Nonaqueous Capillary Electrophoretic Separation of Analogs of (24R)-1,24-Dihydroxyvitamin D3 Derivative as Predicted by Quantum Chemical Calculations.

Nonaqueous capillary electrophoretic (NACE) separation was obtained of analogs of (24R)-1,24-dihydroxyvitamin D3 derivative (calcipotriol) as predicted by quantum chemical calculations supported by the density functional theory (DFT). Among the key electronic properties investigated, absolute values of the dipole polarizability and energy gap between HOMO and LUMO molecular orbitals of the analog molecules differ significantly for particular analogs, and there is a direct relationship with their electrophoretic migration time. These differences and relationships suggest that the structurally related analogs should be separable in the electrostatic field. Indeed, the robust, sensitive, and rapid NACE method was first developed for the identification and determination of the anticancer analog of calcipotriol (coded PRI-2205) and its process-related impurities (coded PRI-2201, PRI-2203, and PRI-2204) in organic and aqueous biological solutions. The direct relation between the calculated electronic properties of the analogs and the experimental electrophoretic migration time could be a promising prospect for theoretically predicting the electrophoretic separations.

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3).

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.

In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite.

In vivo metabolism of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in female dogs has been studied thoroughly, and its major bile metabolite identified. After single oral administration of 24,25-(OH)2 [6,19,19-3H]D3 the plasma concentrations of radioactive metabolites were monitored for 504 h, and the metabolites in the bile collected and analyzed. The concentration of 24,25-(OH)2D3 in plasma reached a maximum after 6 h and decayed in two distinct phases; a fast-phase with a half-life of 17 h, followed by a slow-phase with a 17-day half-life. The area under the concentration/time curve (AUC) was 78-84% (0-504 h). The only detectable metabolite in the plasma was 25-hydroxy-24-oxovitamin D3 whose AUC was less than 5%. At 504 h, about 50% of administered radioactivity has been excreted, of which about 90% was found in the feces, indicating most of the administered 24,25-(OH)2D3 to be excreted in bile. A major metabolite, which constituted 23% of the total bile radioactivity at 504 h, was found in the bile. This metabolite was efficiently deconjugated by beta-glucuronidase to afford an aglycone which was identified as 23S,25-dihydroxy-24-oxovitamin D3 (23S,25-(OH)2-24-oxo-D3), by co-chromatography on HPLC with synthetic standards. The glucuronide was isolated from the bile of dogs given large doses of 24,25-(OH)2D3, and the structure determined being 23-(beta-glucuronide) of 23S,25-(OH)2-24-oxo-D3, by analyzing its negative ion mass spectrum and the positive ion mass spectrum of its derivatives. Thus it was concluded that, in dogs, 24,25-(OH)2D3 is a long lasting vitamin D metabolite, is mainly excreted in bile when metabolized to 23S,25-(OH)2-24-oxo-D3 and is conjugated at 23-OH as glucuronide.

Naturally occurring 24,25-dihydroxyvitamin D3 is a mixture of both C-24R and C-24S epimers.

Tritium-labeled 24,25-dihydroxyvitamin D3 was prepared both in vitro, by using chick kidney homogenates, and in vivo in rats from [26,27-methyl-3H]25-hydroxyvitamin D3. These compounds were mixed with synthetic 24(R),25- and 24(S),25-dihydroxyvitamin D3, converted to the corresponding trimethylsilyl ether derivatives, and analyzed by a high-pressure liquid chromatography procedure that separates the derivatized isomers. The tritium-labeled 24,25-dihydroxyvitamin D3 derivatives were found to be a mixture of both the 24(R) and 24(S) epimers; the ratio was found to be 96.4:3.6 in chick kidney homogenates and 96.8:3.2 in the serum of rats under physiological conditions. In addition, nonradioactive 24,25-dihydroxyvitamin D3 isolated from the serum of rats given large doses of vitamin D3 was shown to be an 89.5:10.5 mixture of the 24(R) and 24(S) isomers. When 25-hydroxy-24-oxo-vitamin D3 was utilized as a substrate, it was found to be more selectively reduced to 24(S),25-dihydroxyvitamin D3 than 24(R),25-dihydroxyvitamin D3 by the renal enzyme. The 24(S),25-dihydroxyvitamin D3 has been identified by ultraviolet absorption spectrophotometry, cochromatography with an authentic standard, and mass spectrometry. The reduced metabolites of 25-hydroxy-24-oxo-vitamin D3 were a 1:50 mixture of the 24(R) and 24(S) epimers. There are two known metabolic pathways leading to 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; one is 24(R)-hydroxylation of 25-hydroxyvitamin D3 and the other is reduction of 25-hydroxy-24-oxo-vitamin D3. In contrast, 24(S),25-dihydroxyvitamin D3 is produced only by reduction of 25-hydroxy-24-oxo-vitamin D3 in the kidney. Therefore, naturally occurring 24,25-dihydroxyvitamin D3 is a mixture of the 24(R) and 24(S) isomers, and not just the 24(R) isomer as reported previously.

