Retinol Esters Research Articles

Hepatic lecithin: Retinol acyltransferase activity is induced in vivo by retinoic acid, but not by triiodothyronine, in vitamin A-deficient, hypothyroid rats

The activity of the enzyme lecithin; retinol acyltransferase (LRAT) is extremely low in the liver of vitamin A-deficient rats, but is rapidly induced after administration of retinoic acid (RA). The nuclear receptors for RA are closely related to the receptors for thyroid hormone, and molecular cross-talk between these receptors has been observed in vitro and in cultured cells. Therefore we have examined whether retinoid status and thyroid hormone status interact in the regulation of hepatic LRAT activity in vivo. Vitamin A-deficient male Lewis rats, either euthyroid or made hypothyroid by treatment with propylthiouracil, were treated once or three times with 20 μg of all- trans-RA, 10 μg/100 gm body weight of triiodothyronine (T 3), or both hormones. Hepatic LRAT activity, which was negligible in all retinoid-deficient rats, was induced by RA ( P < 0.0001) regardless of the animal's thyroid hormone status. T 3 by itself had no ability to induce hepatic LRAT activity ( P = 0.42), nor did co-administration of T 3 with RA increase or decrease the response to RA ( P = 0.13). In retinoid-sufficient rats, hypothyroidism did not alter hepatic LRAT activity; however, LRAT activity was reduced by half ( P < 0.05) after three treatments with T 3. Therefore we conclude that thyroid hormone status alone does not regulate LRAT, nor does thyroid status affect the ability of RA to induce hepatic LRAT in retinoid-deficient animals. However, in retinoid-sufficiency, repeated treatment with T 3 may reduce the hepatic esterification of retinol by LRAT.

Beta-Carotene in breast milk and serum is increased after a single beta-carotene dose

Normal lactating mothers were administered a single dose of 60 or 210 mg beta-carotene and changes in serum and milk retinol, alpha-tocopherol, and carotenoids were monitored for 8 d. Average serum beta-carotene concentrations increased 4.1- and 4.0-fold after the 60- and 210-mg doses, respectively. Milk beta-carotene concentrations increased 4.1- and 3.0-fold after the 60- and 210-mg doses, respectively. Maximum serum concentrations were reached 24 h after both supplements, although concentrations of milk beta-carotene continued to rise for 2-3 d. After 8 d, both serum and milk beta-carotene continued to rise for 2-3 d. After 8 d, both serum and milk beta-carotene concentrations remained about twofold higher than baseline concentrations. Increases in serum or milk beta-carotene concentrations were not dose-dependent. Initial serum and milk concentrations of beta-carotene predicted increases after supplementation, and increases in serum beta-carotene concentrations predicted those in milk. Concentrations of milk carotenoids were less than one-tenth their respective concentrations in serum. Lutein, beta-cryptoxanthin, lycopene, alpha-carotene, retinol, and alpha-tocopherol concentrations in serum or milk did not change significantly after beta-carotene supplementation. Retinol esters account for most of the retinol equivalents in the milk of well-nourished mothers. Initial and maximum concentrations of beta-carotene in serum and milk were strongly correlated for individual mothers. Collectively, the data showed that a single 60-mg supplement of beta-carotene sustained elevated beta-carotene concentrations in serum and milk for > 1 wk in normal mothers but did not affect concentrations of other major carotenoids, retinol, or alpha-tocopherol.

Retinyl ester storage is altered in liver stellate cells and in HL60 cells transfected with cellular retinol-binding protein type I

