Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway.

In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.

Identification of 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one as a product of metabolism of 25-hydroxycholecalciferol by liver microsomes.

The ability of liver microsomes, sites of synthesis of 25-hydroxycholecalciferol, to further metabolize 25-hydroxycholecalciferol has been assessed. When liver microsomes were incubated with 25-hydroxycholecalciferol in the presence of cytosol, a metabolite was isolated that comigrated with 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3- one in three different chromatographic systems. The ultraviolet spectrum (220-350 nm) and mass spectrum of the purified metabolite were identical to that of synthetic 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. This study indicates that liver microsomes convert 25-hydroxycholecalciferol to 8 alpha,25-dihydroxy-9,10-seco-4,6,10(19)-cholestatrien-3-one. The significance of this metabolite, which has been shown previously by others to be produced by alveolar macrophages, has yet to be determined.

24-homologated 1,25-dihydroxyvitamin D3 compounds: separation of calcium and cell differentiation activities.

A series of 24-homologated 1,25-dihydroxyvitamin D3 compounds have been chemically synthesized and studied with regard to their activity in inducing differentiation of human promyelocyte HL-60 cells to monocytes and in calcium mobilizing activity in vitamin D deficient rats. Homologation of 1,25-dihydroxyvitamin D3 or its delta 22 analogue by one or two carbons increases by 10-fold and three-carbon homologation reduces by half the activity in causing differentiation of HL-60. On the other hand, homologation causes a substantial decrease in in vivo calcium mobilization activity. The addition of each carbon at the 24-position decreases binding to the HL-60 receptor or rat intestinal receptor by 5-10-fold so that binding affinity of the trihomo compound for the receptors is 130 times less that of 1,25-dihydroxyvitamin D3. Thus, binding affinity for the receptor cannot account for the preferential activity of the 24-homologated compounds in inducing cell differentiation.

Vitamin D metabolites and bioactive parathyroid hormone levels during Spacelab 2.

The purpose of this study was to determine whether plasma levels of the vitamin D hormone and parathyroid hormone (PTH), two potent activators of bone remodeling sites, were altered in four astronauts during the 8-day (d) Spacelab 2 mission (SL2). Increased circulating levels of either hormone could change calcium homeostasis and bone cell activity and, thus, contribute to bone loss in crewmembers in space. The vitamin D hormone was elevated in all astronauts at the end of the first inflight day but returned to normal by the seventh day. Biologically active PTH tended to be normal throughout the mission. Both hormones were within the normal range by the end of the 8-d flight of this SL2 crew. Plasma levels of 25OHD, 24,25(OH)2D, calcium, phosphorus, and albumin were essentially normal during the mission.

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs.

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.

A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase.

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3.

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin 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.

Inaction of saxitoxin-oximes on the sodium channel of frog skeletal muscle fibers.

Three oximes of saxitoxin, saxitoxin oxime, saxitoxin methyloxime, and saxitoxin carboxymethyloxime, were synthesized in which the oxime functions replaced the ketone function on C-12 of saxitoxin. On the voltage-clamped single frog muscle fibers these oximes were very weak or inactive in blocking the sodium channel. The results indicate that the hydrated ketone function in saxitoxin is essential for blockade of the sodium channel, probably through a hydrogen bonding mechanism with some receptor groups.

Studies on the role of 1,25-dihydroxyvitamin D in chick embryonic development.

Vitamin D-deficient laying hens were repleted with 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3. Egg production returned to normal for both groups of hens by the third week. Eggs from hens fed either 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3 contained 1,25-dihydroxy[26,27-3H]vitamin D3. Eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 contained substantial amounts of 25-hydroxy[26,27-3H]vitamin D3, while those from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained none. Plasma from 18-day embryos from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained little or no 1,25-dihydroxy[26,27-3H]vitamin D3, while that from 18-day embryos from hens given 25-hydroxy[26,27-3H]vitamin D3 had normal levels of 1,25-dihydroxy[26,27-3H]vitamin D3. No eggs from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 hatched, while eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 achieved a hatchability of 90%. It appears that embryos from hens maintained on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D are essentially vitamin D deficient.

Synthesis of coenzyme A ester of retinoic acid: intermediate in vitamin A metabolism.

Coenzyme A esters of all-trans- and 13-cis-retinoic acid were synthesized for use in studying vitamin A metabolism. The esters were obtained by two different synthetic methods starting from retinoic acids, which were converted to activated succinimidyl esters or anhydrides. These in turn were coupled with coenzyme A to form their respective thioesters. The retinoyl coenzyme A esters were purified by reverse-phase high performance liquid chromatography.

Purification of human serum vitamin D-binding protein by 25-hydroxyvitamin D3-Sepharose chromatography.

