Early activation of hepatic stellate cells induces rapid initiation of retinyl ester breakdown while maintaining lecithin:retinol acyltransferase (LRAT) activity.

Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release.

Vitamin D Maintains Growth and Bone Mineral Density against a Background of Severe Vitamin A Deficiency and Moderate Toxicity in a Swine Model.

Excessive vitamin A (VA) negatively impacts bone. Interactions between VA and vitamin D (VD) in bone health are not well-understood. This study used a traditional two-by-two factorial design. Pigs were weaned and randomized to four treatments (n = 13/group): -A-D, -A+D, +A-D, and +A+D for 3 and 5 wk. Serum, liver, kidney, adrenal glands, spleen, and lung were analyzed by ultra-performance LC. Growth was evaluated by weight measured weekly and BMD by DXA. Weights were higher in -A+D (18.1 ± 1.0 kg) and +A+D (18.2 ± 2.3 kg) at 5 wk than in -A-D (15.5 ± 2.1 kg) and +A-D (15.8 ± 1.5 kg). Serum retinol concentrations were 0.25 ± 0.023, 0.22 ± 0.10, 0.77 ± 0.12, and 0.84 ± 0.28 µmol/L; and liver VA concentrations were 0.016 ± 0.015, 0.0065 ± 0.0035, 2.97 ± 0.43, 3.05 ± 0.68 µmol/g in -A-D, -A+D, +A-D, and +A+D, respectively. Serum 25(OH)D3 concentrations were 1.5 ± 1.11, 1.8 ± 0.43, 27.7 ± 8.91, and 23.9 ± 6.67 ng/mL in -A-D, +A-D, -A+D, +A+D, respectively, indicating a deficiency in -D and adequacy in +D. BMD was highest in +D (p < 0.001). VA and the interaction had no effect on BMD. Dietary VD influenced weight gain, BMD, and health despite VA status.

Repeated High-Dose Vitamin A Supplements, Standard of Care for Treating Xerophthalmia, Leads to Hypervitaminosis A in Piglets.

BACKGROUND: Vitamin A (VA) deficiency and excess negatively affect development, growth, and bone health. The World Health Organization's standard of care for xerophthalmia due to VA deficiency, is 3 high-dose VA supplements of 50,000-200,000 IU, based on age, which may cause hypervitaminosis A in some individuals. OBJECTIVES: This study measured VA status following 3 VA doses in 2 piglet studies. METHODS: In Study 1, 5 groups of piglets (n = 10/group) were weaned 10 d postbirth to VA-free feed and orally administered 0; 25,000; 50,000; 100,000; or 200,000 IU VA ester on days 0, 1, and 7. On days 14 and 15, the piglets underwent the modified relative dose-response (MRDR) test for VA deficiency, and were killed. Tissues were collected for high-pressure liquid chromatography analysis. Study 2 used the same design in 3 groups (n = 13/group) weaned at 16 d and administered 0; 25,000; and 200,000 IU doses. RESULTS: In Study 1 (final weight: 3.6 ± 0.7 kg), liver VA concentration was hypervitaminotic in 40%, 90%, and 100% of 50,000; 100,000; and 200,000 IU groups, respectively. The 25,000 IU group was 100% adequate, and the placebo group was 40% deficient. In Study 2 (final weight: 8.7 ± 0.8 kg), where 200,000 IU could be prescribed to infants with a similar body weight, 31% of the piglets were hypervitaminotic, the 25,000 IU group was 100% VA adequate, and the placebo group was 100% deficient. The MRDR test measured deficiency in 50% and 70% of the placebo group in each study but had 3 false positives among hypervitaminotic piglets in Study 1. CONCLUSIONS: Repeated high-dose VA may cause hypervitaminosis, indicating dose sizes may need reduction. The MRDR resulted in false positives in a hypervitaminotic state during malnutrition and should be paired with serum retinyl ester evaluation to enhance VA status assessment in populations with overlapping interventions.

