This research proposes to develop an LC/MS assay to distinguish, identify, and quantitate endogeneous retinoids in biological tissues. Despite the importance of retinoic acid (RA) in development, differentiation, vision, reproduction, and immune response, no sensitive assay exists capable of measuring endogeneous levels. HPLC with various detection schemes lacks sensitivity, GC/MS requires derivitization and cannot resolve geometric isomers of RA, and LC/MS assays to date have not significantly improved detection limits of RA and/or have not been developed as an analytically vigorous assay. The measurement of endogeneous levels of RA and investigation of the hypothesis that RA will colocalize with enzymes that synthesize RA is of great interest towards the understanding of RA function.
Specific aims are (1) to develop and validate a sensitive, specific assay for RA and its isomers in biological samples, (2) to demonstrate the utility of the assay by applying it to animal tissue studies consisting of (a) the measurement of retinoids in wt and CRBP -/- mice, (b) the assessment of the impact of ethanol intoxication on all-trans RA levels in wt mice, and (c) the investigation of whether IRA concentration colocalizes with the enzymatic apparatus that synthesizes it in mouse brain and established cell lines.
