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.

Spectral and mass characterization of kinetic conversion from retinoids to retinoic acid in an in vitro 3-D human skin equivalent model.

To investigate the effect of retinoids, such as retinol (ROL), retinal (RAL), and retinyl palmitate (RP), on epidermal integrity, skin deposition, and bioconversion to retinoic acid (RA). 3-D human skin equivalent model (EpiDermFT™) was used. Epidermal cellular integrity measured by TEER values was significantly higher for a topical treatment of ROL and RAL than RP (p < 0.05). The skin deposition (µM) of ROL and RAL was approximately 269.54 ± 73.94 and 211.35 ± 20.96, respectively, greater than that of RP (63.70 ± 37.97) over 2 h incubation. Spectral changes were revealed that the CO maximum absorbance occurred between 1600∼1800 cm-1 and was greater from ROL than that from RAL and RP, indicating conjugation of R-OH to R-CHO or R-COOH could strongly occur after ROL treatment. Subsequently, a metabolite from the bioconversion of ROL and RAL was identified as RA, which has a product ion of m/z 283.06, by using liquid a chromatography-mass spectrometry (LC-MS) - total ion chromatogram (TIC). The amount of bioconversion from ROL and RAL to RA in artificial skin was 0.68 ± 0.13 and 0.70 ± 0.10 µM at 2 h and 0.60 ± 0.04 and 0.57 ± 0.06 µM at 24 h, respectively. RA was not detected in the skin and the receiver compartment after RP treatment. ROL could be a useful dermatological ingredient to maintain epidermal integrity more effectively, more stably deposit on the skin, and more steadily metabolize to RA than other retinoids such as RAL and RP.

Controlled release of vitamin A palmitate from crosslinked cyclodextrin organic framework for dry eye disease therapy.

Controlled release drug delivery systems of eye drops are a promising ophthalmic therapy with advantages of good patient compliance and low irritation. However, the lack of a suitable drug carrier for ophthalmic use limits the development of the aforementioned system. Herein, the crosslinked cyclodextrin organic framework (COF) with a cubic porous structure and a uniform particle size was synthesized and applied to solidify vitamin A palmitate (VAP) by using the solvent-free method. The VAP@COF suspension eye drops were formulated by screening co-solvents, suspending agents, and stabilizing agents to achieve a homogeneous state and improve stability. According to the in vitro release study, the VAP@COF suspension exhibited a controlled release of VAP within 12 h. Both the ex vivo corneal contact angle and in vivo fluorescence tracking indicated that the VAP@COF suspension prolonged the VAP residence time on the ocular surface. This suspension accelerated the recovery of the dry eye disease (DED) model in New Zealand rabbits. Furthermore, the suspension was non-cytotoxic to human corneal epithelial cells and non-irritation to rabbit eyes. In summary, the particulate COF is an eye-acceptable novel carrier that sustains release and prolongs the VAP residence time on the ocular surface for DED treatment.

Enhancing the Solubility, Stability, and Bioavailability of Retinol Acetate through Mechanochemistry.

This study utilizes mechanochemistry to prepare retinol acetate (RA) solid dispersion (RA-sodium starch octenyl succinate (SSOS)), resulting in improved solubility, stability, and bioavailability compared with raw RA and commercial RA microcapsules. RA, poloxamer 188, SSOS, and milling beads (8 mm) were mixed in a ratio of 2:1:8:220 (w/w) and ball-milled at 100 rpm for 3 h. RA-SSOS exhibited a solubility of 1020.35 µL/mL and a 98.09% retention rate after aging at 30 °C. Rats fed with RA-SSOS showed an ∼30% increase in organ RA content. Characterization analysis attributed the solubility and stabilization of RA-SSOS to hydrogen bonding between RA and SSOS, along with an amorphous state. RA-SSOS offers significant advantages for the pharmaceutical and food industries, leveraging mechanochemistry to enhance solid dispersions for hydrophobic compounds and optimize drug delivery.

A promising food-grade protector for Retinyl acetate emulsions with fibrillated egg white.

This work presents a novel use of fibrous egg white protein (FEWP) in food preservation and nutraceutical applications. In this study, food-grade FEWP was used as an encapsulating material, along with chitosan (CS), to stabilize emulsions. The emulsion system was then used as a delivery system to improve the stability of retinyl acetate (RA). The structural and functional properties, as well as the stability and rheological behavior of the FEWP/CS copolymer, was investigated. The stability of RA-enriched emulsions was also evaluated. FEWP and CS stabilized emulsions exhibited smaller particle size and enhanced stability against different ionic strengths and storage periods. Additionally, RA-encapsulated emulsions stabilized by FEWP:CS (25:1 w/w) effectively inhibited apple browning. This study provides a promising strategy for delivering antioxidant components, highlighting its potential in food preservation and nutraceutical applications.

