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The cytochrome P-450 mediated activation of paracetamol (PAR) to the reactive electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI) has been studied by use of SV 6-31G ab initio energy calculations and spin distributions. A simplified model for cytochrome P-450 has been used by substituting the proposed biologically active ferric-oxene state of cytochrome P-450 by a singlet oxygen atom. The results indicate that an initial hydrogen abstraction from the phenolic hydroxyl group is favored by 30.1 kcal/mol over an initial hydrogen abstraction from the acetylamino nitrogen atom. Metabolic activation of PAR via primary formation of a phenoxy radical seems the most likely mechanism. The calculated ab initio spin densities indicate that the radical formed by hydrogen abstraction from the phenolic hydroxyl group stays predominantly localized at the phenolic oxygen. A second hydrogen abstraction from the acetylamino nitrogen atom, giving rise to the reactive intermediate NAPQI, is then favored in terms of energy differences. The unpaired electron of the phenoxy radical was found to delocalize only to a small extent toward the carbon atoms at the ortho and para positions relative to the hydroxyl-containing ring carbon, but nevertheless a recombination reaction between a hydroxyl radical and these radicalized carbon atoms at the ortho or para positions could explain the formation of the minor metabolites 3-hydroxy-PAR and p-benzoquinone plus acetamide.

From the hepatic cytochrome P-450 isozymes b and c isolated from rats treated with phenobarbital and 3-methylcholanthrene respectively, only cytochrome P-450c was found to be active in the oxidation of paracetamol, in the presence of glutathione ultimately leading to the formation of the 3-glutathionyl conjugate. Paracetamol interacted with both cytochrome P-450b and c, as shown by difference spectrophotometry. Cytochrome P-450b was found to have a higher affinity for paracetamol than cytochrome P-450c and demonstrated a type I spectral change, whereas in the case of cytochrome P-450c a reverse type I spectral change was observed. Proton n.m.r. longitudinal relaxation rate measurements revealed that in the case of cytochrome P-450c, paracetamol was orientated with its phenolic hydroxyl group in closest proximity to the central haem iron ion. In the case of cytochrome P-450b, the acetylamino group of paracetamol most closely approached the haem iron ion.

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