Liver Retinyl Ester Research Articles

Retinyl Ester Storage Is Normal in Transgenic Mice with Enhanced Expression of Cellular Retinol-Binding Protein Type I

This report describes the production and characterization of transgenic mice with high expression of human cellular retinol-binding protein type I [hCRBP(I)]. In initial experiments, overexpression of hCRBP(I) was driven by the strong promoter SRα, but no transgenic offspring were produced. When we used the less efficient mouse metallothionein I promoter fused to the hCRBP(I) cDNA for microinjection, we obtained 12% transgenic offspring. Two of these transgenic mice (409/1 and 401/2) expressed mRNA and immunoreactive hCRBP(I) in several organs. Both lines had relatively high contents of hCRBP(I) in intestine, testis and epididymis. On the other hand, only 401/2 transgenic mice had high contents of hCRBP(I) in kidney. Effects on storage of vitamin A were studied by measuring the concentration of retinyl esters in different organs. The concentrations of retinyl esters in liver, lung and kidney did not significantly differ between transgenic and control mice, and the concentration of total retinol in plasma was within the normal range in transgenic mice. Furthermore, feeding mice a diet with high or low concentrations of vitamin A for 2 wks resulted in no marked differences in the concentrations of retinyl esters in liver, kidney, lung, intestine and testis in transgenic mice compared with control mice. Therefore, in spite of high expression of hCRBP(I) in several organs, the transgenic mice had normal storage of retinyl esters in all organs studied. The present in vivo study indicates that the CRBP(I) content alone does not control retinyl ester storage.

Altered vitamin A metabolism in human liver slices corresponds to fibrogenesis.

All‐trans‐retinoic acid (atRA), the active metabolite of vitamin A, has antifibrogenic properties in vitro and in animal models. Liver vitamin A homeostasis is maintained by cell‐specific enzymatic activities including storage in hepatic stellate cells (HSCs), secretion into circulation from hepatocytes, and formation and clearance of atRA. During chronic liver injury, HSC activation is associated with a decrease in liver retinyl esters and retinol concentrations. atRA is synthesized through two enzymatic steps from retinol, but it is unknown if the loss of retinoid stores is associated with changes in atRA formation and which cell types contribute to the metabolic changes. The aim of this study was to determine if the vitamin A metabolic flux is perturbed in acute liver injury, and if changes in atRA concentrations are associated with HSC activation and collagen expression. At basal levels, HSC and Kupffer cells expressed key genes involved in vitamin A metabolism, whereas after acute liver injury, complex changes to the metabolic flux were observed in liver slices. These changes include a reproducible spike in atRA tissue concentrations, decreased retinyl ester and atRA formation rate, and time‐dependent changes to the expression of metabolizing enzymes. Kinetic simulations suggested that oxidoreductases are important in determining retinoid metabolic flux after liver injury. These early changes precede HSC activation and upregulation of profibrogenic gene expression, which were inversely correlated with atRA tissue concentrations, suggesting that HSC and Kupffer cells are key cells involved in changes to vitamin A metabolic flux and signaling after liver injury. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Vitamin A is metabolized in the liver for storage as retinyl esters in hepatic stellate cell (HSCs) or to all‐trans‐retinoic acid (atRA), an active metabolite with antifibrogenic properties. Following chronic liver injury, vitamin A metabolic flux is perturbed, and HSC activation leads to diminished retinoid stores. WHAT QUESTION DID THIS STUDY ADDRESS? Do changes in the expression of vitamin A metabolizing enzymes explain changes in atRA concentrations and the regulation of fibrosis following acute liver injury? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? In healthy liver, both HSC and Kupffer cells may mediate vitamin A homeostasis. Following acute liver injury, complex changes in metabolizing enzyme expression/activity alter the metabolic flux of retinoids, resulting in a transient peak in atRA concentrations. The atRA concentrations are inversely correlated with profibrogenic gene expression, HSC activation, and collagen deposition. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Improved understanding of altered vitamin A metabolic flux in acute liver injury may provide insight into cell‐specific contributions to vitamin A loss and lead to novel interventions in liver fibrosis.

