-
Photochemistry and Photobiology 2015Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between...
Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8842 μW cm(-2)); time periods of 0-60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.
Topics: Animals; Cattle; Dose-Response Relationship, Radiation; Eye Proteins; Light; Photolysis; Radiation-Protective Agents; Retina; Retinaldehyde; Retinol-Binding Proteins; Vitamin A
PubMed: 25565073
DOI: 10.1111/php.12416 -
The Journal of Biological Chemistry Jul 2012In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde all-trans-retinal that is released by photoactivated...
In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde all-trans-retinal that is released by photoactivated rhodopsin, to all-trans-retinol (vitamin A). Previous studies have shown that RDH8 localizes to photoreceptor outer segments and is a strong candidate for performing this role. However, RDH12 function in the photoreceptor inner segments is also key, because loss of function mutations cause retinal degeneration in some forms of Leber congenital amaurosis. To investigate the in vivo roles of RDH8 and RDH12, we used fluorescence imaging to examine all-trans-retinol production in single isolated rod cells from wild-type mice and knock-out mice lacking either one or both RDHs. Outer segments of rods deficient in Rdh8 failed to reduce all-trans-retinal, but those deficient in Rdh12 were unaffected. Following exposure to light, a leak of retinoids from outer to inner segments was detected in rods from both wild-type and knock-out mice. In cells lacking Rdh8 or Rdh12, this leak was mainly all-trans-retinal. Wild-type rods incubated with all-trans-retinal reduced moderate loads of retinal within the cell interior, but this ability was lost by cells deficient in Rdh8 or Rdh12. Our findings are consistent with localization of RDH8 to the outer segment where it provides most of the activity needed to reduce all-trans-retinal generated by the light response. In contrast, RDH12 in inner segments can protect vital cell organelles against aldehyde toxicity caused by an intracellular leak of all-trans-retinal, as well as other aldehydes originating both inside and outside the cell.
Topics: Alcohol Oxidoreductases; Animals; Light; Mice; Mice, Knockout; Photoreceptor Cells, Vertebrate; Pyridines; Retinal Dehydrogenase; Retinal Rod Photoreceptor Cells; Retinoids; Vitamin A
PubMed: 22621924
DOI: 10.1074/jbc.M112.354514 -
The Journal of Biological Chemistry Dec 1994The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol...
Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes.
The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol uptake and metabolism and the mechanism of retinol-induced activation of gene transcription. Human keratinocytes converted all-trans retinol predominantly to retinyl esters, which accounted for 60 and 90% of cell-associated radiolabel after a 90-min pulse and a 48-h chase, respectively. Human keratinocytes also metabolized all-trans retinol to low levels of all-trans retinoic acid (11.47-131.3 ng/mg of protein) in a dose-dependent manner, between 0.3 and 10 microM added retinol. Small amounts of 13-cis retinoic acid (5.47-8.62 ng/mg of protein) were detected, but 9-cis retinoic acid was detected only when keratinocytes were incubated with radiolabeled retinol. There was no accumulation of the oxidized catabolic metabolites 4-hydroxy- or 4-oxoretinoic acid; however, 5,6-epoxy retinoic acid was detected at pharmacological levels (10 and 30 microM) of added retinol. Biological activity of retinol was assessed through analysis of two known retinoic acid-mediated responses: 1) reduction of type I epidermal transglutaminase and 2) activation of a retinoic acid receptor-dependent reporter gene, beta RARE3-tk-CAT. Both all-trans retinol and all-trans retinoic acid reduced type I epidermal transglutaminase in a dose-dependent manner; however, the ED50 for all-trans retinol (10 nM) was 10 times greater than for all-trans retinoic acid (1 nM). All-trans retinol also stimulated beta RARE3-tk-CAT reporter gene activity in a dose-dependent manner. Half-maximal induction was observed at 30 nM retinol, which was again 10-fold greater than observed with all-trans retinoic acid. Cotransfection of human keratinocytes with expression vectors for dominant negative mutant retinoic acid and retinoid X receptors reduced retinol-induced beta RARE3-tk-CAT reporter gene activation by 80%. Inhibition of conversion of all-trans retinol or all-trans retinaldehyde to all-trans retinoic acid by citral reduced beta RARE3-tk-CAT activity 98 and 86%, respectively. These data demonstrate that retinol-induced responses in human keratinocytes are mediated by its tightly regulated conversion to retinoic acid, which functions as a ligand to activate nuclear retinoic acid receptors.
