Recent advances in the in vivo and in vitro metabolism of retinoic acid.

Retinoic acid, a natural metabolite of retinol, has previously been shown to be capable of supporting growth and maintaining proper differentiation in epithelial tissues. Recently, investigation into the in vivo and in vitro metabolism of retinoic acid in hamsters, using both tracheal organ culture and subcellular preparations derived from intestinal mucosa, liver, and testis, has revealed the production of several metabolites more polar than the parent compound. Two of the early products of this metabolic pathway have been identified as 4-hydroxy- and 4-keto-retinoic acid. The formation of these metabolites is maximal in vitamin A-deficient hamsters that have been pretreated with retinoic acid and in vitamin A-normal animals. This fact, together with the decreased biological activity of the two compounds relative to retinoic acid in a tracheal organ culture assay, suggested that oxidative attack at carbon-four of the cyclohexenyl ring may be the first step in the elimination of retinoic acid from tissues. In addition, observations both in vivo and in vitro indicate that all-trans- and 13-cis-retinoic acid at low concentrations may be sharing a common metabolic pathway that includes an isomer of 4-keto-retinoic acid.     

In vitro metabolism and biological activity of all-trans-retinoic acid and its metabolites in hamster trachea

The in vitro metabolism of all-trans-[11,12-3h]retinoic acid to several more polar compounds has been demonstrated in a hamster tracheal organ culture system. The production of these metabolites is dependent on the presence of tissue. The physiological significance of these compounds is shown by the cochromatography of several of the in vitro formed metabolites synthesized from [carboxy-14C]retinoic acid with metabolites isolated from the intestine and urine of hamsters previously injected with 0.1 to 1.5 microgram of [3H]retinoic acid. One of the metabolites shows about one-tenth the biological activity of all-trans-retinoic acid when tested in a hamster tracheal organ culture assay. This biologically active metabolite is converted by the hamster trachea in vitro to a biologically inactive metabolite.     

Metabolism of all-trans-retinol and all-trans-retinoic acid in rabbit tracheal epithelial cells in culture

As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is inhibited by retinoic acid and retinol; retinoic acid is about 100 times more effective than retinol. To examine the metabolism of these agents in this in vitro model system, RTE cells were grown in the presence of all-trans-[3H]retinol or all-trans-[3H]retinoic acid and their metabolites analyzed by high-pressure liquid chromatography. RTE cells converted most of the retinol to retinyl esters, predominantly retinyl palmitate. A small fraction was metabolized to polar compounds, one of which coeluted with retinoic acid. After methylation this compound eluted as 13-cis-methyl retinoate and as all-trans-methyl retinoate. Conversion to 13-cis-retinol was also observed. All-trans-retinoic acid was rapidly taken up by RTE cells and converted to more polar (peak 1) and less polar (peak 3) metabolites. A proportion of all-trans-[3H]retinoic acid was metabolized to 13-cis-[3H]retinoic acid. These metabolic reactions appeared to be constitutive and were not induced by pretreatment with retinoic acid. The peak 1 metabolites were rapidly secreted into the medium whereas the peak 3 metabolites were retained by the cells and were not detected in the medium. Alkaline hydrolysis of the metabolites in peak 3 yielded retinoic acid, indicating the formation of retinoyl derivatives. Our results establish that RTE cells can convert all-trans-retinol to 13-cis-retinol and retinoic acid. RTE can metabolize all-trans-retinoic acid to 13-cis-retinoic acid and to an unidentified ester of retinoic acid.     

Metabolism and transactivation activity of 13,14-dihydroretinoic acid

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230-50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.     

Isolation by high pressure liquid chromatography and characterization of benzo(a)pyrene-4'5-epoxide as a metobolite of benzoapyrene.

