Synthesis of 25-hydroxy[26,27-3h]vitamin D3 with high specific activity.

A synthesis of radiochemically pure 25-hydroxy[26,27-³H]vitamin D₃ with a specific activity of 160 Ci/mmol is reported. The structure and biological activity of the radiolabeled compound was verified by comigration on high-pressure liquid chromatography with synthetic 25-hydroxyvitamin D₃ to constant specific activity, and by conversion in vitro to 1α,25-dihydroxy[26,27-³H]vitamin D₃ with the chick kidney 1α-hydroxylase.     

Syntheses of 24R,25-dihydroxy-[6,19,19-3H]vitamin D3 and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3

24R,25-Dihydroxy-[6,19,19-3H]vitamin D3 with a specific activity of 54 Ci/mmol and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3 with 2.6 deuterium atoms/mol were synthesized in four steps starting from 24R,25-Dihydroxyvitamin D3 via its sulfur dioxide adduct.     

Synthesis of 25-hydroxy-[6,19,19'-2H3]vitamin D3 and 1 alpha,25-dihydroxy-[6,19,19'-2H3]vitamin D3.

Synthesis of polydeuterated analogs of 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxy vitamin D3 are described. These analogs, containing stable isotope atoms at metabolically stable positions, are potentially useful in studies involving catabolism of hydroxylated metabolites of vitamin D3.     

Synthesis of 25-hydroxy-[26,27-3H]vitamin D2, 1,25-dihydroxy-[26,27-3H]vitamin D2 and their (24R)-epimers

Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.     

Synthesis of 1alpha-hydroxy [6-3H]vitamin D3 and its metabolism to 1alpha, 25-dihydroxy [6-3H]vitamin D3 in the rat.

1alpha-Hydroxy [6-3H]vitamin D3 has been synthesized with a specific activity of 4 Ci/mmol, and its metabolism in rats has been studied. It is rapidly converted to 1alpha,25-dihydroxy [6-3H]vitamin D3 in vivo. Following an intravenous or oral dose, a maximal concentration of 1alpha,25-dihydroxy [6-3H]vitamin D3 is found 2 and 4 hours, respectively, before the maximal intestinal calcium transport response is observed. Similarly, 1alpha,25-dihydroxy[6-3H]vitamin D3 accumulation in bone precedes the bone calcium mobilization response. It appears, therefore, that the biological activity of 1alpha-hydroxyvitamin D3 is largely, if not exclusively, due to its conversion to 1alpha,25-dihydroxy[6-3H]vitamin D3 1alpha-Hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appear in intestine equally well after an oral or an intravenous dose of 1alpha-hydroxy[6-3H]vitamin D3. However, much less of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 appears in bone and blood after an oral than after an intravenous dose. A much reduced bone calcium mobilization response is also noted following an oral dose as compared to an intravenous dose of 1alpha-hydroxyvitamin D3, suggesting that oral 1alpha-hydroxyvitamin D3 is not utilized as well as intravenously administered material.     

Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester

A number of hexadeuterated brassinosteroids (BS) containing a hydroxy group at C-22 or a 22R,23R-diol function were prepared starting from 23,24-bisnorcholenic acid methyl ester for biosynthetic studies. Synthesis of the cyclic part was accomplished via the initial hydroboration-oxidation of Delta(5)-double bond. The key step in the synthesis of the side chain involved addition of (2S)-[3,4-(2)H(6)]2,3-dimethylbutylphenyl sulfone to the corresponding C-22 aldehydes.     

Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate]

It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.     

Serum concentrations of 25-hydroxy vitamin D in Europeans and Asians after oral vitamin D3.

Serum concentrations of 25-hydroxy vitamin D (25-OHD3) were measured in seven Asians of Indian extraction and eight Europeans before and at intervals after taking 1 mg vitamin D3 by mouth. In all subjects the concentrations rose in the 24 hours after ingestion. There was little change over the next nine days in the concentrations in the Europeans but those in the Asians continued to rise until about day 10. Subsequent rates of fall in 25-OHD3 were similar in the two groups. Our observations suggest that the low serum concentrations of 25-OHD3 found in Asians are not caused by either impaired intestinal absorption of vitamin D or rapid clearance of 25-ODH3 from the plasma.     

