Metabolism and excretion of a glutathione conjugate of acetaminophen in the isolated perfused rat kidney.

Acetaminophen-glutathione (APAP-GSH) is the initial sulfur-containing metabolite of APAP produced by the liver. However, little, if any, APAP-GSH is found in the urine of intact animals. Rather, the cysteine (APAP-CYS) and N-acetylcysteine (APAP-NAC) conjugates are the predominant sulfur-containing metabolites of APAP excreted in the urine. To define more precisely the role of the kidney in total body disposition of APAP, the metabolism and excretion of each of these metabolites was quantified in the isolated perfused rat kidney (IPK). With perfusate concentrations of 0.031, 0.125 and 0.250 mM APAP-GSH, the IPK metabolized APAP-GSH to APAP-CYS rapidly. Further metabolism of APAP-CYS to APAP-NAC proceeded at a much slower rate. Consequently, at 0.031 mM APAP-GSH, negligible amounts of APAP-CYS were found in the urine. However, as the concentration of APAP-GSH was increased so did the excretion of APAP-CYS. In contrast, the excretion of APAP-NAC did not exhibit dependence on APAP-GSH concentration. APAP-NAC was excreted by a probenecid sensitive transport mechanism whereas APAP-CYS excretion appeared to be related only to glomerular filtration. In addition, the disappearance of APAP-GSH was much greater than could be accounted for by glomerular filtration. These data indicate that the IPK is an effective model for the study of metabolism and excretion of xenobiotics that have undergone conjugation with GSH.

Metabolism of acetaminophen by the isolated perfused kidney.

Acetaminophen (APAP) produces proximal tubular necrosis in the Fisher 344 rat. This lesion may result from the covalent binding of reactive intermediates of APAP to cellular macromolecules when glutathione (GSH) is sufficiently depleted. Experiments were designed to evaluate the ability of the kidney to convert APAP to reactive electrophilic metabolites capable of depleting renal GSH by quantifying GSH concentrations in isolated perfused kidneys perfused with APAP. Perfusion without APAP reduced (3 X 10(-5) -3 X 10(-5) M) to the perfusion medium further reduced renal GSH content. Treatment of rats with polybrominated biphenyls enhanced the ability of 3 X 10(-8) M APAP to deplete GSH. In contrast, treatment with piperonyl butoxide reduced the depletion of GSH produced by 3 X 10(-5) M APAP. At 3 X 10(-5) M APAP, the glucuronic acid, sulfate and the mercapturic acid conjugates were excreted by the isolate perfused kidneys. After treatment with polybrominated biphenyls, mercapturic acid excretion increased 4-fold, whereas the glucuronic acid and sulfate conjugate excretions were unaffected. These data suggest that the kidney can produce an electrophilic metabolite of APAP which can combine with and deplete renal GSH. An electrophilic metabolite of APAP produced by the kidney may initiate APAP induced renal necrosis.

Formation and disposition of the minor metabolites of acetaminophen in the hamster.

The urinary metabolites of acetaminophen and N-hydroxyacetaminophen were studied in the hamster over a wide dose range and with pretreatments designed to modify drug metabolism. Attention was focused on the origin and disposition of the minor metabolites. The sum of the 3-thio adducts, rather than just the 3-mercapturic adduct, is considered the better index of the formation of the reactive immediate precursor, presumably N-acetyl-p-benzoquinoneimine. At low dosage this amounts to 33% of the administered dose in this species. There is a major contribution from the 3-methylthio adduct, the magnitude of which has not been previously recognized. The 3-methylthio and the 3-methylsulfoxide derivates of acetaminophen are secondarily derived from the 3-glutathione adduct within the enterohepatic circulation, as indicated by their late appearance in the urine, the effect of common bile duct ligation and the metabolism of the minor metabolites when they themselves are administered. Following the administration of N-hydroxyacetaminophen this was excreted in the urine along with its phenolic conjugates, but no urinary N-hydroxyacetaminophen was detectable after the administration of acetaminophen itself. Of particular interest to the pathogenesis of analgesic nephropathy was the detection in the urine of small amounts of p-aminophenol, a known nephrotoxic agent, following dosage with acetaminophen. This metabolite has not been previously detected.

