Production Of Retinoids Research Articles

The Origin of Teratogenic Retinoids in Cyanobacteria.

Although information about the occurrence and distribution of retinoids in the environment is scarce, cyanobacterial water blooms have been identified as a significant source of these small molecules. Despite the confirmed presence of retinoids in the freshwater blooms dominated by cyanobacteria and their described teratogenic effects, reliable identification of retinoid producers and the mechanism of their biosynthesis is missing. In this study, the cultures of several taxonomically diverse species of axenic cyanobacteria were confirmed as significant producers of retinoid-like compounds. The consequent bioinformatic analysis suggested that the enzymatic background required for the biosynthesis of all-trans retinoic acid from retinal is not present across phylum Cyanobacteria. However, we demonstrated that retinal conversion into other retinoids can be mediated non-enzymatically by free radical oxidation, which leads to the production of retinoids widely detected in cyanobacteria and environmental water blooms, such as all-trans retinoic acid or all-trans 5,6epoxy retinoic acid. Importantly, the production of these metabolites by cyanobacteria in association with the mass development of water blooms can lead to adverse impacts in aquatic ecosystems regarding the described teratogenicity of retinoids. Moreover, our finding that retinal can be non-enzymatically converted into more bioactive retinoids, also in water, and out of the cells, increases the environmental significance of this process.

Molecular Properties of β-Carotene Oxygenases and Their Potential in Industrial Production of Vitamin A and Its Derivatives.

β-Carotene 15,15′-oxygenase (BCO1) and β-carotene 9′,10′-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. β-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli.

Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15′-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5’-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid–producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli.

Field cyanobacterial blooms producing retinoid compounds cause teratogenicity in zebrafish embryos.

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an invivo zebrafish embryo test, an invitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity invitro, as well as high degrees of teratogenicity invivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant invitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these invitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected invitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.

Widespread occurrence of retinoids in water bodies associated with cyanobacterial blooms dominated by diverse species

Cyanobacterial blooms represent a worldwide problem in freshwater as well as marine ecosystems as producers of various toxic compounds. This study provides environmentally important information about the common presence of mixtures of retinoids in various water bodies associated with the occurrence of cyanobacterial blooms dominated by many different species. The study documents, for the first time, that retinoids are produced by environmental cyanobacterial blooms dominated by species belonging to different genera such as Microcystis, Dolichospermum, Planktothrix, Woronichinia, Pseudanabaena and others. Samples of biomass of cyanobacterial blooms and their surrounding water were collected from seventeen independent freshwater bodies across the Czech Republic during summer 2015. Retinoid-like activity was detected by an in vitro reporter gene bioassay in water samples from 8 out of 17 localities with a maximal activity of 263 ng all-trans retinoic acid equivalent (REQ)/L. In comparison, in vitro assessment of biomass extracts documented retinoid-like activity at 11 out of 17 localities with a maximal retinoid-like activity of 867 ng REQ/g dry mass (dm). Individual retinoids were detected by chemical analyses in all water samples and in 16 out of 17 biomass samples with 4keto-retinal and all-trans 5,6epoxy retinoic acid being detected in aquatic ecosystems for the first time. Further, all-trans 4keto retinoic acid and retinal were the most commonly detected compounds in both types of samples. With respect to retinoid-like activity, a large proportion was explained in some samples by contributions of individual detected retinoids calculated from their concentrations and relative potencies. However, results also indicate that other unknown compounds with a retinoic acid receptor-mediated mode of action were present. The revealed widespread production of retinoids by cyanobacterial blooms dominated by diverse species across various aquatic ecosystems and their common presence in both biomass and surrounding water raises concern namely because some retinoids belong to the most potent teratogens. These compounds need to be taken into consideration in the assessment of risks associated with massive cyanobacterial blooms.

Molecular Basis for Vitamin A Uptake and Storage in Vertebrates.