Fatty acid/monoglyceride type and amount modulate fat-soluble vitamin absorption from mixed assemblies in mice.

We investigated the effects of fatty acid/ monoglyceride type and amount on the absorption of fat-soluble vitamins. Micelles or vesicles made with either caprylic acid (CA) + monocaprylin (MC) or oleic acid (OA) + monoolein (MO) at low or high concentrations were infused in bile duct-ligated mice. Retinol + retinyl ester and γ-tocopherol intestinal mucosa contents were higher in mice infused with CA + MC than with OA + MO (up to + 350 % for vitamin A and up to + 62 %, for vitamin E; p < 0.05). Cholecalciferol intestinal mucosa content was the highest in mice infused with micelles with CA + MC at 5 mg/mL (up to + 105 %, p < 0.05). Retinyl ester plasma response was higher with mixed assemblies formed at low concentration of FA + MG compared to high concentration (up to + 1212 %, p < 0.05), while no difference in cholecalciferol and γ-tocopherol plasma responses were measured. No correlation between size or zeta potential and vitamin absorption was found. The impact of FA and MG on fat-soluble vitamin absorption thus differs from one vitamin to another and should be considered to formulate adequate vitamin oral or enteral supplements.

Retinyl Ester Analysis by Orbitrap Mass Spectrometry.

Retinoids are light-sensitive molecules that are normally detected by UV absorption techniques. Here we describe the identification and quantification of retinyl ester species by high-resolution mass spectrometry. Retinyl esters are extracted by the method of Bligh and Dyer and subsequently separated by HPLC in runs of 40 min. The retinyl esters are identified and quantified by mass spectrometry analysis. This procedure enables the highly sensitive detection and characterization of retinyl esters in biological samples such as hepatic stellate cells.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as ß-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15'-monooxygenase (BCO1), to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.

ß-Carotene Bioavailability and Conversion Efficiency Are Significantly Affected by Sex in Rats: First Observation Suggesting a Possible Hormetic Regulation of Vitamin A Metabolism in Female Rats.

SCOPE: To study the effect of variation in dietary vitamin A (VA) content on its hepatic and intestinal metabolism. METHODS AND RESULTS: Adult female and male rats are fed with diets containing 400, 2300, or 9858 IU kg-1 VA for 31-33 weeks. VA concentrations are measured in plasma and liver. Bioavailability and intestinal conversion efficiency of ß-carotene to VA are assessed by measuring postprandial plasma ß-carotene and retinyl palmitate concentrations after force-feeding rats with ß-carotene. Expression of genes involved in VA metabolism, together with concentrations of RBP4, BCO1, and SR-BI proteins, are measured in the intestine and liver of female rats. Plasma retinol concentrations are lower and hepatic free retinol concentrations are higher in females than in males. There is no effect of dietary VA content on ß-carotene bioavailability and its conversion efficiency, but bioavailability is higher and conversion efficiency is lower in females than in males. The expression of most genes exhibited a U-shaped dose response curve depending on VA intake. CONCLUSIONS: ß-Carotene bioavailability and conversion efficiency to VA are affected by the sex of rats. Results of gene expression suggest a hormetic regulation of VA metabolism in female rats.

Vitamin A Deficiency during the Perinatal Period and First Weeks of Life Modifies Vitamin A and Lipid Postprandial Metabolism in Both Female and Male Young Rats.

