Retinol Esters Research Articles

Overview of retinoid metabolism and function

Retinoids (vitamin A) are crucial for most forms of life. In chordates, they have important roles in the developing nervous system and notochord and many other embryonic structures, as well as in maintenance of epithelial surfaces, immune competence, and reproduction. The ability of all-trans retinoic acid to regulate expression of several hundred genes through binding to nuclear transcription factors is believed to mediate most of these functions. The role of all-trans retinoic may extend beyond the regulation of gene transcription because a large number of noncoding RNAs also are regulated by retinoic acid. Additionally, extra-nuclear mechanisms of action of retinoids are also being identified. In organisms ranging from prokaryotes to humans, retinal is covalently linked to G protein-coupled transmembrane receptors called opsins. These receptors function as light-driven ion pumps, mediators of phototaxis, or photosensory pigments. In vertebrates phototransduction is initiated by a photochemical reaction where opsin-bound 11-cis-retinal is isomerized to all-trans-retinal. The photosensitive receptor is restored via the retinoid visual cycle. Multiple genes encoding components of this cycle have been identified and linked to many human retinal diseases. Central aspects of vitamin A absorption, enzymatic oxidation of all-trans retinol to all-trans retinal and all-trans retinoic acid, and esterification of all-trans retinol have been clarified. Furthermore, specific binding proteins are involved in several of these enzymatic processes as well as in delivery of all-trans retinoic acid to nuclear receptors. Thus, substantial progress has been made in our understanding of retinoid metabolism and function. This insight has improved our view of retinoids as critical molecules in vision, normal embryonic development, and in control of cellular growth, differentiation, and death throughout life.

Chlormethiazole treatment prevents reduced hepatic vitamin A levels in ethanol-fed rats.

Chronic ethanol intake results in decreased hepatic vitamin A levels through both enhanced degradation of vitamin A via a cytochrome P450 enzyme (CYP)-dependent process and increased mobilization of vitamin A from the liver into the circulation. This study investigated whether treatment with chlormethiazole, a CYP inhibitor, restores vitamin A in the livers of ethanol-fed rats. Ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole (10 and 100 mg/kg body weight) for 1 month. Liver and plasma levels of retinol and retinyl palmitate were analyzed by high-performance liquid chromatography. Expressions of hepatic lecithin:retinol acyltransferase (LRAT) and cellular retinol-binding protein were analyzed with reverse transcription-polymerase chain reaction. Hepatic retinol esterification by LRAT was examined by using incubations of the microsomal fractions of livers with exogenous sources of retinol. Ethanol-feeding in rats for a month resulted in lower hepatic levels of retinol and retinyl palmitate than those found in controls and the occurrence of several polar retinoid metabolites. In contrast, treatment with chlormethiazole at two different doses in ethanol-fed rats completely blocked the formation of hepatic retinoid polar metabolites and restored hepatic levels of retinol and hepatic retinyl palmitate in a dose-dependent manner. Furthermore, increased plasma concentrations of retinyl palmitate in rats fed with ethanol, which indicate increased mobilization of vitamin A, were partially inhibited by chlormethiazole treatment. However, neither ethanol nor chlormethiazole treatment altered the expression and activity of LRAT in the liver of rats. Hepatic expression of cellular retinol-binding protein increased significantly in ethanol-fed rats with or without chlormethiazole treatment compared with control rats. These data suggest that chlormethiazole can restore both hepatic retinol and retinyl ester concentrations to normal levels in ethanol-fed rats through blocking enhanced both degradation of vitamin A and mobilization of vitamin A from the liver into the circulation.

Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle

Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo.

Disruption of the Lecithin:Retinol Acyltransferase Gene Makes Mice More Susceptible to Vitamin A Deficiency

Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin A deficiency and should be an excellent animal model of vitamin A deficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.

A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.

Retinoid Absorption and Storage Is Impaired in Mice Lacking Lecithin:Retinol Acyltransferase (LRAT)

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ∼85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ∼64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.