The ability to store and distribute vitamin A inside the body is the main evolutionary adaptation that allows vertebrates to maintain retinoid functions during nutritional deficiencies and to acquire new metabolic pathways enabling light-independent production of 11-cis retinoids. These processes greatly depend on enzymes that esterify vitamin A as well as associated retinoid binding proteins. Although the significance of retinyl esters for vitamin A homeostasis is well established, until recently, the molecular basis for the retinol esterification enzymatic activity was unknown. In this review, we will look at retinoid absorption through the prism of current biochemical and structural studies on vitamin A esterifying enzymes. We describe molecular adaptations that enable retinoid storage and delineate mechanisms in which mutations found in selective proteins might influence vitamin A homeostasis in affected patients.

Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus.

A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)(+) We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD(+) converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)(+) as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Biochemical properties of retinoid-converting enzymes and biotechnological production of retinoids.

Retinoids are a class of compounds that are forms of vitamin A and include retinal, retinol, retinoic acid, and retinyl ester. Retinal is involved in visual cycle, retinol has anti-infective, anticancer, antioxidant, and anti-wrinkle functions, and retinoic acid is used to treat acne and cancer. Retinol, retinoic acid, and retinyl ester are used in cosmetic and pharmaceutical industries. In this article, the biochemical properties and active sites and reaction mechanisms of retinoid-converting enzymes in animals and bacteria, including retinol dehydrogenase, alcohol dehydrogenase, aldo-keto reductase, and aldehyde dehydrogenase, are reviewed. The production of retinoids, using retinoid-producing enzymes and metabolically engineered cells, is also described. Uncharacterized bacterial proteins are suggested as retinoid-converting enzymes, and the production of retinoids using metabolically engineered cells is proposed as a feasible method.

Retinoid-like activity and teratogenic effects of cyanobacterial exudates

Retinoic acids and their derivatives have been recently identified by chemical analyses in cyanobacteria and algae. Given the essential role of retinoids for vertebrate development this has raised concerns about a potential risk for vertebrates exposed to retinoids during cyanobacterial blooms. Our study focuses on extracellular compounds produced by phytoplankton cells (exudates). In order to address the capacity for the production of retinoids or compounds with retinoid-like activity we compared the exudates of ten cyanobacteria and algae using in vitro reporter gene assay. Exudates of three cyanobacterial species showed retinoid-like activity in the range of 269–2265ng retinoid equivalents (REQ)/L, while there was no detectable activity in exudates of the investigated algal species. The exudates of one green alga (Desmodesmus quadricaudus) and the two cyanobacterial species with greatest REQ levels, Microcystis aeruginosa and Cylindrospermopsis raciborskii, were selected for testing of the potential relation of retinoid-like activity to developmental toxicity in zebrafish embryos. The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, spine and mouth deformation) and interference with growth in zebrafish embryos, while such effects were not observed for the alga. Fish embryos were also exposed to all-trans retinoic acid (ATRA) in a range equivalent to the REQ concentrations detected in exudates by in vitro bioassays. Both the phenotypes and effective concentrations of exudates corresponded to ATRA equivalents, supporting the hypothesis that the teratogenic effects of cyanobacterial exudates are likely to be associated with retinoid-like activity. The study documents that some cyanobacteria are able to produce and release retinoid-like compounds into the environment at concentrations equivalent to those causing teratogenicity in zebrafish. Hence, the characterization of retinoid-like and teratogenic potency should be included in the assessment of the potential adverse effects caused by the release of toxic and bioactive compounds during cyanobacterial blooms.

