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Metabolism of dihydrotachysterol and 5,6-trans-cholecalciferol in the chick and the rat.

ABSTRACT: Dihydrotachysterol and 5,6-trans-cholecalciferol are biologically active analogues of cholecalciferol (vitamin D) with a similarity in steric structure to 1,25-dihydroxycholecalciferol, the active form of the vitamin. The question arises as to the nature of the active form of these analogues. High specific radioactivity (14)C- and (3)H-labelled forms of dihydrotachysterol and 5,6-trans-cholecalciferol and its 25-hydroxy derivative were synthesized and their metabolism was studied in chicks and rats. All these steroids were very rapidly metabolized compared with cholecalciferol; 20% of the dihydrotachysterol dose was excreted in bile in the first 24h, about 50% as a carboxylic acid derivative. Although polar metabolites were detected in tissues, no 1-hydroxy form was observed. Larger proportions of the parent steroid and its 25-hydroxy metabolite were detected in tissues compared with cholecalciferol, but no single metabolite was detected at the intracellular site of action of cholecalciferol. It is suggested that analogues of cholecalciferol will be biologically active if they possess a hydroxyl group in the same steric position as that at C-1 of cholecalciferol, with the greatest activity shown by those that also have a C-25 hydroxyl group. The implication of these findings for the chemical features necessary for binding to receptor proteins are briefly discussed.

SUBMITTER: Lawson DE

PROVIDER: S-EPMC1168208 | biostudies-other | 1974 Jul

REPOSITORIES: biostudies-other

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Project description:5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.

Project description:1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.

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Metabolism of dihydrotachysterol and 5,6-trans-cholecalciferol in the chick and the rat.

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