Retinal metabolism in humans induces the formation of an unprecedented lipofuscin fluorophore 'pdA2E'.

Toxic lipofuscin in the RPE (retinal pigment epithelium) is implicated in blindness in AMD (age-related macular degeneration) or recessive Stargardt's disease patients. In the present study, we identified a novel fluorescent lipofuscin component in human and bovine RPEs. Using 1D and 2D NMR and MS, we confirmed the structure of this pigment and called it pdA2E. It exhibits absorbance maxima at 492 and 342 nm, and is susceptible to photocatalytic isomerization and oxidation. This fluorophore was also detected in the eyecup extracts of Abca4(-/-)Rdh8(-/-) (Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargardt's disease model. Excess amassing of pdA2E within RPE cells caused significant cell viability loss and membrane damage. The formation of pdA2E occurred when atRAL (all-trans-retinal) reacted with excess ethanolamine in the absence of acetic acid, and the process is likely to involve the participation of three atRAL molecules. Our findings suggest that endogenous pdA2E may serve as a sensitizer for yielding singlet oxygen and a singlet oxygen quencher, as well as a by-product of retinal metabolism, and its complete characterization facilitates the understanding of biosynthetic pathways by which adverse RPE lipofuscin constituents form.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (ß-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of ß-CD. Importantly, ß-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of ß-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of ß-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Comparison of extraction phases for a two-phase culture of a recombinant E. coli producing retinoids.

To prevent degradation of intracellular retinoids through in situ extraction from the cells, a two-phase culture system was performed. Several organic solvents, including n-alkanes, mineral oils and cosmetic raw materials, were applied as the extraction phase. Of the n-alkanes, n-decane had the highest retinoid production as 134 mg/l after 72 h. For mineral oil, light and heavy mineral oil gave retinoid productions of 158 and 174 mg/l after 96 h, respectively. Of other materials, isopropyl myristate gave the highest retinoid production of 181 mg/l. These results indicate that many types of oils can be applied for retinoid production, and optimization of the in situ extraction process will lead to further improve of economical production for the industrial purpose.

Rapid purification method for vitamin A-derived aging pigments A2E and iso-A2E using cation exchange resin.

A2E, known to be involved in the pathogenesis of age-related macular degeneration (AMD), is one of the major compounds that accumulate as fluorescent pigments in retinal pigment epithelial (RPE) cells with age and in some retinal disorders. While the biomimetic synthesis of A2E and its cis-isomer, iso-A2E is as simple as 'one-pot' reaction, the purification of these amphiphillic compounds has been a bottleneck for the mass production of these pathophysiologically important eye pigments. In order to provide a new method of rapid purification of A2E and iso-A2E, we employed a cation exchange resin for the separation of these pigments from crude reaction mixture. The reaction mixture was loaded on a weak acid resin and was eluted with 80% methanol with sodium hydroxide (pH 12), 100% methanol, and 100% methanol with 0.1% trifluoroacetic acid (TFA) in sequence. A2E and isoA2E were eluted only with 100% methanol solution containing TFA. Most of unreacted starting materials and intermediates were removed with 80% methanol containing sodium hydroxide. The new method can be used as a relatively simple and economic way to purify A2E and iso-A2E compared to conventional HPLC technique.

Tissue-specific distribution patterns of retinoids and didehydroretinoids in rainbow trout Oncorhynchus mykiss.

Retinoids (vitamin A) are known to be involved in many key biological functions in mammals, such as embryonic development, reproduction or vision. Besides standard vitamin A forms, freshwater fish tissues contain high levels of didehydroretinoids or vitamin A(2) forms. However, the tissue distribution, metabolism and function of both standard and particularly the didehydroretinoids are still poorly known in fish. In this study, we have quantified the levels of retinoids, including retinol, retinaldehyde, retinyl palmitate and their corresponding didehydro forms, as well as the levels of the active polar retinoids all-trans-, 9-cis- and 13-cis-retinoic acid in distinct tissues of juvenile rainbow trout. Our results indicate that the liver is clearly the main retinoid storage tissue in juvenile rainbow trout. Didehydroretinoids were dominant over retinoids in all analyzed tissues with the exception of plasma. Additionally, significant differences among tissues were observed between retinoids and didehydroretinoids, such as differences in the ester profiles and the proportions between free and esterified forms, suggesting that mechanisms that favor the utilization or storage of one of the other groups of compounds might exist in fish. Our data also show the presence of polar retinoids in different tissues of fish at the fmol/g scale. Overall, this study clearly demonstrates the presence of tissue-specific patterns of accumulation of both polar and nonpolar retinoids in fish tissues. The biological relevance of these findings should be the focus of future studies.

