Radiolabeled retinoids

The synthetic chemistry efforts in the retinoid field have been dominated for the most part by two large research groups Hoffmann-La Roche (Mayer and Isler, 1971) and Badische Anilin und Sodafabrik (BASF) (Pommer, 1960). These investigators have published numerous papers and patents as a result of industrially oriented research on the practical total synthesis of retinol and its analogs. Selected portions of this work can be adapted to the synthesis of radiolabeled retinoids. 

UV spectroscopy is a sensitive tool for the differentiation of stereoisomers and has been used extensively in the characterization of retinol and its analogs. Consequently, a wealth of spectral data exists for these compounds. Therefore, characterization of radiolabeled retinoids should include a comparison of the UV spectra with those of standard compounds. 

In general, recrystallization (when feasible) is the best method of purifying radiolabeled retinoids. Perry et aL (1982) found that when all-tran -retinoic acid was tritiated at very high specific activity, it had to be extensively purified by repeated recrystallization before it could be stored for any period of time. On the other hand, tritiated all-rran -retinaldehyde could not be obtained isomerically pure by recrystallization alone (Kaegi et aL, 1982a). 

Radiolabeled retinoids are exposed to the additional hazard of their own radiation. Therefore, it is very imporant to minimize all the effects leading to decomposition of labeled compounds. This is usually attempted by dispersal of the labeled molecules, cooling to low temperatures, and addition of free-radical scavengers. Retinoids are best stored at low temperature (—60 to —80 C) in dilute toluene solution containing an antioxidant like 2,5-di-f rt-butylhydro-... 

Efficiency and specificity of the prepared probe (e.g., ADAM-3) for the binding to RAR are crucial. These fractions can be evaluated by ligand competition assay using radiolabeled retinoids and RAR samples. As RAR samples, RARs extracted from various cells or tissues, including cells (mammalian, E. coli, and so on) transfected with an RAR-expression vector, can be used. (1-3,5,7,9-11) (see Note 3). 

Incubate an RAR sample (500 iL) with 1-4 aM of radiolabeled retinoid in the incubation buffer in the presence or absence of competitors (ADAM-3 and other retinoids, 0 I- to 1000-fold) at 4°C for 9-24 h... 

TLC is still useful for rapid assessment of HPLC methods, because it provides rapid analyses with different mobile phases in much less time than required for reequilibration of HPLC columns. TLC is also practical for rapid qualitative analysis of reaction products from synthetic or semisynthetic reactions and for confirming purity of concentrated solutions, in which impurities might escape detection by HPLC. TLC is in fact preferable to HPLC for analysis of radiochemical impurities (degradation products) in radiolabeled retinoids and carotenoids, since in HPLC some impurities might not be eluted and detected radioactivity on thin-layer plates may be detected by autoradiography (exposure of the plate to x-ray film (98)) or by cutting the TLC plate into short sections which can be counted individually in vials by liquid scintillation. 

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. 

Cells or tissue Radiolabeled precursor Retinoid" Reference... 

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