This work was supported by NIH-NEI grants (EY030889 and 3R01EY030889-03S1) and in part by the University of Minnesota start-up funds to G.P.L. We would also like to thank the National Eye Institute for providing us with the 11-cis-retinal standard used in this manuscript.

NOTE: All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota (protocol # 2312-41637A) and performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Perform all extractions in the dark, under a dim red light for illumination. Be aware of residual light emitted by instrument displays and accessory LEDs.
1. Spectrophotometric retinoid standard generation and external stand...

Vitamin A Metabolite Analysis in Murine Tissue for Rhodopsin Function Studies

In this method, normal-phase HPLC is used to detect and quantify relevant retinoids, including retinyl esters, retinaldehydes, and retinols. Given the importance of 11-cis-retinal as the critical chromophore in the activation of the rhodopsin GPCR, a method that can detect the metabolites that is related to the production of 11-cis-retinal is critical to the study of overall visual function. The main advantage of this method is that all relevant isomers of both retinaldehydes and retinols can be simulta...

G protein-coupled receptors (GPCRs) are one of the most studied and characterized superfamily of proteins known. In its most well-known function, GPCRs serve as a cell surface receptor in signal transduction, initializing intracellular responses upon binding with a specific ligand. GPCRs are characterized by seven transmembrane (TM) helical domains and six total loop domains. Of the six loops, three loops are oriented extracellularly to facilitate ligand binding, while the other three intracellular loops are coupled to a heterotrimeric G protein consisting of the G&#945;, G&#946;, and G&#947; subunits1,2.
GPCRs are classified into several classes, including Class A Rhodopsin-Like, Class B Secretin Receptor family, Class C Glutamate, Class D Fungal Mating Pheromone Receptors, Class E Cyclic AMP receptors, and Class F Frizzled/Smoothened3,4. As its name suggests, the GPCR rhodopsin-like Class A subclass includes rhodopsin, the critical GPCR responsible for phototransduction and visual function. Rhodopsin contains all the pertinent key characteristics and structural elements that are found in the canonical model of GPCRs, including the previously mentioned seven TM helical domains, the six extracellular and intracellular loops, and association with a heterotrimeric G protein, also known as transducin (Gt) in photoreceptors1,5,6,7. Within the binding pocket of rhodopsin, 11-cis-retinal, the light-sensitive chromophore ligand, binds to rhodopsin on lysine 296 through a covalent Schiff base linkage, thus forming 11-cis-retinylidene1,8. Upon absorption of a photon, 11-cis-retinylidene photoisomerizes into all-trans-retinylidene, inducing a conformational change within rhodopsin. Therefore, the 11-cis-retinal ligand is critical to the function of the rhodopsin GPCR, and a robust and efficient supply of 11-cis-retinal must be continuously maintained to overcome the high turnover rate within photoreceptors.
Retinaldehydes such as 11-cis-retinal belong to a group of molecules collectively called retinoids, and biologically relevant retinoids are more widely referred to as vitamin A. Retinoids are characterized by a cyclic end group connected to a conjugated polyene chain, with a polar end group at the other end. Retinaldehydes and associated vitamers of vitamin A are no exception to this characterization, which contain the &#946;-ionone ring as the cyclic end group, a diterpene polyene chain, and a differing polar end group depending on the vitamer, that is, aldehyde group for retinaldehydes, hydroxyl group for retinols, carboxyl group for retinoic acids, ester bond for retinyl esters, etc (Figure 1)9,10.
Mammals cannot synthesize vitamin A de novo, but plants can; therefore, all retinoids within mammalian systems must originate from the diet of plant-based producers to the consumers in the food chain. In the canonical model of vitamin A metabolism, &#946;-carotene, the archetypal plant provitamin A, is absorbed into the intestinal enterocyte through the scavenger receptor class B, member 1 (SCARB1), cleaved into two molecules of all-trans-retinal by &#946;-carotene oxygenase 1 (BCO1/BCMO1), which binds to retinaldehyde binding protein 2 (RBP2) and is reduced to all-trans-retinol by retinol dehydrogenases (RDH), converted into retinyl esters by lecithin retinol acyltransferase (LRAT), and then sent to the bloodstream in chylomicrons11,12,13,14. Retinyl esters, such as retinyl palmitate, on the other hand, serve as the predominant provitamin A from animal sources. Retinyl palmitate from the intestinal lumen is hydrolyzed into all-trans-retinol by carboxylesterase 1 (CES1) and diffuses into the intestinal enterocyte15. The liver is the primary storage and homeostatic organ for vitamin A homeostasis, which absorbs the retinyl esters within these chylomicrons, which are hydrolyzed into all-trans-retinol bound to cellular retinol-binding protein 1 (CRBP1) by retinyl ester hydrolases, enters hepatic stellate cells and is converted back into retinyl esters by LRAT for storage13,16,17. To maintain a homeostatic level of vitamin A in the organism, the liver releases vitamin A in the form of all-trans-retinol bound to a serum transport complex, consisting of retinol-binding protein 4 (RBP4) and transthyretin (TTR)15,18,19. This complex will be referred to as holo-RBP4 in this manuscript.
To use this systemic vitamin A supply in the blood, systemic tissues, including ocular tissue where a robust source of vitamin A is maintained, must have a method to absorb holo-RBP4 into tissue. Within the photoreceptor-rich retina in ocular tissue, the membrane receptor stimulated by retinoic acid 6 (STRA6) is the transporter implicated in this function. In mechanistic studies, STRA6 has been shown to be capable of facilitating the intake of extracellular all-trans-retinol from holo-RBP4 into the RPE20. This imported all-trans-retinol will then enter the visual cycle, which is the process by which all-trans-retinol is converted into 11-cis-retinal within the RPE and the photoreceptor outer segment, thereby facilitating visual function when bound to rhodopsin9,21.
Once all-trans-retinol from circulatory holo-RBP4 crosses the blood-retina barrier into the RPE within ocular tissue through STRA6, all-trans-retinol in the RPE is first esterified into retinyl esters by LRAT, then hydrolyzed into 11-cis-retinol by retinal pigment epithelium-specific 65 kDa protein (RPE65). 11-cis-retinol is then converted into 11-cis-retinal by the retinol dehydrogenase 5. This 11-cis-retinal is then carried into the photoreceptor&#39;s outer segment (OS) by the interphotoreceptor retinoid-binding protein (IRBP)9,21. Within the endoplasmic reticulum that surrounds the photoreceptor nucleus within the outer nuclear layer (ONL), the opsin GPCRs are synthesized and transported across the connecting cilium (CC). The motor proteins that are involved in this transport across the CC are contentious, but current hypotheses implicate kinesin and dynein-based intraflagellar transport (IFT) or myosin-based transport as being likely facilitators of this process14,22,23,24,25,26. Once these two components meet within the membranous disks within the OS, 11-cis-retinal and opsin form 11-cis-retinylidene through a Schiff base covalent linkage at lysine 196 on rhodopsin, ready for phototransduction8.
