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 standard curve generation
NOTE: Prepare a dry ice container for temporary storage of retinoids before analysis with the HPLC.
<ol>
	<li>Weigh an arbitrary but appropriate quantity of retinoids and dissolve them in an appropriate solvent for quantification through spectrophotometry. 
	NOTE: The solvent of choice used in this study is ethanol, and the molar absorptivity values of retinoids dissolved in ethanol are detailed in Table 1. Anhydrous, HPLC-grade ethanol must be used to dissolve standards and samples. Ethanol purified solely through distillation forms an azeotropic mixture with water, containing approximately 4% water by volume. Water is immiscible with the hexanoic mobile phase and will result in the hydration of the silica stationary phase, leading to eventual degradation of the column.</li>
	<li>Utilizing the Beer-Lambert Law and the molar absorptivity of the pertinent retinoid, quantify the concentration of the generated standard (Table 1). 
	Absorbance = Molar Absorbtivitty (&#949;)&#160;&#215; Molar Concentration&#160;&#215; Path Length</li>
	<li>Perform serial dilutions to create concentrations that can generate a standard curve within the range of quantitation for the desired tissue. 
	NOTE: Be aware of the fundamental limitations of the Beer-Lambert Law, such as its lack of validity with high concentrations of analyte. To avoid this issue, the diluted retinoid standards should avoid generating absorbance values greater than 1. For our applications in retinol and retinaldehyde quantification in murine organs, we found that a calibration curve with a range of 1-10 ng was able to cover the typical quantities found. For retinyl palmitate quantification from murine liver, we found that a calibration curve with a range of 20-80 &#181;g was able to cover the typical quantities found.</li>
	<li>By incrementally altering the injection volume from the previously created stock solution, thereby incrementally altering the injected amount of retinoid, integrate the peaks to generate an external standard curve suitable for retinoid quantification where peak integration is directly proportional to the amount of retinoid injected.</li>
</ol>
2. Tissue harvest and sample collection
NOTE: Prepare a dry ice container for temporary storage of tissue before tissue homogenization and retinoid extraction. Recommended tissue harvest amounts are detailed in Table 2. To account for retinoid variations due to variations in blood content for each tissue, tissue extraction should be done on fully perfused mice, and blood extraction should be completed on separate mice.
<ol>
	<li>Euthanize the mice following the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) dictated protocol (CO2 asphyxiation here).</li>
	<li>Blood: Immediately following euthanasia, decapitate the mice with a pair of scissors, and drain the blood from the main trunk of the mice into a 1.5 mL tube.</li>
	<li>Eye: Using a pair of forceps, remove the eyes from the decapitated head.</li>
	<li>Brain: Using a pair of small scissors, cut into the decapitated head and remove the brain using a pair of forceps.</li>
	<li>Kidney, liver, spleen, heart, and lung: Using a pair of small scissors, make an incision into the abdomen, cut in the superior direction along the midline, and cut through the sternum and ribcage. Remove the exposed tissue using a pair of forceps.</li>
</ol>
3. Tissue homogenization
NOTE: If analysis of smaller partitions of organs is desired, such as in larger organs (e.g., liver or lung tissue), the whole organ should be homogenized to avoid differences in retinoid content in different parts of the tissue. Instead, partition the homogenate if smaller quantities of tissue are desired. A schematic for the protocol is detailed in Figure 2. This modified protocol was adapted from Kane and Napoli44.
<ol>
	<li>Place the tissue in the tissue grinder tube, along with 50% ice-cold saline (0.9%) and 50% methanol. See Table 2 for the volume used for each tissue type.</li>