Beta-Carotene Reduces Body Adiposity of Mice via BCMO1

Evidence from cell culture studies indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15′-oxygenase (Bcmo1) and the BC-9′,10′-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into β-10′-apocarotenal and β-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1 -/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and β-10′-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite β-10′-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARγ activity in adipocytes

Male‐specific expression of Aldh1a1 in mouse and chicken fetal testes: Implications for retinoid balance in gonad development

Balanced production and degradation of retinoids is important in regulating development of several organ systems in the vertebrate embryo. Among these, it is known that retinoic acid (RA), and the retinoid-catabolyzing enzyme CYP26B1 together regulate the sex-specific behavior of germ cells in developing mouse gonads. We report here that the gene encoding a cytosolic class-1 aldehyde dehydrogenase, ALDH1A1, a weak catalyst of RA production, is strongly expressed in a male-specific manner in somatic cells of the developing mouse testis, beginning shortly after Sry expression is first detectable. This expression pattern is conserved in the developing male gonad of the chicken and is dependent on the testis-specific transcription factor SOX9. Our data suggest that low levels of RA may be required for early developmental events in the testis, or that Aldh1a1 expression in the fetus may prefigure a later requirement for ALDH1A1 in regulating spermatogenesis postnatally.

Dissection of the pathway required for generation of vitamin A and for Drosophila phototransduction

Dietary carotenoids are precursors for the production of retinoids, which participate in many essential processes, including the formation of the photopigment rhodopsin. Despite the importance of conversion of carotenoids to vitamin A (all-trans-retinol), many questions remain concerning the mechanisms that promote this process, including the uptake of carotenoids. We use the Drosophila visual system as a genetic model to study retinoid formation from β-carotene. In a screen for mutations that affect the biosynthesis of rhodopsin, we identified a class B scavenger receptor, SANTA MARIA. We demonstrate that SANTA MARIA functions upstream of vitamin A formation in neurons and glia, which are outside of the retina. The protein is coexpressed and functionally coupled with the β, β-carotene-15, 15′-monooxygenase, NINAB, which converts β-carotene to all-trans-retinal. Another class B scavenger receptor, NINAD, functions upstream of SANTA MARIA in the uptake of carotenoids, enabling us to propose a pathway involving multiple extraretinal cell types and proteins essential for the formation of rhodopsin.

Retinoic Acid Combined with Vitamin A Synergizes to Increase Retinyl Ester Storage in the Lungs of Newborn and Dexamethasone-Treated Neonatal Rats

Background: Retinyl esters (REs), the major storage form of vitamin A (retinol), provide substrates for the production of bioactive retinoids, including retinoic acid (RA), which are known to promote lung development and maturation. We previously showed that the nutrient-metabolite combination VARA (molar ratio 10 vitamin A to 1 RA), synergistically increased REs in the lungs of 1-week-old rats, compared to vitamin A or RA alone. Objectives: To test the hypotheses, first, that VARA is more effective in increasing lung RE than is vitamin A in newborn rats prior to alveolarization, and, second, that the effect of VARA is maintained during concurrent treatment with the glucocorticoid, dexamethasone (Dex). Methods: Newborn rats were treated with VARA, vitamin A alone, or oil (C) on postnatal days (P) 1–3, and RE in the lungs was quantified on P4, and again on P8 to assess retention. Additionally, neonatal rats were treated on P5–7 with VARA with and without Dex, and the lung and liver REs were quantified on P8. Results and Conclusions: Lung RE was nearly 8-fold higher in VARA compared to vitamin A-treated rats on P4 (p < 0.01) and 2.5-fold higher on P8. In neonates co-treated with Dex and VARA on P5–7, the elevation in lung RE on P8 by VARA was not antagonized by Dex, although Dex reduced growth. Lung morphology and development were not significantly altered. The VARA combination may significantly increase lung RE content even during concurrent Dex therapy. Because lung retinoids are important for lung maturation and repair, increasing lung RE may possibly have clinical benefit.

Lycopenoids: Are lycopene metabolites bioactive?

