Quantitative Analyses of Naturally Occurring Retinoids

It is widely believed that the concentrations of retinoids that activate the two classes of nuclear-retinoid receptors are crucial to the effecis of the receptors, and ultimately the actions of the retinoid-humoral system. It is therefore truly important to know the precise concentrations and exact nature of the retinoids present at definite times and specific loci during development, and indeed, during any event mediated by retinoids. To determine conclusively the mechanisms of retinoid action, it is not sufficient to localize only the receptors; eventually we must establish which retinoids are present and the concentrations in which they are present, because several naturally occurring retinoids can stimulate receptor action, albeit with different ED₅₀ values. For example, retinoids that can activate retinoic acid (RA) receptors (RARs) include, in addition to all-trans RA and 9-cis RA, 4-hydroxy RA, 4-oxo RA, and 1%hydroxy RA. 13-cis RA also binds to RARs, but with a K _d value higher than the former group. Retinol can also induce responses in RA-sensitive systems; generally at doses ~200-fold higher than RA. In RA-dependent responses, retinol probably functions through conversion into RA (both enzymatic and artifactual conversion may contribute, depending on the circumstances). For example, uses of exogenous retinol, especially in higher concentrations (mM ) could generate small amounts of retinoids by oxidation or by the actions of enzymes that normally are denied access to retinol in vivo. The techniques described in this chapter offer sensitive (<2 pmol), specific quantification of a variety of retinoids. A gas chromatography/mass spectrometrc (GC-MS) system has been described previously with greater sensitivity (~0.25 pmol), which can be coupled with high-performance liquid chromatography (HPLC) to enhance specificity (1 –3 ). Additional HPLC systems for quantifying RA in blood have been summarized previously (4 , 5 ).