It is suggested that cellular retinol-binding proteins are important for intracellular metabolism of retinol. Retinol bound to cellular retinol-binding proteins may be esterified with long chain fatty acids by the enzyme lecithin: retinol acyltransferase or may be oxidized to retinoic acid metabolites used in the mechanism of action of vitamin A. The aim of this present report was to determine whether altered levels of cellular retinol-binding protein type I influenced retinol storage and activation. Two different cell types have been examined after transfection with vectors producing sense or antisense mRNA for cellular retinol-binding protein type 1. When HL60 cells were transfected with the expression vector for sense cellular retinol-binding protein type I high amounts of cellular retinol-binding protein type I mRNA and protein were produced. We observed that HL60 cells esterified less retinol than control cells without cellular retinol-binding protein type I. Cellular retinol-binding protein type I had, however, no effects on the proliferation or differentiation of HL60 cells by retinoids. Liver stellate cells transfected with the vector for sense cellular retinol-binding protein type I esterified more retinol than cells transfected with the expression vector for antisense cellular retinol-binding protein type I, while retinol esterification in control cells was intermediate. In conclusion, our data show that cellular retinol-binding protein type I influences retinol esterification both in liver stellate cells and in HL60 cells.

Effects of all-trans-retinoyl-beta-D-glucuronide and all-trans-retinoic acid on chondrogenesis and retinoid metabolism in mouse limb bud mesenchymal cells in vitro.

Retinoids, derivatives of vitamin A, are essential for many vertebrate functions. Furthermore, several drugs of this class of compounds are valuable in the treatment of certain forms of skin disorders and cancer. However, the therapeutic application of retinoids is limited by their teratogenic potency. The limbs are important sites of retinoid-induced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratogenic potency of retinoids. We have now applied this system to compare the effects of all-trans-retinoyl-beta-D-glucuronide (all-trans-RAG) with those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscopic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid concentrations in the cultured mesenchymal limb bud cells were established. While all-trans-RA inhibited chondrogenesis at 10(-7) M by ca. 50%, tenfold higher concentrations of all-trans-RAG were necessary to obtain the same effect. This difference reflects the ratio of RA isomers which were found in the medium after incubation with either all-trans-RAG or all-trans-RA. A pulse experiment (10(-5) M all-trans-RAG or all-trans-RA for the first 2 h of a 6-day incubation period) demonstrated inhibition of chondrogenesis with all-trans-RA, but not with all-trans-RAG. The data indicate that RAG inhibits chondrogenesis upon hydrolysis to RA. Surprisingly, the rather polar RAG isoforms were extensively accumulated in the limb bud mesenchymal cells when compared to the medium. Both all-trans-RAG and all-trans-RA also induced a large increase of retinyl ester concentrations in the chondrocytes compared to vehicle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of the esterification of retinol.

Extraction of Human Epidermis Treated with Retinol Yields Retro-Retinoids in Addition to Free Retinol and Retinyl Esters

Vitamin A, all-trans-retinol, is metabolized to retinoic acid in vivo by a tightly controlled two-step conversion. Retinoic acid then binds to nuclear receptors and modulates cellular proliferation and differentiation. Because only a small fraction of retinol applied topically can be metabolized to retinoic acid, alternative pathways of retinol metabolism in skin were investigated. Retinol (0.4%) was applied to adult human skin under occlusion for 6 h to 4 d. The conversion of retinol into various metabolites such as 14-hydroxy-4,14-retro-retinol, anhydroretinol, 4-oxo-retinol, retinyl esters, and retinyl glucuronides was investigated. The level of 14-hydroxy-retro-retinol was increased from undetectable at time 0 to 326 ng/g wet weight of tissue at 6 h (6% of the retinol level) and maintained approximately the same concentration at 24 h to 409 ng/g wet weight (1.9% of the retinol level); it decreased to 48 ng/g wet weight of tissue (12% of its maximum level) by 4 d. Anhydroretinol was undetectable at time 0, increased only slightly at 6 h, and remained at the same level. We did not detect 4-oxo-retinol. Because 14-hydroxy-retro-retinol was found in the retinol-treated areas, its effects on epidermis were compared with those of retinol. Topical application of trans-retinol (0.3%) significantly increased both epidermal thickness and cellular retinoic acid binding protein II mRNA, whereas 14-hydroxy-4,14-retro-retinol (0.3%) did not increase either of these well-characterized cutaneous retinoid responses. Retinol, when applied topically in pharmacologic doses to human epidermis, remained as free retinol, was metabolized primarily to retinol ester, and was metabolized to a lesser extent to retro-retinoids and didehydroretinol.

