The long-term goal of this research is to provide an in-depth knowledge of vitamin A homeostasis, especially as it is affected by diet. The rationale is that such data: is essential to understanding the relationship between vitamin A status and risk of cancer; may elucidate causal relationships between abnormalities in vitamin A metabolism and disease; and would be useful in evaluating the impact of the lont-term use of synthetic retinoids as chemopreventive agents. The hypothesis being tested in: vitamin A homestasis is modulated by vitamin A and/or its metabolites (endogenus retinoids) and by vitamin E through their effects on retinyl palmitate hydrolase (RPH), a key and perhaps regulatory enzyme of vitamin A metabolism.
The specific aims are: 1) purify neutral RPH from rat liver with a combination of conventional techniques, affinity chomatography, and immunoadsorbent chromatography; 2) characterize RPH with respect to (a) the in vitro effects of Alpha-tocopherol, retinol, and retinoic acid on its activity, (b) the effects of phosphorylation/dephosphorylation on its activity, (c) its specificity as a retinyl ester hydrolase, (d) whether a specific interaction between RPH and serum retinol binding protein (RBP) accelerates retinyl ester hydrolysis; and 3) determine the tissue distribution of RPH in rat, the proportion of neutral retinyl ester hydrolase activity accounted for by RPH, and how RPH concentrations and/or activity in vivo are affected by Alpha-tocopherol, retinol, retinyl palmitate, and retinoic acid concentrations in tissues. To accomplish the last specific aim an enzyme-linked immunoabsorbent assay (ELISA) will be used to quantify RPH in tissues, and HPLC will be used to assay the lipids of interest in tissues. Thus this proposal aims to relate the activity/amounts of the enzymes catalyzing vitamin A metabolism to the in vivo concentrations fo the lipids modulating and/or affected by the enzymes.
| Napoli, J L (1986) Quantification of physiological levels of retinoic acid. Methods Enzymol 123:112-24 |
| Napoli, J L (1986) Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line. J Biol Chem 261:13592-7 |
