Retinoid analysis

In describing widely used methods for retinoid analysis, the discussion of methodological aspects of topics primarily treated elsewhere in this volume has been avoided. The preparation and analysis of radiolabeled compounds, for example, is considered in Chapter 3, biological methods for analyzing retinoids are addressed in Chapter 5, and specific binding proteins for retinoids are described in Chapters 8 and 9. Although the major emphasis of this chapter is placed on the measurement of retinol (Al), retinaldehyde (Cl), and retinoic acid (Dl), the same or similar methods have been, or could be, used for other retinoids. Carotenoids and apocarotenoids are not discussed. To keep the reference list within bounds, many older citations are omitted. 

Straight-Phase HPLC Systems Used in Retinoid Analysis... 

Infrared spectroscopy (IR) has not been extensively used in retinoid analysis (17,18). However, the newer techniques of Resonance Raman and infrared-difference spectroscopy have been applied to retinal proteins m rhodopsin and bacteriorhodopsin. By use of these techniques, it is possible to determine the structures of the chromophores m the visual pigments and in the intermediates of their photoreactions Also, it is possible to study the interactions between the chromophores and the protein, and the structural changes evoked in the protein by the photoreaction (1,19). 

Retinoids—Laboratory manuals. 2. Retinoids— Analysis—Laboratory manuals. I. Redfem, Christopher P. F. 11. Series Methods in molecular biology (Totowa, NJ) 89. 

A wide variety of liquid chromatography-mass spectrometry (LC-MS) techniques have been reported for retinoid analysis including direct liquid introduction with Cl (281,282,299-301), particle beam (302-304), thermospray (305-307), electrospray (308,309), and atmospheric pressure chemical ionization (APCI) (310). Because many of the early LC-MS applications to retinoids carried out hydrolysis of retinyl esters and then derivatization of retinoic acid and related retinoids, Wyss (141) predicted in a review of retinoid analysis that derivatization of retinoids would be necessary for all LC-MS techniques, even the anticipated application of atmospheric pressure chemical ionization (APCI). Recently, LC-MS analyses of retinoids have been carried out using APCI (310) and electrospray (308,309), and highly sensitive LC-MS analyses of retinoic acid and retinyl esters were achieved without hydrolysis or derivatization. 

Owing to the light and air sensitivity of the carotenoids and retinoids, sample handling is a critical issue. It is recommended to conduct extraction of these materials with peroxide-free solvents, to store biological samples at —70° C under argon and in the dark, to perform the analysis under yellow light, and to use reference compounds of high purity (57). 

Rhodopsin is a seven ot-helix trans-membrane protein and visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In this photoreceptor, retinal is the chromophore bound by opsin protein, covalently linked to Lys296 by a Schiff base linkage. Kpega et al.64 have studied NMR spectra of Schiff bases being derivatives of all-frans retinal and amino-p-cyclodextrins as a model of rhodopsin, where p-cyclodextrin plays a role of a binding pocket. On the basis of analysis of the chemical shift differences for the model compound in the presence and in the absence of adamantane carboxylate, it has been shown that the derivative of 3-amino-p-cyclodextrin forms dimer in water and retinoid is inserted into p-cyclodextrin cavity [31]. 

Retinoids The challenge in fat-soluble vitamins analysis is to separate them from the lipid fraction that contains interferents. Alkaline hydrolysis, followed by LLE, is widely applied to remove triglycerides. This technique converts the vitamin A ester to all-trani-retinol. A milder process, which does not hydrolyze vitamin A ester, is alcoholysis carried out with metha-nolic KOH solution under specific conditions that favor alcoholysis rather than saponification. A more accurate explanation of this technique is reported in the book Food Analysis by FIPLC [409]. For some kind of matrices a simple liquid extraction can be sufficient with [421-423] or without [424,425] the purification... 

Chambon P The retinoid signaling pathway molecular and genetic analysis. Cell Biol. 5 115-125,1994. 

Barua, A.B. 2001. Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues. J. Chromatogr. A 936 71-82. 

Barua, A.B. Olson, J.A. 1998. Reversed-phase gradient high-performance liquid chromatographic procedure for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal and plant samples. J. Chromatogr. B 707 69-79. 

The ligand binding pocket of USP is filled by a fortuitous phospholipid co-purified and co-crystallized with the USP LBD that was fiirther characterized by mass-spectroscopic and chemical analysis [57]. In a similar way, recent crystallographic investigations of the retinoid-acid related orphan receptor (3 (ROR (3) [58] and of the heterodimeric complex RARa/RXRa [30] revealed an E.coli endogeneous fatty acid in the ROR (3 and in the RXRa subunit, respectively. 

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