Culture of highly differentiated human retinal pigment epithelium for analysis of the polarized uptake, processing, and secretion of retinoids.

The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensory retina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes of this versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-cis-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediated process that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channel that is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membrane of the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-step conversion to 11-cis-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portion of the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visual cycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequences of these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to total blindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offers one the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process in health and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.

Quantification of endogenous retinoids.

Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimer's disease, type 2 diabetes, obesity, and cancer.

HPLC/MS(N) analysis of retinoids.

This protocol describes a highly sensitive and selective method to quantify retinoids using normal-phase HPLC with online APCI MS(N). The retinoids are key regulators of gene expression, retinol being oxidized via a retinaldehyde intermediate to retinoic acid (RA) which activates specific nuclear receptors, the signalling of which is turned off by oxidative inactivation of the ligand to 4-oxo-RA and other metabolites. Many of these retinoids are present only transiently at low concentrations in tissues and during analysis are labile to heat, light, and oxygen. HPLC with online APCI MS(N) provides a rapid technique to quantify these retinoids simultaneously. Techniques to extract the retinoids and prevent their degradation are described, with an emphasis on transcriptionally active RA. RA controls patterning of gene expression in the embryo, organizing embryonic morphology in the central nervous system. Similarly, a patterned distribution of RA controls function of the adult CNS, a tissue particularly difficult to analyse for RA because of its high lipid content. To understand how these patterns are organized in the brain and change over time, it is essential to determine the concentration of RA in small areas of tissues, and techniques of exquisite sensitivity are indispensable.

Molecular biology and analytical chemistry methods used to probe the retinoid cycle.

The retinoid (visual) cycle is a complex enzymatic pathway essential for regeneration of the visual chromophore, 11-cis-retinal, a component of rhodopsin that undergoes activation by light in vertebrate eyes. Pathogenic mutations within genes encoding proteins involved in the retinoid cycle lead to abnormalities in retinoid homeostasis and numerous congenital blinding diseases of humans. Thus, elucidation of disease-specific changes in enzymatic activities and retinoid content of the retina can provide important insights into the mechanisms of disease initiation and progression. Here, we use the protein RPE65 as an example to describe generally applicable methods for determining the stability and enzymatic activity of proteins and their mutants involved in retinoid metabolism. Additionally, we introduce a range of analytical techniques involving high-performance liquid chromatography and mass spectrometry to detect and quantify retinoids and their derivatives in eye extracts. Biochemical protocols combined with advanced mass spectrometry should facilitate fundamental biological studies of vision.

Downregulation of Fzd6 and Cthrc1 and upregulation of olfactory receptors and protocadherins by dietary beta-carotene in lungs of Bcmo1-/- mice.

An ongoing controversy exists on beneficial versus harmful effects of high beta-carotene (BC) intake, especially for the lung. To elucidate potential mechanisms, we studied effects of BC on lung gene expression. We used a beta-carotene 15,15'-monooxygenase 1 (Bcmo1) knockout mouse (Bcmo1(-/-)) model, unable to convert BC to retinoids, and wild-type mice (Bcmo1(+/+)) mice to dissect the effects of intact BC from effects of BC metabolites. As expected, BC supplementation resulted in a higher BC accumulation in lungs of Bcmo1(-/-) mice than in lungs of Bcmo1(+/+) mice. Whole mouse genome transcriptome analysis on lung tissue revealed that more genes were regulated in Bcmo1(-/-) mice than Bcmo1(+/+) mice upon BC supplementation. Frizzled homolog 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1) were significantly downregulated (fold changes -2.99 and -2.60, respectively, false discovery rate < 0.05) by BC in Bcmo1(-/-). Moreover, many olfactory receptors and many members of the protocadherin family were upregulated. Since both olfactory receptors and protocadherins have an important function in sensory nerves and Fzd6 and Cthrc1 are important in stem cell development, we hypothesize that BC might have an effect on the highly innervated pulmonary neuroendocrine cell (PNEC) cluster. PNECs are highly associated with sensory nerves and are important cells in the control of stem cells. A role for BC in the innervated PNEC cluster might be of particular importance in smoke-induced carcinogenesis since PNEC-derived lung cancer is highly associated with tobacco smoke.