The Effect of Chronic High Dose Vitamin a Supplementation on Lipid Metabolism in Adipose Tissue (P02-013-19)

ObjectivesThe objective of this study was to assess the impact of high-dose vitamin A (VA) on lipid metabolism. Previously, VA has been shown to enhance fat mobilisation, leading to a reduction in body fat. We hypothesise that hypervitaminosis A will increase expression of genes associated with lipid catabolism. MethodsTo induce chronic hypervitaminosis A, two groups of pigs (n = 8) were fed a commercial diet. The treatment group was additionally dosed, daily, with an oral supplement of retinyl propionate of 10,000 μg/KgBW for 17 weeks. To assess the impact of VA on lipid metabolism, a microarray analysis was performed to identify gene expression in adipose tissue. Differentially expressed transcripts and pathways were identified using Genespring and mapped to human orthologues for Ingenuity Pathway Analysis (IPA); gene fold changes were confirmed using qRT-PCR. Additionally, an untargeted UPLC-MS lipidomic analysis was carried out in serum samples to identify changes in lipd classes and their metabolites. ResultsIn dosed animals, significant increases in plasma retinol (0.66 μmol/L) and liver retinyl ester concentrations (11.98 μmol/g both P < 0.001), as well as an increase in serum NEFA of 92.84 μmol/L (P = 0.001) were observed. Gene expression fold changes in subcutaneous adipose tissue were related to mitochondrial dysfunction and lipid metabolism, including increased expression of MT-CYTB (↑4.78x, P < 0.05) and ATP5A1 (↑3.13x, P < 0.05). Metabolomics confirmed changes in lipids and their metabolites relevant to adipose tissue in blood (P = 0.05), namely a decrease in triacylglyceride concentration and increases in acyl carnitine and cardiolipin concentrations. ConclusionsAn integrated pathway is suggested to explain the role of vitamin A in leading to increased lipolysis, β-oxidation and oxidative phosphorylation, but when in excess, markers of mitochondrial dysfunction were observed. Funding SourcesFunded by the Bill and Melinda Gates foundation.

Effects of vitamin A deficiency in the postnatal mouse heart: role of hepatic retinoid stores.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Inhibition of Retinoic Acid Biosynthesis by the Bisdichloroacetyldiamine WIN 18,446 Markedly Suppresses Spermatogenesis and Alters Retinoid Metabolism in Mice

Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

Chronic effects of lead (Pb) on bone properties in red deer and wild boar: Relationship with vitamins A and D3

Here we study the occurrence of abnormalities on bone tissue composition and turnover mechanisms through the Pb-mediated disruption of vitamins A and D in wild ungulates living in a lead (Pb)-polluted mining area. Red deer (Cervus elaphus) and wild boar (Sus scrofa) from the mining area had significantly higher liver and bone Pb levels than controls, which were associated with the depletion of liver retinyl esters and the corresponding increase of free retinol levels both in deer and boar from the mining area. Pb-exposed adult deer had lower carbonate content in bone mineral than controls, which was associated with the increased free retinol percentage. In wild boar, the degree of bone mineralization was also positively associated with higher burdens of retinyl esters. These results suggest that Pb-associated changes in bone composition and mineralization is likely influenced by the depletion of vitamin A in wildlife exposed to environmental Pb pollution.

Enhanced fatty acid flux triggered by adiponectin overexpression.

Adiponectin overexpression in mice increases insulin sensitivity independent of adiposity. Here, we combined stable isotope infusion and in vivo measurements of lipid flux with transcriptomic analysis to characterize fatty acid metabolism in transgenic mice that overexpress adiponectin via the aP2-promoter (ADNTg). Compared with controls, fasted ADNTg mice demonstrated a 31% reduction in plasma free fatty acid concentrations (P = 0.008), a doubling of ketones (P = 0.028), and a 68% increase in free fatty acid turnover in plasma (15.1 ± 1.5 vs. 25.3 ± 6.8 mg/kg · min, P = 0.011). ADNTg mice had 2-fold more brown adipose tissue mass, and triglyceride synthesis and turnover were 5-fold greater in this organ (P = 0.046). Epididymal white adipose tissue was slightly reduced, possibly due to the approximately 1.5-fold increase in the expression of genes involved in oxidation (peroxisome proliferator-activated receptor α, peroxisome proliferator-activated receptor-γ coactivator 1α, and uncoupling protein 3). In ADNTg liver, lipogenic gene expression was reduced, but there was an unexpected increase in the expression of retinoid pathway genes (hepatic retinol binding protein 1 and retinoic acid receptor beta and adipose Cyp26A1) and liver retinyl ester content (64% higher, P < 0.02). Combined, these data support a physiological link between adiponectin signaling and increased efficiency of triglyceride synthesis and hydrolysis, a process that can be controlled by retinoids. Interactions between adiponectin and retinoids may underlie adiponectin's effects on intermediary metabolism.

