Activation of retinoic acid by coenzyme A for the formation of ethyl retinoate.

All-trans-retinoic acid is metabolized to a less polar metabolite in rat liver microsomes. This metabolite was proven to be ethyl retinoate by cochromatography on high-performance liquid chromatography, base hydrolysis to all-trans-retinoic acid, and gas chromatography/mass spectral analysis. The formation of ethyl retinoate is a specific enzymatic process; the apparent Km for all-trans-retinoic acid is 9.8 microM. The production of ethyl retinoate is greatly stimulated by the addition of coenzyme A, suggesting the formation of a retinoic acid-coenzyme A intermediate (retinoyl-coenzyme A).     

Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids.

An unstable intermediate was detected in the transformation of arachidonic acid into 5,6-dihydroxyicosatetraenoic acids (two isomers) and 5,12-dihydroxyicosatetraenoic acids (three isomers) in rabbit peritoneal (glycogen-induced) polymorphonuclear leukocytes. Addition of 10 vol of methanol, ethanol, or ethylene glycol to short-term incubations (30-45 sec) led to the formation of the corresponding 12-O-alkyl derivatives of the 5,12-dihydroxy acids. The time for 50% disappearance of the intermediate (37 degrees C), as measured by formation of 5-hydroxy-12-O-methylicosatetraenoic acids (two isomers) upon trapping with methanol, was about 1 min in live cell preparations (pH 7.4) and about 4 min in water/acetone (1:1), pH 7.4. At pH 6.0 or below, the hydrolysis of the intermediate was too rapid to be measured by the method employed. Data supporting both enzymatic and nonenzymatic hydrolysis of the intermediate into dihydroxy acids are presented. Incubation of the cells with arachidonic acid under an atmosphere of 18O2 led to incorporation of 18O into the 5,6-dihydroxy acids and 5,12-dihydroxy acids only at C-5. The 5-hydroxyicosatetraenoic acid was also labeled at C-5. Considering the chemical reactivity of the intermediate and the structures of the derivatives obtained, it is proposed that the intermediate is 5(6)-oxido-7,9,11,14-icosatetraenoic acid.     

Identification of 5,8-oxyretinoic acid isolated from small intestine of vitamin A-deficient rats dosed with retinoic acid.

A retinoid was isolated by a multistep procedure from the small intestines of vitamin A-deficient rats given a single dose of retinoic acid. The compound, designated 8II, was pure, as demonstrated by four high-pressure liquid chromatographic procedures. It was positively identified as 5,8-oxyretinoic acid by ultraviolet spectrophotometry, mass spectrometry, and spectral and chromatographic comparison to known compounds. It is probable that 5,8-oxyretinoic acid was produced from 5,6-epoxyretinoic acid under the acidic conditions used in the isolation. It is highly probable, therefore, that the natural product is 5,6-epoxyretinoic acid.     

Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor.

High-level expression of the full-length human retinoic acid receptor (RAR) alpha and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RAR alpha protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kd of 2.1 x 10(-10) M.     

Abnormal vitamin D metabolism in cirrhosis.

Vitamin D metabolism was investigated in 10 patients with cirrhosis. Mean plasma 25 hydroxycholecalciferol (25 OHD) centration in alcoholic cirrhosis was lower than in controls but the difference was not significant. In three patients restudied after the summer, plasma 25 OHD had risen. In contrast to the finding in normal subjects, the half-life of intravenously administered 3H cholecalciferol was short in cirrhotics and showed no correlation with plasma 25 OHD. Furthermore, the appearance of 3H 25 OHD from 3H cholecalciferol was reduced compared to the control group four hours after injection. Increased rate of metabolism of cholecalciferol and deficient production of 25 ohd contribute to vitamin D deficiency in liver disease.