Detection of endogenous retinoids in the molluscan CNS and characterization of the trophic and tropic actions of 9-cis retinoic acid on isolated neurons.

Retinoic acid (RA) is an active metabolite of Vitamin A that plays an important role in the growth and differentiation of many cell types. All-trans RA (atRA) is the retinoic acid isomer that has been most widely studied in the nervous system, and can induce and direct neurite outgrowth from both vertebrate and invertebrate preparations. The presence and role of the 9-cis-RA isomer in the nervous system is far less well defined. Here, we used high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to show for the first time, the presence of both atRA and 9-cis-RA in the CNS of an invertebrate. We then demonstrated that 9-cis-RA was capable of exerting the same neurotrophic and chemotropic effects on cultured neurons as atRA. In this study, significantly more cells showed neurite outgrowth in 9-cis-RA versus the EtOH vehicle control, and 9-cis-RA significantly increased the number and length of neurites from identified neurons after 4 d in culture. 9-cis-RA also extended the duration of time that cells remained electrically excitable in culture. Furthermore, we showed for the first time in any species, that exogenous application of 9-cis-RA induced positive growth cone turning of cultured neurons. This study provides the first evidence for the presence of both atRA and 9-cis-RA in an invertebrate CNS and also provides the first direct evidence for a potential physiological role for 9-cis-RA in neuronal regeneration and axon pathfinding.

[Advances in new technique pseudophase biochromatography].

The current status and latest advances in new technique pseudophase biochromatography are reviewed. After brief introduction to the principle of new technique pseudophase biochromatography, the nature and various influence factors including the compositions, the types of new technique pseudophase biochromatography system are summarized in detail and the aspects of the future applications biochromatography in life science are described.

Partially saturated canthaxanthin purified from Aspergillus carbonarius induces apoptosis in prostrate cancer cell line.

A mutant Aspergillus carbonarius selected for temperature tolerance after UV treatment, when grown in shake flasks, produced mycelia bearing yellow pigment. Since the mutant was affected in sterol biosynthetic pathway, the pigment was apparently produced to maintain membrane fluidity and rigidity for growth sustenance in low-pH culture broth. Nuclear magnetic resonance analyses characterizing the pigment as a partially saturated canthaxanthin, containing beta-ionone end rings, suggested its application as a retinoid. When tested for this property in retinoic acid receptor expressing prostate cancer cell line, LNCaP, the fungal partially saturated canthaxanthin induced apoptosis. Low apoptosis percentage in DU145 prostrate cancer cells that does not express functional retinoic acid receptor-beta (RAR-beta) suggested binding specificity of the partially saturated canthaxanthin for RAR-beta.

Structure elucidation of retinoids in biological samples using postsource decay laser desorption/ionization mass spectrometry after high-performance liquid chromatography separation.

Retinoids [retinol (vitamin A) and its metabolites] function in the visual cycle, embryonic development, cellular differentiation, and tissue homeostasis. Notwithstanding pivotal roles of retinoids in mammals, the limited number of commercially available retinoid standards is a major roadblock to identifying and studying retinoids in biological samples. Therefore, a need exists for improved methods to identify retinoid metabolites. We analyzed polar and nonpolar retinoids, including retinoic acid, retinol, retinyl acetate, and other retinyl esters, using postsource decay laser desorption/ionization mass spectrometry (PSD-LDI MS). PSD analysis was employed to examine the PSD fragmentation patterns of retinoids, as these patterns can be used for the characterization of retinoids from biological samples without the need for matching retention time with a commercially available or synthetic retinoid. Mechanisms for the formation of these PSD fragment ions are proposed. The feasibility of employing PSD after HPLC separation was demonstrated by characterizing the endogenous retinoids in canine kidney epithelial cell extracts and in mouse lung. We show that the PSD-LDI MS approach described here can facilitate the identification and characterization of retinoids from mammalian cells and tissues.

A2-rhodopsin: a new fluorophore isolated from photoreceptor outer segments.