SCOPE: The effect of vitamin A deficiency on vitamin A and lipid postprandial metabolism in young rats is addressed, considering the effect of sex. METHODS AND RESULTS: Sprague-Dawley rats are fed either 400 UI.kg-1 vitamin A diet (vitamin A-deficient (VAD) diet) or 2300 UI.kg-1 vitamin A (control diet), before being mated. Mothers receive the same VAD or control diet during gestation and lactation. Offspring receive the same diet than mothers until 8 weeks of age. VAD diet-fed female and male offspring display a severe vitamin A deficiency with no body weight or glucose tolerance defects. Fasting plasma triglyceride concentrations are decreased in VAD diet-fed animals compared to controls (p < 0.05). Retinyl ester postprandial responses after vitamin A gavage, expressed as area under the curves, are not different in VAD diet-fed and control animals, although retinyl ester postprandial peak is significantly delayed (p < 0.05) in VAD diet-fed rats. Lipids also accumulate in the distal part of the intestine after gavage and [1-13 C]-oleate postprandial response is decreased in VAD diet-fed males. CONCLUSION: Vitamin A deficiency modulates both vitamin A absorption rate and lipid postprandial metabolism, which can partly explain the altered fasting lipid status observed in VAD diet-fed offspring.

Vitamin A deficiency has declined in Malawi, but with evidence of elevated vitamin A in children.

BACKGROUND: Reduction of vitamin A deficiency (VAD) in Malawi coincided with introduction of vitamin A-fortified staple foods, alongside continued biannual high-dose vitamin A supplementation (VAS). OBJECTIVE: We describe coverage of vitamin A interventions and vitamin A status in the 2015-2016 Malawi Micronutrient Survey. METHODS: Food samples and biospecimens were collected within a representative household survey across 105 clusters. Retinol was measured using ultraviolet excitation fluorescence (sugar) and photometric determination (oil). Preschool children (PSC, aged 6-59 mo, n = 1102), school-age children (SAC, aged 5-14 y, n = 758), nonpregnant women (n = 752), and men (n = 219) were initially assessed for vitamin A status using retinol binding protein (RBP) and modified relative dose response (MRDR). Randomly selected fasted MRDR participants (n = 247) and nonfasted women and children (n = 293) were later assessed for serum retinol, retinyl esters, and carotenoids. Analyses accounted for complex survey design. RESULTS: We tested sugar and oil samples from 71.8% and 70.5% of the households (n = 2,112), respectively. All of the oil samples and all but one of the sugar samples had detectable vitamin A. National mean retinol sugar and oil contents were 6.1 ± 0.7 mg/kg and 6.6 ± 1.4 mg/kg, respectively. Receipt of VAS in the previous 6 mo was reported by 68.0% of PSC. VAD prevalence (RBP equivalent to <0.7µmol retinol/L) was 3.6% in PSC, and <1% in other groups. One woman and no children had MRDR ≥0.060 indicating VAD. Among fasted PSC and SAC, 18.0% (95% CI: 6.4, 29.6) and 18.8% (7.2, 30.5) had >5% of total serum vitamin A as retinyl esters, and 1.7% (0.0, 4.1) and 4.9% (0.0, 10.2) had >10% of total serum vitamin A as retinyl esters. Serum carotenoids indicated recent intake of vitamin A-rich fruits and vegetables. CONCLUSIONS: Near elimination of VAD in Malawi is a public health success story, but elevated levels of vitamin A among children suggests that vitamin A interventions may need modification.

Vitamin A Absorption Efficiency Determined by Compartmental Analysis of Postprandial Plasma Retinyl Ester Kinetics in Theoretical Humans.

