Role of Lipids in the Control of Sex Differences in the Phase I Metabolism of Drugs

Hepatic microsomal preparations from male and female rats were delipidated by column chromatography following cholate solubilisation. The enzyme activity was assayed using lignocaine as the substrate for the mixed function oxidase. The N-deethylation of lignocaine catalysed by delipidated microsomal proteins from male and female rat liver is greater when reconstituted in microsomal lipid than in dilauroylphosphatidylcholine (DLPC). The 3-hydroxylation of lignocaine is unaffected by this treatment. The above effect is mimicked by incorporation of dilauroylethanolamine (DLPE) into the DLPC vesicles with male- but not the female-derived enzymes. Microsomal lipids derived from the male were more effective than female-derived lipids in reconstituting enzyme activities with both male- and female-derived enzymes. There is, thus, a sex- and pathway dependent effect of the lipids: the male-specific N-deethylase pathway is more affected by lipid composition and then more so in the male-derived enzyme. It is possible, therefore, that some of the sex differences in drug metabolism may be related to changes in lipid composition. In addition, the sex-dependence of the metabolism of lignocaine was maintained in the reconstituted system, indicating that this is a suitable system for investigating the role of lipids in maintaining sex-specific drug metabolism. We have extended this work with the isolation of the isozyme of cytochrome P-450 responsible for the N-deethylation of lignocaine. The male-specific cytochrome P-450 isozyme, lignocaine N-deethylase, was purified to electrophoretic homogeneity from liver microsomes of untreated male rats with high retention of bioactivity. The purified N-deethylase was an efficient catalyst of the 16alpha-hydroxylation of androst-4 ene3,17-dione (specific activity of 10 nmole product/min/ nmole cytochrome P-450). The isozyme, together with purified NADPH-cytochrome P-450 reductase, was reconstituted using known lipids and the drug-metabolising activity assayed using lignocaine as substrate. The results showed that the purified isozyme only N-deethylated lignocaine and led to the following conclusion, (I) Mixed dilauroylphosphatidylcholine (DLPC)/dilauroylphosphatidylethanolamine (DLPE) vesicles gave a higher N-deethylase activity than DLPC vesicles. (II) The N-deethylation of lignocaine catalysed by a purified male-specific cytochrome P-450 from rat liver is greater when reconstituted in microsomal lipid than in DLPC. (III) Microsomal lipids derived from male were more effective than female-derived lipids in reconstituting enzyme activities. These data indicated that it is a direct interaction of the lipid with the enzyme (s) or an alteration of the protein-protein interactions caused by the lipid which leads to the change in enzyme activity and that it is the isozyme responsible for the N-deethylation that is particularly affected in this way. In order to assess which portion of the microsomal lipid causes these changes, the microsomal lipid has been fractionated into phospholipid and neutral lipid. Using delipidation and subsequent relipidation of microsomal lipid preparation, we have confirmed that the phospholipid is the most effective portion for the maintenance of the drug metabolism and the sex differences in drug metabolism. It is clear that the male-derived phospholipid was more efficient than the female-derived phospholipid when incorporated into both male- and female-derived delipidated microsomes. The mixture of phospholipid and neutral lipid fractions was very effective, indeed it restored the N-deethylase activity to the level of the microsomes. Experiments were carried out using the male-specific isozyme responsible for the N-deethylation of lignocaine and again it was shown that the phospholipid fraction was the most effective fraction when reconstituted with this isozyme, but it was less effective than the whole microsomal lipid. It is only when the combination of phospholipid and neutral lipid was used that the control activity was achieved. Again the male derived phospholipid was more effective than the female-derived phospholipid. We analysed the phospholipid composition and our analysis shows sex differences in microsomal phospholipid composition and in fatty acid acyl chain of the microsomal phospholipid. Also the analysis of the phospholipid from the diabetic animals showed that the streptozotocin treatment does alter the phospholipid composition and the ratio of acyl chains found. All these changes might be associated with differences in enzyme activity. (Abstract shortened by ProQuest.)