Identification of a Novel Keratinocyte Retinyl Ester Hydrolase as a Transacylase and Lipase

Retinoic acid influences epidermal morphology and function through its ability to control transcription. Because the circulation presents the epidermis with micromolar amounts of retinol that can be converted to retinoic acid, regulating retinol access is imperative. In keratinocytes the majority of retinol is sequestered as long chain fatty acid esters. Although much has been learned about the major esterifying enzyme, little is known about the hydrolase that accesses retinol from its storage depot. Murine carboxylesterases and hormone sensitive lipase have been shown to have this activity. We found that their in vitro sensitivity to bis-p-nitrophenyl phosphate (BNPP), however, was not shared by the epidermal hydrolase activity. We therefore produced and screened two keratinocyte cDNA expression libraries and identified a previously sequenced gene (GS2) as a keratinocyte retinyl ester (RE) hydrolase insensitive to BNPP. The enzyme also catalyzes fattyacyl CoA-dependent and -independent retinol esterification. The hydrolysis reaction is greater at neutral pH, whereas the esterification reaction is greater at acidic pH. These activities are consistent with the increased RE content that accompanies epidermal maturation. In addition, this enzyme utilizes triolein as substrate and generates diacylglyceride and free fatty acid.

The Roles of Vitamin A for Cytoplasmic Maturation of Bovine Oocytes

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.

Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.

Cellular retinol-binding protein-1 in hepatocellular carcinoma correlates with β-catenin, Ki-67 index, and patient survival

The cellular retinol-binding protein-1 (CRBP-1) plays a key role in the esterification and intercellular transfer of retinol. By in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP-1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP-1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP-1 expression in MFs was down-regulated in 83%. Patients with high CRBP-1 expression in MFs had a significantly higher 2-year survival as compared with patients with low CRBP-1 expression (52% vs. 29%, respectively; P = .034). An aberrant nuclear CRBP-1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP-1 staining correlated positively with a favorable tumor stage (Okuda stage I; P = .01) and negatively with the Ki-67 + proliferation fraction (PF). A Ki-67 + PF of ≥10% was associated with a lower 2-year survival probability as compared with patients with a Ki-67 + PF of <10% (12% vs. 40%, respectively; P = .015). Prognosis did not correlate with the nuclear β-catenin expression. There was, however, a close correlation between nuclear CRBP-1 inclusions and nuclear β-catenin staining in HCCs ( P = .008), suggesting a cross talk between CRBP-1 and the Wnt/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP-1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP-1 expression in HCCs contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis. (H epatology 2003;38:470-480.)

Retinoid Production and Catabolism: Role of Diet in Regulating Retinol Esterification and Retinoic Acid Oxidation

Retinoic acid (RA), a transcriptionally active metabolite of vitamin A (retinol), activates two families of nuclear retinoid receptors that have the potential to regulate the expression of a large number of genes. Although it may be presumed that the concentration of RA is closely regulated, the mechanisms underlying such regulation are not well understood. Our research has examined the expression and function of two enzymes, lecithin:retinol acyltransferase (LRAT) and a cytochrome P450, CYP26, in the liver and lung of rats and mice, over a wide range of vitamin A status or after treatment of vitamin A-deficient animals with exogenous RA. LRAT expression at both the mRNA and protein activity levels and CYP26 mRNA are regulated by dietary vitamin A in a steady-state model and are acutely regulated by RA in an acute repletion model. In the liver, the level of expression of LRAT and CYP26 is as follows: vitamin A deficient < vitamin A marginal < vitamin A adequate < vitamin A supplemented < RA treated. The regulation of LRAT shows strong tissue specificity (highly regulated in liver and lung but not in small intestine), whereas CYP26 is strongly regulated in the liver, lung, testis and intestine. RA may function as a signal of the body's vitamin A adequacy. The regulated expression of LRAT, CYP26 and other genes by RA may provide a sensitive response mechanism that overall serves to adjust the metabolism of vitamin A to maintain retinoid homeostasis and prevent retinoid excess.