BACKGROUND: Better methods are needed for determining vitamin A absorption efficiency in humans to support development of dietary recommendations and to improve the accuracy of predictions of vitamin A status. OBJECTIVES: We developed and evaluated a method for estimating vitamin A absorption efficiency based on compartmental modeling of theoretical data on postprandial plasma retinyl ester (RE) kinetics. METHODS: We generated data on plasma RE and retinol kinetics (30 min to 8 h or 56 d, respectively) after oral administration of labeled vitamin A for 12 theoretical adults with a range of values assigned for vitamin A absorption (55-90%); we modeled all data to obtain best-fit values for absorption and other parameters using Simulation, Analysis, and Modeling software. We then modeled RE data only (16 or 10 samples), with or without added random error, and compared assigned to predicted absorption values. We also compared assigned values to areas under RE response curves (RE AUCs). RESULTS: We confirmed that a unique value for vitamin A absorption cannot be identified by modeling plasma retinol tracer kinetics. However, when RE data were modeled, predicted vitamin A absorptions were within 1% of assigned values using data without error and within 12% when 5% error was included. When the sample number was reduced, predictions were still within 13% for 10 of the 12 subjects and within 23% overall. Assigned values for absorption were not correlated with RE AUC (P = 0.21). CONCLUSIONS: We describe a feasible and accurate method for determining vitamin A absorption efficiency that is based on compartmental modeling of plasma RE kinetic data collected for 8 h after a test meal. This approach can be used in a clinical setting after fasting subjects consume a fat-containing breakfast meal with a known amount of vitamin A or a stable isotope label.

Vitamin A Status and Deposition in Neonatal and Weanling Rats Reared by Mothers Consuming Normal and High-Fat Diets with Adequate or Supplemented Vitamin A.

The circulating level of vitamin A (VA; retinol) was reported to be lower in obese adults. It is unknown if maternal obesity influences the VA status of offspring. The objective of the study was to determine the VA status and deposition of neonatal and weanling rats reared by mothers consuming a normal or high-fat diet (NFD or HFD) with or without supplemented VA. Pregnant Sprague-Dawley rats were randomized to an NFD or HFD with 2.6 mg/kg VA. Upon delivery, half of the rat mothers in the NFD or HFD cohort were switched to an NFD or HFD with supplemented VA at 129 mg/kg (NFD+VA and HFD+VA group). The other half remained on their original diet (NFD and HFD group). At postnatal day 14 (P14), P25, and P35, pups (n = 4 or 3/group/time) were euthanized. The total retinol concentration in the serum, liver, visceral white adipose tissue (WAT), and brown adipose tissue (BAT) was measured. At P14, the HFD+VA group showed a significantly lower serum VA than the NFD+VA group. At P25, both the VA concentration and total mass in the liver, WAT, and BAT were significantly higher in the HFD+VA than the NFD+VA group. At P35, the HFD group exhibited a significantly higher VA concentration and mass in the liver and BAT compared with the NFD group. In conclusion, maternal HFD consumption resulted in more VA accumulation in storage organs in neonatal and/or weanling rats, which potentially compromised the availability of VA in circulation, especially under the VA-supplemented condition.

Mamey sapote fruit and carotenoid formulations derived thereof are dietary sources of vitamin A - A comparative randomized cross-over study.

Mamey sapote is a fruit rich in specific keto-carotenoids, namely sapotexanthin and cryptocapsin. Their chemical structure suggests their provitamin A activity, although their absorption and conversion to vitamin A remained to be demonstrated in humans. Besides structure-related factors, the fruit matrix might also hamper absorption and conversion efficiency. Therefore, we monitored carotenoid and vitamin A levels in triacylglycerol-rich lipoprotein (TRL) fractions in plasma of human participants after consumption of fresh sapote and a carotenoid-rich "matrix-free" formulation derived thereof. A randomized 2-way cross-over study was conducted to compare the post-prandial bioavailability of 0.8 mg sapotexanthin and 1.2-1.5 mg cryptocapsin from the above-mentioned test meals. Seven blood samples were drawn over 9.5 h after test meal consumption. Carotenoids and retinoids were quantitated in TRL fractions using HPLC-DAD. Sapotexanthin was absorbed by all participants from all meals, being ca. 36% more bioavailable from the "matrix-free" formulation (AUCmedian = 73.4 nmol∙h/L) than from the fresh fruit (AUCmedian = 54.0 nmol∙h/L; p ≤ 0.001). Cryptocapsin was only absorbed by 4 of 13 participants. The appearance of retinyl esters was observed in all participants independent of the test meal. Although the fruit matrix hampered carotenoid in vivo-bioavailability from sapote, the fruit clearly represents a valuable source of vitamin A for humans.