Topics: Cell Nucleus; Cells, Cultured; Epidermis; Humans; Keratinocytes; Receptors, Retinoic Acid; Transcription, Genetic; Transglutaminases; Tretinoin; Tritium; Vitamin A
PubMed: 7806506
DOI: No ID Found -
Investigative Ophthalmology & Visual... Apr 2016Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown...
PURPOSE
Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp(-/-) retina.
METHODS
Wild-type and Irbp(-/-) mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer.
RESULTS
Content of atRAL in Irbp(-/-) retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP.
CONCLUSIONS
Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the cells.
Topics: ADAM Proteins; ADAM17 Protein; Animals; Cell Survival; Chromatography, High Pressure Liquid; Dark Adaptation; Eye Proteins; Immunoblotting; In Situ Nick-End Labeling; Light; Membrane Potential, Mitochondrial; Mice; Microscopy, Confocal; Mitochondrial Diseases; Nitric Oxide; Oxidative Stress; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retinal Degeneration; Retinol-Binding Proteins; Tumor Necrosis Factor-alpha; Vitamin A
PubMed: 27046120
DOI: 10.1167/iovs.15-18551 -
Experimental Biology and Medicine... May 2021Vitamin A is a fat-soluble vitamin involved in essential functions including growth, immunity, reproduction, and vision. The vitamin A Dietary Reference Intakes (DRIs)... (Review)
Review
Vitamin A is a fat-soluble vitamin involved in essential functions including growth, immunity, reproduction, and vision. The vitamin A Dietary Reference Intakes (DRIs) for North Americans suggested that a minimally acceptable total liver vitamin A reserve (TLR) is 0.07 µmol/g, which is not explicitly expressed as a vitamin A deficiency cutoff. The Biomarkers of Nutrition for Development panel set the TLR cutoff for vitamin A deficiency at 0.1 µmol/g based on changes in biological response of several physiological parameters at or above this cutoff. The criteria used to formulate the DRIs include clinical ophthalmic signs of vitamin A deficiency, circulating plasma retinol concentrations, excretion of vitamin A metabolites in the bile, and long-term storage of vitamin A as protection against vitamin A deficiency during times of low dietary intake. This review examines the biological responses that occur as TLRs are depleted. In consideration of all of the DRI criteria, the review concludes that induced biliary excretion and long-term vitamin A storage do not occur until TLRs are >0.10 µmol/g. If long-term storage is to continue to be part of the DRI criteria, vitamin A deficiency should be set at a minimum cutoff of 0.10 µmol/g and should be set higher during times of enhanced requirements where TLRs can be rapidly depleted, such as during lactation or in areas with high infection burden. In population-based surveys, cutoffs are important when using biomarkers of micronutrient status to define the prevalence of deficiency and sufficiency to inform public health interventions. Considering the increasing use of quantitative biomarkers of vitamin A status that indirectly assess TLRs, i.e. the modified-relative-dose response and retinol-isotope dilution tests, setting a TLR as a vitamin A deficiency cutoff is important for users of these techniques to estimate vitamin A deficiency prevalence. Future researchers and policymakers may suggest that DRIs should be set with regard to optimal health and not merely to prevent a micronutrient deficiency.
Topics: Biomarkers; Humans; Liver; Reference Values; Vitamin A; Vitamin A Deficiency
PubMed: 33765844
DOI: 10.1177/1535370221992731 -
Methods in Molecular Biology (Clifton,... 2010The mobility of all-trans-retinol makes a crucial contribution to the rate of the reactions in which it participates. This is even more so because of its low aqueous...