While the expression of STRA6 within the RPE of the retina helps facilitate the intake of all-trans-retinol from holo-RBP4, STRA6 was not found to be expressed in the liver, despite its role as the main homeostatic organ for vitamin A and exhibiting capabilities in intaking all-trans-retinol from holo-RBP415,19,27,28,29,30,31. Eventually, an analogous receptor called retinol-binding protein 4 receptor 2 (RBPR2) was discovered, exhibiting the capability to intake all-trans-retinol from holo-RBP4, much like STRA6, but is expressed in hepatic tissue32.
Therefore, a complete understanding of the role of rhodopsin in visual function necessitates an understanding of the biological processes that culminate in the regeneration of the visual pigment. This is, in turn, intimately related to the previously described processes, including the metabolism of provitamin A precursors, storage within the liver, release of holo-RBP4 by the liver, and eventual uptake of holo-RBP4 through STRA6 and RBPR2 membrane receptors. As mentioned above, animal models such as mice remain one of the premier models in the study of such processes. Hence, we would like to present an extraction method for retinoids in murine tissue, as well as a normal-phase high-performance liquid chromatography (HPLC) method that can detect and quantify these retinoids. Using these methods, the important retinoids described above, such as the 11-cis-retinal rhodopsin ligand or the main transport retinoid all-trans-retinol, can be analyzed in ocular, hepatic, and systemic organs. By assessing retinoid supply in murine tissue, our understanding of the disease states and pathologies related to the logistical supply of retinoids can be further advanced.
Besides functioning as a chromophore in visual function through association with opsin GPCRs, retinoids also play a major role in mammalian cell signaling through retinoic acid signaling, facilitated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), that bind directly to DNA and regulated gene transcription33. These two families or receptors both utilize retinoids in the form of retinoic acids as the ligand. RARs have been shown to have affinity for both all-trans-retinoic acid and 9-cis-retinoic acid, whereas RXRs express affinity for only 9-cis-retinoic acid34,35. Retinoic acids in uncontrolled quantities are teratogenic, and retinoic acid signaling must be extremely tightly controlled36. Production of retinoic acids for signaling must occur locally and at very specific time points for the proper development of tissues, such as in hindbrain and limb development, but countless other examples utilize retinoic acid signaling37,38. Within cells participating in retinoic acid signaling, retinoic acids are synthesized by two groups of enzymes, alcohol/retinol dehydrogenases (ADHs/RDHs) that facilitate the oxidation of retinols taken in by STRA6 or RBPR2 to retinaldehydes, and retinaldehyde dehydrogenases (RALDHs) that facilitate oxidation of retinaldehydes to retinoic acids39. While not participating in GPCR signaling per se, retinoic acids nonetheless present as a crucial retinoid that also functions as a ligand for signaling receptors.
While not described in detail here, we would like to acknowledge the previously established methods for retinoid detection using HPLC across various contexts, such as in food research and the study of microbial rhodopsin. These methods employ different goals and approaches to retinoid detection, including the use of reverse-phase techniques that require less volatile and hazardous mobile phases40,41,42, the detection of retinoic acids and their associated isomers40,41, and purification and extraction from different biological sources43. Our method focuses specifically on the detection of retinyl palmitate, retinaldehyde isomers, and retinol isomers from mammalian tissue. Different protocols should be considered if the intended use case differs from this specific application.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Octanol, suitable for HPLC, &#8805;99.5%</td>
			<td>Sigma-Aldrich, Millipore Sigma</td>
			<td>203-917-6</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dioxane, suitable for HPLC, &#8805;99.5%</td>
			<td>Sigma-Aldrich, Millipore Sigma</td>
			<td>204-661-8</td>
			<td></td>
		</tr>
		<tr>
			<td>11-cis-retinal</td>
			<td>National Eye Institute</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>11-cis-Retinol</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R252105</td>
			<td></td>
		</tr>
		<tr>
			<td>13-cis-retinal</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R239900</td>
			<td></td>
		</tr>
		<tr>
			<td>13-cis-retinol</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R252110</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans-Retinal</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R240000</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans-Retinol</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R252002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate, suitable for HPLC, &#8805;99.7%</td>
			<td>Sigma-Aldrich, Millipore Sigma</td>
			<td>205-500-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC Grade</td>
			<td>Fisher Scientific, Spectrum Chemical</td>
			<td>18-610-808</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (HPLC)</td>
			<td>Fisher Scienctific</td>
			<td>A452SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinyl Palmitate</td>
			<td>Toronto Research Chemicals</td>
			<td>TRC-R275450</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (Crystalline/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity II Analytical Fraction Collector</td>
			<td>Agilent</td>
			<td>G1364F</td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity II Binary Pump</td>
			<td>Agilent</td>
			<td>G7112B</td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity II Diode Array Detector</td>
			<td>Agilent</td>
			<td>G7115A</td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity II Multicolumn Thermostat</td>
			<td>Agilent</td>
			<td>G7116A</td>
			<td></td>
		</tr>
		<tr>
			<td>1260 Infinity II Vialsampler</td>
			<td>Agilent</td>
			<td>G7129A</td>
			<td></td>
		</tr>
		<tr>
			<td>ST40R Refrigerated Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>TSST40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacufuge Plus Centrifuge Concentrator</td>
			<td>Eppendorf</td>
			<td>22820168</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Amber Screw Top Vials</td>
			<td>Agilent</td>
			<td>5188-6535</td>
			<td></td>
		</tr>
		<tr>
			<td>Crimp Cap with PTFE/red rubber septa, 11 mm</td>
			<td>Agilent</td>
			<td>5183-4498</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Glass Conical Centrifuge Tubes</td>
			<td>Millipore Sigma</td>
			<td>CLS9950215</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 mL</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial insert, 150 &#181;L, glass with polymer feet</td>
			<td>Agilent</td>
			<td>5183-2088</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of dietary vitamin a metabolites in murine ocular and non-ocular tissues using high-performance liquid chromatography