Retinoid metabolism (LRAT, REH) in the yolk-sac membrane of Japanese quail eggs and effects of mono- ortho-PCBs

Retinoids stored in the avian egg are essential for normal development, however, laboratory and field experiments suggest that they are affected by environmental contaminants. Lecithin:retinol acyltransferase (LRAT) activity was detected in the microsomal fraction of the yolk-sac membrane of the Japanese quail at day 6 of development. LRAT activity was maximal at pH 7.0 having apparent kinetic parameters of K m=1.35 μM and V max=0.21 nmol/mg protein/h and was inhibited by the sulfhydryl modifying agent N-ethyl-maleimide. Retinol ester hydrolase (REH) activity in the microsomal fraction of the yolk-sac membrane was stimulated by the bile salt analogue 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate and was maximal at pH 9.0 with apparent K m=77 μM and V max=34.3 nmol/mg protein/h. Injection of the PCB congener 2,3,3′,4,4′-pentachlorobiphenyl increased both REH and LRAT activities, whereas 2,3,3′,4-tetrachlorobiphenyl stimulated LRAT. Yolk retinol concentration and the molar ratio retinol:retinyl palmitate were lower in the exposed eggs. Yolk retinol concentration decreased as LRAT increased ( R 2=0.89) suggesting that certain PCB congeners may affect vitamin A mobilization in ovo by increasing LRAT activity in the yolk-sac membrane.

Treatment of the rat hepatic stellate cell line, PAV-1, by retinol and palmitic acid leads to a convenient model to study retinoids metabolism

The main site of vitamin A storage in the liver is the hepatic stellate cells (HSC). Involvement of HSC in vitamin A metabolism has mainly been studied using primary culture, which represents the most physiological model but technically suffers several drawbacks (yield, low reproducibility, etc.). To circumvent these problems, we have previously established and characterised an immortalised rat HSC line named PAV-1. This study aimed to investigate in PAV-1 and in primary HSC (i) the incorporation of retinol and its esterification, (ii) the cellular retinol-binding protein (CRBP) content, (iii) the acid retinyl ester hydrolase activity (aREH), (iv) the thermal susceptibility and (v) the lipid composition of the membranes, which may play a crucial role in retinol transport across cellular membrane. In routine conditions of culture, the rate of retinol esterification in PAV-1 was low (5.2%) compared to that obtained with primary HSC (69.9%). Retinol pre-treatment doubled this esterification rate (10.7%) and the CRBP content in PAV-1. The co-incubation with retinol and palmitic acid enabled PAV-1 to esterify retinol with a rate close to that of primary HSC (66.2% vs. 69.9%) and with similar retinyl ester profiles. aREH activity was higher in primary HSC than in PAV-1. Thermal susceptibility and phospholipid composition of membranes in PAV-1 treated cells were similar to those of primary HSC. In conclusion, our study shows that PAV-1 cells treated with retinol and palmitic acid is a sound and convenient model for studying vitamin A mobilisation, a fundamental physiological event occurring in HSC.

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and α-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and α-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic α-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and α-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when α-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and α-SM actin expression. In both cell populations, transforming growth factor-β1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.

Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study

A collaborative study was conducted for the determination of vitamins A and E. Existing AOAC liquid chromatographic (LC) methods are suited for specific vitamins A and E analytical applications. This method differs from existing methods in that it can be used to assay samples in all 9 sectors of the food matrix. Standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters and tocopherol esters to retinol and tocopherol, respectively. Retinol and alpha-tocopherol are quantitated on separate LC systems, using UV detection at 313 or 328 nm for retinol, and fluorescence detection (excitation 290 nm, emission 330 nm) for alpha-tocopherol. Vitamin concentrations are calculated by comparison of the peak heights or peak areas of vitamins in test samples with those of standards.

Effects of season on vitamins A and E contents and colour of Turkish butter.