Specific estimation of 24,25-dihydroxyvitamin D in plasma by gas chromatography-mass spectrometry.

This paper describes a specific mass-fragmentographic method, involving a stable-isotope-labeled internal standard, for measurement of 24,25-dihydroxyvitamin D in human plasma. Vitamin D metabolites were rapidly extracted from plasma by using Sep-Pak C18 cartridges and separated into fractions on Sep-Pak SIL cartridges. The polar fraction, containing the dihydroxylated metabolites, was further purified by "high-performance" liquid chromatography on Zorbax SIL. The fraction containing 24,25-dihydroxyvitamin D was collected, evaporated, and converted to the 24:25-cyclic n-butyl boronate-3-trimethylsilyl ether derivative before analysis by gas chromatography-mass spectrometry. The intensity of the mass fragment (m/z 449, m/z 455 for the hexadeuterated internal standard) arising from the loss of one of the angular methyls and the 3-silanol group [( M-90-15]+) was monitored. The minimum limit of detection for this method is about 0.1 microgram/L. Inter- and intra-assay reproducibility was acceptable, and analytical recovery of added 24,25-dihydroxyvitamin D3 over the concentration range 1.0 to 5.0 micrograms/L was quantitative. Concentrations of 24,25-dihydroxyvitamin D3 in plasma of 21 apparently healthy volunteers were between 0.55 and 5.39 micrograms/L, higher values being obtained after prolonged exposure to the sun. No 24,25-dihydroxyvitamin D2 could be detected in any plasma sample examined.

24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.

We describe a precise, specific method for measuring 24,25-dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by "high-performance" liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.

Natural 25,26-dihydroxyvitamin D3 is an epimeric mixture.

Radiolabeled 25,26-dihydroxyvitamin D3 was prepared in vitro by using chicken kidney homogenates and in vivo in rats from [23,24-3H]-25-hydroxyvitamin D3. These compounds were mixed with synthetic (25S)- and (25R)-25,26-dihydroxyvitamin D3, converted to the corresponding (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl esters, and subjected to high-performance liquid chromatography that separates the derivatized epimers. The radiolabeled 25,26-dihydroxyvitamin D3 derivatives were a 1:1 mixture of the 25S and 25R isomers. Similarly unlabeled 25,26-dihydroxyvitamin D3 isolated from the plasma of rats given large amounts of vitamin D3 was shown to be a 1:1 mixture of the S and R isomers. Therefore, naturally occurring 25,26-dihydroxyvitamin D3 is a mixture of the 25R and 25S isomers and not just the S isomer reported previously.

Chromatographic separation of 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone using a cyano-bonded phase packing.

A high-performance liquid chromatographic system is described for the baseline resolution of 25-hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, the three principal circulating metabolites of vitamin D3 in the vitamin D-replete animal. The system is based upon a cyano-bonded phase packing and the solvent hexane--isopropanol--methanol (94:5:1). Of particular interest is the strong retention of carbonyl-containing vitamin D metabolites. The new system can be used for unequivocal analysis of vitamin D metabolites in plasma samples from clinical and animal studies and in the separation and identification of renal metabolites generated in vitro.

23,25-Dihydroxy-24-oxovitamin D3: a metabolite of vitamin D3 made in the kidney.

Kidney homogenates of rats produced a new metabolite of 25-hydroxyvitamin D3 which has been isolated in pure form after five column chromatographic steps. It was identified as 23,25-dihydroxy-24-oxovitamin D3 by means of ultraviolet and infrared absorption spectrophotometry, mass spectrometry, and proton nuclear magnetic resonance spectrometry. The stereochemistry at the C-23 position is as yet unknown. 25-Hydroxy-24-oxovitamin D3, which also has been isolated in pure form from this system, was found to be the precursor of the new metabolite in vitro. The production of the new metabolite was induced by two different methods: (a) perfusion of the kidneys with 1,25-dihydroxyvitamin D3 contained in the perfusate and (b) injection of 1,25-dihydroxyvitamin D3 in the intact animal. 23,25-Dihydroxy-24-oxovitamin D3 was not biologically active in an assay for intestinal calcium transport and bone calcium mobilization in the vitamin D deficient chick at a dose level of 5.3 nmol. A metabolic pathway is proposed to describe the results; it leads from 25-hydroxyvitamin D3 leads to 24(R),25-dihydroxyvitamin D3 leads to 25-hydroxy-24-oxovitamin D3 leads to 23,25-dihydroxy-24-oxovitamin D3.

Presence of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 24-hydroxylase in vitamin D target cells of rat yolk sac.