Vitamin A and Retinoic Acid Act Synergistically to Increase Lung Retinyl Esters During Normoxia and Reduce Hyperoxic Lung Injury in Newborn Mice

We have shown that vitamin A (VA) and retinoic acid (RA) synergistically increase lung retinyl ester content in neonatal rats. To confirm whether this biochemical synergism attenuates early neonatal hyperoxic lung injury in mice, we exposed newborn C57BL/6 mice to 95% O2 or air from birth to 4 d. The agent [vehicle, VA, RA, or the combination vitamin A+retinoic acid (VARA)] was given orally daily. Lung and liver retinyl ester content was measured, and lung injury and development were evaluated. We observed that lung, but not liver, retinyl ester levels were increased more by VARA than by VA or RA alone. Hyperoxic lung injury was reduced by VA and RA, and more so by VARA. VARA attenuated the hyperoxia-induced increases in macrophage inflammatory protein (MIP)-2 mRNA and protein expression, but did not alter hyperoxia-induced effects on peptide growth factors (PDGF, VEGF, and TGF-beta1). The 4-d exposure to hyperoxia or retinoids did not lead to observable differences in lung development. We conclude that the VARA combination has synergistic effects on lung retinyl ester concentrations and on the attenuation of hyperoxia-induced lung injury in newborn mice, possibly by modulation of inflammatory mediators.

A gene knockout corroborates the integral function of cellular retinol-binding protein in retinoid metabolism.

Continually expanding evidence has moved inexorably toward establishing key functions for cellular retinol-binding protein (CRBP) in retinoid metabolism. These experimental data integrate into a model of CRBP as a chaperone that protects retinol from the cellular milieu and interacts with certain retinoid-metabolizing enzymes. Mutant mice with an inactivated CRBP gene show decreased liver retinyl ester storage, a shorter elimination half-life of liver retinoids, and predisposition to vitamin A deficiency. No morphologic phenotype was observed until vitamin A was exhausted. Although the mechanisms underlying diminished vitamin A in the CRBP-null mice have not been elucidated, the observations support the model of CRBP as a chaperone of retinoid metabolism.

Studies of adipose tissue vitamin A storage and metabolism in mutant mice

Work by Yang et al. (Nature 2005 436: 356– 362) indicates that retinol-binding protein (RBP) synthesized in adipose tissue may be an adipokine acting in the causation of diabetes. We demonstrated that adipose tissue vitamin A storage and metabolism involves different enzymes and processes than in the liver, the major site of vitamin A storage in the body (O'Byrne et al. J. Biol. Chem. 2005 280:35647-35657). Using mutant mice lacking lecithin:retinol acyltransferase (LRAT-/-), RBP (RBP-/-) and LRAT−/−/RBP−/ − double knockout mice, we carried out studies of adipose vitamin A storage and metabolism to define the enzymes and processes needed for this. LRAT−/ − mice show elevated (by 2- to 3-fold) retinyl ester levels in adipose tissue but totally lack any retinyl esters in liver, lung and kidney. Gavage studies of wild type, LRAT−/ − and RBP−/ − mice suggest that RBP contributes a significant proportion of the vitamin A stored in adipose. This notion is supported by comparison of LRAT−/ − mice with LRAT−/ −/RBP−/ − mice. The absence of RBP in double knockouts greatly diminishes (by approximately 3-fold) the amount of postprandial vitamin A that is accumulated by adipose tissue. This suggests that newly absorbed dietary vitamin A passes initially through the liver before transfer via RBP to adipose stores. Finally, our in vitro data suggest that diacylglycerol acyltransferase 1 (DGAT1) can act as an acyl-CoA:retinol acyltransferase (ARAT) in physiologic situations. Using LRAT−/ − and LRAT−/ −/DGAT1−/ − mice we are assessing the physiologic significance of DGAT1 as an ARAT in adipose tissue. (Supported by NIH grant R01 DK068437)

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.