Auto-regulation of Retinoic Acid Biosynthesis through Regulation of Retinol Esterification in Human Keratinocytes

In this report, we describe an auto-regulatory loop in human keratinocytes, whereby all-trans retinoic acid (retinoic acid) regulates its own biosynthesis from all-trans retinol (retinol) through regulation of retinol esterification. Retinol esterification activity was low in normal proliferating human keratinocytes, cultured in retinoid-free media. Treatment of keratinocytes with retinoic acid induced retinol esterifying activity (8-fold). Induction of retinol esterifying activity was blocked by either actinomycin D or cycloheximide. Based on substrate specificity and inhibitor sensitivity, lecithin:retinol acyltransferase (LRAT) was identified as the retinoic acid-inducible retinol esterifying enzyme. Induction of LRAT by retinoic acid reduced conversion of retinol to retinoic acid by 50%. This reduction in retinoic acid synthesis resulted from sequestration of retinol as retinyl esters, since inhibition of LRAT restored retinoic acid synthesis to control levels. In normal human skin, undifferentiated keratinocytes, in the lowest cell layer, esterified retinol 4 times greater, than differentiating keratinocytes, in upper cell layers, reflecting an induced state, under conditions of retinol sufficiency. Regulation of LRAT activity by retinoic acid provides a novel mechanism through which retinoic acid can regulate its own level by controlling availability of retinol for conversion to retinoic acid. In human skin in vivo, retinyl esters synthesized in basal keratinocytes could undergo hydrolysis during differentiation and thus serve as a source of retinol for keratinocytes in upper layers of skin.

Early effects of high doses of retinol (vitamin A) on the in situ cellular metabolism in rat liver.

Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.

Retinoids in clinical cancer therapy.

Retinoids constitute a class of naturally occurring and synthetic compounds that are structurally related to vitamin A. The chemical structure of retinoids consists of a cyclic end group (trimethylcyclohexenyl ring) and a dimethyl substituted tetraene chain, ending in a polar hydroxyl, aldehyde, or carboxyl group. Naturally occurring retinoids include vitamin A (all-trans retinol) and its oxidative metabolites: retinaldehyde, all-trans-retinoic acid (tRA), and 13-cis-retinoic acid (13-cRA). Four additional naturally occurring retinoids have been newly discovered in the past 5 years: 9-cis-retinoic acid (9-cRA) [1,2], 3,4-didehydroretinoic acid [3], 14-hydroxy retro-retinol [4], and more recently, 9-cis, 13-cis-retinoic acid [5,6]. The potential for the existence of 11-cis and 15-cis-retinoic acid is very high [7]. Retinoic acid isomers are generated by changes in the position and chemical nature of the double bonds in the tetraene chain (Figure 1). The clinical use of retinoids in oncology has been dominated by tRA, 13-cRA, and more recently 9-cRA, which are the focus of this discussion. Esters of all-trans retinol (vitamin A), particularly retinol palmitate, have also been used as cancer chemopreventive or chemotherapeutic agents [8–10] but will not be discussed in this presentation.KeywordsRetinoic AcidAcute Promyelocytic LeukemiaRetinoic Acid ReceptorPromyelocytic Leukemia Zinc FingerSynthetic RetinoidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Lack of lecithin: retinol acyltransferase activity in chick lungs.