Disturbed Vitamin A Metabolism in Non-Alcoholic Fatty Liver Disease (NAFLD).

Vitamin A is required for important physiological processes, including embryogenesis, vision, cell proliferation and differentiation, immune regulation, and glucose and lipid metabolism. Many of vitamin A's functions are executed through retinoic acids that activate transcriptional networks controlled by retinoic acid receptors (RARs) and retinoid X receptors (RXRs).The liver plays a central role in vitamin A metabolism: (1) it produces bile supporting efficient intestinal absorption of fat-soluble nutrients like vitamin A; (2) it produces retinol binding protein 4 (RBP4) that distributes vitamin A, as retinol, to peripheral tissues; and (3) it harbors the largest body supply of vitamin A, mostly as retinyl esters, in hepatic stellate cells (HSCs). In times of inadequate dietary intake, the liver maintains stable circulating retinol levels of approximately 2 µmol/L, sufficient to provide the body with this vitamin for months. Liver diseases, in particular those leading to fibrosis and cirrhosis, are associated with impaired vitamin A homeostasis and may lead to vitamin A deficiency. Liver injury triggers HSCs to transdifferentiate to myofibroblasts that produce excessive amounts of extracellular matrix, leading to fibrosis. HSCs lose the retinyl ester stores in this process, ultimately leading to vitamin A deficiency. Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is a spectrum of conditions ranging from benign hepatic steatosis to non-alcoholic steatohepatitis (NASH); it may progress to cirrhosis and liver cancer. NASH is projected to be the main cause of liver failure in the near future. Retinoic acids are key regulators of glucose and lipid metabolism in the liver and adipose tissue, but it is unknown whether impaired vitamin A homeostasis contributes to or suppresses the development of NAFLD. A genetic variant of patatin-like phospholipase domain-containing 3 (PNPLA3-I148M) is the most prominent heritable factor associated with NAFLD. Interestingly, PNPLA3 harbors retinyl ester hydrolase activity and PNPLA3-I148M is associated with low serum retinol level, but enhanced retinyl esters in the liver of NAFLD patients. Low circulating retinol in NAFLD may therefore not reflect true "vitamin A deficiency", but rather disturbed vitamin A metabolism. Here, we summarize current knowledge about vitamin A metabolism in NAFLD and its putative role in the progression of liver disease, as well as the therapeutic potential of vitamin A metabolites.

Vitamin A supplementation redirects the flow of retinyl esters from peripheral to central organs of neonatal rats raised under vitamin A-marginal conditions.

Background: Vitamin A (VA; retinol) supplementation is used to reduce child mortality in countries with high rates of malnutrition. Existing research suggests that neonates (<1 mo old) may have a limited capacity to store VA in organs other than the liver; however, knowledge about VA distribution and kinetics in individual, nonhepatic organs is limited.Objective: We examined retinol uptake and turnover in nonhepatic organs, including skin, brain, and adipose tissue, in neonatal rats without and after VA supplementation.Design: Sprague-Dawley neonatal rats (n = 104) were nursed by mothers fed a VA-marginal diet (0.35 mg retinol/kg diet) and treated on postnatal day 4 with an oral dose of either VA (6 µg retinyl palmitate/g body weight) or canola oil (control), both containing 1.8 µCi of [3H]retinol. Subsequently, pups (n = 4 · group-1 · time-1) were killed at 13 different times from 30 min to 24 d after dosing. The fractional and absolute transfer of chylomicron retinyl esters (CM-REs), retinol bound to retinol-binding protein (RBP-ROH), and total retinol were estimated in WinSAAM software.Results: VA supplementation redirected the flow of CM-REs from peripheral to central organs and accumulated mainly in the liver. The RBP-ROH released from the liver was acquired mainly by the peripheral tissues but not retained efficiently, causing repeated recycling of retinol between plasma and tissues (541 compared with 5 times in the supplemented group and control group, respectively) and its rapid turnover in all organs, except the brain and white adipose tissue. Retinol stores in the liver lasted for ∼2 wk before being gradually transferred to other organs.Conclusions: VA supplementation administered in a single high dose during the first month after birth is readily acquired but not retained efficiently in peripheral tissues of neonatal rats, suggesting that a more frequent, lower-dose supplementation may be necessary to maintain steady VA concentrations in rapidly developing neonatal tissues.