	<li>Place the pestle into the grinder tube, slowly and gently perform five full rotations with the pestle to obtain a homogenate.</li>
	<li>Transfer samples into 15 mL tubes immediately after homogenization.</li>
	<li>Add 2 mL of methanol and let sit for 15 min at room temperature. 
	NOTE: If analysis of retinaldehyde oxime derivatives is desired, add 1 mL of 0.1 M hydroxylamine hydrochloride in 0.1 M HEPES (pH 6.5) (Figure 1).</li>
</ol>
4. Retinoid extraction
CAUTION: Hexane is highly flammable, highly volatile, and highly toxic. National Institute for Occupational Safety and Health (NIOSH)-approved respirators, eye protection, butyl gloves, and a fume hood must be used when handling hexane. When evaporating hexane from samples, some form of enhanced air circulation apparatus is recommended to prevent solvent fume buildup, for example, a snorkel suction apparatus.
<ol>
	<li>Add 10 mL of hexane to the homogenate and vortex mix the tube horizontally for at least 10 s. 
	NOTE: It is critical that the phases mix fully.</li>
	<li>Centrifuge the homogenate/hexane mixture for 3 min at 1,000 &#215; g to facilitate phase separation.</li>
	<li>Conduct the extraction 2x to ensure total extraction of retinoids from the homogenate. Repeat steps 4.1 and 4.2.</li>
	<li>Draw off the hexane layer using a pipette and place the hexane layer into a separate set of glass 15 mL tubes for vacuum evaporation. 
	NOTE: For evaporation, use GLASS 15 mL tubes to avoid adhesion of retinoids to the walls of the tube.</li>
	<li>Using a vacuum centrifuge, completely evaporate the hexane.</li>
</ol>
5. Resuspension and HPLC analysis
NOTE: Since the HPLC system used in this manuscript was a binary pump system, the four-component mobile phase was premixed into a singular bottle prior to operation.
CAUTION: All four organic solvents used in this method are highly flammable, highly volatile, and highly toxic. 1,4-dioxane is susceptible to explosive peroxide formation upon exposure to oxygen. Keep all vessels containing 1,4-dioxane closed when not in use. National Institute for Occupational Safety and Health (NIOSH)-approved respirators, eye protection, butyl gloves, and a fume hood must be used when handling these solvents. While running these solvents in an HPLC, some form of enhanced air circulation apparatus is recommended to prevent solvent fume buildup, for example, a snorkel suction apparatus.
<ol>
	<li>Resuspend the dried 15 mL tube with 100 &#181;L of hexane; vortex well to make sure all retinoids are dissolved.</li>
	<li>Pipette all 100 &#181;L of hexane into a single glass insert for HPLC analysis.</li>
	<li>Set up the HPLC run (adapted from Landers and Olson45): mobile phase: 85.4% hexane (v/v), 11.2% ethyl acetate (v/v), 2% dioxane (v/v), 1.4% 1-octanol (v/v); column: two 4.6 mm ID x 250 nm, 5 &#181;m columns, connected in series; a multicolumn Thermostat Temperature: 25 &#176;C; injection volume: 100 &#181;L; flow rate: 1 mL/min; run time: 40 min. Use UV spectrum absorbance detection; keep the option checked in to acquire UV spectrum from 200 nm to 400 nm.</li>
</ol>
6. Peak Identification and Integration
<ol>
	<li>Identify peaks using retention time and UV spectra of each retinoid of interest as observed from analysis of retinoid standards (Figure 3, Table 3, and Table 4).</li>
	<li>Using the chromatographic data system of the chosen HPLC system, integrate the identified peaks. The integration, or area under the curve is directly proportional to the amount of analyte. Reference the external standard curve generated in step 1 to quantify the analyte.
	<ol>
		<li>For analysis of chromatograms generated from biological tissue, use manual integration over automatic integration offered by typical chromatographic data systems, since variabilities in parameters such as retention time are often observed in such samples.</li>
		<li>Ensure that chromatograms do not exhibit irregularities that might indicate issues during the run, such as noisy baselines or non-Gaussian peaks. These issues indicate contaminants in the HPLC or wear on columns and should be rectified for valid analysis.</li>
	</ol>
	</li>
</ol>

