Metabolite Of Retinol Research Articles

Absorption and Metabolism of (all-trans-) Retinol Acetate, Retinol, and Retinoic Acid in the Single-Pass, Albumin-Perfused Rabbit Ear

Background: Retinoic acid, a metabolite of retinol, is formed in the skin of various species. Purpose: Formation rates have not been determined in a dynamic skin perfusion model which may show the dehydrogenation of retinol to be rate limited. Methods: (all-trans-) Retinol acetate, retinol, or retinoic acid was applied dermally with isopropyl myristate. The ears were single-pass perfused with 6% hetastarch or 5% bovine serum albumin in the buffer solution. The effluent was analyzed by HPLC for these substances. Result: No ester hydrolysis of retinol acetate was observed, nor did this substrate appear in the effluent. Retinol or retinoic acid were detected in the effluent. The absorption rate was linearly related to the dermally applied concentration density. The absorption rate of retinoic acid was 70 times larger than that for retinol. The formation rate of retinoic acid from retinol was rate limited (apparent V_max: 0.08 pmol/min/cm² skin). Conclusion: Since retinoic acid is a recognized pharmacologically active ingredient in the skin, the dehydrogenation of retinol or retinal was also observed in the effluent of this skin perfusion model. The percutaneous retinol adds to the endogenous pool of retinoic acid by cytosolic dehydrogenation or microsomal oxidation elsewhere. The steady-state absorption rate of 0.1% retinoic acid in isopropyl myristate, referred to 500 cm² of the human skin, would contribute about 20% of the daily requirement of vitamin A, without increase in endogenous retinoic acid plasma concentration.

Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway.

In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.

Beta-lactoglobulin suppresses melanogenesis in cultured human melanocytes.

The effects of whey proteins from bovine milk on melanogenesis in cultured human melanocytes were examined. Among the major protein components of milk whey including beta-lactoglobulin (BLG), alpha-lactalbumin, serum albumin, and IgG, only BLG exhibited the depigmenting effect at a concentration of 1 mg/ml. Also, BLG suppressed the activity of tyrosinase in these cells. Retinol, to which BLG is known to bind, slightly increased the pigmentation of the cells at concentrations in the range of 1-100 nM, and retinoic acid, a metabolite of retinol, exhibited a strong pigmentation-promoting effect within the same concentration range. Treatment of the cells with 1 mg/ml BLG completely abrogated the pigmentation induced by these A vitamins. These results demonstrate a novel biological activity of BLG and suggest that this activity is dependent on its ability to bind retinol.

Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-β in liver stellate cells

Liver stellate cells (SCs) play central roles in both the storage of retinol and the development of liver fibrosis. The present study is aimed to understand the mechanism by which retinoic acid (RA, an active metabolite of retinol) enhances hepatic fibrosis in rats. We tested the effect of 9-cis-RA on several aspects in vitro rat SC cultures, including the activity of cellular plasminogen activator (PA), messenger RNA (mRNA), and protein levels of transforming growth factor-β (TGF-β) mRNA level of type-I procollagen, and the activity of type-I collagenase. Employing the rat liver fibrosis model produced by porcine serum, we also estimated the effect of oral administration of a stable RA analog on the progression of the fibrosis, as well as on hepatic TGF-β contents. In vitro SC cultures, 9-cis-RA enhanced cellular PA and plasmin levels thereby induced plasmin-mediated activation of latent TGF-β. Active TGF-β generated self-stimulated its synthesis as well as that of collagen and suppressed the production of collagenase in an autocrine manner. In in vivo rat models, an RA analog accelerated the porcine serum-induced fibrosis by enhancing TGF-β contents and, thus, collagen levels in the liver, although the RA analog alone was not fibrogenic. These results suggest that RA exacerbated liver fibrosis, at least in part, by inducing the activation and production of latent TGF-β in liver SCs.

Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-beta in liver stellate cells

Liver stellate cells (SCs) play central roles in both the storage of retinol and the development of liver fibrosis. The present study is aimed to understand the mechanism by which retinoic acid (RA, an active metabolite of retinol) enhances hepatic fibrosis in rats. We tested the effect of 9-cis-RA on several aspects in vitro rat SC cultures, including the activity of cellular plasminogen activator (PA), messenger RNA (mRNA), and protein levels of transforming growth factor-beta (TGF-beta) mRNA level of type-I procollagen, and the activity of type-I collagenase. Employing the rat liver fibrosis model produced by porcine serum, we also estimated the effect of oral administration of a stable RA analog on the progression of the fibrosis, as well as on hepatic TGF-beta contents. In vitro SC cultures, 9-cis-RA enhanced cellular PA and plasmin levels thereby induced plasmin-mediated activation of latent TGF-beta. Active TGF-beta generated self-stimulated its synthesis as well as that of collagen and suppressed the production of collagenase in an autocrine manner. In in vivo rat models, an RA analog accelerated the porcine serum-induced fibrosis by enhancing TGF-beta contents and, thus, collagen levels in the liver, although the RA analog alone was not fibrogenic. These results suggest that RA exacerbated liver fibrosis, at least in part, by inducing the activation and production of latent TGF-beta in liver SCs. (Hepatology 1997 Oct;26(4):913-21)

