Kinetic Characterization of Na,K-ATPase Inhibition by the Acetaminophen Metabolite N-Acetylbenzoquinoneimine

Jeff B Helms,Jeremy P Holden,Craig Gatto,Charles J Costa,Lauren P Saunders,Matthew Corbitt,Nick Williford,Jamie Meyer,Eric Plowman

doi:10.4236/ojmip.2015.51001

Abstract

N-acetylbenzoquinoneimine (NABQI) is a toxic metabolite of the common analgesic acetaminophen (APAP). NABQI is an electrophilic intermediate formed via the oxidation of APAP within the cytochrome P450 system. Within the normally recommended low-dose use of APAP, NABQI is a minor metabolite which is either quickly reduced back to APAP or conjugated to Glutathione (GSH) producing an innocuous by-product. However, with overdose or prolonged high-dose usage of acetaminophen, GSH levels can become depleted and the bioactive NABQI is thought to form adducts with proteins and oxidize protein sulfhydryls producing intra- and intermolecular disulfide bridges in proteins. In this work we investigated the effect of NABQI on purified kidney Na,K-ATPase to see if the clinical renal insufficiencies seen in APAP overdose may be linked to inhibition of the Na,K-ATPase. Our work has shown that NABQI does indeed inhibit the Na,K-ATPase in a dose dependent (IC50 = 19.8 ± 2.9 μM) and irreversible manner. Interestingly, brief storage of NABQI at -20°C eliminates the irreversible effects of the compound, and leads to a product that remains a potent reversible inhibitor of the Na,K-ATPase (IC50 = 58.7 ± 19.5 μM). Further, the reversible inhibition produced by stored NABQI competes with para-nitrop.

Highlights

Acetaminophen (APAP) is a common and widely used mild analgesic and antipyretic drug
Since it has been reported that both APAP [1] [2] [5] and its reactive metabolite NABQI [6] [7] may affect Na,K-ATPase activity in situ, our investigation focused on elucidating the direct nature of these effects in un-sided preparations of Na,K-ATPase
We directly tested the toxicity of the predominant metabolite of APAP, NABQI on Na,K-ATPase function, an enzyme that plays a critical role in the transport processes of liver and kidney

Summary

Introduction

Acetaminophen (APAP) is a common and widely used mild analgesic and antipyretic drug. APAP in therapeutic doses is metabolized primarily by glucuronidation and sulfation (30% and 55% respectively) [3] [4], potentially toxic metabolic products result in [1]. One such metabolic product is N-acetylbenzoquinoneimine (NABQI). With high doses of APAP available GSH is rapidly depleted and bioactive NABQI has been shown to form adducts with proteins which can subsequently lead to the formation of intra- and intermolecular disulfide bridges in proteins [9]. Histopathological study of nephrotoxicity has shown damage to the proximal tubule, which is an area that relies on the function of Na,K-ATPase to drive transport in the kidney [15]

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Conclusion