Vitamin A Metabolite Analysis in Murine Tissue for Rhodopsin Function Studies

The authors declare no conflict of interest.

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 simultaneously detected and quantified with a single run. Given how scarce viable experimental tissues are, this method allows for efficient use of tissue in retinoid detection. While the use of hexane and ethyl acetate-based mobile phases has been reported in the separation of retinoids49, the addition of 1,4-dioxane and 1-octanol improves the resolution of retinoid isomers. 1,4-dioxane is necessary for the resolution of 13-cis-retinol and 11-cis-retinol50, while long-chain alcohols such as 1-octanol further improve the separation of retinol isomers45,51,52.
For quantification of retinaldehyde isomers, prior conversion to the corresponding retinaldehyde oximes with hydroxylamine is required, since the reactive aldehyde group is prone to react with other biomolecules such as proteins and lipids45,53,54. Retinaldehyde oximes are more readily extracted and cause retinaldehyde peaks to elute later in the run, thus preventing peak co-elution with the unknown group of peaks and the retinyl palmitate peak that elutes at ~5-8 min for biological tissues. Treatment of retinaldehydes with hydroxylamine also prevents co-elution of retinaldehydes with similar elution times45. The conversion to retinaldehyde oximes generates both syn and anti isomers, which elute at two different times with two different peaks for each retinaldehyde isomer. The integration for both peaks must be summed to properly quantify each retinaldehyde isomer. Besides retention time, the UV spectra provide another metric that can be utilized in peak identification. Each retinoid exhibits a slightly different UV absorbance maximum. When taken into consideration along with retention time, these two metrics allow for the accurate identification of peaks (Table 3 and Table 4).
While the presence of canonical retinaldehyde isomers such as 11-cis-retinal and all-trans-retinal are expected in ocular tissue, with 11-cis-retinal being the critical rhodopsin ligand chromophore and all-trans-retinal being the photoisomerized ligand, other non-canonical retinaldehyde isomers are detected with this method. In particular, 13-cis-retinal was readily detected in HPLC runs of murine ocular tissue during our analysis (Figure 4A-D). The appearance of these non-canonical retinaldehydes is not completely unexpected however, both 13-cis-retinal and 9-cis-retinal are known byproducts resulting from light-induced stress upon the retina55,56. These retinaldehyde isomers do not participate in the known canonical phototransduction cascade pathway and their metabolic fates remain relatively unknown. However, 9-cis-retinal has been shown to also stably bind to the binding pocket of rhodopsin, forming isorhodopsin. The role of isorhodopsin has yet to be fully characterized, but studies have shown that isorhodopsin does exhibit photosensitive capabilities much like the 11-cis-retinal based rhodopsin57,58. At the very least, the capability of 9-cis-retinal to stably form isorhodopsin and photoisomerize into all-trans-retinal provides a reasonable explanation for its absence in our HPLC analysis56. 13-cis-retinal, on the other hand, has not been shown to participate in any known ocular biochemical pathway, and its accumulation within ocular tissue might result in oxidative stress within the retina55,59,60.
Although 9-cis-retinal is typically not found in murine tissue, it should be mentioned that the hydroxylamine-treated method is not a viable method for the separation and quantification of 9-cis-retinal. The peaks for syn 9-cis-retinal and syn 13-cis-retinal will coelute at 11.2 min. In cases where significant amounts of 9-cis-retinal are expected, such as when it is applied exogenously, only quantification of combined 9-cis-retinal and 13-cis-retinal will be possible. For qualitative identification, performing the method without treating the tissue with hydroxylamine with allow for the complete separation of 13-cis-retinal and 9-cis-retinal peaks.
Besides ocular tissue, this method can also be readily applied to systemic tissue. While retinaldehydes in their various isomeric forms are typically not found in systemic tissue, all-trans-retinol bound to retinol-binding protein 4 (holo-RBP4) is the main transport form of vitamin A in mammalian organisms and all-trans-retinol can be readily detected in the blood using this method. Besides the blood, all-trans-retinol can also typically be detected in all systemic tissues. Hence, using this method, a complete systemic tissue-wide retinoid profile can be created using this method.
Since retinaldehydes, especially the critical rhodopsin ligand 11-cis-retinal, are extremely susceptible to photoisomerization, it is critical that preventative measures are taken to prevent accidental exposure to light during any of the described steps above. Besides the obvious sources from room lighting and instrument panels, we have found that errant light from accessory LEDs in various instruments, such as those found behind instruments, including the HPLC itself, to be major sources of accidental light exposure (Figure 6). Additionally, we have found that a dedicated dark room was vital to the proper handling of retinaldehyde isomers and experimental tissues. Besides light minimization, we recommend that tissue is swiftly and promptly processed following euthanization and harvest since degradation of retinaldehyde isomers occurs even in the absence of light. Ideally, tissue harvest, extraction, and HPLC analysis should all be completed on the same day.
Tissue that is not processed immediately must be stored in the dark at -80 &#176;C and should not be stored for more than a week before processing. As was mentioned above, all solvents used in the mobile phase and in the extraction steps in the above method are highly toxic, volatile, and flammable. Proper procedures and equipment must be used when handling these hazardous solvents. Proper personal protective equipment (PPE) such as butyl gloves, NIOSH-approved respirators, and eye protection must be donned prior to solvent handling, and all solvent transfers must be done inside a fume hood. Be aware that 1,4-dioxane is susceptible to explosive peroxide formation upon exposure to oxygen and keep all solvent containers with 1,4-dioxane tightly sealed from air.
If a normal-phase HPLC method is unviable due to inadequate materials or equipment, more typical reverse-phase methods with less volatile solvents such as acetonitrile and water do exist to detect and quantify retinaldehydes and retinols. However, these methods are not able to separate isomers and are only able to detect total retinaldehydes or total retinols44,54.