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon association with its ligand. In all mammalian vision, rhodopsin is the GPCR responsible for the initiation of the phototransduction cascade. Within photoreceptors, rhodopsin is bound to its chromophore 11-cis-retinal and is activated through the light-sensitive isomerization of 11-cis-retinal to all-trans-retinal, which activates the transducin G protein, resulting in the phototransduction cascade. 
While phototransduction is well understood, the processes that are involved in the supply of dietary vitamin A precursors for 11-cis-retinal generation in the eye, as well as diseases resulting in disruption of this supply, are not yet fully understood. Once vitamin A precursors are absorbed into the intestine, they are stored in the liver as retinyl esters and released into the bloodstream as all-trans-retinol bound to retinol-binding protein 4 (RBP4). This circulatory RBP4-retinol will be absorbed by systemic organs, such as the liver, lungs, kidney, and eye. Hence, a method for the quantification of the various metabolites of dietary vitamin A in the eye and systemic organs is critical to the study of proper rhodopsin GPCR function. 
In this method, we present a comprehensive extraction and analytical method for vitamin A analysis in murine tissue. Through normal-phase, high-performance liquid chromatography analysis, all relevant isomers of retinaldehydes, retinols, and retinyl esters can be detected simultaneously through a single run, which allows for the efficient use of experimental samples and increases internal reliability across different vitamin A metabolites within the same sample. With this comprehensive method, investigators will be able to better assess systemic vitamin A supply in rhodopsin GPCR function.