A high-pressure liquid chromatography (HPLC) system is described that separates at least nine benzo(a)pyrene metabolites including an epoxide. The epoxide metabolite has been isolated and characterized as benzo(a)pyrene-4,5-epoxide by comparison of its HPLC retention times, ultraviolet and mass spectral analysis with synthetic benzo(a)pyrene-4,5-epoxide and its conversion by liver microsomes to benzo(a)pyrene-4,5-dihydrodiol.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.     

Metabolism of retinoids by embryonal carcinoma cells

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

The biological activity of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid in the rat vaginal smear assay

The biological activity of a series of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid has been evaluated in the vaginal smear assay carried out in vitamin A-deficient rats. These analogs were designed to probe the role of the 13-cis isomer in the actions of the parent all-trans-retinoic acid by blocking the interconversion of these two compounds. Although relatively less active, the potency of some of the cyclopropyl analogs suggests that 13-cis-retinoic acid is a fully active metabolite of all-trans-retinoic acid. Since 13-cis-retinoic acid represents a small percentage of the retinoic acid metabolites, the physiological significance of this activity is still unclear. Possible reasons for the reduced activity of the cyclopropyl analogs, as well as an aromatic analog of retinoic acid, are discussed.     

Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.     

Determination of uric acid in biological fluids by high-pressure liquid chromatography

A high-pressure liquid chromatographic assay for uric acid in biological fluids has been developed. Blood uric acid can be analyzed in as little as 20 μl of plasma. The mean and range of plasma uric acid concentrations in healthy adults determined by high-pressure liquid chromatography were similar to these obtained by enzymatic analysis. One of the advantages of the present method is that naturally occurring metabolites in biological fluids or drugs do not interfere with the analysis. Data are presented for blood and urine specimens obtained from mice fed a known uricase inhibitor, potassium oxonate. Comparisons are made between the present method and methods previously employed for uric acid determination.     

Mercapturic acid conjugates of 4-hydroxy-2-nonenal and 4-oxo-2-nonenal metabolites are in vivo markers of oxidative stress

Oxidative stress-induced lipid peroxidation leads to the formation of cytotoxic and genotoxic 2-alkenals, such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). Lipid-derived reactive aldehydes are subject to phase-2 metabolism and are predominantly found as mercapturic acid (MA) conjugates in urine. This study shows evidence for the in vivo formation of ONE and its phase-1 metabolites, 4-oxo-2-nonen-1-ol (ONO) and 4-oxo-2-nonenoic acid (ONA). We have detected the MA conjugates of HNE, 1,4-dihydroxy-2-nonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), the lactone of HNA, ONE, ONO, and ONA in rat urine by liquid chromatography-tandem mass spectrometry comparison with synthetic standards prepared in our laboratory. CCl(4) treatment of rats, a widely accepted animal model of acute oxidative stress, resulted in a significant increase in the urinary levels of DHN-MA, HNA-MA lactone, ONE-MA, and ONA-MA. Our data suggest that conjugates of HNE and ONE metabolites have value as markers of in vivo oxidative stress and lipid peroxidation.     

Oxygenation of arachidonic acid by hepatic microsomes of the rabbit. Mechanism of biosynthesis of two vicinal dihydroxyeicosatrienoic acids

[1-14C] Arachidonic (eicosatetraenoic) acid was incubated at 37 degrees C for 15 min with rabbit liver microsomes fortified with NADPH (1 mM). The products were purified by high-pressure liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry. Based on polarity on reversed phase HPLC, the metabolites could be divided into three groups. The major metabolites of lowest polarity were 19- and 20-hydroxyarachidonic acid and 19-oxoarachidonic acid. The major metabolites of medium polarity were two diols, 14,15-dihydroxy-5,-8,11-eicosatrienoic acid and 11,12-dihydroxy-5,8,14-eicosatrienoic acid. Microsomal incubation under atmospheric isotopic oxygen led to incorporation of only one 18O molecule in each diol, indicating that the diols could originate from breakdown of 14(15)-oxido-5,8,11-eicosatrienoic acid and 11(12)-oxido-5,8,14-eicosatrienoic acid, respectively. Major metabolites in the most polar group were 14,15,19- and 14,15,20-trihydroxy-5,8,11-eicosatrienoic acid. 11,12,19- and 11,12,20-trihydroxy-5,8,14-eicosatrienoic acid and 11,12-dihydroxy-19-oxo-5,8,-14-eicosatrienonic acid. About 0.5% of exogenous radioactively labelled arachidonic was covalently bound to microsomal proteins. The metabolites and the protein-bound products were formed in considerably smaller amounts by non-fortified microsomes. Carbon monoxide inhibited this pathway of arachidonic acid metabolism, indicating that these reactions might be catalyzed by the cytochrome P-450-linked monooxygenase systems.     