In vitro lycopene is the most potent antioxidant among carotenoids. While antioxidant function may be relevant to health, we hypothesize that metabolites of lycopene may be bioactive and responsible for the beneficial effects of tomato product consumption. We term these metabolites “lycopenoids,” which we believe may be produced from carotenoid monooxygenase (CMO) II, paralleling the production of retinoids from β-carotene by CMO I. We present evidence suggesting that tomato carotenoid metabolites may be responsible for the reduced risk of prostate cancer seen in men consuming high levels of tomato products. Finally, we identify gaps in knowledge in this evolving area of carotenoid research.

Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in a synergistic manner in neonatal rats

Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5-7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs.

Human choriocarcinoma cell line JEG-3 produces and secretes active retinoids from retinol.

Vitamin A (retinol) and its active derivatives (the retinoids) are essential for growth and development of the mammalian fetus. Maternally-derived retinol has to pass through the placenta to reach the developing fetus. Despite its apparent importance, little is known about placental metabolism of retinol, and particularly placental production and/or secretion of active retinoids. It has been previously considered that retinoids are recruited from the uterine environment to influence placental development and function during gestation. We have studied retinoid metabolism in the human choriocarcinoma cell line JEG-3 and demonstrate, for the first time, that active retinoids are produced endogenously by the JEG-3 cell line from retinol. These retinoids induce gene expression from a retinoic acid-responsive enhancer element reporter plasmid and modulate placental transglutaminase activity. Furthermore, retinoids are secreted from JEG-3, as shown by the activation of retinoic acid-responsive beta lacZ reporter cells grown in conditioned media. These results suggest that there could be an active role for trophoblast-derived retinoids during human development.

The Effects of 3,4,3′-Triiodo-[formula omitted]-Thyronine on Didehydroretinol Synthesis by Isolated Coho Salmon Retinal Pigment Epithelial Cells

The effects of thyroid hormone on the production of vitamin A2 retinoids from vitamin A1 precursor were investigated in retinal pigmented epithelial (RPE) cells obtained from juvenile coho salmon. Specifically, changes in the production of both the storage and the free forms of radiolabeled 3,4-didehydroretinoids were measured after 24 and 48 h incubation with [11,12-3H]all-trans-retinol with (or in the absence of) exogenous 3,4,3′-triiodo-l-thyronine (T3). RPE cells incubated with radiolabeled all-trans-retinol showed double the production of radiolabeled 3,4-didehydroretinoids (both storage and free forms) after 48 h compared to 24 h incubation. The addition of T3 had no significant effect on the production of didehydroretinyl esters (storage form) after 24 h incubation, but did have a significant effect after 48 h, with a ca. 50% reduction in the amount of labeled didehydroretinyl produced compared to the amount produced by the controls after 48 h. Essentially the same observation was made for the production of radiolabeled didehydroretinol (free form) where, after 48 h of incubation with T3, there was a ca. 50% reduction in the amount of labeled didehydroretinol produced compared to the amount produced by the controls after 48 h. The reduction in both the storage and the free forms of didehydroretinoids indicates that T3 probably had the effect of altering the activity of the “terminal ring dehydrogenase” enzyme which acts on the outer ring of retinoids. The scope of this experiment, however, does not indicate whether the production of didehydroretinoids is specific to the free or to the storage forms as retinoids within the RPE can shuttle between both forms. The results of this experiment do indicate that the in vitro observations of this experiment may be extended to in vivo and field observations. In vitro, T3 by itself altered the production of didehydro-derivatives of retinoids. The changes in visual pigment complement seen in salmonids, which are due to changes in relative proportions of 11-cis–retinal and 11-cis-3,4-didehydroretinal in the photoreceptors, may also be the result of T3 acting by itself on the RPE cells.

