RESUMEN

OBJECTIVES: For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated. DESIGN AND METHODS: After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi-reaction monitoring is used for analyte detection. RESULTS: Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-265.3 nmol/L for 25OH-D(3), 3.9-183.6 nmol/L for 25OH-D(2), 2.0-133.8 nmol/L for 3-epi-25OH-D(3) and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D(3) (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D(3), 3.9 nmol/L for 25OH-D(2), 2.0 nmol/L for 3-epi-25OH-D(3) and 2.8 nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3). CONCLUSIONS: This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3). Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).

Asunto(s)

RESUMEN

In a previous report, we indicated that 1alpha,23(S), 25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S), 25(OH)(3)-24-oxo-D(3)], a natural metabolite of 1alpha, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is almost equipotent to 1alpha,25(OH)(2)D(3) in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1alpha,23(S),25(OH)(3)-24-oxo-D(3) has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D(3) analogs structurally related to 1alpha,23(S),25(OH)(3)-24-oxo-D(3) may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1alpha,25(OH)(2)D(3) showed that the removal of carbon-19 (C-19) reduces the calcemic activity of 1alpha, 25(OH)(2)D(3.) Therefore, in an attempt to produce vitamin D(3) analogs with a better therapeutic index, we synthesized C(23) epimers of 1alpha,23,25(OH)(3)-24-oxo-19-nor-vitamin D(3) [1alpha,23, 25(OH)(3)-24-oxo-19-nor-D(3)]. The two epimers were compared to 1alpha,25(OH)(2)-19-nor-D(3) and 1alpha,25(OH)(2)D(3) in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) was as potent as 1alpha, 25(OH)(2)-19-nor-D(3) but was less potent than 1alpha,25(OH)(2)D(3). In the same assay 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) exhibited greater potency than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) was less potent than 1alpha,25(OH)(2)-19-nor-D(3) but was equipotent to 1alpha, 25(OH)(2)D(3). More importantly, in the same assays, 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was more potent than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) and was equipotent to 1alpha, 25(OH)(2)-19-nor-D(3). Also, the vitamin D receptor-mediated transcriptional activity of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was almost equal to that of 1alpha, 25(OH)(2)-19-nor-D(3), but higher than that of 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). This finding explains in part the greater in vitro biologic activities of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3). In summary, our results indicate that 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) and to a lesser extent 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) are potent 19-nor vitamin D(3) analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro.

Asunto(s)

RESUMEN

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

Asunto(s)

RESUMEN

A specific and sensitive assay for the measurement of the concentration of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D2 and 25,26-dihydroxyvitamin D2 in a single plasma sample is described, using stable isotope dilution mass fragmentography. After addition of appropriate deuterium-labelled internal standards, plasma samples were treated with acetonitrile to precipitate protein, and vitamin D metabolites were extracted on prepacked microparticulate reverse-phase cartridges. Further purification was achieved using straight-phase cartridges and high-performance liquid chromatography. Gas chromatography-mass spectrometry was carried out after appropriate derivatisation of samples and standards. The method has been evaluated in terms of specificity, recovery of added standards, and reproducibility.

Asunto(s)

Asunto(s)

RESUMEN

By cochromatography, mass spectrometry, and chemical derivatization, we have shown that a metabolite isolated from the perfused rat kidney incubated with 24-(R),25-dihydroxyvitamin D3 is indistinguishable from chemically synthesized 24,25,26,27-tetranor-23-hydroxyvitamin D3. The new metabolite is also produced from 24-oxo-25-hydroxyvitamin D3 but not from 23(S),25-dihydroxyvitamin D3. Enzymes required for the synthesis of the new metabolite are absent in the vitamin D deplete animal but are induced along with the 25-hydroxyvitamin-D3 24-hydroxylase by vitamin D repletion. The pathway of 24,25-dihydroxyvitamin D3 metabolism in the perfused kidney is stimulated by pre-treatment of the rat with large doses of vitamin D3, suggesting that the pathway is a degradative one.

Asunto(s)

RESUMEN

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

Asunto(s)

RESUMEN

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.

Asunto(s)

Asunto(s)

RESUMEN

Extraction, lipid-reduction, and chromatographic methods suitable for the resolution and subsequent quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxy-vitamin D3 from human milk are described. This procedure utilizes a methanol:methylene chloride extraction, precipitation of unwanted lipids with cold methanol and ether, backwash with alkaline buffer, silica Sep-Pak preparative chromatography, normal- and reverse-phase high-performance liquid chromatography with final quantitation of the antirachitic sterols by competitive protein binding assay. The described assay was used to determine these antirachitic sterols in milk from women receiving various supplements of vitamin D or undergoing ultraviolet phototherapy.