Butter is a product that consists of roughly the same constituents asmilk, but their distribution is different. In buttermaking, the fat contentof the milk is concentrated approximately 20 times. Milk and butter aregood sources of the fat solublevitamins A, D and E.Vitamin A (retinol) is a primary alcohol derivative of a polymer con-sisting of four isoprene units. Vitamin A is present in cow’s milk as reti-nol, retinol esters and carotenes. The content of vitamin A dependsstrongly on the amount of carotenoids in the feed. The ratio of retinol tocarotene in cow’s milk varieswith breed [1–3].Vitamin E consists of a group of tocopherols. The most potent ofthese and the principal one in milk is alpha-tocopherol. This is a strongreductant and serves as an antioxidant protecting lipids from oxidation.It is stable to heat but may be partly destroyed by intensive illuminationin the presence of O

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [ 3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT’s activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.

Nutritional assessment of serum and hepatic vitamin A levels in patients with cirrhosis

BACKGROUND Serum vitamin A (retinol) levels may not correlate with hepatic vitamin A stores in patients with cirrhosis; thus, supplementation of vitamin A based on serum levels may have a detrimental effect. Our aim was to determine whether serum levels correlate with hepatic stores in cirrhotic patients. METHODS A prospective study of patients with cirrhosis undergoing orthotopic liver transplantation was completed. Serum and hepatic levels of vitamin A were measured by high-performance liquid chromatography. Statistical analysis was performed using rank sum tests and Spearman rank correlation coefficients. RESULTS Fifty cirrhotic patients (33 men and 17 women, mean age 53 years) were compared with a control group (25 men and 25 women, mean age 47 years) of liver donors. Median serum levels of retinol were 259 microg/L in controls and 166 microg/L in cirrhotic patients (p < .001). Median hepatic levels of retinol were 25 microg/g in controls and 27.5 +/- g/g in cirrhotic patients (p not significant). Total hepatic vitamin A levels (retinol plus retinyl esters) were 471 microg/g in controls and 244 microg/g in cirrhotic patients (p = .028). Serum retinol did not correlate with total hepatic vitamin A stores in cirrhotic patients (rs = .10, p = .332). CONCLUSIONS Serum retinol and total hepatic vitamin A stores are lower in cirrhotic patients than in controls. However, because levels of serum retinol do not correlate with hepatic vitamin A levels, the decision to prescribe vitamin A replacement for patients with cirrhosis should not be made solely on the basis of serum retinol levels.

17β-Hydroxysteroid dehydrogenase type 9 and other short-chain dehydrogenases/reductases that catalyze retinoid, 17β- and 3α-hydroxysteroid metabolism

Subgroups of related short-chain dehydrogenase/reductase (SDR) family members serve as retinoid/androgen/estrogen metabolizing enzymes. These include retinol dehydrogenases (RoDHs) 1–3, cis-retinol/androgen dehydrogenase 1 and 2 (CRAD), retSDRs1–4, 9/11- cis-retinol dehydrogenase, and 17β-hydroxysteroid dehydrogenase (17β-HSD) types 6 and 9. Interaction with cellular retinol-binding protein (CRBP), the major physiological form of retinol, led to the identification and cDNA cloning of RoDH1. Probes for RoDH1 contributed to cDNA cloning many of the others. Some of these SDRs show specificity with all- trans-retinol (RoDH, retSDR, 17β-HSD6 and 9) and others with 9 and/or 11- cis-retinol (CRAD, 9/11- cis-retinol dehydrogenase). Many have 3α-HSD activities with 3α-androstandiol as the most efficiently used substrate, followed by androsterone. In addition to 3α-HSD activity, CRAD2 shows relatively weak 17β-HSD activity with testosterone. Rat 17β-HSD6 and mouse 17β-HSD9, which are not interspecies homologs, have efficient 17β-HSD activities. 17β-HSD6 has ∼50% greater 17β-HSD activity with estradiol than with 3α-androstandiol. With 3α-androstandiol, 17β-HSD9 operates equally efficiently as a 17β-HSD or a 3α-HSD. The multi-substrate nature of these SDRs allows for retinoid/steroid interactions. The ability of some these SDRs to access retinol bound with CRBP provides specificity in retinoid metabolism and allows retinoic acid biosynthesis and retinol esterification to continue, as CRBP protects retinol from the general cellular milieu.