A novel method for the separation and quantitation of benzene metabolites using high-pressure liquid chromatography

A method for the separation of benzene metabolites using reverse-phase high-pressure liquid chromatography is described. The antoxidant, ascorbic acid is added to an aqueous mixture of 1,2,4-benzenetriol, hydroquinone, catechol, and phenol, to prevent autooxidation. The eluting solvents are equilibrated with nitrogen, degassed, and maintained under a nitrogen atmosphere during the analysis. A highly resolved and reproducible profile of the metabolites is achieved under these conditions. This method should prove useful in a number of pharmacokinetic studies where the biotransformation of the parent compound to autooxidizable species such as polyphenols and quinones precludes analysis under aerobic conditions.     

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.     

Simultaneous analysis of homovanillic acid, 5-hydroxyindoleacetic acid, 3-methoxy-4-hydroxy-phenylethylene glycol and vanilmandelic acid in plasma from alcoholics by high-performance liquid chromatography with electrochemical detection Critical comparison of solid-phase and liquid—liquid extraction methods

A method is described for the simultaneous determination of vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, and homovanillic acid in a human plasma sample using reversed-phase high-performance liquid chromatography with column switching and amperometric detection. Two methods of sample preparation were tested. Liquid—liquid extraction yields better recoveries, is more selective and precise than solid-phase extraction and allows a shorter time of chromatographic analysis. Estimated plasma values of the metabolites from healthy controls are in good agreement with previously reported levels. Studies of alcoholics at the beginning of the delirium tremens provided different plasma levels of the metabolites, dependent on the different duration — and hence the severity — of the delirium.     

Metabolism of retinoic acid in vivo in the vitamin A-deficient rat.

Sample preparation and high-pressure liquid-chromatography separation methods useful for the study of retinoic acid metabolism are reported. The sample preparation procedure does not cause significant degradation of retinoic acid, and the gradient high-pressure liquid-chromatography separation method gives excellent separation of the major metabolites of retinoic acid. These methods were used to examine the metabolites of retinoic acid in blood, trachea and lung, testes, kidneys and small intestine of vitamin A-deficient rats dosed subcutaneously with 2 micrograms of [11,12-3H] retinoic acid. At 6h after dosing, a total of eight metabolites of retinoic acid produced in vivo were found in the tissues examined. Of these, four were found in most of the epithelial tissues examined, and therefore may be of interest as possible active metabolites in the epithelial functions of vitamin A.     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Microsomal activation of estrogens and binding to nucleic acids and proteins.

Natural and synthetic estrogens can be activated by rat liver microsomes to bind covalently to polyguanylic acid, single-stranded DNA, nucleotides and proteins. Incubation of polyG, estrone and liver microsomes (0.5 nmole cytochrome P-448 or P-450) from 3-methylcholanthrene-induced, phenobarbital-induced or control rats showed that the former microsomes gave better $\text{net}$ binding of estrogens to polyG than the other two. Estradiol incubated with 3MC-induced microsomes did bind to DNA but marginally to polyG. Mestranol and estrone sulfate, both constituents of oral contraceptive formulations, bound to polyG whereas progesterone and cholesterol did not bind. We present also preliminary data on the characterization of estrogen-nucleic acid interactions using nucleases, proteinase K and high-pressure liquid chromatography.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.