Asunto(s)

RESUMEN

A new vitamin D3 metabolite was isolated in pure form (18.2 micrograms) from the serum of rats given large doses (two doses of 26 mumol/rat) of vitamin D3. The new metabolite has been unequivocally identified as 3 beta, 25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-peroxylactone by ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, mass spectrometry, field desorption mass spectrometry, and specific chemical reaction with triphenyl phosphine. The stereochemical configuration at the C-23 and c-25 positions of the 25-hydroxyvitamin D3-26-23-peroxylactone was definitely determined to be the 23(S)25(R),25-hydroxyvitamin D3-26,23-peroxylactone is suggested for this metabolite. The isolation involved chloroform-methanol extraction and four column chromatographic procedures. The metabolite purification and elution position on these columns were followed by UV measurement at 264 nm. This metabolite was ultimately resolved from the previously known 25-hydroxyvitamin D3-26,23-lactone by high pressure liquid chromatography using a Zorbax Sil column. The 25-hydroxyvitamin D3-26,23-peroxylactone was converted upon storage at room temperature or -20 degrees C into the 25-hydroxyvitamin D3-26,23-lactone. Since under the conditions of this isolation only the 26,23-peroxylactone and no 26,23-lactone of 25-hydroxyvitamin D3 was present in the rat serum, this suggests that the 25-hydroxyvitamin D3-26,23-peroxylactone is the naturally occurring metabolite.

Asunto(s)

Asunto(s)

RESUMEN

Two new vitamin D metabolites were isolated in pure form from incubations of 53 nM 1,25-dihydroxyvitamin D3 with homogenates of small intestinal mucosa of vitamin D-replete chicks. The birds were injected intravenously with 8 to 9 nmol of 1,25-dihydroxyvitamin D3/100 g body weight 5 to 8 h before death. The isolation involved methanol-chloroform extraction and four successive chromatographic procedures (Sephadex LH-20 and high performance liquid chromatography). Chemical structures of the metabolites are proposed on the basis of (a) their chromatographic behavior, (b) their mass spectra, and (c) ultraviolet absorption spectra. They are identified as 1 alpha,25-dihydroxy-23-oxo-vitamin D3 and 1 alpha,25,26-trihydroxy-23-oxo-vitamin D3. Neither of the two new metabolites is produced by the intestinal mucosa when 1,25S,26-trihydroxyvitamin D3 is used as a substrate.

Asunto(s)

RESUMEN

A simple method has been developed using 'SEP-PAK' disposable silica cartridges to separate the major endogenous vitamin D metabolites, namely vitamin D3, 25-hydroxy vitamin D3 (25OHD3), 1,25 dihydroxy vitamin D3 (1.25 (OH)2D3) and 24,25 dihydroxyvitamin D3 (24,25 (OH) 2D3). After extraction of plasma in isopropanol-toluene (25:75) the dried extract is reconstituted in hexane; this is applied to a SEP-PAK column, and stepwise elution carried out under gravity with 0.1 divided by isopropanol in hexane (neutral lipids), 1% isopropanol in hexane (D3), 3 divided by isopropanol in hexane (25OHD3), 3.125 divided by ethanol in dichloromethane (24,25 (OH) 2D3) and 50 divided ethanol in toluene (1, 25(OH) 2D3). Complete separation of these D3 metabolites is achieved by this process and up to 40 samples can be handled at one time. If combined with a suitable ligand binding assay, the system appears to be suitable for preparation of samples prior to the routine assay of vitamin D metabolites.

Asunto(s)

RESUMEN

A procedure for the biosynthesis and purification of 1 alpha, 24(R),25-trihydroxy[26,27-methyl-3H]-vitamin D3 (1,24,25-(OH)3[3H]D3) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was used for C-24-hydroxylation of 1 alpha, 25-dihydroxy[26,27-methyl-3H]-vitamin D3 (1,25-(OH)2[3H]D3), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24-25-(OH)3[3H]D3 with a specific radioactivity equivalent to the initial substrate (166 Ci/mmol). The authenticity of the generated metabolite was assessed by co-migration with synthetic 1,24,25-(OH)3D3 on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH)3D3 for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH)2D3 for hormone binding sites. Further analysis of 1,24,25-(OH)3D3-receptor interaction reveals a high-affinity, saturable binding with an apparent K4 of 2.2 x 10(-9) M. These studies demonstrate that although slightly less active than 1,25-(OH)2D3, 1,24,25-(OH)3D3 is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.

Asunto(s)

RESUMEN

A new metabolite of vitamin D was isolated from the blood plasma of chicks given large doses of vitamin D3. The isolation involved methanol-chloroform extraction and four column chromatographic steps. The metabolite was identified by high- and low-resolution mass spectroscopy, chemical derivatization, an comigration with authentic standard as 3 beta, 24(R)-dihydroxy-9,10-seco-5,7,10(19)-cholestatriene [24(R)-hydroxyvitamin D3]. No detectable 24-(R)-hydroxyvitamin D3 was recovered from 16 L of plasma from chicks receiving physiologic levels of vitamin D3.

Asunto(s)

RESUMEN

A metabolite of 25-hydroxyvitamin D3 has been isolated in pure form from incubation mixtures containing kidney homogenates of chicks given large doses of vitamin D3. The isolation involved methanol--chloroform extraction and six steps of column chromatography. The metabolite was identified as 25-hydroxy-24-oxovitamin D3 by means of ultraviolet absorption spectrometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions. Use of a sensitive in situ technique revealed that 25-hydroxy-24-oxovitamin D3 enhances intestinal calcium transport in rats approximately as effectively as 24,25-dihydroxyvitamin D3 does. In contrast, 25-hydroxy-24-oxovitamin D3 appeared to be less active than 24,25-dihydroxyvitamin D3 in chicks 24 h after intravenous injection.

Asunto(s)