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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Octanol, suitable for HPLC, &amp;ge;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, &amp;ge;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, &amp;ge;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>&lt;strong&gt;Instruments&lt;/strong&gt;</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>&lt;strong&gt;Consumables&lt;/strong&gt;</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 &amp;micro;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.

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 were harvested for retinoid extraction and subsequent HPLC analysis. For ocular tissue, one eye was extracted without the addition of hydroxylamine, while the other was subjected to hydroxylamine treatment. For liver and blood, hydroxylamine treatment was not needed since retinaldehydes are not typically detected in these tissues.
In the chromatogram of the mouse eye not treated with hydroxylamine, 13-cis-retinal, one of the two non-canonical retinaldehyde isomers, was identified. Additionally, both 11-cis-retinal and all-trans-retinal, the two canonical retinaldehyde isomers, were identified. In both chromatograms, the visual cycle intermediate 11-cis-retinol, as well as the main vitamin A transport form all-trans-retinol, were both identified (Figure 4A).
In the chromatogram of the mouse eye treated with hydroxylamine, all previously mentioned retinaldehyde isomers were still present. However, the retention times for these isomers were significantly increased. Moreover, these retinaldehyde isomers now present as both syn and anti isomers. During quantification through peak integration, both the syn and anti peaks must be summed to gain an accurate integration value. Both 11-cis-retinol and all-trans-retinol were still present (Figure 4B).
In the chromatogram of mouse hepatic tissue, retinyl palmitate and all-trans-retinol were identified within these tissues. Retinyl palmitate, a retinyl ester, serves as the main storage form of vitamin A in mammals and can be found in significant quantities within mammalian hepatic tissue. The liver releases vitamin A into systemic tissues as holo-RBP4, containing all-trans-retinol within the RBP4-transthryetin complex. Subsequently, a large all-trans-retinol peak can be identified within this chromatogram (Figure 4C).
In the chromatogram of mice blood, a large all-trans-retinol peak was identified. Given that holo-RBP4 from the liver is released to systemic tissues through the circulatory system, this is as expected (Figure 4D). The characteristic UV spectrum absorbance (~250-400 nm) of the retinoid isoforms on the time-dependent resolved HPLC peaks can be helpful in initial cross-confirming the quality of the isoform presence in a particular respective peak (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67034/67034fig01.jpg" /> 
Figure 1: Chemical structures of retinoids. (A)&#160;These retinoids are typically found in extracted murine tissue. Note the differing polar end groups, as well as the differing locations of cis double bonds in retinoid isomers. (B)&#160;Conversion of retinaldehydes to retinal oximes increases extraction efficiency, prevents co-elution of peaks with retinyl palmitate, as well as co-elution within retinaldehyde isomers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67034/67034fig02.jpg" /> 
Figure 2: Workflow for tissue harvest, homogenization, retinoid extraction and analysis. Retinas and tissue samples from dark-adapted mice were homogenized and extracted. Step 1: After euthanizing mice according to guidelines, blood and various tissues are collected. Steps 2-4: Tissue homogenization is then done with saline and methanol, with hydroxylamine hydrochloride for quantitative analyses. Steps 5-7: Retinoids are treated using hexane, followed by evaporation. Steps 8,9: Resuspended with 100 &#181;L of hexane to make sure all retinoids are dissolved. Step 10: Finally, samples are analyzed using HPLC. Abbreviation: HPLC = High-performance Liquid Chromatography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67034/67034fig03.jpg" /> 
Figure 3: Chromatogram of retinoid standards and hydroxylamine-treated retinoid standards. (A)&#160;This chromatogram contains retinyl palmitate, 13-cis-retinal, 11-cis-retinal, all-trans-retinal, 11-cis-retinol, 13-cis-retinol, and all-trans-retinol standards. (B) This chromatogram contains retinyl palmitate, 13-cis-retinal, 11-cis-retinal, all-trans-retinal, 11-cis-retinol, 13-cis-retinol, and all-trans-retinol standards. This chromatogram contains the syn and anti oxime retinaldehyde isomers, in addition to retinol isomers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67034/67034fig04.jpg" /> 
Figure 4: Representative Chromatograms. (A)&#160;Representative chromatogram of mouse ocular tissue.This chromatogram contains retinoids typically found in murine ocular tissue, including retinaldehyde isomers, in addition to retinol isomers. (B) Representative chromatogram of hydroxylamine-treated mouse ocular tissue. Note that in lieu of the non-chemically modified retinaldehydes found in the previous chromatograms (Figure 4A), this chromatogram contains the syn and anti oxime retinaldeyde isomers, in addition to retinol isomers. (C) Representative chromatogram of mouse liver tissue.This chromatogram contains retinoids typically found in murine hepatic tissue, including retinyl palmitate and all-trans-retinol. (D) Representative chromatogram of mouse blood.&#160;This chromatogram contains retinoids typically found in murine blood, including all-trans-retinol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67034/67034fig05.jpg" /> 
