Effects of N–(4–hydroxyphenyl) Retinamide on Vitamin A Homeostasis and A2E Biosynthesis in abcr Null Mutant Mice

Abstract

Abstract: : Purpose: In order to explore the relationship between serum retinol and biosynthesis of the lipofuscin fluorophore (A2E), N–(4–hydroxyphenyl)retinamide (HPR) was administered chronically to abcr –/– mutant mice. Methods: HPR was delivered to abcr –/– mice daily for a period of 28 days (20 mg/kg in DMSO, i.p.). Control abcr –/– mice received only DMSO for the same period. Animals were sacrificed at 1–week intervals for serum and eye collection. All–trans retinol and HPR were extracted from serum into acetonitrile and lipid soluble constituents (i.e., A2E and precursors) were extracted from eye tissue using chloroform/methanol/water phase partitioning. Effects of HPR on enzymes and proteins of the visual cycle (lecithin retinol acyl transferase (LRAT), isomerase (IS), 11–cis retinol dehydrogenase (11cRDH), cellular retinol binding protein (CRBP) and cellular retinaldehyde binding protein (CRALBP)) were determined using in vitro enzyme assays and fluorescence quenching spectroscopy. Results:Administration of HPR caused immediate and profound reductions in serum retinol (40% reduction 2 hrs post–injection). Chronic HPR administration was associated with a 53% decrease in the A2E precursor (A2PE–H2) following 14 days of treatment and a 60% decrease in A2E at the conclusion of the trial. Serum retinol was reduced by 72% at the end of the study period. In vitro assays revealed a competitive inhibition of LRAT activity by HPR, no direct effect on IS or 11cRDH was observed. Data from binding protein assays indicated that HPR is able to displace retinol from RBP at physiological temperature. Although HPR was observed to bind CRALBP at high concentrations (2–fold molar excess relative to [CRALBP]), no displacement of 11–cis retinal was detected. No binding of HPR to apo–CRBP was observed. Conclusions: The data clearly demonstrate a strong correlation between decreases in serum retinol and decreases in A2E biosynthesis. The fact that HPR also manifests a specific intracellular effect on LRAT makes it a favorable candidate drug for modulation of retinoid concentration in the eye and treatment for lipofuscin–based retinopathies.

Keywords: retinoids/retinoid binding proteins • degenerations/dystrophies • drug toxicity/drug effects