The mobility of all-trans-retinol makes a crucial contribution to the rate of the reactions in which it participates. This is even more so because of its low aqueous solubility, which makes the presence of carrier proteins and the spatial arrangement of cellular membranes especially relevant. In rod photoreceptor outer segments, all-trans-retinol is generated after light exposure from the reduction of all-trans-retinal that is released from bleached rhodopsin. The mobility of all-trans-retinol in rod outer segments was measured with fluorescence recovery after photobleaching (FRAP), using two-photon excitation of its fluorescence. The values of the lateral and axial diffusion coefficients indicate that most of the all-trans-retinol in rod outer segments move unrestricted and without being aided by carriers.
Topics: Animals; Anura; Cell Separation; Diffusion; Fluorescence Recovery After Photobleaching; Movement; Photons; Rod Cell Outer Segment; Vitamin A
PubMed: 20552425
DOI: 10.1007/978-1-60327-325-1_6 -
Nutrients Feb 2023The prenatal period is critical for auditory development; thus, prenatal influences on auditory development may significantly impact long-term hearing ability. While...
The prenatal period is critical for auditory development; thus, prenatal influences on auditory development may significantly impact long-term hearing ability. While previous studies identified a protective effect of carotenoids on adult hearing, the impact of these nutrients on hearing outcomes in neonates is not well understood. The purpose of this study is to investigate the relationship between maternal and umbilical cord plasma retinol and carotenoid concentrations and abnormal newborn hearing screen (NHS) results. Mother-infant dyads ( = 546) were enrolled at delivery. Plasma samples were analyzed using HPLC and LC-MS/MS. NHS results were obtained from medical records. Statistical analysis utilized Mann-Whitney U tests and logistic regression models, with ≤ 0.05 considered statistically significant. Abnormal NHS results were observed in 8.5% of infants. Higher median cord retinol (187.4 vs. 162.2 μg/L, = 0.01), maternal -β-carotene (206.1 vs. 149.4 μg/L, = 0.02), maternal -β-carotene (15.9 vs. 11.2 μg/L, = 0.02), and cord -β-carotene (15.5 vs. 8.0 μg/L, = 0.04) were associated with abnormal NHS. Significant associations between natural log-transformed retinol and β-carotene concentrations and abnormal NHS results remained after adjustment for smoking status, maternal age, and corrected gestational age. Further studies should investigate if congenital metabolic deficiencies, pesticide contamination of carotenoid-rich foods, maternal hypothyroidism, or other variables mediate this relationship.
Topics: Pregnancy; Infant, Newborn; Infant; Adult; Female; Humans; Vitamin A; beta Carotene; Vitamins; Nutritional Status; Chromatography, Liquid; Tandem Mass Spectrometry; Carotenoids
PubMed: 36839158
DOI: 10.3390/nu15040800 -
Cells Feb 2022Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding...
Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.
Topics: Breast Neoplasms; Cell Proliferation; Collagen; Epithelial Cells; Female; Gene Expression; Humans; Retinoids; Retinol-Binding Proteins, Cellular; Tretinoin; Tumor Microenvironment; Vitamin A
PubMed: 35269414
DOI: 10.3390/cells11050792 -
The Journal of Biological Chemistry Aug 2017Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the...
Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all--retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on / ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.
Topics: Biocatalysis; Cytochrome P450 Family 27; Diterpenes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Humans; Hydrogenation; Hydroxylation; Isoenzymes; Kinetics; Mitochondria; Molecular Structure; Organ Specificity; Oxidation-Reduction; Peptide Fragments; Proteolysis; Proteomics; Skin; Stereoisomerism; Substrate Specificity; Vitamin A
PubMed: 28701464
DOI: 10.1074/jbc.M116.773937 -
The Journal of Biological Chemistry May 2006
Review
Topics: Alcohol Oxidoreductases; Animals; Multigene Family; Oxidation-Reduction; Protein Structure, Tertiary; Substrate Specificity; Vitamin A
PubMed: 16428379
DOI: 10.1074/jbc.R500027200