Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
James A. Olson
A statistical optimization technique, based on estimating a three-dimensional response surface for resolution over a range of mobile-phase compositions, was applied to the separation of isomers of retinol in normal-phase HPLC (high-performance liquid chromatography). The optimum solvent mixture for resolution of the 11-cis from the 13-cis isomer was very different from the optimum mixture for resolution of the 9-cis from the all-trans isomer. The effect of solvent selectivity on resolution was found to be much greater for resolution of different retinoid classes than for resolution of isomers within a class;No isomerization of all-trans retinal, retinyl palmitate or retinol in solution was observed on extended exposure to gold fluorescent lamps or in the dark, but extensive isomerization was seen on exposure to white light, particularly in polar solvents. The 9-cis isomer resulted when retinol and retinyl palmitate dissolved in polar solvents were exposed to white light, while exposure in hexane solution produced much less isomerization and produced primarily the 13-cis isomer. No evidence was found for free-radical or acid-catalyzed mechanisms for the observed isomerization in white light;A method was developed for the resolution of thirteen geometric isomers of retinol, retinal, and retinal oxime in a single chromatographic run on two 4 x 250 mm 5-[mu]m Lichrosorb Si-60 columns in series, by using 11.2% ethyl acetate, 1.4% 1-octanol and 2% dioxane in hexane as the mobile phase. All of the 11-cis and all-trans isomers of retinol and retinal oxime were completely resolved from each other and from the 9-cis and 13-cis isomers;In bleached bovine eye-cups dark-incubated either at 30°C or on ice, endogenous 11-cis retinal increased and all-trans retinol decreased in the retinas at 30°C relative to those on ice, each by approximately 5 nanomoles/eye. Retinyl esters and 11-cis retinol also decreased, but to a smaller degree, in the warm-incubated retinas. No significant changes in retinoids were noted in the retinal pigment epithelium. The addition of a supplement, containing pyruvate, L-glutamate, succinate, and glucose, to the medium enhanced isomerase activity, and a strong correlation (r = 0.95) between isomerase activity and oxidation of retinol to retinal was observed.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Gary Marshall Landers
Landers, Gary Marshall, "11-Cis retinoids: methods of analysis and handling, and regeneration in excised bovine eyes " (1987). Retrospective Theses and Dissertations. 8671.