Impact of MB-001 treatment on the production of 11-cis-retinol by ground squirrel RPE. (A–D) Examples of high-performance liquid chromatography separation of retinoids obtained from vehicle-treated squirrels (A), squirrels treated with MB-001 at doses of 8 mg/kg/day (B) or 80 mg/kg/day (C), and untreated bovine RPE (D). Blue and red traces show separations from assays performed only with added vehicle (dimethylformamide) or with all-trans-retinol delivered in dimethylformamide, respectively. Retinoids are labeled as follows: 1, all-trans-retinyl esters; 2, 11-cis-retinol; 3, all-trans-retinol; and 4, 13-cis-retinol. The insets in A and B show absorbance spectra for peaks 2 and 3. (E) Quantification of 11-cis-retinol production by the indicated RPE samples. The different vehicle and 8 mg/kg MB-001 samples are biological replicates. Each sample was assayed in duplicate. The results are presented as means, and the error bars represent SDs. An RPE65 immunoblot of the samples loaded at the same relative amounts as used in the assay demonstrates that the difference in isomerase activity between the vehicle and drug-treated samples is not attributable to large differences in RPE65 levels. Only a single band at ∼65 kD corresponding to RPE65 was visualized on the immunoblot; hence, the image was cropped to the area surrounding this band. DMF, dimethylformamide.