On the role of cytochrome P450 3A4 in the metabolism of cholesterol and bile acids

Abstract

The origin of 4beta-hydroxycholesterol, a major oxysterol in human circulation, has previously been unknown. In this thesis, the formation and elimination of this compound are investigated by various techniques. The biological effects of 4beta-hydroxycholesterol are also discussed.

Patients treated with cytochrome P450 3A4 (CYP3A4) inducers, were found to have markedly elevated plasma levels of 4beta-hydroxycholesterol. Carbamazepine-induced patients had up to 20 times higher levels of 4beta-hydroxycholesterol than healthy volunteers. In contrast, patients treated with valproate, an antiepileptic drug that does not induce CYP3A4, had plasma levels of 4beta-hydroxycholesterol similar to those of healthy volunteers. Recombinant human cytochrome P450s were used to demonstrate that CYP3A4 catalyzes the 4betahydroxylation of cholesterol.

The increased levels of 4beta-hydroxycholesterol found in carbamazcpine treated patients are due to increased biosynthesis rather than impaired elimination, since the elimination rates were approximately the same in healthy volunteers and in a carbamazepine treated volunteer. The rates of elimination were determined by injecting deuterium-labeled 4beta-hydroxycholesterol intravenously. The rate of elimination of 4beta-hydroxycholesterol was found to be extremely slow compared to other oxysterols, due to slow conversion into bile acids. This was also demonstrated by incubations of 4beta-hydroxycholesterol with primary human hepatocytes. The slow formation of bile acids may partially be explained by a slow 7alpha-hydroxylation, the rate-limiting step in bile acid biosynthesis. One additional reason for a slow elimination may be due to a reduced rate of transfer over lipophilic membranes. 4betaHydroxycholesterol was thus found to have a slow rate of transfer from erythrocyte membranes to plasma lipoproteins compared to other oxysterols. The rate of transfer in vitro of 4beta-hydroxycholesterol and other oxysterols was found to correlate with their rate of elimination in vivo.

The physiological effects of elevated 4beta-hydroxycholesterol levels in humans is as yet unknown. However, previous studies have shown that 4beta-hydroxycholesterol is a relatively potent liver X receptor alpha (LXRalpha) agonist in cell-based transactivation studies. The nuclear receptor LXRalpha is an important regulator of genes involved in lipid metabolism. Thus, disturbances in lipid homeostasis seen in patients treated with antiepleptic drugs may partly be due to LXRalpha activation.

In addition to cholesterol, bile acids may also be substrates for CYP3A4. CYP3A4 was found to metabolize bile acids into 1beta-hydroxy- and 22-hydroxy bile acids. 1beta-Hydroxy-bile acids are known to appear in connection with cholestatic liver diseases and in infancy. CYP3A4 also oxidized the 3alpha-hydroxy group in the bile acids into a 3-oxo group. 3-Oxo bile acids have been shown to be ligands for nuclear receptors pregrane X receptor (PXR) and vitaminD receptor (VDR), which regulate the expression of CYP3A4. Thus, formation of 3-oxo bile acids by CYP3A4 suggests a metabolic link between bile acid homeostasis and CYP3A4 expression.