Retinoic acid synthesis by cytosol from the alcohol dehydrogenase negative deermouse.

Cytosolic alcohol dehydrogenase in the deermouse is coded by a single genetic locus and a strain of the deermouse which is alcohol dehydrogenase negative exists. These two strains of the deermouse were used to extend insight into the role of cytosolic alcohol dehydrogenases in the conversion of retinol into retinoic acid. Retinoic acid synthesis from physiological concentrations of retinol (7.5 microM) with cytosol from the alcohol dehydrogenase negative deermouse was 13% (liver), 14% (kidney), 60% (testes), 78% (lung), and 100% (small intestinal mucosa) of that observed with cytosol from the positive deermouse. The rates in the negative strain ranged from 0.3 to 0.7 nmol/h/mg protein: sufficient to fulfill cellular needs for retinoic acid. Ten millimolar 4-methylpyrazole inhibited retinoic acid synthesis 92, 94, 26, and 30% in kidney, liver, lung, and testes of the positive deermouse, respectively, but only 50, 30, 0, and 0% in the same tissues from the negative deermouse. Ethanol (300 mM) did not inhibit retinoic acid synthesis in kidney cytosol from the negative strain. Therefore multiple cytosolic dehydrogenases, including alcohol dehydrogenases, contribute to retinol metabolism in vitro. The only enzyme(s) likely to be physiologically significant to retinoic acid synthesis in vivo, however, is the class of dehydrogenase, distinct from ethanol dehydrogenase, that is common to both the positive and the negative deermouse. This conclusion is supported by the data described above, the kinetics of retinoic acid synthesis and retinal reduction in kidney cytosol from the negative deermouse, and the very existence of the alcohol dehydrogenase negative deermouse. This work also shows that microsomes inhibit the cytosolic conversion of retinol into retinoic acid and that the synthesis of retinal, a retinoid that has no known function outside of the eye, does not reflect the ability or capacity of a sample to synthesize retinoic acid.

Retinoic acid regulates growth hormone gene expression.

Vitamin A is required for normal growth and development, and retinoic acid (RA) may be the active metabolite in this process. Recent evidence indicates that RA acts through binding to a nuclear receptor which belongs to the steroid/thyroid hormone receptor superfamily. The receptors seem to associate with hormone-response elements in the target genes resulting in the activation (or inhibition) of transcription. Although no interaction of RA-receptor complex with specific DNA sequences has yet been reported, the homology of the different receptors suggests their mechanisms of action are similar. We therefore examined whether the effects of RA on growth could be related to changes in the expression of the growth hormone gene which is known to be transcriptionally regulated by both thyroid and glucocorticoid hormones. Our results show that RA controls growth hormone production in pituitary GH1 cells and that its effect is synergistic with that caused by these hormones.

Biogenesis of retinoic acid from beta-carotene. Differences between the metabolism of beta-carotene and retinal.

The ability of beta-carotene to serve as precursor to retinoic acid was examined in vitro with cytosol prepared from rat tissues. The rate of retinoic acid synthesis from 10 microM beta-carotene ranged from 120 to 224 pmol/h/mg of protein with intestinal cytosol, and from 344 to 488 pmol/h/mg of protein with cytosols prepared from kidney, lung, testes, and liver. Retinol generated during beta-carotene metabolism was not the major substrate for retinoic acid synthesis. At low substrate concentrations (2.5 microM), the rates of retinoic acid synthesis in intestinal cytosol from beta-carotene or retinol were equivalent, and at higher concentrations (10 microM) the rates of retinoic acid synthesis from beta-carotene or retinol in intestine, testes, lung, and kidney were comparable. Thus, beta-carotene metabolism may be an important source of retinoic acid in retinoid target tissues, particularly in species such as humans that are capable of accumulating high concentrations of tissue carotenoids. Retinal, considered an initial retinoid product of beta-carotene metabolism, was not detected as a product of beta-carotene metabolism in vitro. A ratio of retinol and retinoic acid different from that observed during beta-carotene metabolism in vitro was observed with incubations of retinal under identical conditions. These data indicated that beta-carotene metabolism is not merely a simple process of producing retinal and releasing it into solution to be metabolized independently.

