A fluorescence-based method for analyzing retinoic acid in biological samples

Abstract

Retinoic acid (RA) modulates the rates of transcription of numerous genes and thus plays key roles in multiple biological processes and is used in therapy of a number of diseases. However, RA therapy is often confounded by toxicity, raising the need for methodologies for its ready quantitation in biological samples. We describe a fluorescence-based method for quantitating RA that takes advantage of the high affinity and selectivity of the intracellular lipid-binding protein termed CRABP-I and CRABP-II and that uses them as RA sensors. L28C CRABP mutants were generated, and the inserted cysteine was covalently labeled with an environmentally sensitive fluorescent probe. The label was introduced into a region of the protein that undergoes a conformational shift on ligation. Consequently, RA binding resulted in distinct changes in the fluorescence of the protein-bound probe, allowing direct quantitation of RA. We show that the method can be used to monitor the biosynthesis of RA from its precursor retinal in cultured mammalian cells as well as the detection of exogenous RA in serum. The assay provides ease of use and sensitivity that enable quantitation of RA in biological samples of limited size, and it should prove to be useful in a variety of research and clinical applications.