A2E and iso-A2E are fluorescent amphiphilic pyridinium bisretinoids involved in age-related macular degeneration (AMD). It is now shown that the presence of high exogenous concentrations of all-trans-retinal in photoreceptor outer segments leads to the formation of A2-rhodopsin (A2-Rh), an unprecedented fluorescent rhodopsin adduct which consists of bisretinoids (A2) linked to each of three lysine residues in rhodopsin (Rh) and which exhibits an emission spectrum similar to A2E. The fluorophore to protein ratio was determined by MALDI-TOF-MS and UV-VIS spectroscopy. Enzymatic degradation with thermolysin and cathepsin D showed that two of the A2 moieties were located in the region of the third cytoplasmic loop and 8th helix of Rh. Examination of A2-Rh and A2-PE (the precursor of A2E) fluorescence in relation to all-trans-retinal concentration indicated that whereas A2-PE formation is favored over that of A2-Rh, for a single rhodopsin molecule only one phosphatidylethanolamine molecule is available to react with all-trans-retinal; this phosphatidylethanolamine is probably tightly associated with the protein.

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.

Characterization of a dehydrogenase activity responsible for oxidation of 11-cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene. A model for the human hereditary disease fundus albipunctatus.

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.

Identification of effective retinoids for inhibiting growth and inducing apoptosis in bladder cancer cells.

PURPOSE: Retinoids modulate the growth and differentiation of normal and malignant epithelial cells in vitro and in vivo, and inhibit bladder carcinogenesis in animal models. Retinoid analogs have been used in several clinical chemoprevention trials of superficial bladder cancer recurrence. There is a clear need to identify new effective retinoids and develop novel approaches for the chemoprevention and treatment of superficial bladder cancer. We investigated the effects of various retinoids on growth inhibition and apoptosis induction in bladder cancer cell lines. MATERIALS AND METHODS: Ten grades 1 to 3 bladder cancer cell lines and the 4 retinoids all-trans-retinoic acid, 9-cis retinoic acid, 4-(N-hydroxyphenyl) retinamide (4HPR) and LGD1069 were used in the study. We compared the ability of these retinoids to inhibit growth, induce apoptosis, affect the expression of nuclear retinoid receptors and modulate apoptosis related genes. RESULTS: Most bladder cancer cell lines did not express retinoic acid receptor beta and were resistant to the effect of all-trans-retinoic acid and 9-cis retinoic acid on growth inhibition and apoptosis induction, even at a concentration of 10(-5) M. The 2 cell lines that expressed retinoic acid receptor beta were constitutively sensitive to the growth inhibitory effect of all-trans-retinoic acid. 4HPR inhibited cell growth by about 90% in all but 1 cell line and induced apoptosis at a concentration of 10(-5) M after a 24-hour treatment. LGD1069 had virtually no effect. All-trans-retinoic acid and 4HPR induced retinoic acid receptor beta expression in 1 bladder cancer cell line. However, the effect of 4HPR on cell growth and apoptosis were not related to the constitutive expression of retinoic acid receptor beta. 4HPR decreased bcl-2 expression in 6 of 8 bladder cancer cell lines but did not change p53 gene expression. CONCLUSIONS: The results demonstrate that 4HPR is the most potent growth inhibitor and apoptosis inducer of the retinoids tested. Lack of retinoic acid receptor beta expression may be responsible for cell resistance to all-trans-retinoic acid but not to the other retinoids.

Application of micellar electrokinetic chromatography for the separation of retinoids.

The applicability of micellar electrokinetic chromatography (MEKC) using sodium dodecylsulphate (SDS) as pseudo-stationary phase for the separation of five retinoids (retinol, retinal, retinyl acetate, retinyl palmitate, retinoic acid), was investigated. The effects of the acetonitrile content, the SDS concentration, the pH and the addition of Brij 35 to the background electrolyte on the migration behaviour of the retinoids were determined. It was found that the effective mobilities of retinol, retinal and retinyl acetate could be easily regulated through the ACN content and the SDS concentration of the BGE. The electrophoretic behaviour of the very hydrophobic retinyl palmitate was abnormal. Under various conditions this compound showed up as a late, very sharp peak. A strong indication was found that the retinyl palmitate forms a stable, charged complex with SDS during sample preparation. The mobility of the retinyl palmitate peak could be regulated, independently from the other peaks, through the Brij concentration of the BGE. Using a running buffer consisting of Tris buffer (pH 8), 20 mmol l(-1) SDS, 1 mmol l(-1) Brij 35 and 35% (v/v) acetonitrile, a complete separation of the five retinoids could be realised in less than 20 min.