Figure 5: UV absorbance spectrum of retinaldehyde isomers from murine ocular tissue. Absorbance spectra of (A) 13-cis-retinal, (B) 11-cis-retinal, (C) 9-cis-retinal, (D) all-trans-retinal, (E)&#160;syn 11-cis-retinal oxime, (F)&#160;anti 11-cis-retinal oxime. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67034/67034fig06.jpg" /> 
Figure 6:&#160;Overview of HPLC modules, mobile phase flow, and other instrumentation. Mobile phase flow begins from the prepared solvent bottles and is pumped at high pressure by the binary pump into the autosampler, where the analyte is incorporated into the solvent flow. The mobile phase and analyte reach the temperature-regulated column compartment, and finally, into the diode array detector for measurement. (A) Red tinted monitor screen minimizing photoisomerization of retinoids. (B) Binary pump. (C) Vial sampler. (D) Multicolumn thermostat. (E) Diode array detector. (F) Analytical fraction collector. (G) High-density poly waste container. (H) From left to right, aluminum crimp cap, 250 &#956;L insert with polymer feet, amber tinted crimp top vial, final assembly of injection vial. (I) Left, refrigerated centrifuge. Right, centrifuge concentrator. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67034/67034fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Molar Absorptivity (&#949;)</td>
			<td>Solvent</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Retinyl Palmitate</td>
			<td>49260</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
		<tr>
			<td>13-cis-Retinal</td>
			<td>35500</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
		<tr>
			<td>11-cis-Retinal</td>
			<td>24935</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
		<tr>
			<td>9-cis-Retinal</td>
			<td>36100</td>
			<td>Ethanol</td>
			<td>Robeson et al.51</td>
		</tr>
		<tr>
			<td>All-trans-Retinal</td>
			<td>42880</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
		<tr>
			<td>11-cis-Retinol</td>
			<td>34890</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
		<tr>
			<td>13-cis-Retinol</td>
			<td>48305</td>
			<td>Ethanol</td>
			<td>Robeson et al.50 and Robeson et al.51</td>
		</tr>
		<tr>
			<td>All-trans-Retinol</td>
			<td>52770</td>
			<td>Ethanol</td>
			<td>Hubbard et al.49</td>
		</tr>
	</tbody>
</table>
Table 1: Molar absorptivity of retinoids in ethanol. Molar absorptivity values of retinoids were gathered from Kane and Napoli44, Hubbard et al.46,Robeson et al.47, and Robeson et al.48. The molar absorptivity depends on the solvent. The values listed here are specific to retinoids dissolved in ethanol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue</td>
			<td>Tissue Harvest Amount</td>
			<td>Volume for Homogenization</td>
		</tr>
		<tr>
			<td>Blood</td>
			<td>75 &#181;L</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Liver</td>
			<td>0.2 g&#160;</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Kidney</td>
			<td>2 Whole Kidneys</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Skin</td>
			<td>0.2 g</td>
			<td>1 mL &#8211; 1.5 mL</td>
		</tr>
		<tr>
			<td>Spleen</td>
			<td>1 Whole Spleen</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Eye</td>
			<td>Both Retina, pool 4 Retina</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Brain</td>
			<td>1 Whole Brain&#160;</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Heart</td>
			<td>1 Whole Heart</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Lung</td>
			<td>0.2 g</td>
			<td>2 mL</td>
		</tr>
	</tbody>
</table>
Table 2: Recommended tissue quantity per analysis. Tissue quantity per analysis and volume of 50% ice-cold saline (0.9%) and 50% methanol for tissue homogenization, as recommended by Kane and Napoli44.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Retention Time (min)</td>
			<td>UV Absorbance Maxima (nm)</td>
		</tr>
		<tr>
			<td>Retinyl Palmitate</td>
			<td>5.177</td>
			<td>326</td>
		</tr>
		<tr>
			<td>13-cis-Retinal</td>
			<td>7.268</td>
			<td>366</td>
		</tr>
		<tr>
			<td>11-cis-Retinal</td>
			<td>7.691</td>
			<td>366</td>
		</tr>
		<tr>
			<td>9-cis-Retinal</td>
			<td>7.931</td>
			<td>364</td>
		</tr>
		<tr>
			<td>All-trans-Retinal</td>
			<td>8.993</td>
			<td>370</td>
		</tr>
		<tr>
			<td>11-cis-Retinol</td>
			<td>17.003</td>
			<td>318</td>
		</tr>
		<tr>
			<td>13-cis-Retinol</td>
			<td>17.83</td>
			<td>328</td>
		</tr>
		<tr>
			<td>All-trans-Retinol</td>
			<td>23.933</td>
			<td>326</td>
		</tr>
	</tbody>
</table>
Table 3: Retention times and maximum absorbance wavelengths of retinoids. These values were observed when used with the described method and mobile phase, as detected with the referenced diode array detector.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Retention Time (min)</td>
			<td>UV Absorbance Maxima (nm)</td>
		</tr>
		<tr>
			<td>Syn 11-cis-Retinal Oxime</td>
			<td>9.59</td>
			<td>346</td>
		</tr>
		<tr>
			<td>Syn All-trans-Retinal Oxime</td>
			<td>10.6</td>
			<td>356</td>
		</tr>
		<tr>
			<td>Syn 13-cis-Retinal Oxime</td>
			<td>11.3</td>
			<td>352</td>
		</tr>
		<tr>
			<td>Anti 13-cis-Retinal Oxime</td>
			<td>13</td>
			<td>356</td>
		</tr>
		<tr>
			<td>Anti 11-cis-Retinal Oxime</td>
			<td>15.1</td>
			<td>350</td>
		</tr>
		<tr>
			<td>Anti All-trans-Retinal Oxime</td>
			<td>19.7</td>
			<td>360</td>
		</tr>
	</tbody>
</table>
Table 4: Retention times and maximum absorbance wavelengths of hydroxylamine-treated retinaldehydes, resulting in syn and anti oxime isomers of each retinaldehyde. These values were observed when used with the described method and mobile phase, as detected with the referenced diode array detector.