All-trans-Retinoic Acid Regulates Retinol and 3,4-Didehydroretinol Metabolism in Cultured Human Epidermal Keratinocytes

The potential for all-trans-retinoic acid to regulate the metabolism of 3H-retinol and 3H-3,4-didehydroretinol was examined in cultured human epidermal keratinocytes. Confluent cultures were treated daily with medium containing 5% fetal bovine serum or the same medium supplemented with nanomolar concentrations of all-trans-retinoic acid for up to 3 d. During the last 24 of treatment, cells were incubated with 3H-retinol or 3H-3,4-didehydroretinol for 24 h (isotopic steady state) to label the endogenous retinoids. After the labeling period, one group of cells was harvested and another group was allowed to incubate for an additional 24 h in the absence of medium retinol for the determination of endogenous 3H-retinoid utilization. The 3H-retinoids present in cells were extracted and quantitated by reverse-phased high-pressure liquid chromatography. Keratinocytes treated with retinoic acid and labeled with 3H-retinol exhibited time- and concentration-dependent (i) increases in retinyl ester mass, (ii) increases in the rate of retinyl ester synthesis, (iii) decreases in retinyl ester utilization, and (iv) decreases in the cellular concentrations of retinoic and 3,4-didehydroretinoic acids. There was no effect of exogenous retinoic acid on its own metabolism. Cells labeled with 3H-3,4-didehydroretinol exhibited exclusive labeling of vitamin A2-related retinoids suggesting that the A1 to A2 conversion is not reversible. Treatment of cells with low nanomolar concentrations of retinoic acid decreased the utilization of 3,4-didehydroretinyl esters, decreased the production of 3,4-didehydroretinoic acid but had no effect on the synthesis of 3,4-didehydroretinol or its esters. The results demonstrate that keratinocytes respond to extracellular retinoic acid by decreasing endogenous production of active retinoids, sequestering extracellular substrate retinol as retinyl ester, and decreasing ester utilization.

Characterization of retinol metabolism in cultured human epidermal keratinocytes

Retinoids present in cultured human epidermal keratinocytes maintained in medium containing 25 nM retinol were identified and quantitated utilizing reverse-phase gradient high performance liquid chromatography. Total cell retinoid mass and composition averaged 12.59 pmol/mg cell protein +/- 1.86, (mean +/- S.D., n = 5) between strains and was constant with culture age. Long chain fatty acid esters of retinol and 3,4-didehydroretinol comprised greater than 95% of total retinoid, each contributing 77 and 23% of total esters, respectively. In mature stratified cultures, less abundant retinoids included unesterified retinol, 1%, and 3,4-didehydroretinol, 0.5%, retinoic acid, 0.5%, and a retinoid not previously reported in mammalian cells, 3,4-didehydroretinoic acid, 0.3%. This absolute and relative retinoid composition was essentially constant with culture age except that 3,4-didehydroretinoic acid was not detected in 3-4-day-old cultures (16-32 cell colonies), suggesting that keratinocyte differentiation may be related to endogenous changes in production of "active" retinoids. Separate pools of retinyl and 3,4-didehydroretinyl esters served as predominant sources of substrate for production of retinoic and 3,4-didehydroretinoic acids, respectively, suggesting that their production may be regulated independently.

19] Retinoic acid synthesis from β-carotene in Vitro

It is clear that both retinol and β -carotene serve as initial substrates for retinoic acid synthesis and several pathways of retinoic acid synthesis exist, at least in vitro . One pathway, the conversion of β -carotene to retinoic acid, is discussed in this chapter. It describes high-performance liquid chromatography (HPLC) assays to study the conversion of β -carotene to retinoids. Retinoic acid supports vitamin A-dependent differentiation through modulating gene transcription. As far as the mechanism of the production of retinoids from β -carotene is concerned, the difference between the products of β -carotene and retinal metabolism in vitro is inconsistent with an exclusive “central cleavage” mechanism. Two types of experiments illustrate this point and indicate that β -carotene conversion to retinoids in vitro is not a simple case of generating retinal and releasing it into solution to diffuse to enzymes that convert it to retinol and retinoic acid. The first type is based on the observation that the relative amounts of retinol and retinoic acid generated from β -carotene are different from those generated from retinal. With retinal as substrate, the major product in vitro is retinoic acid. In the absence of cofactor, little or no retinol is detected.