The efficacy of a modified aluminosilicate as a detoxifying agent in Fusarium toxin contaminated maize containing diets for piglets

Two feeding experiments with female weaned piglets were carried out applying a complete two by two factorial design to investigate the effects of the dietary inclusion of 500 g/kg Fusarium toxin contaminated maize (8.6 mg/kg deoxynivalenol (DON); 1.2 mg/kg zearalenone (ZON)) and of 4 g/kg aluminosilicate (AS) as a detoxifying agent. The resulting four diets were fed ad libitum to a total of 80 piglets (20 piglets per group, allotted to a total of 20 pens) covering a live weight range of 10.5 +/- 1.3 to 27.5 +/- 4.4 kg in experiment 1, and to a total of 48 piglets (12 piglets per group, allotted to 12 pens) covering a live weight range of 9.7 +/- 1.8 to 21.4 +/- 4.8 kg in experiment 2. The animals of experiment 1 were slaughtered on days 34-36 of feeding the experimental diets. The mycotoxin analyses revealed that the control maize also contained considerable concentrations of Fusarium toxins, but the differences in DON and ZON concentrations between control and contaminated diets were sufficiently high to demonstrate both dose-related toxin effects. Voluntary feed intake and live weight gain of the animals were significantly reduced by the inclusion of Fusarium toxin contaminated maize into the diets in both experiments, while a significantly decreased feed to gain ratio was found in experiment 1. Furthermore, the relative weight of the uterus, stomach and heart of the animals fed the contaminated maize containing diets were significantly increased. Serum albumin concentrations and the activity of GLDH were significantly reduced by the inclusion of the contaminated maize. The addition of AS to the Fusarium toxin contaminated diets did not prevent or alleviate any of the mentioned effects. Moreover, the feed intake tended to be decreased by this supplementation in both experiments, while a significantly decreased feed to gain ratio was indicated for this factor in experiment one as well. The serum concentration of albumin and the activities of ASAT and gammaGT were significantly increased if AS was present in the diets while serum concentration of cholesterol and alpha-tocopherol were decreased significantly or in tendency, respectively. The concentrations of retinol and retinyl esters in liver and serum were not altered by the treatments. The analysed concentrations of zearalenone (ZON) and its metabolites in the bile fluid clearly indicated the differences in dietary ZON concentrations and showed that AS was ineffective in preventing the absorption of the toxin from the gastrointestinal tract. Also, serum concentrations of DON reflected the DON intake prior to sampling. However, there were no differences between groups fed diets with or without AS which also suggests the inefficacy of the tested AS in preventing the DON absorption. The present investigations failed to demonstrate a detoxifying capacity of the tested additive and emphasize the general necessity for a critical verification of detoxifying agents in vivo.

Opposing actions of cellular retinol-binding protein and alcohol dehydrogenase control the balance between retinol storage and degradation.

Vitamin A homoeostasis requires the gene encoding cellular retinol-binding protein-1 (Crbp1) which stimulates conversion of retinol into retinyl esters that serve as a storage form of vitamin A. The gene encoding alcohol dehydrogenase-1 (Adh1) greatly facilitates degradative metabolism of excess retinol into retinoic acid to protect against toxic effects of high dietary vitamin A. Crbp1-/-/Adh1-/- double mutant mice were generated to explore whether the stimulatory effect of CRBP1 on retinyl ester formation is due to limitation of retinol oxidation by ADH1, and whether ADH1 limits retinyl ester formation by opposing CRBP1. Compared with wild-type mice, liver retinyl ester levels were greatly reduced in Crbp1-/- mice, but Adh1-/- mice exhibited a significant increase in liver retinyl esters. Importantly, relatively normal liver retinyl ester levels were restored in Crbp1-/-/Adh1-/- mice. During vitamin A deficiency, the additional loss of Adh1 completely prevented the excessive loss of liver retinyl esters observed in Crbp1-/- mice for the first 5 weeks of deficiency and greatly minimized this loss for up to 13 weeks. Crbp1-/- mice also exhibited increased metabolism of a dose of retinol into retinoic acid, and this increased metabolism was not observed in Crbp1-/-/Adh1-/- mice. Our findings suggest that opposing actions of CRBP1 and ADH1 enable a large fraction of liver retinol to remain esterified due to CRBP1 action, while continuously allowing some retinol to be oxidized to retinoic acid by ADH1 for degradative retinoid turnover under any dietary vitamin A conditions.

Content of retinol and retinyl esters in blood plasma, liver, kidney and reproductive organs of Japanese quails.