Our previous study revealed that no retinyl esters were detectable in chick and hen lungs, suggesting that the retinol esterification system may be absent in these tissues. This possibility encouraged us to investigate whether chick lungs exhibit the activity of a retinol esterifying enzyme, i.e., lecithin: retinol acyltransferase (LRAT). The LRAT activity was assayed with dilauroyl phosphatidylcholine and either complex of retinol-cellular retinol-binding protein, type two or retinol-cellular retinol-binding protein in microsomal preparations of lung, duodenum and liver of 7-day-old chicks. Relatively high levels of LRAT activity were present in the duodenum and the liver of chicks as well as in the rat lung. However, the chick lung exhibited no LRAT activity. The lungs of both rat and chick showed similar and low levels of acyl-CoA: retinol acyltransferase (ARAT) activity, but only rat lung, but not chick lung, contained a detectable amount of retinyl esters. Thus, the retinyl ester storage in the lung seems to depend on the presence of LRAT activity in the lung, but it is independent of the presence of ARAT activity in the lung. The absence of LRAT activity and retinyl esters in the chick lung suggests that the retinol in the chick lung may not be provided from retinyl ester storage, and the retinol transferred directly from serum should be utilized to generate retinoic acid.

Application of a simple serology based genotyping assay in a prospective cohort of patients with chronic hepatitis C: [formula omitted] California Pacific Medical Center, San Franscio, CA; Section of Hepatobiliary Diseases 1, University of Florida, Gainesville, FL, and Murex Diagnostics 2, Kent, UK

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.

Evaluation of mucosal blood flow in portal hypertensive gastropathy using endoscopic laser-doppler flowmetry: [formula omitted] Department of Internal Medicine and Gastroenterology, University of Bologna — Italy

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.

Simultaneous determination of retinyl esters and retinol in human livers by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method for the simultaneous determination of retinol and retinyl esters in human liver samples is presented. The free retinol and the prevalent retinyl esters (retinyl palmitate, oleate and stearate) are resolved within less than 30 min, using an octasilyl (C8)-substituted column and an isocratic elution with methanol-water as mobile phase. This method allows to determine in duplicate all retinyl ester concentrations in small liver samples (3-10 mg of fresh tissue). The results obtained from thirteen patients without liver disease are described.

Metabolic Regulation of Active Retinoid Concentrations in Cultured Human Epidermal Keratinocytes by Exogenous Fatty Acids

Retinol metabolism was examined in cultured human epidermal keratinocytes incubated with medium containing physiological concentrations of bovine serum albumin-free fatty acid complexes and [3H]retinol. Unsaturated 16- and 18-carbon fatty acids: (1) promoted up to a 50% increase in the steady state total cell retinoid mass which was accounted for as retinyl ester corresponding to the added fatty acid, (2) decreased the rate of endogenous retinyl ester utilization up to 80%, (3) decreased the steady state cellular concentrations of retinol, 3,4-didehydroretinol and their respective carboxylic acids up to 80%, and (4) did not alter the rate of retinoic acid metabolism. Increasing the medium retinol concentration augmented the fatty acid-stimulated increase in esters and blunted the extent of decrease in alcohols and acids. Saturated fatty acids with 14, 16, 17, and 18 carbons also reduced the rate of retinyl ester utilization and the cellular concentrations of the retinoid alcohols and acids but had little to no effect on retinyl ester synthesis. The data demonstrate that exogenous fatty acids possess the capacity to regulate the cellular concentrations of substrate retinoid alcohols, active retinoid acids, and the overall rate of retinol metabolism via substrate-mediated stimulation of retinol esterification and decreasing retinyl ester utilization. Fatty acids thus possess the potential to play a physiological role in modulating retinoid signaling in keratinocytes by regulating cellular concentrations of active retinoids.

Simultaneous Determination of Retinol Esters and Tocochromanols in Foods Using Nitro‐Column HPLC

AbstractAll food components with vitamin A and E activity, i.e. all‐trans retinol. its cis isomers and their esters as well as the four tocopherols. the four tocotrienols, and α‐tocopheryl acetate, were separated by HPLC using a nitro‐column and a three‐step gradient elution with n‐hexane and terα‐butylmethylether mixtures. β‐carotene and the retinol isomers were determined with an UV/Vis detector, all other compounds with a fluorescence detector connected in series. Fats, oils, and lipid extracts from foods can he injected onto the HPLC column without any clean‐up.