Author Spotlight: Investigating Physiological Functions of Vitamin A Transporters Using HPLC-Based Vitamin A Profiling

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

Our lab investigates two critical vitamin A transporters, STRA6 and Rbpr2. These membrane proteins facilitate the intake of vitamin A in the blood into tissues such as the eye and liver. Through HPLC we can investigate the physiological functions of these transporters by making a systemic vitamin A profile in transgenic mice.Another technique used in our field is Surface Plasmon Resonance, SPR which examinees the binding affinities and kinetics of a protein to its liking and we have applied SPR to investigate the binding between STRA6 or Rbpr2, the RBP4 retinol binding complex. One current challenge of this method is sample throughput. The solvent evaporation step is a major bottleneck within this protocol, and future investigators should experiment with drying methods such as nitrogen blow down evaporation, which have a far greater throughput when compared to vacuum evaporation.We have determined the role of Rbpr2 as an important facilitator in maintaining the systemic vitamin A homeostasis through its function in the liver. And disruption of the Rbpr2 expression in transgenic mice has led to the significant disruption in vitamin A levels across all the examine systemic tissues as quantified through HPLC. Most other protocols in HPLC based vitamin A detection utilize reverse face methods and typically do not allow for retinoid isomers to resolve separately.Our normal face protocol allows for the resolution of both retinaldehyde and retinal isomers, allowing for us to create a detailed vitamin A profile, which is critical given the importance of vitamin A photoisomerization in the phototransduction cascade.