Due to its importance in many physiological processes such as cell proliferation and differentiation, vitamin A plays a key role in reproduction. The present study examines the content and distribution of retinol and retinyl esters in the blood plasma, liver, kidney, ovary and oviduct (infundibulum, magnum, isthmus and uterus) of the laying Japanese quail. (1) The results show that the stage of egg laying had no influence on the level of vitamin A (retinol or retinyl esters) in plasma, kidney and liver. (2) The results further indicate that in the oviduct there are quantitative and qualitative differences in the concentration of retinol and retinyl esters, but that these differences are not altered by the stage of egg formation. (3) The highest levels of vitamin A in the isthmus and uterus were associated with a predominance of retinyl esters (palmitate and stearate); sections with lower total levels of vitamin A (infundibulum, magnum) had retinol as the more dominant form of vitamin A. (4) Changes in the ratio of retinol to retinyl esters in the various sections of the avian oviduct might point to metabolic differences. The storage of vitamin A might therefore be the predominant function of the uterus and isthmus; in the infundibulum and magnum, where vitamin A is predominantly present as retinol, vitamin A serves rather as a precursor for the modulation of the cellular metabolism of these structures.

Inhibition of purified pig and human liver retinyl ester hydrolase by pharmacologic agents.

Identification of inhibitors of retinyl ester hydrolase (REH) would help to elucidate its role in vitamin A metabolism in vivo. By using standard incubation conditions, the effects of 215 drugs as potential inhibitors of purified pig and human liver REH when acting on micellar substrate retinyl palmitate were evaluated at 16.7, 167, and 1670 microM. Out of the compounds tested, 103 were inhibitors of the pig liver enzyme. The most potent compounds, in order of decreasing activity, were chloral hydrate, lovastatin, phytomenadione, alimemazine, physostigmine, thioridazine, phenoxybenzamine, probucol, cinnarizine, cyclandelate, amiodarone, flupenthixol, and naftidrofuryl; this order is roughly similar to that of their inhibition of human liver REH. Of the 10 tricyclic ring-containing drugs tested, alimemazine was the most potent enzyme inhibitor. The concentrations necessary for 50% enzyme inhibition ranged from <2.6 up to >540 microM. Moreover, inhibitory kinetic studies showed that at least two pharmaceuticals, chloral hydrate and amiodarone, are potent REH inhibitors at therapeutically achievable serum concentrations. First-pass metabolites were inactive as REH inhibitors compared to that of the parent compounds, in the cases of chloral hydrate, lovastatin, and cyclandelate.

Retinol and retinyl ester concentrations in rat tissues during total parenteral nutrition

We have previously observed that continuous total parenteral nutrition (TPN) that supplies retinyl palmitate induces a strong decrease of the circulating retinol, which is associated with an impaired hepatic production of retinol-binding protein. We have investigated the effect of 7 days of TPN on the retinol and retinyl ester concentrations in rat tissues, relative to the vitamin A status [ n = 30 for vitamin A-sufficient (A +) and n = 30 for deficient (A −)]. Rats were cannulated for intravenous feeding ( n = 12 for TPN-A +, n = 12 for TPN-A −) and were compared with their per os pair-fed counterparts ( n = 12 for O-A + and n = 12 for O-A −). Retinol and retinoic acid in serum and retinol and retinyl ester concentrations in liver, kidney, lung, heart, and testis were measured by high performance liquid chromatography. TPN induced a dramatic decrease in circulating retinol of A + rats, whereas retinoic acid concentration in serum was unchanged. When TPN was given to A − rats, retinol concentration in serum remained low. Lower retinol and retinyl ester concentrations were measured in the livers of TPN rats compared with orally pair-fed rats, no matter the initial vitamin A status ( P < 0.02 for A + and P < 0.01 for A −). By contrast, in extrahepatic tissues, retinol and retinyl ester concentrations were similar in TPN rats compared with orally pair-fed rats. Our results indicate that TPN induced a decrease of retinol in serum and retinyl esters in liver. However, TPN was able to maintain retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-sufficient rats and to restore retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-deficient rats, albeit a low circulating retinol.

Development of a compartmental model describing the dynamics of vitamin A metabolism in men.

