UBC Theses and Dissertations
Kinetics and cellular control mechanisms for imipramine metabolism in the isolated perfused rat liver Moldowan, Mervin John
An investigation was undertaken to study the kinetics and possible cellular control mechanisms for imipramine HCl metabolism in the isolated perfused rat liver. The isotope ¹⁴C-imipramine was used and quantification was done by liquid scintillation counting. Analysis for imipramine (IMI), desmethylimipramine (DMI), free hydroxy (OH), glucuronide (G) and N-oxide (N-0) metabolites was done on the perfusate, bile and liver. The rate of IMI metabolism was found to be dependent on two major enzymatic routes, N-demethylation (formation of DMI) and aromatic hydroxylation (formation of G, OH) of imipramine and one minor enzymatic route, N-oxidation (N-O). The rate of aromatic hydroxylation of IMI was found to be inhibited after thirty minutes, with IMI concentration 2 X 10 ⁻⁵M. This inhibition of aromatic hydroxylation could not be detected if the perfusate half-life for IMI (t½=18.5 minutes) was the only parameter monitored. After incubation periods of fifteen, thirty and sixty 80 per cent and the remainder of IMI was in the perfusate. The dose of IMI was varied (0.5 X 10⁻⁵ M, 1 X 10⁻⁵ M and 2 X 10⁻⁵ M) for metabolism by the perfused rat liver. The incubation time was kept constant at fifteen minutes. The rate of imipramine metabolism (formation of DMI and GOH) followed first order kinetics when the dose of IMI was 0.5 X 10⁻⁵ M or 1 X 10⁻⁵ M. Increasing the dose of IMI to 2 X 10⁻⁵ M slightly suppressed the formation of DMI and the formation of GOH followed zero order kinetics. It was found that the endogenous DMI formed from IMI metabolism inhibited the formation of GOH after fifteen minutes and thirty minutes of IMI metabolism as shown by the following results. DMI (1.65, 3.33, 6.66 or 13.32 X 10⁻⁶ M) was preincubated prior to addition of IMI. DMI (1.65 or 3.33 X 10⁻⁶ M) was found to specifically inhibit aromatic hydroxylation of IMI. Higher concentration of DMI (6.66 or 13.32 X 10⁻⁶ M) inhibited the formation of GOH and DMI. Ethyl alcohol (1 mM) preincubated prior to addition of 1 X 10⁻⁵ M of IMI specifically inhibited DMI formation. No inhibition of GOH occurred. Ethyl alcohol (1 mM) caused inhibition of formation of DMI from IMI metabolism when the dose of IMI was 2 X 10⁻⁵ M. The incubation time for IMI metabolism was fifteen and sixty minutes. With this decrease of DMI formation, the formation of GOH increased after fifteen or sixty minutes of incubation time. From these experiments it was concluded that suppression of aromatic hydroxylation of imipramine was due to the formation of endogenous DMI formed from IMI metabolism. Optimal conditions were found to study possible cellular control mechanisms for IMI metabolism in the isolated perfused rat liver. The dose of IMI was 1 X 10⁻⁵ M and the incubation time was fifteen minutes. Dibutyryl cyclic AMP (2 X 10⁻⁶ M) caused inhibition of IMI metabolism. DMI formation was inhibited 28 per cent while GOH formation was inhibited 29 per cent. NADPH (1.1 X 10⁻⁶ M) or NADH (1.3 X 10⁻⁶ M) was found to inhibit imipramine metabolism. GOH and DMI were both inhibited. Succinic acid (1.6 X 10⁻³ M) was found to inhibit DMI formation but not GOH.
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