S. J. Hart

Paracetamol and the isolated perfused kidney: Metabolism and functional effects

1. Renal metabolism of paracetamol has been studied in the isolated perfused rat kidney. 2. The major metabolites of paracetamol normally observed in vivo were present in low concentrations in the urine of the isolated perfused rat kidney. 3. Paracetamol was bound covalently to kidney protein in a linear relationship to dose up to 31.9 mM paracetamol in the perfusate. 4. Using Michaelis-Menten kinetics, the apparent Km of 5.5 mM and Vmax of 139 nmol/h/g wet wt. for renal paracetamol oxidation were observed. 5. Concn. of paracetamol greater than 14.2 mM induced immediate diuresis and diminished sodium reabsorption. Lower concn. were without effect on function.

The metabolism and toxicity of paracetamol in Sprague-Dawley and wistar rats

SummaryUrinary paracetamol metabolites from Sprague-Dawley and Wistar rats were analysed by reversed-phase HPLC. Variations in the metabolic profile were observed as a function of dose, age, sex, species and route of administration. In addition the effect of 3-methylcholanthrene as an inducer of cytochrome P450 mixed function oxidase on paracetamol metabolism was also studied. Increased oxidative metabolism which lead to the formation of 3-thiomethylparacetamol conjugates along with paracetamol mercapturic acid could be correlated with increased susceptibility to hepatic demage. Furthermore it appears that the length of time taken for excretion and the level of free drug excreted may be involved in the aetiology of chronic renal damage.

Induction of paracetamol metabolism in the isolated perfused kidney

1. At a perfusate concn. of 3.5-4.0 mM, 59 plus or minus 9 nmol of paracetamol h per g wet wt. were oxidized by isolated rat kidney. 2. Approx. half the paracetamol undergoing oxidation was converted to a mercapturic acid metabolite and the remainder was covalently bound to kidney protein. 3. Addition of GSH to the perfusate decreased the level of covalent binding. Depletion of cellular GSH, by prior administration of diethyl maleate, significantly decreased formation of the mercapturic acid metabolite. 4. The metabolic pathways of glucoronylation, sulphation and mercapturic acid formation were induced either by 3-methylcholanthrene pretreatment or by prolonged feeding of aspirin or paracetamol; covalent binding of paracetamol to kidney protein was not increased.

Complete separation of urinary metabolites of paracetamol and substituted paracetamols by reversed-phase ion-pair high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic procedure has been developed for the separation of thirteen urinary metabolites of the analgesic drug paracetamol. The method involved the use of radially compressed columns packed with octadecylsilica with a particle diameter of 5 micron. Metabolites were chromatographed by linear gradient elution using an ion-pair solvent system composed of tetrabutylammonium hydroxide and Tris buffered to pH 5.0 with phosphoric acid, and acetonitrile as the organic solvent. Analyses can be performed at the rate of three per hour. This method enables the direct identification of sulphate and glucuronide conjugates of 3-thiomethylparacetamol and 3-thiomethylparacetamol sulphoxide which have only previously been detected following enzyme hydrolysis of urine samples. The application of this fully optimised separation to the study of the metabolism of substituted paracetamols is also discussed.

Hepatotoxicity of Phenacetin and Paracetamol in the Gunn Rat

&NA; Both phenacetin and paracetamol produce acute centrilobular liver necrosis in the homozygous Gunn rat. Paracetamol is more hepatotoxic than phenacetin, and both are more hepatotoxic to the homozygous Gunn rat than to the heterozygous Gunn rat or to the albino rat. These findings have relevance to the role of the compounds in the clinical syndromes of paracetamol induced liver necrosis and analgesic nephropathy.

3-Thiomethylparacetamol sulphate and glucuronide: metabolites of paracetamol and N-hydroxyparacetamol

1. Two new metabolites of paracetamol, 3-thiomethylparacetamol sulphate and glucuronide, have been isolated and identified. 2. The metabolites occurred in both rat and mouse urine after administration of either paracetamol of N-hydroxyparacetamol. The amount excreted increased proportionally with the dose.

Metabolism of paracetamol by the isolated perfused kidney of the homozygous Gunn rat

1. Isolated kidneys from homozygous Gunn rats were perfused with paracetamol in concentrations lower and higher than Km for paracetamol oxidation in the albino rat kidney. 2. Glucuronylation of paracetamol was not detected at either concentration. 3. An increase in oxidative metabolism at the higher concentration, similar to that seen with the Sprague-Dawley rat kidney, did not occur with kidneys from homozygous Gunn rats. 4. This finding does not support the hypothesis that the enhanced nephrotoxicity of paracetamol observed in the homozygous Gunn rat in vivo is due to increased intrarenal formation of reactive metabolites.

Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially compressed columns.

Methods have been adapted for the high-performance liquid chromatographic (HPLC) analysis of urinary paracetamol metabolites on radial compression columns. Enhanced resolution and decreased analysis time were two major advances. Various modifications to existing methods were made to counter the effect of the different C18 surface. Thus in ion suppression HPLC the addition of triethylamine at pH 3.0 (phosphate buffer) was necessary to block residual hydroxyl sites, while in ion-pair HPLC a higher tetrabutyl-ammonium hydroxide concentration of 0.01 M at pH 5.0 was used to enhance selectivity. The methods were successfully applied to the study of the metabolism of paracetamol, its glutathione conjugate and 3-thiomethylparacetamol in Sprague-Dawley rats. 3-Thiomethyl-paracetamol sulphoxide and its glucuronide and sulphate conjugates were shown to be metabolites of both 3-thiomethylparacetamol and paracetamol. 3-Thiomethylparacetamol sulphate was unresolved from the sulphates of paracetamol and 3-methoxyparacetamol in ion-pair HPLC. This raises a previously unrecognised problem in which the peak normally attributed to paracetamol sulphate contains metabolites arising from an oxidative metabolic pathway. Elevated levels of 3-methoxyparacetamol conjugates were found in human overdose urine and to some extent in analgesic nephropathy.