Vitamin A in serum is a survival factor for fibroblasts.

Murine 3T3 cells arrest in a quiescent, nondividing state when transferred into medium containing little or no serum. Within the first day after transfer, fibroblasts can be activated to proliferate by platelet-derived growth factor (PDGF) alone; cells starved longer than 1 day, however, are activated only by serum. We demonstrate that endogenous vitamin A (retinol) or retinol supplied by serum prevents cell death and that retinol, in combination with PDGF, can fully replace serum in activating cells starved longer than 1 day. The physiological retinol derivative 14-hydroxy-4, 14-retro-retinol, but not retinoic acid, can replace retinol in rescuing or activating 3T3 cells. Anhydroretinol, another physiological retinol metabolite that acts as a competitive antagonist of retinol, blocks cell activation by serum, indicating that retinol is a necessary component of serum. It previously has been proposed that activation of 3T3 cells requires two factors in serum, an activation factor shown to be PDGF and an unidentified survival factor. We report that retinol is the survival factor in serum.

Retinoid X receptors

Retinoids are metabolites of retinol (vitamin A), which act as signalling molecules in embryogenesis and as stimulators of cellular differentiation. The potency of retinoids as differentiating agents has led to their successful use in treating skin diseases and some forms of cancer. The retinoid X receptors (RXRs) are receptors for the vitamin A metabolite 9- cis retinoic acid, and they are members of the steroid/thyroid hormone receptor superfamily of DNA binding nuclear hormone receptors. The RXRs are also cofactors required for transcription activated by some other members of the steroid/thyroid hormone receptor superfamily, including the all- trans retinoic acid, thyroid hormone, vitamin D and peroxisome proliferator-activated receptors. The biological importance of the RXRs has been demonstrated in recent genetic studies which have shown that some RXR null mutations in mice have phenotypic effects similar to vitamin A deficiency.

Tissue concentrations of retinol, retinyl esters, and retinoic acid in vitamin A deficient rats administered a single dose of radioactive retinol

The tissue concentration of retinol and its metabolites was determined in a group of vitamin A deficient rats after a single dose of 53 micrograms of [11,12-3H]retinol. The sensitive technique of high performance liquid chromatography was used to analyse the metabolites of retinol. The analysis of the metabolites in tissues at different days after the administration of radioactive retinol showed a rapid decrease in the amount of retinol and retinyl esters in the liver tissue, accompanied by an increase in the retinol and retinyl ester values in the kidney. In addition, the content of retinoic acid was higher in liver and kidney compared with intestine, testis, and blood. It reached maximum at 4 and 11 days, respectively. After 17 days the retinoid(s) concentrations decreased markedly in all tissues studied; yet the kidney showed higher concentrations of retinoic acid and retinyl esters. These studies indicate that the kidney retains more vitamin A as vitamin A becomes depleted in the body, probably as a reserve for the production of the active metabolite retinoic acid, needed for the growth and differentiation.

Topical Retinaldehyde Increases Skin Content of Retinoic Acid and Exerts Biologic Activity in Mouse Skin