The protective effect of vitamin A palmitate eye gel on the ocular surface during general anaesthesia surgery: a randomized controlled trial.

PURPOSE: To investigate the change in tear production associated with general anesthesia and the protective effect of vitamin A palmitate eye gel on the ocular surface during general anesthesia. METHODS: This double-blind, randomized clinical trial included patients undergoing non-ophthalmic surgery under general anesthesia who randomly received vitamin A palmitate eye gel and taping for one eye (Group A, n = 60) or taping alone for the other eye (Group B, n = 60). Symptom assessment in dry eye (SANDE) score, tear film break-up time (TBUT), corneal fluorescein staining (CFS) score, and Schirmer tear test I (STT-1) were analyzed under a hand-held slit lamp before anesthesia (T0), 0.5 h postoperatively (T1), and 24 h postoperatively (T2). RESULTS: At 0.5 h postoperatively, an increase in CFS score was observed in both groups (P < 0.05 in Group A and P < 0.01 in Group B), and the participants in Group A had less corneal abrasions than those in Group B. STT-1 significantly increased in Group A (P < 0.05), while it significantly decreased in Group B (P < 0.001). The changes between the two groups were statistically significant (P < 0.001). At 24 h postoperatively, both CFS score and STT-1 almost returned to baseline levels in the two groups. In both groups, the SANDE score and TBUT showed little change at 0.5 h and 24 h postoperatively (all P > 0.05). CONCLUSION: Vitamin A palmitate eye gel effectively protected the ocular surface and aqueous supplementation during general anesthesia. TRIAL REGISTRATION: This study was registered in the Chinese Clinical Trial Registry (ChiCTR2100052140) on 20/10/2021.

LX-2 Stellate Cells Are a Model System for Investigating the Regulation of Hepatic Vitamin A Metabolism and Respond to Tumor Necrosis Factor α and Interleukin 1ß.

Hepatic stellate cells (HSCs) are the major site of vitamin A (retinol) esterification and subsequent storage as retinyl esters within lipid droplets. However, retinyl esters become depleted in many pathophysiological states, including acute and chronic liver injuries. Recently, using a liver slice culture system as a model of acute liver injury and fibrogenesis, a time-dependent increase and decrease in the apparent formation of the bioactive retinoid all-trans-retinoic acid (atRA) and retinyl palmitate was measured, respectively. This coincided with temporal changes in the gene expression of retinoid-metabolizing enzymes and binding proteins, that preceded HSC activation. However, the underlying mechanisms that promote early changes in retinoid metabolism remain unresolved. We hypothesized that LX-2 cells could be applied to investigate differences in quiescent and activated HSC retinoid metabolism. We demonstrate that the hypermetabolic state of activated stellate cells relative to quiescent stellate cells may be attributed to induction of STRA6, RBP4, and CYP26A1, thereby reducing intracellular concentrations of atRA. We further hypothesized that paracrine and autocrine cytokine signaling regulates HSC vitamin A metabolism in both quiescent and activated cells. In quiescent cells, tumor necrosis factor α dose-dependently downregulated LRAT and CRBP1 mRNA, with EC50 values of 30-50 pg/mL. Likewise, interleukin-1ß decreased LRAT and CRBP1 gene expression but with less potency. In activated stellate cells, multiple enzymes were downregulated, suggesting that the full effects of altered hepatic vitamin A metabolism in chronic conditions require both paracrine and autocrine signaling events. Further, this study suggests the potential for cell type-specific autocrine effects in hepatic retinoid signaling. SIGNIFICANCE STATEMENT: HSCs are the major site of vitamin A storage and important determinants of retinol metabolism during liver fibrogenesis. Here, two LX-2 culture methods were applied as models of hepatic retinoid metabolism to demonstrate the effects of activation status and dose-dependent cytokine exposure on the expression of genes involved in retinoid metabolism. This study suggests that compared to quiescent cells, activated HSCs are hypermetabolic and have reduced apparent formation of retinoic acid, which may alter downstream retinoic acid signaling.

[Influence of chronic immobilization stress on vitamin status in rats fed different diets].