Hepatic Detoxification of Bisphenol A is Retinoid-Dependent.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl) propane) is a widely used industrial chemical. The extensive distribution of BPA in the environment poses risks to humans. However, the molecular mechanisms underlying BPA toxicity as well as its effective detoxification and elimination are not well understood. We have investigated specifically for BPA the notion raised in the literature that the optimal sensing, detoxification, and elimination of xenobiotics requires retinoid (natural derivatives and synthetic analogs of vitamin A) actions. The objective of the study was to explore how retinoids, both those stored in the liver and those originating from recent oral intake, help maintain an optimal xenobiotic detoxification response, affecting mRNA expression and activities of elements of xenobiotic detoxification system upon BPA administration to mice. Wild-type and mice lacking hepatic retinoid stores (Lrat-/-) were acutely treated with BPA (50 mg/kg body weight), with or without oral supplementation with retinyl acetate. Hepatic mRNA expression levels of the genes encoding nuclear receptors and their downstream targets involved in xenobiotic biotransformation, phase I and phase II enzyme activities, and levels of oxidative damage to cellular proteins and lipids in hepatic microsomes, mitochondria and cytosol, were assessed. BPA treatment induced hepatic activities needed for its detoxification and elimination in wild-type mice. However, BPA failed to induce these activities in the livers of Lrat-/- mice. Oral supplementation with retinyl acetate restored phase I and phase II enzyme activities, but accelerated BPA-induced oxidative damage through enhancement of non-mitochondrial ROS production. Thus, the activities of the enzymes involved in the hepatic elimination of BPA require hepatic retinoid stores. The extent of hepatic damage that arises from acute BPA intoxication is directly affected by retinoid administration during the period of BPA exposure and hepatic retinoid stores that have accumulated over the lifetime of the organism.

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT.

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC-MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT-/- HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT-/- HSCs (1080nm) is significantly smaller than in wild type HSCs (1618nm). This is a consequence of an altered lipid droplet size distribution with 50.5±9.0% small (≤700nm) lipid droplets in LRAT-/- HSCs and 25.6±1.4% large (1400-2100nm) lipid droplets in wild type HSC cells. Upon prolonged (24h) incubation, the amounts of small (≤700nm) lipid droplets strongly increased both in wild type and in LRAT-/- HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.

Vitamin A and retinoic acid combined have a more potent effect compared to vitamin A alone on the uptake of retinol into extrahepatic tissues of neonatal rats raised under vitamin A-marginal conditions.

