All-trans Retinol Research Articles

Fluorometric Determination of All-trans Retinol in Rat Serum

A rapid, specific, and sensitive fluorometric assay for retinol in rat serum is reported. Serum retinol is extracted into cyclohexane after dilution of the serum sample with aqueous acetonitrile. Sample volumes as small as 0.2ml can be used with a limiting detectable concentration of 2.5 × 10-8 M for retinol.

A microspectrophotometric study of normal and artificial visual pigments in the photoreceptors of Xenopus laevis

Absorbance spectra of visual pigments in the Xenopus retina were obtained by microspectrophotometry. Principal rods contained a pigment with peak absorbance of 524 ± 2nm. A minority of rods contained a pigment with λ max at 445 ± 6nm; single cone spectra peaked at 611 ± 4nm. Xenopus tadpoles were maintained on a Vitamin A-free diet for 6–8 weeks, then injected systemically with all-trans retinol or 9-cis retinal. In the principal rod, these resulted in pigments peaking at 511 ± 3nm and 498 ± 2nm, respectively. The normally encountered Vitamin A 2-based pigment of the principal rod had a broader half-width than its Vitamin A 1-based analog. In single cones, all-trans retinol led to the formation of a pigment with λ max 578 ± 7nm. The substantial shifts seen in the wavelengths of peak absorbance as well as in the shapes of the spectral curves following administration of retinol or 9-cis retinal indicate that at least most of the normally encountered Vitamin A 2-based pigment was replaced by a Vitamin A 1-based pigment.

Retinoid inhibition of superoxide anion radical production by human polymorphonuclear leukocytes stimulated with tumor promoters

All-trans retinol, retinyl acetate and retinoic acid were found to be effective inhibitors of O 2 − • polymorphonuclear leukocytes stimulated with the tumor promoter phorbol myristate acetate. Retinol similarly inhibited cells stimulated with mezerein or teleocidin B. No-effect concentrations of the protease inhibitor antipain potentiated the inhibitory effect of low levels of retinol. Higher concentrations of retinol and antipain resulted in an additive or less than additive inhibitory effect.

The membrane bound retinol dehydrogenase from bovine rod outer segments

Retinol dehydrogenase from bovine rod outer segments was solubilized in detergent and partially purified 25-fold through a combination of hydroxyapatite and retinyl-Sepharose chromatography. Alltrans retinol solubilized in protein solutions of bovine serum albumin or β-lactalbumin was a better substrate for the enzyme than retinol solubilized in detergents or suspended in buffer. Retinol dehydrogenase was sensitive to the carbonyl reagent pyridoxal-5′-phosphate but was not inhibited by retinal followed by reduction with NaBH 4. The solubilized enzyme requires phospholipids to maintain enzymatic activity, as was evidenced by the inactivating effect of phospholipase A 2 on the partially purified enzyme.

Kinetic and mechanistic studies of oxidation of vitamin A alcohol to vitamin A aldehyde by horse liver alcohol dehydrogenase. The inhibition by ethanol and pyrazole.

The present investigation shows that all-trans retinol (Vitamin A alcohol), an alcohol of great physiological importance, is efficiently oxidized to all-trans retinaldehyde by the enzyme horse liver alcohol dehydrogenase. We observe a Km retinol value of 145 microM and a turnover number of 0.45s-1 for the oxidation of all-trans retinol in the presence of Triton X-100, a surfactant used as a solubilizer. Over the concentration range of surfactant used (up to 0.1% Triton X-100) our studies on the oxidation of ethanol and all-trans retinol show that turnover numbers for both reactions remain constant as does the value for Km ethanol. On increasing the concentration of Triton X-100 from 0.025% to 0.10%, however, the Km retinol value increases by a factor of two. This behavior for retinol oxidation can be attributed to the partitioning of retinol between enzyme and surfactant. Pyrazole, a known inhibitor of alcohol oxidation by horse liver alcohol dehydrogenase, is a competitive inhibitor of both all-trans retinol and ethanol, with observed Ki values of 3.3 X 10(-7) M and 3.9 x 10(-7) M, respectively. We also find that ethanol inhibits all-trans retinol oxidation in a complex fashion, an observation which may have important consequences in view of the physiological role of retinol and its oxidation products. Our present studies indicate that all-trans retinol binds in the same region of the enzyme as does ethanol and is oxidized with an efficiency approaching that of ethanol itself.

Interaction between prealbumin and retinol-binding protein studied by affinity chromatography, gel filtration and two-phase partition.

The interaction between prealbumin and apo or holo retinol-binding proteins has been studied by affinity chromatography, gel filtration and two-phase partition. At physiological ionic strength apo and holo retinol-binding protein form 1:1 molar complexes with prealbumin. Mean dissociation constants for the prealbumin compex with apo retinol-binding protein and holo retinol-binding protein with all-trans retinol, retinoic acid, retinal and retinyl acetate were calculated from the partition data as 0.33 +/- 0.11 x 10(-6) M and 0.075 +/- 0.015 x 10(-6) M respectively (mean +/- S.E.M.). The difference was statistically significant. Quantitative data on the amount of retinol, retinol-binding protein and prealbumin in plasma and urine were in good agreement with the ratio of the dissociation constants for the complexes of apo and holo retinol-binding proteins with prealbumin as determined in the partition experiment. The magnitude of the dissociation constants was compatible with previously published data on the turnover of retinol-binding protein.

Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

Spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells) displayed an increased adhesion when cultured in the presence of 10(-6) M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [15-(14)C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [(14)C]retinol was taken up by the cells. The hydrocarbon [(14)C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T12 cells was also active in synthesizing [(14)C]anhydroretinol and [(14)C]metabolite-310 from [(14)C]retinol. Moreover incubation of metabolite-310 with the 3T12 microsomes yielded anhydroretinol (40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [(14)C]anhydroretinol incubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.-Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

Fluorescence studies of retinol and retinyl acetate

Abstract The polarized emission study of all-trans retinol and retinyl acetate in solid films at room temperature has been carried out. The shape of the absorption and fluorescence is typical of Franck-Condon forbidden bands. The high positive polarization of the fluorescence excitation and the emission (when excited into main absorption band due to 1Bu ← 1Ag transition) suggests that the lowest excited singlet state of retinols has a large amount of 1Bu character. The explanation for the high positive polarization involving strong vibronic interactions suggesting 1Ag - (a very weakly allowed state) being the lowest excited singlet has also been considered.

Natural Occurrence and Biological Activity of Vitamin a Derivatives in Rat Bile

A study was made to determine the biological activity of retinoyl β-glucuronide, a major biliary excretion product of administered retinoic acid in the rat, to evaluate the significance of the biliary excretion of vitamin A derivatives under normal nutritional conditions, to analyze the rate of excretion of vitamin A derivative, and to determine the chemical nature of the excreted products. Retinoyl β-glucuronide has a biopotency 30 to 100% as great as all-trans retinol or as all-trans retinoic acid. A more accurate estimate cannot be given because of the unknown effect of the isolation procedure on the glucuronide's biopotency. Similarly, a nonpolar retinoate derivative in bile (fraction I), which presumably is methyl retinoate, has a biopotency 50 to 200% as great as all-trans retinoate by like criteria. The biliary secretion of vitamin A derivatives, presumably glucuronides, appears to be a normal physiological process, inasmuch as labeled compounds were secreted at the rate of about 0.3 µg/ml in the bile of vitamin A-depleted rats from 6 to 32 days after the administration of labeled retinol. Finally, the rate of excretion of labeled retinoic acid derivatives in the excreta closely paralleled the rate of their secretion into bile. Most of the radioactivity appeared in the feces, mainly as retinoic acid but with a lesser amount as the glucuronide. A labeled derivative, presumably the glucuronide, appeared earlier in the urine but in much smaller amounts.

On the biochemical mechanism of the visual process

Evidence is presented supporting an ionic mechanism for the stimulation of the photoreceptor by light. Illumination of a rhodopsin monolayer at an air-water interface causes expansion of the layer, which can be interpreted as the result of penetration of the monolayer by retinal. Illumination of a rod outer segment suspension causes efflux of K ions and an equivalent influx of Na ions. The same effect, qualitatively and quantitatively, occurs upon addition of all-trans retinal to the suspension. All-trans retinal added to artificial phosphatidyl-ethanolamine membranes greatly increases cation leakage through these micelles, but has no effect on lecithin micelles. All-trans retinol has a small effect on both types of micelles. These findings are interpreted as suggesting that upon photolysis of rhodopsin all-trans retinal would be transferred from opsin to a Schiff base link with the amino group of phosphatidylethanolamine or phosphatidylserine present in the rod sac membrane. Blocking of the amino group would make the membrane more negatively charged and thus more cation-permeable. The resulting cation exchange would cause a depolarizing current stimulating the synapse with the bipolar cell.

Kinetics of Rhodopsin Regeneration in the Eye of the Frog

RHODOPSIN, the red photosensitive pigment of rod vision, consists of the colourless protein, rod opsin, carrying 11-cisretinal (formerly retinene) as chromophore1. In the light, rhodopsin bleaches through orange-red intermediates (lumi- and meta-rhodopsin) into the yellow mixture of free all-trans retinal and opsin2. The enzyme alcohol dehydrogenase and DPNH2 in the rod outer segments reduce the retinal to retinol (formerly vitamin A)3. The all-trans retinol migrates from the rod outer segments into the pigment epithelium, where, most likely, an energy-utilizing process isomerizes the all-trans retinol into 11-cis retinol4. The exact material for the isomerization is, however, uncertain. This process supplies the pigment epithelium both in light- and dark-adapted frogs' eyes with a relatively great amount of 11-cis retinol. About 95 per cent of the retinol in the eye of the frog (Rana pipiens) is in the form of fatty acid-esters4.

POLAROGRAPHIC INVESTIGATION OF CONJUGATED FAT-SOLUBLE VITAMINS.

The half-wave potentials of all-trans beta-carotene, all-trans retinol, 13-cis retinol, all-trans retinyl acetate, all-trans retinal, and vitamins D(2) and D(3) were related to the number of double bonds in conjugation. A minimum of three double bonds in conjugation was required before reduction took place at the dropping-mercury electrode. As the number of conjugated bonds increased in the fat-soluble vitamins, the initial reduction took place at a lower half-wave potential. All of the waves were linearly proportional to the concentration of the vitamins in the concentration range studied.