Synthesis of 24S and 24R-hydroxy-[24-3H]vitamin D3 and their metabolism in rachitic rats.

An epimeric mixture of 24-hydroxy-[24-₃H]vitamin D₃ was synthesized by the reduction of 24-ketovitamin D₃ by sodium borotritide. The epimeric mixture was converted to the trimethylsilylether derivatives and subjected to high-pressure liquid chromatography using silica gel columns to separate the 24-hydroxy-[24-₃H]vitamin D₃ isomers. The 24R-hydroxy-[24-₃H] vitamin D₃ induced calcification in rachitic rats while the 24S-hydroxy-[24-₃H] vitamin D₃ had little or no such activity. As both isomers of 24-hydroxy-vitamin D₃ are metabolized to 24,25-dihydroxyvitamin D³, it appears that the 24-hydroxyvitamin D₃-25-hydroxylase does not discriminate between the isomers. Only the R-isomer of 24-hydroxyvitamin D₃ is metabolized to 1,24-dihydroxyvitamin D₃, although only trace amounts of this compound were found 2 days after the administration of 24-hydroxyvitamin D₃. The striking difference in the metabolism of the isomers is the high selectivity of the 1-hydroxylase for R-isomer. It is suggested that the high specificity of biological activity for the R-isomer of 24-hydroxyvitamin D₃ is because of the specificity of the 1-hydroxylation of 24,25-dihydroxyvitamin D₃ for the R configuration.     

Assay of 25-hydroxy vitamin D3-1 alpha-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry

An assay of 1 alpha-hydroxylation of 25-hydroxy vitamin D3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D3 with the mitochondrial fraction. The incubation extracts were purified by high-performance liquid chromatography. After formation of the trimethylsilyl derivative, the product was quantitated by mass fragmentography using the ion at m/z 452 and m/z 455. With the use of this assay it was found that formation of 1,25-dihydroxy vitamin D3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 microM of 25-hydroxy vitamin D3. The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein X minute.     

Comparative studies on the contents of vitamin D3, 25-hydroxy vitamin D3 and 7-dehydrocholesterol in fish liver

• 1.1. The contents of vitamin D₃, 25-hydroxyvitamin D₃ (25-OH-D₃) and 7-dehydrocholesterol (7-DHC) in 22 kinds of fish liver samples were determined by a high-performance liquid chromatographic (HPLC) method.
• 2.2. Vitamin D₃ was detected in all fish liver samples, but its contents varied from 84 to 264,000 ng/g wet tissue. The liver of fish belonging to Carangidae and Scombridae contained large amounts of the vitamin and therefore we deduced that vitamin D₃ levels in liver might have some relations with taxonomical positions of fishes.
• 3.3. 25-OH-D₃ was detected in 7 out of 22 kinds of fish liver samples, while 7-DHC was in 14 out of 22. The contents of the two sterols were generally much lower than those of vitamin D₃ and there was no special relationship between the contents of the sterols and the vitamin.

     

Synthesis of [3 beta-3H]-3-epivitamin D3 and its metabolism in the rat

3-Epivitamin D3, the 3 alpha epimer of vitamin D3, was synthesized, and its biological activity in the rat was evaluated. It was found to be approximately 4 times less active on a weight basis than vitamin D3 with respect to intestinal calcium transport, bone calcium mobilization, and calcification score as determined by the line-test assay. Tritiated 3-epivitamin D3 was prepared, and its metabolism in the rat was compared with that of vitamin D3 to investigate the reasons for this diminished activity. 3-Epivitamin D3 was converted to two polar metabolites, for which the chromatographic properties and the origin of biosynthesis (in the liver and kidney, respectively) correspond to 25-hydroxy-3-epivitamin D3 and 1 alpha,25-dihydroxy-3-epivitamin D3. The fact that the concentration of 1 alpha,25-dihydroxy-3-epivitamin D3 in the intestine is half that of 1 alpha,25-dihydroxyvitamin D3 may be one explanation for the reduced biological activity of this epimer.     

