroxylases/antagonists & inhibitors , Vitamin D3 24-HydroxylaseABSTRACT
The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into calcitroic acid through the carbon 24 (C-24) oxidation pathway. It is now well established that the C-24 oxidation pathway plays an important role in the target tissue inactivation of 1alpha,25(OH)2D3. Recently, we reported that 1alpha,25(OH)2D3 is also metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] through the carbon 3 (C-3) epimerization pathway in human keratinocytes, human colon carcinoma cells (Caco-2), and bovine parathyroid cells. In a previous study, it was demonstrated that 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 was less active in stimulating intestinal calcium absorption, calcium mobilization from bone, and induction of calbindin D28k. These findings suggest that the C-3 epimerization pathway, like the C-24 oxidation pathway, may play a role in the target tissue inactivation of 1alpha,25(OH)2D3. In this study, we determined the relationship between the C-24 oxidation and the C-3 epimerization pathways by investigating the metabolism of 1alpha,25(OH)2D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8). These two cell lines differ from each other in their ability to metabolize 1alpha,25(OH)2D3 through the C-24 oxidation pathway. It has been previously reported that the C-24 oxidation pathway is expressed only in UMR 106 cells but not in ROS 17/2.8 cells. The results of our present study provide new evidence that both cell lines possess the ability to metabolize 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 through the C-3 epimerization pathway. Our results also reconfirm the findings of previous studies indicating that UMR 106 cells are the only ones which express the C-24 oxidation pathway out of the two cell lines studied. Furthermore, this study reveals for the first time that the C-3 epimerization pathway may become an alternate metabolic pathway for the target tissue inactivation of 1alpha,25(OH)2D3 in some cells, such as ROS 17/2.8, in which the C-24 oxidation pathway is not expressed.
Subject(s)
Bone Neoplasms/metabolism , Calcitriol/biosynthesis , Osteosarcoma/metabolism , Animals , Calcitriol/analysis , Gas Chromatography-Mass Spectrometry , Kidney/cytology , Kidney/metabolism , Male , Oxidation-Reduction , Perfusion , Rats , Rats, Sprague-Dawley , Stereoisomerism , Tumor Cells, CulturedABSTRACT
We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 as a major in vitro metabolite of 1alpha,25-dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1alpha,25-dihydroxy-3-epi-vitamin D3 from the serum of rats treated with pharmacological doses of 1alpha,25-dihydroxyvitamin D3. 1alpha,25-dihydroxy-3-epi-vitamin D3 was identified through its co-migration with synthetic 1alpha,25-dihydroxy-3-epi-vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1alpha,25-dihydroxy-3-epi-vitamin D3, other previously known metabolites, namely, 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxo-vitamin D3 and 1alpha,25-dihydroxyvitamin D3-26,23-lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1alpha,25-dihydroxy-3-epi-vitamin D3 is an in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats.
Subject(s)
Cholecalciferol/metabolism , Vitamin D/analogs & derivatives , Animals , Cholecalciferol/analogs & derivatives , Chromatography, Gas , Chromatography, High Pressure Liquid , Lipid Metabolism , Male , Mass Spectrometry , Rats , Rats, Sprague-Dawley , Time Factors , Vitamin D/metabolismABSTRACT
Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1, 25(OH)2D3 on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1, 25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1, 25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3 in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.
Subject(s)
Calcitriol/pharmacology , Cyclic AMP/physiology , Cytochrome P-450 Enzyme System , Gene Expression Regulation, Enzymologic/physiology , Kidney Tubules/enzymology , Nephrons/enzymology , Parathyroid Hormone/physiology , Steroid Hydroxylases/genetics , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Base Sequence , Cells, Cultured , Cyclic AMP/pharmacology , Enzyme Induction , Gene Expression Regulation, Enzymologic/drug effects , Kidney Tubules, Distal/enzymology , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Rats , Recombinant Fusion Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Steroid Hydroxylases/biosynthesis , Teriparatide/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects , Transfection , Vitamin D3 24-HydroxylaseABSTRACT
Vitamin D2 is less toxic in rats when compared to vitamin D3. Our laboratory has been involved in research projects which were directed towards identifying the possible mechanisms responsible for the toxicity differences between vitamins D2 and D3 in rats. The present research project was designed to isolate and identify new metabolites of vitamin D2 from serum of rats which were fed toxic doses of vitamin D2. Hypervitaminosis D2 was induced in 30 rats by feeding each rat with 1000 nmol of vitamin D2/day x 14 days. The rats were sacrificed on the 15th day and obtained 180 ml of serum. The lipid extract of the serum was directly analyzed by a straight phase HPLC system. The various vitamin D2 metabolites were monitored by their ultraviolet (UV) absorbance at 254 nm. One of the UV absorbing peaks did not comigrate with any of the known vitamin D2 metabolites. This unknown metabolite peak was further purified by HPLC and was then subjected to UV absorption spectrophotometry and mass spectrometry. The structure assignment of the new metabolite was established to be 4,25-dihydroxyvitamin D2 [4,25(OH)2D2] by the techniques of UV absorption spectrophotometry and mass spectrometry and by the new metabolite's susceptibility to sodium metaperiodate oxidation. At present the biological activity of this unique 'A-ring' hydroxylated vitamin D2 metabolite is not known. As this new metabolite is isolated from the serum of rats intoxicated with vitamin D2, we speculate that 4,25(OH)2D2 may be playing an important role in the deactivation of vitamin D2.
