11] Identification and quantification of retinoic acid and other metabolites from β-carotene excentric cleavage in human intestine in vitro and ferret intestine in vivo

Abstract

This chapter discusses the identification and quantification of retinoic acid and other metabolites from β-carotene excentric cleavage in human intestine in vitro and ferret intestine in vivo. The identification of metabolites from β-carotene is based on high-performance liquid chromatography (HPLC), ultra-violet (UV) spectrum, chemical derivatization, and gas chromatography/mass chromatography (GC/MS). Individual carotenoids and retinoids are initially identified by coelution with standards, and quantified relative to the internal standard (γ-carotene for carotenoids and retinyl acetate for retinoids) by determining peak areas calibrated against known amounts of standards. An additional Waters 994 programmable photodiode array detector is used for measurement of absorption spectra. To obtain a more detailed analysis of the retinoic acid isomers formed during the incubations, the retinoic acid fraction from the HPLC chromatogram is collected and dried under the N2. The residue is dissolved in 3 ml peroxide-free diethyl ether and derivatized with diazomethane to form the methyl retinoates. Authentic 9-cis-retinoic acid and all-trans-retinoic acid, as well as the retinoic acid fraction from either the incubation of 9-cis-β-carotene with human intestinal mucosa fraction or the perfusion of β-carotene with ferret intestine, are reanalyzed using the same HPLC system.