The influence of a stress factor, widespread in modern conditions, on the vitamin status has not been studied enough. At the same time, the negative stress impact can be aggravated against the background of unhealthy nutrition, which in turn affects the vitamin status of the organism. In this regard, the goal of the research was to evaluate the effect of chronic restrict stress on the vitamin supply in rats fed a diet with adequate and increased content of fat, sugar and cholesterol. Material and methods. The experiment was carried out on 37 growing male Wistar rats (initial body weight of 45±5 g) divided into 4 groups. Animals of the 1st (control) and the 2nd groups received a complete semi-synthetic diet (CSSD) (20% protein, 10% fat, 58% carbohydrates in the form of starch, 384 kcal/100 g) for 92 days. The levels of all vitamins and mineral elements in the rats' diets were adequate for growing rats. Rats of the 3rd and the 4th groups were fed a high-calorie, high-fat high-carbohydrate diet (HFHCD) (20% protein, 28% fat, 2% cholesterol, 18% carbohydrates in the form of starch, 20% sucrose, 511 kcal/100 g). Animals of groups 2 and 4 were subjected to daily 90-minute immobilization. The concentration of vitamins A (retinol and retinol palmitate) and E (α-tocopherol) in the blood serum and liver were determined by high-performance liquid chromatography, vitamins B1 and B2 in the liver and urine, as well as riboflavin in the blood serum and 4-pyridoxic acid (4-PA) in urine were determined by fluorimetric methods. Biochemical parameters of blood serum were determined on a biochemical analyzer; the total content of fat, triglycerides (TG) and cholesterol (CH) was determined in the liver. Results. Replacing CSSD with HFHCD, both under restraint stress and without, was accompanied by an increase in liver weight by 1.8-2.0 fold, in its fat content by 2.6-3.3 fold, cholesterol by 32.6-35.3 fold and TG - by 33.0-57.6 fold (p=<0.001). An increase in alanine aminotransferase (ALT) activity by 1.7-2.0 fold (p=<0.01), in low-density lipoprotein (LDL) cholesterol level by 5.4 fold (p=<0.05) and the atherogenic coefficient by 2.5 fold (p<0.01) as well as a decrease in creatinine and urea level (p=<0.05) in blood serum were revealed. Immobilization was accompanied by a decrease in body weight, liver and liver fat in rats fed both CSSD and HFHCD (p<0.05), but didn't affect the blood serum biochemical parameters, with the exception of an increase in ALT activity. If the activity of alkaline phosphatase (ALP) did not change during immobilization of rats fed the CSSD, then in animals fed the high-calorie diet it decreased by 37.5% (p=<0.05 from the control) under its increase against the background of restrict stress by 78.7% (p=<0.01) compared to the indicator of rats of the 3rd group. Immobilization of rats treated with CSSD was accompanied by an increase in both absolute serum α-tocopherol level and concentration correlated with the level of cholesterol and triglycerides by 26.0-57.5% (p<0.05), with a simultaneous decrease in its content in the liver per 1 g of wet tissue by 22.1% (p=0.041) relative to the indicators of intact animals. Immobilization reduced the level of retinol palmitate in the liver by 2.3 times (p<0.01), but did not affect retinol level in the blood serum. At the same time, indicators of B vitamin status (the content of vitamins B1 and B2 in the liver per 1 g of wet tissue and per organ, blood serum riboflavin level, urinary excretion of riboflavin and 4-PA) did not change, with the exception of thiamine urinary excretion, which reduced compared to the control by 38.8%. In rats fed HFHCD, immobilization had no additional effect on the supply with vitamins A and E. The content of vitamins B1 and B2 in the liver in terms of the whole organ was reduced by 14.0-26.7% relative to the indicator in animals of the 3rd group, not subjected to chronic stress, only due to differences in liver weight in animals of these groups. Conclusion. The data obtained indicate that chronic stress has a negative effect on the vitamin status of the body, worsening the supply with vitamins A, E and B1, and substantiate the feasibility of studying the mechanisms of this effect in order to develop effective vitamin complexes for the treatment and prevention of diseases caused by long-term stress.

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.

Time-dependent changes in retinoids content in liver and adipose tissue after feeding of a vitamin A-deficient diet to mice.

Vitamin A is an important nutrient for multiple physiological functions. To elucidate the role of vitamin A in vivo, vitamin A-deficient diets have been often used in mice to establish a vitamin A-deficiency model. However, the information on the appropriate feeding periods and time course of changes in vitamin A content in organs after the start of vitamin A-deficient diet feeding is lacking. This study aimed to assess the retinoids levels in liver and white adipose tissue in mice fed a vitamin A-deficient diet for ≤8 weeks. High-performance liquid chromatography was used to measure the retinoids levels in liver and white adipose tissue every 2 weeks for ≤8 weeks. Vitamin A-deficient diet feeding significantly decreased retinol in the liver over 6 weeks, but retinyl palmitate, a main storage form of vitamin A, was not changed over 8 weeks. The plasma retinol level remained constant throughout the experiment. In white adipose tissue, retinyl palmitate gradually decreased over 8 weeks. These results indicate that vitamin A-deficient diet feeding longer than 6 weeks reduced retinol in liver and retinyl palmitate in white adipose tissue over 8 weeks, although it is not enough for the induction of a whole-body vitamin A deficiency.

