High resolution NMR spectroscopic studies on the metabolism and futile deacetylation of 4-hydroxyacetanilide (paracetamol) in the rat

Abstract

Paracetamol (4-hydroxyacetanilide, acetaminophen) was synthesized with the acetyl group labelled with C 2H 3 (paracetamol-C 2H 3, and dosed to rats i.p. at 25 mg/kg (N = 5) and 40 mg/kg (N = 3) body weight. Paracetamol, with a 13CH 3 in the acetyl group (paracetamol- 13CH 3) was also synthesized and dosed to rats i.p. at 40 mg/kg (N = 3). The metabolism and excretion of the 2H-labelled compound was followed in the rat using 600 MHz 1H and 92.1 MHz 2H NMR spectroscopy of urine collected 0–8, 8–24, 24–32 and 32–48 hr post-dosing. The metabolism of paracetamol- 13CH 3 was also monitored using 600 MHz 1H NMR spectroscopy of urine collected 0–8, 8–24 and 24–48 hr post-dosing. For paracetamol-C 2H 3 the total recovery of the sulphate, glucuronide and N-acetyl cysteinyl metabolites via the urine accounted for 61.2 ± 14.1% of the 25 mg/kg dose and 61.4 ± 8.8% of the 40 mg/kg dose. For paracetamol- 13CH 3 the recovery was 102.7 ± 3.7% indicating that the low % urinary recovery with the C 2H 3-labelled drug is the result of isotope effects on the disposition of paracetamol. In the case of the paracetamol-C 2H 3, quantitative 1H NMR analysis of urine showed that 13.3 ± 0.5 and 10.0 ± 1.2 mole% (25 and 40 mg/kg, respectively) of the urinary paracetamol sulphate recovered following dosing of the deuterium labelled drug had the C 2H 3 acetyl groups replaced by C 1H 3 acetyl groups from endogenous sources. In the case of the paracetamol- 13CH 3 8.9 ± 0.7 mole% of the sulphate conjugate had also been transacetylated to paracetamol- 12CH 3. There was no significant difference between the level of futile deacetylation observed for the deuterated and 13C-labelled drug. Overall these data indicate a high level of deacetylation followed by reacetylation (i.e. futile deacetylation) prior to excretion of paracetamol via the nephrotoxic intermediate 4-aminophenol. The level of deacetylation is much higher than has previously been thought which may cast new light on the role of 4-aminophenol in the development of paracetamol induced nephrotoxicity.