Subject(s)
Ergocalciferols/metabolism , Animals , Chromatography, High Pressure Liquid , Ergocalciferols/blood , Ergocalciferols/chemistry , Ergocalciferols/isolation & purification , Ergocalciferols/poisoning , Male , Mass Spectrometry , Molecular Structure , Pilot Projects , Rats , Rats, Sprague-Dawley , Spectrophotometry, UltravioletABSTRACT
1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1alpha,25(OH)2-20-epi-D3, namely, 1alpha,24(R),25(OH)3-20-epi-D3 and 1alpha,25(OH)2-24-oxo-20-epi-D3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1alpha,25(OH)2D3. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1alpha,25(OH)2-20-epi-D3 by itself is not metabolically stable when compared to 1alpha,25(OH)2D3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1alpha,25(OH)2-20-epi-D3.
Subject(s)
Calcitriol/metabolism , Calcitriol/pharmacology , Animals , Calcitriol/chemistry , Genes, Reporter/drug effects , Humans , In Vitro Techniques , Kidney/metabolism , Ligands , Male , Perfusion , Rats , Rats, Sprague-Dawley , Receptors, Calcitriol/drug effects , Receptors, Calcitriol/metabolism , Stereoisomerism , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
We used the human colon adenocarcinoma-derived cell line Caco-2, which spontaneously differentiates in vitro, as a model system to investigate the metabolism of 1 alpha,25-dihydroxycholecalciferol in colon cancer cells. Subconfluent proliferating and confluent differentiating cells were incubated with 1 microM 1 alpha,25-dihydroxycholecalciferol for a period of 24 to 48 h. HPLC analysis of the lipid extract of both cells and media was performed to isolate and identify the various metabolites of 1 alpha,25-dihydroxycholecalciferol. Undifferentiated, highly proliferating Caco-2 cells metabolized 1 alpha, 25-dihydroxycholecalciferol into several side chain modified metabolites formed through the C-24 oxidation pathway. In contrast, no metabolites of the C-24 oxidation pathway were identified in differentiated Caco-2 cells. However, differentiated cells produced significant amounts of a metabolite which was less polar than 1 alpha, 25-dihydroxycholecalciferol on a straight phase HPLC system. This metabolite was identified as 1 alpha,25-dihydroxy-3alpha-cholecalciferol by comigration with a synthetic standard on two different HPLC systems and gas chromatography--mass spectrometry. Thus, we were able to demonstrate that the state of differentiation has a profound influence on 1 alpha,25-dihydroxycholecalciferol metabolism in colon cancer cells.
Subject(s)
Caco-2 Cells/metabolism , Dihydroxycholecalciferols/metabolism , Alkaline Phosphatase/metabolism , Biological Transport, Active , Caco-2 Cells/drug effects , Caco-2 Cells/pathology , Calcitriol/chemistry , Calcitriol/metabolism , Calcitriol/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Dihydroxycholecalciferols/chemistry , Dihydroxycholecalciferols/pharmacokinetics , Gas Chromatography-Mass Spectrometry , Humans , Thymidine/metabolism , Time Factors , TritiumABSTRACT
We compared the separate effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analog, 1alpha,25-dihydroxy-16ene,23yne-vitamin D3 (1alpha25(OH)2-16ene,23yne-D3), as well as their interactions with 17-beta estradiol (E2) in our human osteosarcoma SaOS-2 cell models representing two stages of differentiation, the SaOS+DEX and SaOS-DEX cells. SaOS+DEX cells have been previously shown to express higher PTH-stimulated adenylate cyclase (PTH-AC) and basal alkaline phosphatase (ALP) activities compared with SaOS-DEX cells. ALP: In SaOS+DEX cells, 0.1 nmol/L analog, but not 1alpha,25(OH)2D3, increased ALP activity 1.7-fold (p < 0.05). Instead, 1 nmol/L 1alpha,25(OH)2D3 increased ALP 1.4-fold (p < 0.05). In these cells, E2 enhanced 1alpha,25(OH)2D3-stimulated ALP activity (ANOVA, F = 51.22, p <0.0001), while inhibiting the effect of the analog. [3H]-Thymidine uptake: In SaOS+DEX cells, 1alpha,25(OH)2D3 had biphasic effects (ANOVA, F = 13.08, p < 0.0001), which were not altered by E2. In contrast, the analog was stimulatory only with E2 (ANOVA, F = 3.59, p < 0.025). Osteocalcin (OC): 1alpha,25(OH)2D3 and its analog stimulated OC production in SaOS-DEX cells with smaller effects in SaOS+DEX cells. In SaOS-DEX cells, E2 enhanced the effect of 1alpha,25(OH)2D3, but not that of the analog. PTH-AC: In SaOS-DEX cells, 100 nmol/L analog inhibited PTH-AC activities by 50% (p < 0.01), whereas 1alpha,25(OH)2D3 had little effect. In SaOS+DEX cells, both compounds inhibited PTH-AC approximately 35%. E2 inhibited the effect of the analog in SaOS-DEX cells, but enhanced the effects of both compounds in SaOS+DEX cells. These results show that the analog 1alpha,25(OH)2-16ene,23yne-D3 was effective in regulating osteoblastic function; its effects were modulated by E2 and dependent upon the stage of osteoblast differentiation.