Retinaldehyde, a natural metabolite of beta-carotene and retinol, has been proposed recently for topical use in humans. Because retinaldehyde does not bind to retinoid nuclear receptors, its biologic activity should result from enzymatic transformation by epidermal keratinocytes into ligands for these receptors, such as all-trans retinoic acid and 9-cis-retinoic acid. In this study, we analyzed by high performance liquid chromatography the type and amounts of tissue retinoids as well as several biologic activities resulting from topical application of either retinaldehyde or all-trans retinoic acid on mouse tail skin. Biologic activities of all-trans retinoic acid and retinaldehyde were qualitatively identical in metaplastic parameters (induction of orthokeratosis, reduction of keratin 65-kDa mRNA, increase in filaggrin and loricrin mRNAs) and hyperplastic parameters (increase in epidermal thickness, increase in bromodeoxyuridine (BrdU)-positive cells, increase in keratin 50-kDa mRNA, and reduction in keratin 70-kDa mRNA). Some quantitative differences, not all in favor of all-trans retinoic acid, were found in several indices. Cellular retinoic acid-binding protein II and cellular retinol-binding protein I mRNAs were increased by both topical retinaldehyde and all-trans retinoic acid. Whereas all-trans retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoic acid were not detectable (limit 5 ng/g) in vehicle-treated skin, 0.05% retinaldehyde-treated skin contained 13 +/- 6.9 ng/g wet tissue of all-trans retinoic acid (mean +/- SD), 12.6 +/- 5.9 ng/g 13-cis-retinoic acid, and no 9-cis-retinoic acid. In contrast, 9-cis-retinoic acid was detectable in 0.05% of all-trans retinoic acid-treated skin, which also contained 25-fold more all-trans retinoic acid and 5-fold more 13-cis-retinoic acid than retinaldehyde-treated skin. Our results show that topical retinaldehyde is transformed in vivo into all-trans retinoic acid by mouse epidermis. The small amounts of ligand for retinoic acid nuclear receptors thus produced are sufficient to induce biologic effects similar to those resulting from the topical application of the ligand itself in much higher concentration.

Recent Advances in the Development of Retinoids

Abstract: Retinoic acid (RA) is a metabolite of retinol (vitamin A) which is necessary for a number of biological functions including the maintenance of growth and epithelial cell differentiation. It appears that most of these functions are mediated through formation of a complex with nuclear retinoid receptors which act in a steroid hormone-like mechanism. RA and its analogs (retinoids) have found use in treating dermatological diseases and show pro- mise as cancer chemopreventive/chemotherapeutic agents because of this impact on epithelial tissue differentiation. RA is extensively metabolized and this has a profound impact on its actions and activity. Thus, a reasonably detailed discussion of the quantitatively and/or biologically important metabolites of RA is provided as well as molecular design efforts to inhibit metabolic inactivation processes and stabilize metabolic activation products. The review is concluded with a description of current efforts in the development of more heavily structurally modified analogs, particularly those with nuclear retinoid receptor or receptor subtype specificity.

Metabolites of All-trans-Retinol in Day 10 Conceptuses of Vitamin A-Deficient Rats

It has been previously demonstrated that vitamin A-deficient rats supplemented with retinoic acid cannot complete gestation. Resorption of fetuses invariably occurs in these animals beginning Day 15 of gestation. Retinol must be administered on or before Day 10 of gestation in order to prevent this phenomenon. The administration of as little as 2 μg on Day 10 is sufficient to allow continuation of gestation through parturition. This study examines the metabolites of all-trans-retinol in the conceptuses of vitamin A-deficient, retinoic acid-supported pregnant animals at Day 10 of gestation. The retinoids recovered in this study could be identified as all-trans-retinol, retinyl palmitate, and other retinyl esters. Virtually no radiolabeled retinoic acid was found in the conceptuses of these animals over the 12-h period examined. This may suggest that retinoic acid that may be required by the fetus can arise directly from maternal sources. Additionally, the isolation of an early appearing, very polar metabolite of retinol is reported. This aqueous-soluble compound accounts for more than 20% of the radioactivity recovered at 1 h post-dose. The amount of this compound increases through 6 h post-dose. This metabolite is not found in urine from the same animals and is not likely to be an excretion product.

Identification of 14-hydroxy-4,14- retro-retinol as an in vivo metabolite of vitamin A

Retinol (vitamin A alcohol) undergoes extensive metabolism in vertebrates. We report here (i) the identification of a yet undescribed in vivo metabolite of retinol as 14-hydroxy-4,14- retro-retinol in pregnant mice, rats and rabbits following dosing with vitamin A, and (ii) the preferential accumulation of 14-hydroxy-4,14- retro-retinol in maternal and embryonic tissues, rather than in material plasma.

Vitamin A down-regulation of IFN-gamma synthesis in cloned mouse Th1 lymphocytes depends on the CD28 costimulatory pathway.

