Metabolism and biological activity of all-trans 4,4-difluororetinyl acetate

All-trans [11-³H]4,4-difluororetinyl acetate was synthesized by treating methyl all-trans [11-³H]4-oxoretinoate with diethylaminosulfurtrifluoride, followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol, difluororetinyl palmitate and related esters, 4-oxoretinol, 4-oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and / or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by ¹⁹F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 ± 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8–3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Preparation,characterization, biological activity and metabolism of all-trans retinoyl fluoride

All-trans retinoyl fluoride was prepared by treating all-trans retinoic acid with diethylaminosulfurtrifluoride. The crystalline product, which was characterized by melting point, infrared, ¹H-NMR, ¹⁹F-NMR and elementary analysis, showed λ_max at 382 nm in hexane (ε = 4.98·10⁴ M^?1·cm^?1) and at 392 nm in methanol (ε = 4.60·10⁴ M^?1·cm^?1). Its biological activity in the rat growth assay, relative to all-trans retinyl acetate, was 22% ± 10%. Upon oral administration for 5 days to vitamin A-depleted rats, retinoyl fluoride (1020 μg) was rapidly metabolized to a polar metabolite fraction and, in the intestine, to an unstable retinol-like metabolite, purpotedly 15-fluororetinol. Upon administering intraperitoneally smaller doses (47–94 μg) of [11-³H]retinoyl fluoride, which was synthesized from [11-³H] retinoic acid, radioactive retinoic acid was noted in the liver and plasma but not in the intestine. As expected, a radioactive polar fraction appeared in the intestine and liver, but radioactive retinol, retinyl ester and some common oxidation products were not detected. Of the administered radioactivity, 72% was excreted in the urine, and only 4% was found in the feces over a 7-day period. Hydrolysis of the urine gave a radioactive fraction with a polarity similar to that of retinoic acid. Retinoyl fluoride also reacts readily with glycine to yield N-retinoyl glycine. Thus, the biological activity of retinoyl fluoride can be attributed to the formation of retinoic acid, probably by way of N-retinoyl derivatives. A possible pathway for its metabolism is presented.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

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 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.     

Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Proteolysis of chloroplast thylakoid membranes. II. Evidence for the involvement of the light-harvesting chlorophyll a/b-protein complex in thylakoid stacking and for effects of magnesium ions on photosystem II-light-harvesting complex aggregates in the absence of membrane stacking

Experimental evidence indicates that the major pathway of retinoic acid metabolism in hamster liver microsomes follows the sequence: retinoic acid → 4-hydroxy-retinoic acid → 4-keto-retinoic acid → more polar metabolites. Using all-trans-[10-³H]retinoic acid, it can be shown by reverse-phase high pressure liquid chromatographic analysis that the first and last steps of this sequence require NADPH, whereas the oxidation of 4-hydroxy to 4-keto-retinoic acid is NAD⁺ (or NADP⁺) dependent. Both NADPH-dependent steps, but not the NAD⁺-dependent dehydrogenase reaction, are strongly inhibited by carbon monoxide. The metabolism of retinoic acid but not of 4-hydroxy-retinoic acid is highly dependent on the vitamin A regimen of the animal. Retinoic acid is rapidly metabolized by liver microsomes either from vitamin A-normal hamsters or from vitamin A-deficient hamsters that have been pretreated with retinoic acid, but not by microsomes from vitamin A-deficient animals; in direct contrast, the rate of metabolism of 4-hydroxy-retinoic acid is equivalent in each of these microsomal preparations. Analysis of the kinetics of these reactions yields the following Michaelis constants with respect to the retinoid substrates: retinoic acid, 1 × 10^?6m; 4-hydroxy-retinoic acid, 2 × 10^?5m; and 4-keto-retinoic acid, 1 × 10^?7m. The 4-hydroxy to 4-keto-retinoic acid oxidation has been shown to be experimentally irreversible, to have a K_mNAD⁺of 2 × 10^?5m, to be strongly inhibited by NADH, and to be unaffected by the presence of retinoic acid or its 4-keto-derivative in an equimolar ratio to the 4-hydroxy-substrate.     

The biological activity of rat intestinal retinoic acid metabolites in the vaginal smear assay

Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-³H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [³H]retinoic acid. The biological activity of all discernible metabolities was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-β-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Binding of all-trans-retinoic acid to MLTC-1 proteins

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 mean ± SD) fmoles/8 × 10⁴ cells at 37 °C. About 90% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.9 μM and 0.65 μM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 μM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 μM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [³H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [³H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells. (Mol Cell Biochem 276: 55–60, 2005)This paper is dedicated to the memory of Prof. E. Quagliariello.     

Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

Background

Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes.

Methodology/Principal Findings

Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, ¹⁸O- and ¹⁶O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA.

Conclusions/Significance

Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.     

Retinoylation Reaction of Proteins in Leydig (TM-3) Cells

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570± 27 fmoles/8×10⁴ cells at 37C. About 95% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.6 and 1.2 M respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 M respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [³H]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 M); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.This paper is dedicated to the memory of Prof. J. A. Olson.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

Induction of Normal Cardiovascular Development in the Vitamin A-Deprived Quail Embryo by Natural Retinoids

The biological activity of various natural retinoids and the time "window" when vitamin A activity is required for normal cardiovascular development were examined in vitamin A-deprived Japanese quail embryos. The administration of 1 μg of retinol at the beginning of incubation resuited in normal cardiovascular development in 97% of embryos; retinoic acid was toxic at this dose level. Treatment of embryos with 0.1 μg of all-trans-retinol or 13-cis-retinoic acid at the beginning of incubation resulted in normal cardiovascular development in 47 and 12% of embryos, respectively; administration of these retinoids at other time points attenuated the percentage of embryos with normal cardiovascular development. Didehydroretinol, 0.1 μg, and 9-cis-retinoic acid, 0.1 μg, were inactive at all time points examined; 9-cis-retinoic acid did not enhance the biological activity of all-trans-retinoic acid. All-trans-retinoic acid, 0.1 μg, administered during 22-28 hr of incubation induced normal cardiovascular development in 20-34% of embryos; biological activity was optimal when it was administered at 24 hr. All retinoids tested were inactive in establishing normal cardiovascular development when administered at 36 hr of incubation or later. The studies suggest that all-trans-retinoic acid is the biologically active form of vitamin A required for normal cardiovascular development in the avian embryo. There is a critical time point within the first 22-28 hr of quail embryogenesis when all-trans-retinoic acid initiates events that lead to normal cardiovascular development.