Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma

Abstract

The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.

Introduction

Esterases in human plasma have an important role in the disposition of drugs. They participate in activation of ester prodrugs, for example, the prodrug bambuterol is converted to the anti-asthma drug terbutaline, and isosorbide-based prodrugs release aspirin. A second role is to inactivate drugs. For example, esterases in plasma inactivate the local anesthetics procaine and tetracaine, the muscle relaxants, succinylcholine and mivacurium, and the analgesics, aspirin, and cocaine. A third role for esterases in plasma is to detoxify natural and synthetic ester-containing poisons, for example, eserine (physostigmine) from the Calabar bean and organophosphorus pesticides are detoxified by hydrolysis or by binding. Table 1 lists drugs modified by the action of esterases in human plasma. Note that aspirin is hydrolyzed by BChE and albumin, and that paraoxon is hydrolyzed by PON1 and albumin. Paraoxon also interacts with BChE, but is not listed in the BChE column because the binding is stoichiometric rather than catalytic. To understand individual variation in response to drugs it is important to know the identity of the esterase responsible for drug hydrolysis. A classic example is the case of the muscle relaxant succinylcholine. The era of pharmacogenetics was born when it was discovered that people who responded abnormally to succinylcholine had an inherited deficiency of butyrylcholinesterase [1], [2]. Today more than 56 mutations in the BChE gene have been identified [3]. People who have two deficient BChE alleles cannot metabolize succinylcholine and therefore are unable to breathe for 2 h from a dose intended to paralyze for 3–5 min.

Knowledge of the identity of the esterases involved in drug hydrolysis can help explain drug response in individuals with disease. For example, diabetics have higher than average BChE levels in their plasma [4], [5], [6] and might therefore require higher doses of aspirin for antiplatelet therapy to prevent stroke and myocardial ischemia.

A drug that requires the action of an esterase could be ineffective if that esterase were inhibited by another drug. For example, echothiophate eyedrops administered for treatment of glaucoma, inhibit plasma BChE [7]. Bambuterol might be ineffective as an asthma drug in a patient receiving echothiophate. Natural toxins present in food may also affect the metabolism of esters, and thus cause side effects. For instance, potato glyco-alkaloids (solanine and chaconine) that reversibly inhibit BChE have been reported to slow the degradation of the myorelaxant mivacurium [8].

While BChE, AChE, and PON1 are well known esterases, albumin is not usually included in the family of esterases. Albumin does not have an enzyme commission number, which signifies that this protein is considered to be inert without catalytic activity. However, albumin has been conclusively proven to be an esterase [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. The active site of human albumin is Tyr 411 and of bovine albumin is Tyr 410 [21], [22]. The active site of bovine albumin was identified by mass spectroscopy after labeling albumin with a biotinylated organophosphorus agent [22]. Although the enzymatic activity of a single molecule of human albumin is low, the concentration of albumin is very high, so that albumin makes a significant contribution to drug metabolism.

Our goal is to identify the esterases in human plasma, and to demonstrate the absence of carboxylesterase (EC 3.1.1.1) in human plasma.