The biosynthesis of retinoic acid from retinol by rat tissues in vitro.

This report shows that a spectrum of vitamin A-dependent tissues can produce retinoic acid by synthesis in situ, indicates that cellular retinol and retinoic acid binding proteins are not obligatory to retinoic acid synthesis, and provides initial characterization of retinoic acid synthesis by rat tissues. Retinoic acid synthesis from retinol was detected in homogenates of rat testes, liver, lung, kidney, and small intestinal mucosa, but not spleen. Zinc did not stimulate the conversion of retinol into retinoic acid by liver homogenates. Retinoic acid synthesis was localized in cytosol of liver and kidney, where its rate of synthesis from retinol was fourfold (liver) and sevenfold (kidney) slower than from retinal. The synthesis of retinoic acid from retinol required NAD and was not supported by NADP. NADH (0.5 mM) reduced retinoic acid synthesis from retinol, supported by NAD (2 mM), by 50-70%, but was fivefold less potent in reducing retinoic acid synthesis from retinal. Dithiothreitol enhanced the conversion of retinol, but not retinal, into retinoic acid. EDTA inhibited the conversion of retinol into retinoic acid slightly (13%, liver; 29%, kidney). A high ethanol concentration (100 mM), relative to retinoid substrate (10 microM), inhibited retinoic acid synthesis from retinol (liver, 54%; kidney, 30%) and from retinal (30%, liver; 9%, kidney). 4'-(9-Acridinylamino)methansulfon-m-anisidine, an inhibitor of aldehyde oxidase, and disulfiram, a sulfhydryl-group crosslinking agent, were potent inhibitors of retinoic acid synthesis at 10 microM or less, and seemed equipotent in liver and kidney. 4-Methylpyrazole, an inhibitor of ethanol metabolism, also inhibited retinoic acid synthesis from retinol, but was less potent than the former two inhibitors, and affected liver to a greater extent than kidney, particularly with retinal as substrate.

Inhibition by chlorogenic acid of haematin-catalysed retinoic acid 5,6-epoxidation.

Chlorogenic acid (3-O-caffeoylquinic acid) inhibited haematin- and haemoglobin-catalysed retinoic acid 5,6-epoxidation. Some other phenol compounds (caffeic acid and 4-hydroxy-3-methoxybenzoic acid) also showed inhibitory effects on the haematin- and haemoglobin-catalysed epoxidation, but salicylic acid did not. Of the above compounds, caffeic acid and chlorogenic acid were potent inhibitors compared with the other two, suggesting that the o-hydroquinone moiety of chlorogenic acid and caffeic acid is essential to the inhibition of the epoxidation. Although caffeic acid inhibited retinoic acid 5,6-epoxidation requiring the consumption of O2, formation of retinoic acid radicals was not inhibited on the addition of caffeic acid to the incubation mixture. The above results suggest that caffeic acid does not inhibit the formation of retinoic acid radicals but does inhibit the step of conversion of retinoic acid radical into the 5,6-epoxide.

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line.

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.

Retinoic acid 5,6-epoxidation by hemoproteins.

Retinoic acid 5,6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy- and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5,6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity. Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70 degrees C) reduced its epoxidase activity with increasing temperature, whereas the activity of hematin was unaffected. Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN- (100 mM), inhibited the epoxidase activities but N3- (100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediate in the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.

Biosynthesis of retinoyl-beta-glucuronide, a biologically active metabolite of all-trans-retinoic acid.

All-trans-retinoic acid is metabolized in vitro to a biologically active metabolite, retinoyl-beta-glucuronide. We have studied the synthesis of this metabolite in vitro. The identity of the product was established by cochromatography on reverse-phase high-performance liquid chromatography, beta-D-glucuronidase hydrolysis, and fast atom bombardment and collisionally activated decomposition/fast atom bombardment mass spectrometry. The formation of retinoyl-beta-glucuronide is catalyzed by a UDP-glucuronosyltransferase with apparent Km's of 54.7 microM for all-trans-retinoic acid and 2.4 mM for UDP-glucuronic acid. The reaction requires enzyme, UDP-glucuronate, and no other factor. It is strongly inhibited by millimolar concentrations of coenzyme A. The specific activity of UDP-glucuronosyltransferase is greatest in the liver and least in the kidney of those tissues examined. The specific activity of the enzyme is increased by vitamin A deficiency. The increased specific activity observed in the vitamin A-deficient rat liver is uncharacteristic of retinoic acid inactivation enzymes; therefore, retinoyl-beta-glucuronide may be of functional importance.