Mechanism of decomposition of N-hydroxyacetaminophen, a postulated toxic metabolite of acetaminophen.

The decomposition of N-hydroxyacetaminophen (N-acetyl-N-hydroxy-p-aminophenol, 2), a postulated toxic metabolite of acetaminophen (N-acetyl-p-aminophenol, 1) in aqueous solution is quantitatively accounted for by the appearance of equimolar amounts of p-nitrosophenol and acetaminophen. The rate of decomposition depends on initial concentration and varies with pH. Antioxidants decrease the rate of decomposition and change the products. In the presence of cysteine, N-acetyl-3-(S-cysteine)-p-aminophenol, an in vivo metabolite of acetaminophen, is a product of decomposition.

Competition for binding to multiple sites of human serum albumin for cholecystographic agents and sulfobromophthalein.

The binding of two cholecystographic agents, iophenoxate and iopanoate, to human serum albumin was studied with 11 putative competitors; the results were qualitatively consistent with competitive binding to common sites. A more precise analysis of competition was achieved with four pairs of compounds for which the free and bound concentration of each was determined. The results were analyzed by a computer program and the dissociation constants calculated for both binder and competitor at specified sites on albumin. With numbering based on the rank order of dissociation constants for iophenoxate, the highest binding of the four compounds occurs at different sites: iophenoxate at site I; iopanoate at site II, sulfobromophthalein at site III; and bromphenol blue at site II. For a given compound, there is close agreement in the calculated affinities at different sites regardless of the competitor.

Synthesis of N-hydroxyacetaminophen, a postulated toxic metabolite of acetaminophen, and its phenolic sulfate conjugate.

The synthesis of N-hydroxyacetaminophen (N-acetyl-N-hydroxy-p-aminophenol, 4), a postulated toxic metabolite of acetaminophen (N-acetyl-p-aminophenol, 3), and its phenolic sulfate conjugate (potassium N-acetyl-N-hydroxy-p-aminophenyl sulfate) (13) is described. Potassium p-nitrophenyl sulfate was reduced to the hydroxylamine, acetylated, and treated with sulfatase to yield N-hydroxyacetaminophen. The structures assigned are supported by the spectral data (IR, UV, MS, 1H NMR, and 13C NMR). N-Hydroxyacetaminophen was found to be moderately unstable at physiological pH and temperature, whereas it phenolic sulfate conjugate was stable.

Covalent binding of metabolites of acetaminophen to kidney protein and depletion of renal glutathione.

Experiments in CD-1 mice and Sprague-Dawley rats were carried out to determine the extent to which biochemical changes described previously for acute acetaminophen-induced hepatotoxicity might be applicable to the kidney. After intraperitoneal injection of acetaminophen, tissue glutathione and covalent binding of tritiated metabolites of acetaminophen to tissue protein were measured for liver, kidney cortex and kidney papilla. Glutathione was reduced more in mice than in rats, and more in liver than in kidney, without appearance of oxidized glutathione in either tissue. Covalent binding was likewise greater in mice than in rats and greater in liver than in kidney. The determination of covalent binding was extremely sensitive to the trace radiochemical impurities of the labeled drug. With prior administration of 3-methyl-cholanthrene, the induced changes were far greater in liver than in kidney, suggesting that the formation of a reactive metabolite from acetaminophen occurred in each organ by slightly different mechanisms. At doses less than those associated with demonstrable acute toxicity, the duration of covalent binding to protein was longer for renal papilla than for renal cortex or for liver. The results may be applicable to the pathogenesis of both acute and chronic nephrotoxicity.