Application of chiral stationary phases for the separation of vitamin A acetate isomers.

The separation of vitamin A acetate isomers is essential for quality assurance of e.g. nutrition supplements, cosmetics, and pharmaceutical ingredients. High performance liquid chromatography (HPLC) is currently the most suitable analytical method for tackling this challenging separation task. However, the existing methods based on normal phase chromatography (NPC) are poorly reproducible due to the typical disadvantages of NPC, such as long equilibration times and fluctuation in retention factors. A new reversed phase method developed in our labs allows the separation of the isomers applying a chiral stationary phase (CSP). This phase consists of an immobilized polysaccharide which can be used in every chromatographic mode. However, they are not typically used in reversed phase mode. Through the screening of various stationary phases with different polysaccharide based chiral selectors, the choice of the ideal stationary phase could be confirmed, allowing to draw conclusions about the retention mechanism. The CSP Chiralpak IG-3 was found to be the most suitable among the examined. Regarding the separation mechanism, the spatial helical structure of the polysaccharide derivatives was confirmed to be of particular significance. In addition to the stationary phase, the mobile phase was tested for optimization regarding composition, gradient parameters as well as temperature using chromatographic method optimization software for the sake of method robustness.

[Efficacy of 0.05% cyclosporine A combined with vitamin A palmitate in the treatment of meibomian gland dysfunction-related dry eye].

Objective: To evaluate the efficacy of 0.05% cyclosporine A eye drops combined with vitamin A palmitate eye gel in the treatment of dry eye associated with meibomian gland dysfunction (MGD). Methods: A single-center, prospective, randomized, parallel controlled trial design was used to include patients diagnosed with MGD-associated dry eye. The patients were randomly divided into three groups and administered with medications binocularly for 12 weeks. The CsA+VA group was given 0.05% cyclosporine A eye drops twice a day and vitamin A palmitate eye gel three times a day. The CsA+HA group was given 0.05% cyclosporine A eye drops twice a day and 0.1% sodium hyaluronate eye drops three times a day. The HA group was given 0.1% sodium hyaluronate eye drops 3 times a day. The OSDI score, tear meniscus height, fluorescein tear break-up time, Schirmer Ⅰ test (without anesthesia), tear film lipid layer thickness, meibomian gland morphology and function examination, and corneal fluorescein sodium staining score were evaluated at baseline, 4, 8, and 12 weeks after the initiation of the treatment, respectively. Results: A total of 120 patients with MGD-related dry eye met the enrollment criteria, but 10 patients were lost to follow-up; 110 patients were finally included for observation, including 36 patients in the CsA+VA group, 38 in the CsA+HA group and 36 in the HA group. The OSDI score, tear meniscus height, fluorescein tear break-up time and meibomian gland secretion of the 3 groups were significantly improved. At the 12th week of the treatment, the differences of the CsA+VA group [25.45±15.11, (0.30±0.13) mm, (3.72±1.40) s, (5.03±2.52) points] and the CsA+HA group [26.98±16.89, (0.27±0.10) mm, (4.34±1.76) s, (5.11±2.39) points] from the HA group [24.57±11.26, (0.24±0.06) mm, (3.18±1.11) s, (9.11±3.34) points] were statistically significant (P<0.05). Compared with the CsA+HA group [(68.39±26.66) nm], the tear film lipid layer thickness in the CsA+VA group [(72.61±23.65) nm] was significantly increased (P<0.05). In the CsA+VA group, the meibomian gland secretion characters and discharge capacity among patients with severe abnormalities [(6.28±2.59) and (5.89±2.77) points at the 12th week of treatment], moderate abnormalities [(4.27±2.02) and (4.64±2.02) points at the 12th week of treatment] and mild abnormalities [(2.80±0.84) and (2.60±0.55) points at the 12th week of treatment] were significantly different (P<0.05). Conclusion: 0.05% cyclosporine A combined with vitamin A palmitate can significantly improve the symptoms and signs of patients with MGD-related dry eye, especially the tear film lipid layer thickness and the meibomian gland secretion characters and discharge capacity in severe cases.