Some infections deplete serum retinol, low retinol reduces immunity, and reduced immunity establishes susceptibility to further infection in a cyclical relationship that is poorly understood. We showed that when retinol was low, there was excessive Th1 cell IFN-gamma synthesis and inadequate Th2 cell IL-4 and IL-5 synthesis. The retinol metabolite retinoic acid inhibited the IFN-gamma stimulatory activity of APCs, enhanced Th2 cell differentiation, and inhibited Th1 cell IFN-gamma synthesis. Here we focus on the mechanism for retinoic acid inhibition of IFN-gamma synthesis in myelin basic protein-specific MM4 Th1 cells. Physiologic amounts of all-trans-retinoic acid directly and specifically down-regulated the MM4 Th1 cell IFN-gamma secretion rate in vitro without affecting cell growth, viability, or overall protein synthesis. All-trans-, 9-cis-, and 13-cis-retinoic acid, and the synthetic retinoid Ch55, inhibited IFN-gamma synthesis effectively, whereas retinaldehyde, retinol, and retinyl acetate did not. This pattern suggests retinoic acid receptor involvement in the inhibition mechanism. Retinoic acid did not inhibit when Th1 cells were activated only through the TCR/CD3 complex, with or without IL-2 costimulation. Retinoic acid inhibited IFN-gamma synthesis when the CD28 costimulatory pathway was activated in addition to the TCR/CD3 pathway, suggesting it blocks some step in the CD28 pathway. Retinoid probably acted to decrease IFN-gamma transcript accumulation by decreasing transcription because it did not decrease transcript stability. We suggest that unrestrained IFN-gamma synthesis is one key immunobiologic mechanism that accounts for poor antibody-mediated immunity in hypovitaminosis A, since IFN-gamma in relatively small amounts can limit Th2 cell growth and interfere with the B cell stimulatory functions of Th2 cell cytokines.

THE EFFECT OF RETINOIC ACID AND DEXAMETHASONE ON THE mRNA EXPRESSION OF THE RETINOIC ACID RECEPTOR-β IN THE RAT. † 1833

Retinol is essential for normal epithelial cell differentiation and maintenance of epithelial cell integrity. Retinoic acid (RA), the active metabolite of retinol, influences gene expression via binding to nuclear retinoic acid receptors (RAR). In vitamin A-deficient adult rats, lung RAR-β gene expression is induced by retinoic acid (Proc Natl Acad Sci 1991; 88:8272). Recently, RAR-β gene expression has been shown to be decreased by dexamethasone (DEX) in neonatal rats (Pediatr Pulmonol 1995; 20:234). We hypothesized that RA and DEX interact to influence RAR-β gene expression. To test this hypothesis, adult, female, vitamin A-sufficient rats were treated with RA 100 μg. DEX 2 μg/g body weight, both RA and DEX, or cottonseed oil or saline as controls. The animals were sacrificed 6 hours after treatment; lung and liver tissue were then obtained for northern blot RNA analysis using 32P labeled cDNA probes specific for RAR-β (two isoforms, β1 = 3.3 kb andβ2 = 3.0 kb) and G3PDH. Densitometry was performed and results are expressed in arbitrary densitometry units. The results (mean ±SD) are shown in the table.

Plasma retinoids after topical use of retinaldehyde on human skin.

Retinaldehyde (RAL), a natural metabolite of beta-carotene and retinol (ROL), is tolerated by human skin after topical application. To see if topical application of a large quantity of RAL on human skin is associated with a detectable alteration of constitutive levels of plasma retinoids resulting from metabolism of RAL in the skin. Plasma retinoids [ROL, all-trans-retinoic acid (all-trans-RA), RAL, retinyl palmitate/oleate, 13-cis-RA and 4-oxo-13-cis-RA] were analyzed by high-pressure liquid chromatography. Determinations were done in 10 healthy male volunteers kept on a vitamin-A-poor diet before, during and after daily topical application of 7 mg of RAL to 40% of the body surface for 14 days. The introduction of a restricted vitamin A diet before RAL application resulted in a decrease in the plasma levels of ROL, all-trans-RA and retinyl palmitate/oleate. Topical application of RAL did not induce an alteration of the plasma levels of retinoid metabolites. No RAL was detectable in any of the plasma samples. The skin metabolism of topically applied RAL does not result in detectable alterations of constitutive levels of plasma retinoids in humans.

Vitamin A, differentiation and cancer

Retinoids, which are derivatives of vitamin A, have a variety of effects on normal cellular differentiation and on the process of carcinogenesis. A number of novel endogenous retinol metabolites have been identified recently. The response of many cell types to retinoid treatment is mediated by retinoid receptors, and involves changes in gene expression, cell growth and cell differentiation. The gene encoding one of the retinoic acid receptors is disrupted by the chromosome translocations associated with acute promyelocytic leukemia, and the expression of another is altered in epithelial tumors; both of these findings have important implications for the use of retinoids as anti-carcinogenic agents. It has been demonstrated recently that certain homeobox genes are regulated by retinoids; these genes may also prove to be useful agents for anti-carcinogenic therapies.