In the pregnant rat, the yolk sac, which possesses true placental functions, is a vitamin D target organ. We tested its ability to hydroxylate 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). 24,25-Dihydroxy- and 1,24,25-trihydroxyvitamin D3 were produced by rat yolk sac homogenates incubated with tritiated 25-OHD3 and 1,25-(OH)2D3. Rat yolk sac homogenates also formed small amounts of 25,26-dihydroxyvitamin D3. These newly synthesized metabolites were isolated and identified by Sephadex LH-20 chromatography, high performance liquid chromatography, and periodate cleavage. Yolk sac 25-OHD3- and 1,25-(OH)2D3-24-hydroxylases were present in mitochondria and were of a mixed function oxidase nature. They were detected in the yolk sac as early as day 12 in the embryonic period and until the end of gestation. No hydroxylation occurred in maternal liver, amnion, fetal brain, or skin homogenates. Both 24-hydroxylases were detected in pure isolated rat yolk sac endodermal cells. This may be of physiological importance, since they are the 1,25-(OH)2D3 target cells in the yolk sac. Injection of 1,25-(OH)2[3H]D3 into rat yolk sac vitelline veins strongly suggested that the yolk sac vitelline veins strongly suggested that the yolk sac produced 1,24,25-(OH)3D3 in vivo. We conclude that the yolk sac and more precisely its endodermal cells may help to control vitamin D metabolism within the fetoplacental unit.

A simple method for the isolation of vitamin D metabolites from plasma extracts.

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.

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3.

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.

Purification of plasma vitamin D metabolites for radioimmunoassay.

The ability of four solvent systems to extract tritiated 25-hydroxy vitamin D3, 24,25-dihydroxy vitamin D3, 25,26-dihydroxy vitamin D3 and 1 alpha, 25-dihydroxy vitamin D3 from plasma was compared. Diethyl ether gave the highest yield of metabolites and lowest yield of "lipid" materials, the dihydroxylated metabolites were more readily extracted than 25-hydroxy vitamin D3. Following extraction with ether the vitamin D metabolites could be separated, without prior purification, on a 6.2 mm X 250 mm Zorbax-Sil high pressure liquid chromatography (HPLC) column, a simplification of previous methods. Plasma 1 alpha, 25-dihydroxy vitamin D levels measured after purification by this method were not significantly different from those obtained by an established, more complex method.

A specific competitive protein binding assay for serum 24,25-dihydroxyvitamin D in normal children and patients with nephrotic syndrome.

A specific competitive protein binding assay for 24,25-dihydroxyvitamin D by Sephadex LH-20 column chromatography, followed by high pressure liquid chromatography with normal rat kidney cytosol as the binding protein, was developed. The mean concentrations of serum 24,25-dihydroxyvitamin D of the cord, in newborn infants and in infants under 12 months of age were 0.90 +/- 0.40 (S.D.) ng/ml, 0.52 +/- 0.21 (S.D.) ng/ml and 1.20 +/- 0.38 (S.D.) ng/ml, respectively. These concentrations were significantly lower than those in children aged 1-15 years (1.96 +/- 0.83 (S.D.) ng/ml). The serum levels in the acute stage of the nephrotic syndrome were significantly reduced, and they increased in remission. These results show that patients wioth nephrotic syndrome have low levels of serum 24,25-dihydroxyvitamin D. This is probably due to its loss in the urine.

A specific 1,25-dihydroxyvitamin D3 binding macromolecule in chicken bone.

Cytosol prepared from homogenates of bone from vitamin D3-deficient chicks contains a 3.7 S macromolecule having high affinity and low capacity for 1,25-dihydroxyvitamin D3. Employing 1,25-dihydroxy-[26,27-3H]vitamin D3 (160 Ci/mmol) an apparent Kd has been calculated to be 7.6 x 10(-11) M while the association and dissociation rate constants for the binding process at 25 degrees C were determined to be 9.5 x 10(8) M-1 min-1 and 2.3 x 10(-2) min-1, respectively. A 5.5 S molecule is also present which binds 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 but appears to prefer 25-hydroxyvitamin D3 and is increased by the addition of chick serum to cytosol. The 3.7 S material is neither a serum contaminant nor a component of the 5.5 S molecular species and is likely of intracellular origin. Under low salt conditions the 3.7 S macromolecule migrates to 4.3 S and 5.5 S regions on sucrose gradients suggesting aggregation of the protein. Several vitamin D3 metabolites are capable of specifically binding to the 3.7 S macromolecule. The relative order of potency for several analogs causing displacement of specifically bound 1,25-dihydroxy-[26,27-3H]vitamin D3 is: 1,25-dihydroxyvitamin D3 greater than 1 alpha-hydroxyvitamin D3 greater than or equal to 25-hydroxyvitamin D3 greater 24(R),25-dihydroxyvitamin D3. It is concluded that chick bone cytosol contains a macromolecule of high affinity and low capacity for 1,25-dihydroxyvitamin D3 which may function as a receptor for some physiological events in bone.