Characterization of oxidized and glucuronidated metabolites of retinol in monkey plasma by thermospray liquid chromatography/mass spectrometry

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Abstract
Eight metabolites of retinol were isolated by high-performance liquid chromatography (HPLC) from the plasma of the non-human primate Macaca fasicularis after acute exposure to 150 000 IU of vitamin A per kilogram body weight. After enrichment and further chromatographic purification, the metabolites were reinjected individually into a second HPLC system which was connected on-line by a thermospray interface to a mass spectrometer operated in the positive ionization mode. Six retinoids were identified by (i) a comparison of their retention times with those of appropriate reference compounds in the two chromatographic systems and (ii) by comparison of their mass spectra with those of reference compounds. These retinoids were: 13-cis-4-oxoretinoic acid, all-trans-4-oxoretinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, all-trans-retinoyl β-glucuronide and all-trans-retinyl β-glucuronide. One further metabolite could be identified for the first time as all-trans-4-oxoretinoyl β-glucuronide by its mass spectrum and, after treatment of the unknown metabolite with β-glucuronidase, by its hydrolysis product all-trans-4-oxoretinoic acid. The molecular structure of one metabolite could not be elucidated. A major metabolic pathway of high-dose vitamin A in the non-human primate is apparently the oxidation of the primary alcohol group of retinol resulting in the formation of all-trans-retinoic acid. Subsequently, a broad spectrum of various metabolites of all-trans-retinoic acid, including β-glucuronides and retinoids with a 13-cis configuration, appear in the plasma.