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

974 Views.  University of Minnesota. Ons lab onderzoekt twee cruciale vitamine A-transporters, STRA6 en Rbpr2. Deze membraaneiwitten vergemakkelijken de opname van vitamine A in het bloed in weefsels zoals het oog en de lever. Via HPLC kunnen we de fysiologische functies van deze transporters onderzoeken door een systemisch vitamine A-profiel te maken bij transgene muizen.Een andere techniek die in ons vakgebied wordt gebruikt, is Surface Plasmon Resonance, SPR die de bindingsaffiniteiten...

Video: Auteur in de schijnwerpers: onderzoek naar fysiologische functies van vitamine A-transporters met behulp van op HPLC gebaseerde vitamine A-profilering

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biology

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

GC-MS Analysis of Deuterium Enrichment of Retinol in Serum and Estimation of Vitamin A Total Body Stores

This method describes the determination of deuterium enrichment of retinol in serum and the estimation of vitamin A stores in the body. The process involves extracting retinol from 0.4 mL of serum using 0.5 mL of 0.85% saline solution, 100 &#181;L of internal standard solution, and 5 mL of chloroform-methanol (2:1 v/v) solution. After centrifugation and removal of the lower chloroform layer, the mixture is dried under nitrogen and resuspended in 0.1 mL of ethanol, and the retinol fraction is separated from other constituents using an HPLC system equipped with a PE C18 column. The retinol fraction can be collected manually or with a fraction collector. Subsequently, the retinol fraction is dried under nitrogen and derivatized with O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 10% trimethylchlorosilane. Finally, labeled and non-labeled retinol isotopes are quantified using a GC-MS system equipped with a 19091z-431 HP-1 methyl siloxane capillary column, employing electron capture negative chemical ionization with helium as the carrier gas and methane as the ionization agent. The ratio of labeled to non-labeled retinol is then used in the Olson, Green, or mass balance equations to estimate vitamin A stores.

This method involves extracting retinol from serum, separating it using HPLC, and determining labeled and non-labeled retinol isotopes using GC-MS. The ratio of labeled to non-labeled retinol is used to estimate total body stores of vitamin A.

This method describes the determination of deuterium enrichment of retinol in serum and the estimation of vitamin A stores in the body. The process ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

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

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