Here, we utilized the method described above to detect and quantify retinoids in murine ocular and systemic tissue and generated representative chromatograms. We will additionally give a summary of the typical retinoids that can be detected in these tissues.
At 6 months of age, mice were euthanized through CO2 asphyxiation. To maintain ocular retinoid content, mice were dark-adapted for 2 days prior to euthanization and extraction. Two eyes, 0.2 g of liver, and 75 &#956;L of blood w...

Here, a normal-phase, high-performance liquid chromatography method is described to detect and quantify critical retinoids involved in the facilitation of visual function in both ocular and systemic tissue, in the context of the systemic vitamin A supply to generate the essential photosensitive rhodopsin chromophore 11-cis-retinal.

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon ...

quantitative-analysis-dietary-vitamin-metabolites-murine-ocular-non

Research

JoVE Journal

Biochemistry

Simultaneous Detection of Retinoids in Murine Tissues Using HPLC Analysis to Study Rhodopsin and Vitamin A Pathways

To begin, place the extracted mouse tissues into the tissue grinder tube containing 50%ice cold saline and 50%methanol. Insert the pestle into the grinder tube and gently rotate it five times to homogenize the tissue. Immediately transfer the homogenized tissue into a 15 milliliter tube.Add two milliliters of methanol and one milliliter of 0.1 molar hydroxylamine to the homogenized sample. Allow the mixture to sit for 15 minutes at room temperature. For retinoid extraction, add 10 milliliters of hexane to the homogenate and vortex the tube horizontally for at least 10 seconds.Centrifuge the homogenate hexane mixture for three minutes at 1000 x g to separate the phases. Using a pipette, transfer the hexane layer into a separate 15 milliliter glass tube and place the sample into a vacuum centrifuge to completely evaporate the hexane from the extracted sample. Resuspend the dried retinoids in the 15 milliliter tube with 100 microliters of hexane and vortex well to ensure all retinoids are dissolved.Pipette the entire 100 microliters of hexane into the second glass tube and vortex the tube to dissolve the retinoids. Then pipette the entire 100 microliters of hexane into single glass inert for HPLC analysis. Set up the HPLC run using the appropriate conditions.Identify peaks using retention times and UV spectra for each retinoid of interest based on standards. Using the chromatographic data system, integrate the identified peaks and reference the external standard curve to quantify the analyte. For biological tissue chromatograms, manually integrate the peaks to account for variabilities in retention times.In the chromatogram of the control mouse eye, 13-cis-retinal, 11-cis-retinal, all-trans-retinal, 11-cis-retinol, and all-trans-retinol were identified. In the chromatogram of the mouse eye treated with hydroxylamine, the retention times were increased. Syn and anti isomers of these retinaldehydes were also present.In the chromatogram of mouse hepatic tissue, retinyl palmitate and all-trans-retinol were identified as the main forms of vitamin A, and that in mouse blood showed a trans-retinol peak. The UV absorbent spectra confirmed the presence of retinoid isoforms in the chromatogram peaks.

206 Views.  University of Minnesota.  Here, a normal-phase, high-performance liquid chromatography method is described to detect and quantify critical retinoids involved in the facilitation of visual function in both ocular and systemic tissue, in the context of the systemic vitamin A supply to generate the essential photosensitive rhodopsin chromophore 11-cis-retinal.

Vitamin A Metabolite Analysis in Murine Tissue for Rhodopsin Function Studies

Summary