25-Hydroxyvitamin D3-Sepharose was prepared by coupling 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)-ether to thio-activated Sepharose CL-6B, forming a protease-resistant linkage between the sterol and the matrix. Vitamin D-binding protein from human plasma was obtained 85-92% pure after ligand affinity chromatography. Subsequent hydroxylapatite chromatography provided homogeneous protein. The purified vitamin D-binding protein was fully active in regard to 25-hydroxyvitamin D3 and actin binding capabilities.

Isolation and spectral characteristics of four toxins from the dinoflagellate Ptychodiscus brevis.

Four ichthyotoxins were isolated from crude toxic extracts of Ptychodiscus brevis using a combination of solvent partitioning, thin layer chromatography and reversed phase high pressure liquid chromatography. The toxins were analyzed by mass, infrared, 1H- and 13C-NMR spectroscopy and were found to constitute two structural families of two toxins each: brevetoxins 1 and 2 and brevetoxins 3 and 4. Comparison with literature data indicates that brevetoxins 1 and 2 are identical to the previously described and characterized 11-ring polyether toxins brevetoxins C and B, respectively. The other two compounds (brevetoxins 3 and 4) also represent a structural pair (with chloroacetone and alpha-methylene-propanal side chains, respectively) which has a different, but related, basic ring-structure.

Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein.

3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

1H nuclear magnetic resonance studies of the conformations of vitamin D compounds in various solvents.

Using proton NMR, the solution conformation of the A ring of vitamin D3 and its analogs has been studied by application of the Karplus relation to the observed coupling constants. The A-ring conformations of vitamins D3, D2, and 25-hydroxyvitamin D3 were found to be solvent dependent, with a clear preference for an equatorial hydroxyl group in polar solvents such as methanol and dimethyl sulfoxide. Conversion of the hydroxyl group to an acetate did not affect solution conformation appreciably, but the corresponding t-butyl-dimethylsilyl ether derivative of vitamin D3 showed a strong preference for the 3 beta-equatorial conformer. The A-ring conformation of the active hormone, 1,25-dihydroxyvitamin D3, which has two hydroxyl groups competing for the equatorial position, was found not to be solvent-dependent.

Actions of epimers of 12-(OH)-reduced saxitoxin and of 11-(OSO3)-saxitoxin on squid axon.

The actions of the 12 alpha-saxitoxinol, 12 beta-saxitoxinol and a C-12 ethylene thioketal derivative of saxitoxin, as well as those of 11 alpha-(OSO3)-saxitoxin, 11 beta-(OSO3)-saxitoxin and 11 alpha-(OH)-saxitoxin, have been examined on the isolated squid giant axon. Each of these analogues acted similarly to saxitoxin in blocking specifically the sodium channel. The relative potencies are: STX (1); 11 beta-(OSO3)-STX (gonyautoxin III) (0.42); 11 alpha-(OSO3)-STX (gonyautoxin II) (0.20); 11 alpha-(OH)-STX (0.10); 12 alpha-saxitoxinol (0.0021); 12 beta-saxitoxinol (0.0005). Thus, the presence of a bulky and negatively charged sulphate group on C-11 does not materially affect the biological activity of STX. Hydrogen bonding at the C-12 position is probably an important means of binding of STX to the membrane receptor site. The difference between the epimers of saxitoxinol suggests that the H in one of them may be geometrically better aligned than that in the other, with the hydrogen acceptor group in the receptor.

Distribution of paralytic toxins in California shellfish.

Samples of Saxidomus nuttali and Mytilus californianus collected during the 1981 dinoflagellate bloom at Bodega Bay, California, were analyzed for the presence of paralytic toxins. Neck tissue of S. nuttali contained saxitoxin (STX) and neoSTX (95% of the total toxicity), whereas the bodies contained neoSTX and a mixture of the gonyautoxins. In a sample of M. californianus the presence of neoSTX and the gonyautoxins was demonstrated, whereas a second sample, collected at a different site, contained almost exclusively neoSTX.

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).

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3.

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2-3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3. They metabolize 25-hydroxyvitamin D3 to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D3 and the other as its cis isomer. These two "metabolites" have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D3 on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.

In vitro inhibitor studies of vitamin D 25-hydroxylase in rat liver microsomes.

The ability of four vitamin D analogs to inhibit the liver microsomal vitamin D-25-hydroxylase was determined. 19-Hydroxy-10(S),19-dihydrovitamin D3,25-fluorovitamin D3, 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide and 25-aza-vitamin D3 were competitive inhibitors with apparent KI values of 44, 137, and 870 nM, and 6.4 microM, respectively. The values for the 19-hydroxy-10(S), 19-dihydrovitamin D3, 25-fluorovitamin D3, and 25-aza-vitamin D3 correspond well to other literature reports with respect to their relative in vivo inhibitory properties. 24-Oxovitamin D3 oxime also proved to be a potent inhibitor but a detailed analysis was prohibited by the lack of material. The 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide was also tested in vivo but had no antagonistic activity when provided at a 2000-fold excess over vitamin D3.