The Teratogenic Metabolites of Vitamin A in Women Following Supplements and Liver

Ten healthy female volunteers were given 5 doses of retinol as the palmitate; 50 and 150 mg retinol as an oral supplement, 50 and 150 mg as fried calf liver (50 and 150 g) and 3, 9 or 30 mg by intra-muscular injection. Plasma concentrations of retinyl palmitate were higher after 50 mg retinol given as an oral supplement compared with 50 mg as liver; there was no significant difference between the 150 mg doses. Plasma concentrations of retinol showed only small increases. The peak plasma concentrations (Cmax) of all-transretinoic acid, the principal teratogenic metabolite of retinol, and the area under the concentration-time curve (AUC) were up to 20-times higher after supplements compared to the same dose as liver. Plasma concentrations of all-trans-4-oxo-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid showed smaller differences between supplements and liver. Intra-muscular administration of 30 mg retinol gave retinyl palmitate concentrations similar to those found after the oral doses but did not increase circulating concentrations of the acid metabolites. Based on the formation of all-trans-retinoic acid, liver and supplements are not of equivalent teratogenic potential. Advice to pregnant women on the consumption of liver based on the reported teratogenicity of vitamin A supplements should be reconsidered.

Tretinoin: A review of in vivo pharmacological studies

AbstractTretinoin (all‐trans‐retinoic acid), a metabolite of retinol, has shown efficacy in the treatment of acute promyelocytic leukemia. Preclinical studies in rodents indicated that tretinoin inhibited or prevented chemically‐induced skin papillomas or carcinomas. However, tretinoin had no effect on several types of transplantable tumors. Studies of tretinoin's pharmacodynamic profile indicated a lack of central nervous system or cardiovascular activity. Effects of tretinoin on the immune system were dependent on the animal model employed, both enhancement and suppression being reported. Tretinoin appeared to increase serum triglycerides and lower serum as well as liver cholesterol levels. Additional preclinical studies are needed to establish minimal effective doses and quantitation of the time course of changes induced by this retinoid. © 1993 wiley‐Liss, Inc.

Growth control or terminal differentiation: endogenous production and differential activities of vitamin A metabolites in HL-60 cells.

Vitamin A (retinol) is a prohormone that exerts its pleiotropic biological effects after conversion into multiple metabolites. In this report we describe the identification of three endogenous, retinolderived effector molecules, 14-hydroxy-retro-retinol (14-HRR), anhydroretinol (AR), and retinoic acid (RA) and a putative storage form of retinol, retinylesters (RE) in the human promyelocytic leukemia cell line HL-60. Exogenous application of the retinol metabolites in retinol-depleted serum-free cultures of HL-60 allowed the identification of unique cellular functions for each metabolite: 14-HRR is a growth factor for HL-60. AR is a functional antagonist of 14-HRR with growth-inhibiting activity, and RA is a potent inducer of granulocyte differentiation accompanied by growth arrest. Finally, intracellular RE serves as storage form allowing continuous production of 14-HRR when no external retinol is available.

Defective lens fiber differentiation and pancreatic tumorigenesis caused by ectopic expression of the cellular retinoic acid-binding protein I

All-trans retinoic acid, a metabolite of retinol, is a possible morphogen in vertebrate development. Two classes of cellular proteins, which specifically bind all-trans retinoic acid, are thought to mediate its action: the nuclear retinoic acid receptors (RAR alpha, beta, gamma), and the cytoplasmic binding proteins known as cellular retinoic acid-binding proteins I and II (CRABP I and II). The function of the retinoic acid receptors is to regulate gene transcription by binding to DNA in conjunction with the nuclear retinoid X receptors (RXR alpha, beta, gamma), which in turn have 9-cis retinoic acid as a ligand. Several lines of evidence suggest that the role of the cellular retinoic acid-binding proteins is to control the concentration of free retinoic acid reaching the nucleus in a given cell. Here, we have addressed the role of the cellular retinoic acid-binding protein I in development by ectopically expressing it in the mouse lens, under the control of the alpha A-crystallin promoter. We show that this ectopic expression interferes with the development of the lens and with the differentiation of the secondary lens fiber cells, causing cataract formation. These results suggest that correct regulation of intracellular retinoic acid concentration is required for normal eye development. In addition, the generated transgenic mice also present expression of the transgene in the pancreas and develop pancreatic carcinomas, suggesting that overexpression of the cellular retinoic acid-binding protein is the cause of the tumors. These results taken together provide evidence for a role of the cellular retinoic acid-binding protein in development and cell differentiation. The relevance of these findings to the possible role of the cellular retinoic acid-binding proteins in the transduction of the retinoic acid signal is discussed.