Effect of high fat diet on liver microsomal oxygenations in ferret.

1. Ferret on a high fat diet accumulated large amounts of fat in its liver and had blood acetoacetate and beta-hydroxybutyrate concentrations 250 and 375% of those in control animals. 2. The high fat diet alone increased ferret liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity by 90%, but had no effect on 7-methoxy-, 7-pentoxy-, or 7-benzyloxy-resorufin, O-dealkylase activities. Administration of 3-methylcholanthrene (MC) increased only liver EROD activity, by 5- to 6-fold, in ferret on both high fat and control diets. Induction of EROD, but not MROD activity, in ferret on the high fat diet indicates that P4501A1, but not P4501A2, is induced. 3. Activation of 3H-paracetamol, measured by covalent tissue binding to ferret liver microsomal fractions, was increased three-fold in ferret on the high fat diet, nine-fold by MC administered to ferret on a control diet, and 13-fold by MC given to ferret on the high fat diet. Similar results were obtained with activation of the cooked-food amine, Glu-P-1, by ferret liver microsomes. 4. Western blots with antibodies to rat liver P450s showed that ferret liver contains proteins orthologous with rat liver P4504A1 and bifunctional protein. However, whereas clofibrate, similar to high fat diets, induced these two proteins in rat liver, no increase of these proteins occurred in liver of ferret fed a high fat diet. Western blots also showed that ferret liver contains no P4501A1 or 1A2, and although these two proteins were induced by MC, no induction occurred when ferret was fed the high fat diet alone. Ferret liver microsomes also contain a protein recognized by rat anti-P4502E1 but of a lower molecular weight. 5. Immunosorbent (ELISA) analyses of ferret liver for P4501A1 and 4A1 showed that the high fat diet increased a protein orthologous to rat P4501A1 but did not increase any protein orthologous to rat P4504A1. 6. These findings indicate that the high fat diet does not induce ferret liver bifunctional protein or P4504A1 enzyme protein, but may enhance liver P4501A1 and 1A2 activities through the hyperketonaemia resulting from the high dietary fat. The conflicting P450 results, namely Glu-P-1 activation but no MROD activity for P4501A2, high EROD activity and ELISA quantification of P4501A1, but no positive Western blot, are probably due to differences in substrate specificity and immunological characteristics between rat and ferret enzymes.