Effects of polyhalogenated aromatic hydrocarbons (PHAHs) on biochemical parameters in chicks of the common tern ( Sterna hirundo)

Common tern ( Sterna hirundo) eggs were collected from eight breeding colonies that differed in degree of polyhalogenated aromatic hydrocarbon (PHAH) pollution. The eggs were artificially incubated and the chicks sacrificed within 12 hours after hatching. Plasma thyroid hormones (total thyroxine (TT4), free thyroxine (FT4) and triiodothyronine (TT3)), hepatic T4-glucuronyltransferase (T4-UGT) activity, plasma retinol and yolksac retinoid levels were measured and compared with yolksac PHAH residues and hepatic ethoxyresorufin- and pentoxyresorufin- O-deethylase (EROD and PROD) activities. The yolksac PHAH residues measured were polychlorinated biphenyl (PCB), polychlorinated dibenzofuran (PCDF) and polychlorinated dibenzo- p-dioxin (PCDD). No significant differences were observed between colony average levels of plasma thyroid hormones (TT4, FT4 and TT3) nor plasma retinol levels and T4-UGT activities that could be related to colony average PHAH levels. However, average colony yolksac retinyl ester levels did show significant differences. When correlated for individual terns, significant correlations between all these parameters and PHAH levels or EROD or PROD activities were found. The correlation between hepatic T4-UGT and EROD and PROD activities was very strong, suggesting a concomitant induction of these drug metabolizing enzymes. The PHAH levels were expressed as dioxin equivalents (TEQs) based on toxic equivalence factors (TEFs) as proposed by Safe (1990, 1994). In addition to plasma retinol, another retinoid was found which resembles 3,4-didehydro-retinol (vitamin A2) in chromatographic and spectroscopic behaviour. This putative plasma vitamin A2 was significantly positive correlated with the yolksac TEQs and with the hepatic EROD activity. Esters of both retinol and putative vitamin A2 were detected in the yolksac. The average yolksac vitamin A2 levels of the cleanest Zeewolde colony were significantly higher than the average levels in the other colonies.

Lipoprotein lipase hydrolysis of retinyl ester. Possible implications for retinoid uptake by cells

Adipose tissue contains substantial stores of retinoid (retinol+retinyl ester) that, quantitatively, are second only to retinoid stores in the liver. Our studies show that retinoid levels in adipose tissue are markedly influenced by dietary retinoid intake. Because lipoprotein lipase (LPL) increases the uptake of lipoproteins and lipid emulsion particles by many cell types including adipocytes, we investigated whether LPL also increases retinoid uptake by adipocytes from lipid-containing particles. Addition of LPL (10 micrograms/ml) to BFC-1 beta adipocytes produced a 2-fold increase in cellular uptake of [3H]retinoid from a lipid emulsion containing [3H]retinyl ester. Heparin, which displaces LPL from binding sites on cell surface proteoglycans, increased [3H]retinoid uptake by an additional 2-fold. High performance liquid chromatography analyses showed that greater than 75% of the media and 85% of the cellular radioactivity was present as retinol. The conversion of retinyl ester to retinol by LPL was then assessed using model retinyl ester containing lipid emulsions. Although triglyceride appears to be the preferred substrate for LPL, after greater than 25% of the triglyceride was hydrolyzed, significant amounts of retinyl ester were hydrolyzed by LPL. Retinyl ester hydrolysis was increased approximately 20-fold in the presence of a source of apolipoprotein C-II. The physiologically significant palmitate, stearate, oleate, and linoleate esters of retinol were all hydrolyzed by LPL. When LPL was incubated with [3H]retinyl ester containing rabbit mesenteric chylomicrons and in the presence of heparin and apolipoprotein C-II, the LPL was able to completely hydrolyze the retinyl ester to retinol. Thus, LPL is able to catalyze the hydrolysis of retinyl esters and, through the process of hydrolysis, may facilitate uptake of retinoid by adipocytes.