Model-based compartmental analysis was used with the Simulation, Analysis and Modeling (SAAM) computer programs to analyze data on plasma retinoid kinetics in adult male subjects for 7 d after a single oral dose of 105 mumol of [8,9,19-13C]retinyl palmitate. We present here the data for one subject and discuss in detail the steps taken to develop a physiologically-based compartmental model that describes the dynamic behavior of plasma retinyl esters, [12C]retinol, [8,9,19-13C]retinol, and the sum of [12C] and [13C]retinol. First an absorption model was developed to fit data on the plasma appearance and disappearance of retinyl esters; this was used as input in development of models for labeled and unlabeled retinol. The large oral load of labeled vitamin A perturbed the unlabeled tracee system, and thus parallel models for tracer and tracee were developed; and a time-variant fractional transfer coefficient was incorporated into the tracee model. Following the absorption model, four-compartment models were developed to describe the dynamics of both labeled and unlabeled retinol. These models predict that, in spite of the large vitamin A load, the absorption efficiency was 34%; hepatic (presumably parenchymal cell) processing of the absorbed dose was essentially complete by 24 h; and, by 7 days, approximately 80% of the absorbed dose was in a compartment that presumably represents stored liver retinyl esters. The model also predicts that approximately 50 mumol of retinol passed through the plasma each day, compared to an estimated utilization rate of 4 mumol/day. This project provides unique and important information about whole-body vitamin A dynamic in humans, and presents approaches to specific modeling issues that may be encountered by others.

Multiple retinoids alter liver bile salt-independent retinyl ester hydrolase activity, serum vitamin A and serum retinol-binding protein of rats

Liver bile salt-independent retinyl ester hydrolase (BSI-REH) has been suggested to play a significant role in the hydrolysis of chylomicron derived retinyl esters. Studies were conducted to investigate the individual effects of N-(4-hydroxyphenyl)retinamide (HPR), retinoic acid, 13- cis-retinoic acid, Acitretin and Temarotene on BSI-REH, serum retinol, and serum retinol-binding protein (RBP) concentrations. We have demonstrated that micromolar concentrations of HPR, retinoic acid, 13- cis-retinoic acid or Acitretin significantly reduced the in vitro hydrolysis of retinyl palmitate. In contrast, Temarotene stimulated retinyl palmitate hydrolysis by BSI-REH. Retinoic acid and 13- cis-retinoic acid produced transient, but significant, depressions of both serum retinol and RBP concentrations, when the individual retinoids were administered orally to rats. The duration of the depression was shorter than we previously observed with acute HPR administration. Furthermore, Acitretin appeared to function with bimodal activity, producing significant depressions of serum retinol at 2 h and 24 h. No effect of Acitretin or Temarotene on serum RBP concentration was observed. The alterations observed in BSI-REH activity, serum retinol and RBP concentrations provide evidence that these retinoids can alter liver retinyl ester hydrolysis, but the effects observed on serum retinol concentration can only be partially explained by the BSI-REH activity.

Hydrolysis of retinyl esters in rat liver. Description of a lysosomal activity

When incorporated into liposomes made of phospholipids, retinyl palmitate is an adequate substrate for an acidic REH (aREH). In rat liver, this activity is mainly localized in the lysosomal fraction. Kinetic parameters have been determined for retinyl palmitate ( K m = 315 μM; maximal rate = 22.1 nmol retinol/h per mg protein). The aREH activity is different from the lysosomal acidic cholesteryl ester hydrolase (aCEH): cholesteryl oleate does not inhibit aREH activity, neither do some aCEH specific inhibitors, and aREH does not hydrolyse cholesteryl ester. Involvement of aREH in the hydrolysis of lipid droplets retinyl esters in fat storing cells is discussed.

Retinol and retinyl esters in rabbit bone marrow and blood leukocytes.

Due to the well documented effects of retinoids on growth and differentiation of some leukaemic cells in vivo and in vitro, we measured the amount of retinol and retinyl esters in bone marrow, blood leukocytes, and liver in rabbits fed large doses of retinyl palmitate. Both Chinchilla rabbits and Watanabe Heritable Hyperlipidaemic rabbits which lack functional low density lipoprotein receptors, were fed 26 mumoles (25.000 IU) of retinyl palmitate daily for 8 weeks. The animals stored retinoids in large amounts in the liver, whereas only minor amounts were stored in bone marrow. More than 97% of the retinoids in the liver was esterified, while most of the retinoids in bone marrow were unesterified. We also studied the post-prandial increase in chylomicron associated retinyl esters in rabbit leukocytes in vivo. After administering an oral load of 26 mumoles retinyl palmitate, retinoids increased four-fold in blood leukocytes after 5 h. There was almost no difference in retinoid uptake in leukocytes in Watanabe Heritable Hyperlipidaemic rabbits compared to normal rabbits, suggesting that chylomicron remnant retinyl esters are taken up in peripheral blood leukocytes independently of the low density lipoprotein receptor. In conclusion, bone marrow does not store high amounts of retinoids, and retinyl ester transport and storage appear normal in absence of functional low density lipoprotein receptors.