Subject(s)
Calcitriol/analogs & derivatives , Dihydroxycholecalciferols/pharmacology , Estradiol/pharmacology , Osteoblasts/drug effects , Adenylyl Cyclases/metabolism , Alkaline Phosphatase/metabolism , Calcitriol/pharmacology , Cell Differentiation , Humans , Osteoblasts/metabolism , Osteocalcin/biosynthesis , Osteocalcin/drug effects , Osteosarcoma , Parathyroid Hormone/pharmacology , Thymidine/metabolism , Tumor Cells, CulturedABSTRACT
1alpha,25(OH)2-16-ene-D3, a synthetic analog of the steroid hormone, 1alpha,25(OH)2D3, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1alpha,25(OH)2-16-ene-D3 expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently. 1alpha,25(OH)2-24-oxo-16-ene-D3, an intermediary metabolite of 1alpha,25(OH)2-16-ene-D3 formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1alpha,25(OH)2-24-oxo-D3, the corresponding intermediary metabolite of 1alpha,25(OH)2D3. In a subsequent in vivo study, we also reported that 1alpha,25(OH)2-24-oxo-16-ene-D3 exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1alpha,25(OH)2-24-oxo-16-ene-D3, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1alpha,25(OH)2-16-ene-D3 and 1alpha,25(OH)2D3 are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1alpha,25(OH)2-24-oxo-16-ene-D3 in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1alpha,25(OH)2D3. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1alpha,25(OH)2-16-ene-D3 and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1alpha,25(OH)2D3 itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1alpha,25(OH)2-24-oxo-16-ene-D3, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1alpha,25(OH)2-16-ene-D3.
Subject(s)
Calcitriol/analogs & derivatives , Cell Differentiation/drug effects , Leukemia, Myeloid/drug therapy , Calcitriol/metabolism , Calcitriol/pharmacology , Cell Division/drug effects , Chromatography, High Pressure Liquid , Humans , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Receptors, Calcitriol/drug effects , Receptors, Calcitriol/genetics , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
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)2-24,25,26,27-tetranor D3, and calcitroic acid formed through the C-24 oxidation pathway and 1 alpha, 23(S),25-(OH)3D3 and 1 alpha, 25-(OH)2D3-26,23-lactone formed through the C-23 oxidation pathway were detected under basal conditions. Also, the synthesis of these metabolites was increased significantly when the cells were treated with 1 alpha, 25-(OH)2D3 (50 nM) for 24 h before incubation with the tracer. As 25-hydroxyvitamin D3 (25OHD3) follows similar side-chain modifications as 1 alpha, 25-(OH)2D3, the metabolism of 25OHD3 in normal human osteoblasts was studied under basal conditions. We found that 25OHD3 was also metabolized through both C-24 and C-23 oxidation pathways, resulting in significant synthesis of 24(R),25-(OH)2D3 along with 25OH-24-oxo-D3, 23(S),25-(OH)2-24-oxo-D3, 23(S),25-(OH)2D3, and 25OHD3-26,23-lactone. Under the same experimental conditions, we looked for 1 alpha, 25-(OH)2D3 synthesis, as earlier studies have shown production of 1 alpha, 25-(OH)2D3 in human bone cells. During a time-course study ranging from 1-24 h, we found that by 2 h, the 24(R), 25-(OH)2D3 concentration rose and accumulated considerably during the following 24 h, but 1 alpha, 25-(OH)2D3 did not accumulate at any time. However, other 1-hydroxylated metabolites, 1 alpha, 23(S),25-(OH)3D3, 1 alpha, 23(S),25-(OH)3-24-oxo-D3, as well as 1 alpha, 25-(OH)2D3-26,23-lactone were detected.(ABSTRACT TRUNCATED AT 400 WORDS)