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).

Metabolism of retinoic acid and retinol during differentiation of F9 embryonal carcinoma cells.

Retinol and retinoic acid dose-response curves were obtained for promotion of the differentiation of F9 murine embryonal carcinoma cells with an enzyme-linked immunoadsorbent assay for laminin, a product of differentiated F9 cells. Retinoic acid produced a half-maximum response at 1.3 nM and a maximum response at about 30 nM; retinol was 1/175th as potent. Maximum differentiation required 48 hr of continuous exposure to retinoic acid, whereas retinol required 72 hr of exposure. The half-time of retinoic acid conversion into polar metabolites was 3.5 hr; metabolism was accelerated by pretreating F9 cells with retinoic acid. An inhibitor of oxidative metabolism, ketoconazole, decreased the rate of retinoic acid metabolism and decreased the concentration of retinoic acid required to produce a half-maximum response. Unchanged retinoic acid was the sole compound isolated from nuclei of F9 cells incubated with retinoic acid. Retinol had a half-life approximately 5-fold longer than retinoic acid, attained greater cell concentrations, and was converted into retinoic acid by F9 cells. These data indicate that retinoic acid itself directs the differentiation of F9 cells and may mediate differentiation induced by retinol.

Metabolism in vivo of all-trans-retinoic acid. Biosynthesis of 13-cis-retinoic acid and all-trans- and 13-cis-retinoyl glucuronides in the intestinal mucosa of the rat.

The metabolites of all-trans-[3H]retinoic acid appearing in the intestines of bile duct-cannulated rats were compared to those of similarly treated intact rats. 2.4% of administered radioactivity was found in the small intestines of bile duct-cannulated rats 2 H after dose, while a much larger proportion of the dose (7.2%) was found in the intestines of the intact rats. All-trans- and 3-cis-retinoic acids predominate in the intestinal mucosa of bile duct-cannulated rats shortly after dosing. Retinoyl glucuronide was the major metabolite occurring as a mixture of the all-trans and 13-cis forms. Highly polar metabolites of retinoic acid appear in mucosa at all times in both intact and bile duct-cannulated rats demonstrating rapid metabolism of retinoic acid in intestine. The finding of a similar proportion of the 13-cis isomers in retinoic acids and retinoyl glucuronides suggests that injected all-trans-retinoic acid is isomerized in vivo probably prior to conjugated with glucuronic acid.

In vitro and in vivo metabolism of all-trans- and 13-cis-retinoic acid in hamsters. Identification of 13-cis-4-oxoretinoic acid.

Administration of either all-trans-[3H]- or 13-cis-[3H]retinoic acid to hamsters fed a normal diet results in the formation of a number of polar metabolites. At least one of these metabolites has been shown to be common to both isomers of retinoic acid and can be generated in a hamster liver 10,000 X g supernatant system using 13-cis-retinoic acid as substrate. It has been identified as 13-cis-4-oxoretinoic acid by mass spectral, ultraviolet absorption, and proton NMR characteristics, as well as by its co-migration with synthetic 13-cis-4-oxoretinoic acid in two different high pressure liquid chromatographic systems. In addition, its metabolic precursor, 13-cis-4-hydroxyretinoic acid, has been tentatively identified. These compounds are believed to be early metabolites in the elimination pathway of retinoic acid from the body.

Formation of vitamin A in a freshwater fish. Isolation of retinoic acid.

The intestines of freshly caught Saccobranchus fossilis (a freshwater fish that contains dehydroretinol) became free from carotenoids and from vitamin A when the fish were starved for about 20 days. When beta-carotene was administered to such fish, retinoic acid could be isolated from the intestines after approx. 4h. When lutein was administered to such fish, dehydroretinol and 3-hydroxyretinol could be isolated from the intestines after approx. 5h.