Retinoic acid radiolabeling

Throughout this century, many techniques have been used in an attempt to discover the mechanisms of limb regeneration—denervation, transplantation of blastemas to ectopic sites, organ culture, radiolabeling, gel electrophoresis, administration of compounds, such as retinoic acid, and so forth. However, recently three technical advances have been made that are certain to have an important influence on future discoveries. 

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). 

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. 

Retinoic acid receptors (RARs) have been detected m nuclear extracts from various cell types on the basis of their ability to bind radiolabeled retinoic acid (1 3). Prior to the isolation of RAR cDNAs in 1987 (4,5), biochemical evidence for the existence of nuclear retinoic-acid binding proteins distinct from cytoplasmic CRABP had been obtained (1). The techniques used in these experiments can be used to study the biochemical properties of the RARs and may also be applicable to the study of the related receptors known as RXRs, which bind 9-cis retinoic acid with high affinity (6). 

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

Radiolabeled retinoic acid isomers [11,12 H(N)] a -trans retinoic acid is available commercially from Dupont NEN (Stevenage, Hertfordshire, UK) This material can also be used for preparation of the 13-cis and 9-cis isomers [ H] 9-cis retinoic acid is available from Amersham Life Science (Little Chalfont, Buckinghamshire, UK)... 

Incubation of cells with radiolabeled retinoic acid is best carried out in serum-free medium (see Note 2). 

Remove the medium from each 75-cm flask of cells, and replace with 5 mL of DMEM or an equivalent serum-free culture medium containing radiolabeled retinoic acid (see Note 2). 

Incubate the cells with the radiolabeled retinoic acid for 2-4 h at 37°C It is important that the incubation should not be prolonged, because retinoic acid may be metabolized quite quickly (see Note 3)... 

The specificity of binding of radiolabeled retinoic acid to nuclear proteins can be tested by incubating the cells with the radiolabeled retinoic acid in the presence of 100-fold excess of unlabeled retinoic acid. 

We recommend that the incubation of cells with radiolabeled retinoic acid is performed in the absence of serum Retinoic acid binds with low affinity to serum albumin (11) and this may reduce the concentration of free retinoic acid available for entry into the cells. For studies with [ H] ll-trans retinoic acid (15-50 Ci/ mmol), we have obtained satisfactory results using a final concentration of 20 nM. In the presence of 10% (v/v) fetal calf serum (FCS), we find that it is necessary to increase the retinoic-acid concentration to 0 2 pM to obtain a similar level of binding of retinoic acid to nuclear receptors... 

Ligands Both radiolabeled and cold ligands should be stored at -20°C in the dark in DMSO or ethanol. [ H]All-tra 5-retinoic acid is commercially available from NEN [ H]Am80 was prepared at Amersham (9). 

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