Chemical synthesis of (24R)-24,25-dihydroxy[26,27-3H]vitamin D3 of high specific activity

Chemical synthesis of (24R)-24,25-dihydroxy-[26,27-3H]vitamin D3, and its 24-epimer has been devised that allows introduction of 3H at the terminal step of the synthesis. The epimeric mixture is derivatized as the tris(trimethylsilyl) ethers and resolved by high-performance liquid chromatography. The product has a specific activity of 178 Ci/mmol and is fully active in binding to the rat plasma vitamin D binding protein and in the elevation of serum calcium levels of vitamin D deficient rats. The synthesis begins with the readily available 3 beta-hydroxy-5-cholenic acid methyl ester and involves a Pummerer rearrangement, introduction of the delta 7, irradiation, and isolation of the 26,27-dinor-25-carboxylic acid methyl ester of vitamin D3. This compound is then treated with a Grignard reagent containing 3H (80 +/- 10 Ci/mmol).     

Conjugated forms of [3H] alpha, 25-dihydroxyvitamin D3 in rat bile

When small doses of [3H]D3, [3H]25-OHD3 and [3H]alpha, 25-diOHD3 were administered intravenously to rats 6.3 +/- 1.1% (means +/- SEM, n = 4), 9.7 +/- 0.9% (n = 6) and 12.8 +/- 2.6% (n = 8), respectively, of the administered radioactivity was excreted in bile. The radioactive biliary conjugated metabolites were analysed by ion exchange chromatography: in the case of all 3 substrates about 30% of metabolites were found to be cationic on the basis of their being retained on sulphopropyl-Sephadex G-25 (H+-form) when applied in 70% methanol. The balance of the metabolites were neutral and anionic and were analysed on TEAP-Lipidex: in the case of 1 alpha, 25-diOHD3 the following metabolite classes were detected (on the basis of co-elution with authentic standards) (in order of quantitative importance): taurine conjugates, neutral metabolites, monosulphates, glucuronides, carboxylic acids, glycine conjugates and disulphates. Alkaline hydrolysis of the taurine and glycine conjugates yielded products 60% of which now chromatographed as carboxylic acids. Hydrolysis of the glucuronide and monosulphate fractions indicated significant levels of mixed conjugation yielding some products which now chromatographed as glycine and taurine conjugates, respectively. The nature of the cationic conjugates was not elucidated but they had the following properties: they could be hydrolysed by alkali to yield non-cationic radioactive metabolites (these released metabolites were heterogeneous as judged by TEAP-lipidex chromatography); they were partially hydrolysed to non-cationic forms by beta-glucuronidase; and on reverse-phase HPLC they had an elution profile that was significantly different to that of histidyl-, ornithyl- or lysyl-calcitroic acid.     

Vitamin D supplements and 25-hydroxy vitamin D concentrations in the elderly.

Serial 25-hydroxy vitamin D (25-OHD) concentrations were measured in long-stay geriatric patients treated with vitamin D. Comparison between a treatment and a control group showed that a daily dose of 500 IU vitamin D produced a significant increase in 25-OHD levels by two months. The supplement had a striking effect when the initial 25-OHD level was low and very little effect when it was high. 25-OHD levels in subjects on 2000 IU vitamin D daily were only marginally higher than those in subjects on 500 IU. A dose of 500 IU vitamin D daily should therefore produce adequate blood 25-OHD concentrations in most old people, and probably prevent most cases of osteomalacia in the elderly--though a large-scale study is needed to confirm this.     

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.     

Synthesis of a [1,2-3H]-labeled pregnanolone.

A method is described for the synthesis and purification of 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. [1,2-3H]progesterone (55 Ci/mmol) was incubated with a homogenate of rat brain tissue. The product was purified by Sephadex chromatography and thin-layer chromatography. The identity and purity of the product were established by successive recrystallizations and high-performance liquid chromatography. A 34% portion of the starting material was converted to 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. The final radiopurity of 3 alpha-hydroxy-5 alpha-pregnan-20-one obtained from four independent preparations was 94% to 99%.