BACKGROUND: Vitamin A (VA, retinol) supplementation is widely used to reduce child mortality in low-income countries. However, existing research suggests that supplementation with VA alone may not be optimal for infants. OBJECTIVE: We compared the effect of VA vs. VA combined with retinoic acid (VARA) on retinol uptake and turnover in organs of neonatal rats raised under VA-marginal conditions. METHODS: Secondary analysis was conducted on data obtained from two prior kinetic studies of Sprague-Dawley neonatal rats nursed by mothers fed a VA-marginal diet (0.35 mg retinol equivalents/kg diet). On postnatal d 4, pups had been treated with a single dose of VA (6 µg/g; n = 52; VA study), VA + 10% retinoic acid (6 µg/g; n = 42; VARA study) or placebo (canola oil; n = 94; both studies), all containing ~2 µCi of [3H]retinol as the tracer for VA. Total retinol concentrations and tracer levels had been measured in plasma and tissues from 1 h to 14 d after dosing. Control group data from both studies were merged prior to analysis. Kinetic parameters were re-estimated and compared statistically. RESULTS: VARA supplementation administered to neonatal rats within a few days after birth resulted in a lower turnover of retinol in the lungs, kidneys, and carcass and less frequent recycling of retinol between plasma and organs (100 vs. 288 times in VARA- vs. VA-treated group). Although the VA supplementation resulted in a higher concentration of retinol in the liver, VARA supplementation led to a higher uptake of postprandial retinyl esters into the lungs, intestines, and carcass. CONCLUSIONS: Given the relatively higher retinol uptake into several extrahepatic organs of neonates dosed orally with VARA, this form of supplementation may serve as a targeted treatment of low VA levels in the extrahepatic organs that continue to develop postnatally.

Vitamin A Supplementation Increases the Uptake of Chylomicron Retinyl Esters into the Brain of Neonatal Rats Raised under Vitamin A-Marginal Conditions.

BACKGROUND: The most rapid phase of brain development occurs during the neonatal period. Vitamin A (VA; retinol) is critical for many aspects of this process, including neurogenesis, synaptic plasticity, learning, and memory formation. However, the metabolism of retinol in the neonatal brain has not been extensively explored. OBJECTIVE: We examined the uptake of VA into the brain in neonatal rats raised under VA-marginal conditions (control group) and assessed the effect of VA supplementation on the uptake of VA into the brain. METHODS: Sprague-Dawley neonatal rats (n = 104) nursed by mothers fed a VA-marginal diet were randomly assigned and treated on postnatal day 4 with an oral dose of either VA (6 µg retinyl palmitate/g body weight) or canola oil as the control, both of which contained 1.8 µCi [(3)H]retinol. Pups (n = 4/group at a time) were killed at 13 sampling times from 30 min to 24 d after dosing. The uptake of total retinol, chylomicron-associated retinyl esters (REs), and retinol bound to retinol-binding protein (RBP) was estimated with the use of WinSAAM version 3.0.8. RESULTS: Total retinol mass in the brain was closely dependent on its mass in plasma over time (r = 0.91; P < 0.001). The uptake of retinol into the brain involved both postprandial chylomicrons and RBP, with RBP delivering most of the retinol in the control group [0.27 nmol/d (RBP) compared with 0.01 nmol/d (chylomicrons)]. VA supplementation increased the fractional uptake of chylomicron REs from 0.3% to 1.2% of plasma pool/d, decreased that of RBP retinol from 0.5% to 0.2% of plasma pool/d, and increased the transfer rate of chylomicron REs from nearly zero to 0.7 nmol/d, causing a day-long elevation in the brain mass of total retinol. CONCLUSION: Postprandial chylomicrons may be a primary mechanism for delivering a recently ingested large dose of VA to the brain of neonatal rats raised under VA-marginal conditions.

An HPLC-MS/MS method for the separation of α-retinyl esters from retinyl esters.

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene.

Quantitative profiling of retinyl esters in milk from different ruminant species by using high performance liquid chromatography-diode array detection-tandem mass spectrometry.

An effective high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) analytical approach was developed for retinoid profiling in raw milk samples (cow, buffalo, ewe, and goat). The analytes were isolated by means of liquid-liquid extraction, including a "lipid freezing" step, with yields exceeding 66%. Since the positive atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) detection is not completely selective, a reliable identification has been accomplished by fully separating the analytes on a tandem C18/C30 column system under non-aqueous reversed phase (NARP) chromatography conditions. After validation, different milk varieties obtained from pasture-fed animals were analysed, providing, for the first time, the retinoid composition of both buffalo's and ewe's milk. According to the literature, retinyl palmitate has been found to be the most abundant vitamin A vitamer, but retinyl oleate is the prevalent form in the caprine milk.