Amount and quality of dietary protein regulate lecithin:retinol acyltransferase activity without change in cellular retinol-binding protein, type two in rat jejunum

The effects of amount and quality of dietary protein on the cellular retinol-binding protein (type two) and two enzymes for retinol esterification, i.e., lecithin:retinol acyltransferase and acyl-CoA:retinol acyltransferase in rat jejunum were examined. Three groups of five rats were pair-fed defined diets for 14 days. The control group was fed a diet containing 20% casein; the two experimental groups received diets containing 5% casein or 20% gluten. Feeding the 5% casein diet or the 20% gluten diet decreased serum concentration of retinol and retinol-binding protein and increased liver total retinol and retinol-binding protein contents. The lecithin:retinol acyltransferase activity in jejunal microsomes, which was determined using retinol bound to cellular retinol-binding protein (type two), was significantly reduced in animals fed the 20% gluten diet (by 30%) as compared with animals fed the 20% casein diet. Feeding the 5% casein diet also led to a slight decrease in the segmental lecithin:retinol acyltransferase activity. The acyl-CoA:retinol acyltransferase activity was unaffected by feeding these diets. The jejunal cytosolic cellular retinol-binding protein (type two) content was also unchanged by feeding these diets. Feeding the 20% wheat gluten diet resulted in remarkable low levels of the unesterified retinol (by 64%), retinyl palmitate (by 32%), and protein (by 47%) in the whole jejunal mucosa, when compared with the corresponding values of the 20% casein group. These results suggest that the dietary protein malnutrition will suppress intestinal esterification of retinol absorbed by a decline of lecithin:retinol acyltransferase activity and without change in intestinal cellular retinol-binding protein (type two) level. The results also suggest that retinol uptake may decrease due to an impaired absorption process in protein malnutritional status.

Retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and four retinyl esters in serum determined simultaneously by reversed-phase HPLC with multiwavelength detection

We describe the use of HPLC with multiwavelength detection to measure retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, beta-carotene, and the linoleate, oleate, palmitate, and stearate esters of retinol in a single 200-microL serum sample. The method is sensitive enough to detect individual retinyl esters in fasting serum from a nonhyperlipidemic population and requires only 12 min for each sample. Serum concentration ranges and means are reported for retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and the sum of the retinyl esters from serum analyses of 3480 participants from several different studies.

Regulation of Vitamin A Transport into Cultured Stellate Cells of Rat Liver: Studies by Anchored Cell Analysis and Sorting System

Stellate cells (SC) in the liver store the most retinoid in the body, but the mechanisms of specific retinoid transport into SC remain to be elucidated. In this study, to analyze the retinoid content of cultured SC, we employed an anchored cell analysis and sorting system (ACAS), which provides fluorescence analysis of single cultured cells under the phase-contrast microscope by utilizing a laser. First, we examined the effect of retinol binding protein (RBP) on retinol transport into cultured SC of rat liver. Rat holo-RBP added to the medium inhibited retinol uptake into SC. We also prepared RBP-free human serum by affinity chromatography using conjugated anti-human RBP IgG and compared retinoid fluorescence of SC cultured in human serum with or without RBP. No significant difference in retinoid fluorescence intensity was observed between SC cultured with and without holo-RBP. Second, the removal of cellular retinol by esterification may be important for the continued uptake of retinol. Retinyl esters are stored in lipid droplets of SC. Therefore we examined the relationship between the lipid droplet number and the retinoid fluorescence intensity in SC which were cultured in medium containing retinol for 1-3 days. The increases in lipid droplet number and in retinoid fluorescence in SC were almost parallel. Progesterone, previously shown to increase the esterification of retinol by lecithin:retinol acyltransferase (LRAT) in vitro, was added to the SC medium; progesterone facilitated retinol uptake in cultured SC. In conclusion, RBP did not facilitate specific retinol transport into SC. However, the specific transport of retinol is likely to be dependent on the intracellular esterification of retinol by LRAT in SC.

Retinol and retinyl esters in rabbit bone marrow and blood leukocytes

Retinol and retinyl esters in rabbit bone marrow and blood leukocytes