Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present 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.
Section snippets
Materials
Silent BChE plasma samples were from the United States, India, and France. Silent plasma was stored at −20 °C. Silent BChE samples had no detectable activity with butyrylthiocholine or benzoylcholine. Human serum from people with wild-type BChE contained no anticoagulant and was stored at −80 °C. Serum containing wild-type BChE had an activity of 3–4 μmol/(min ml) (1 mM butyrylthiocholine, 0.1 M potassium phosphate buffer pH 7.0, 25 °C).
All mice were strain 129Sv. Mouse serum was freshly drawn from
Human plasma contains four esterases
The esterases in human plasma are BChE, PON1, albumin, and AChE. The migration of each of these esterases relative to each other on nondenaturing gels is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4. Fig. 1 shows the monomer, dimer, trimer, and tetramer bands of BChE in human serum. Fig. 1 also shows that plasma from six individuals with silent BChE have no BChE activity as the lanes are blank. In Fig. 2, human plasma samples were run on a gel stained with acetylthiocholine, revealing a band of
Three esterases in human plasma
The convention of naming an esterase for the drug or substrate being studied gives the impression that human plasma contains dozens of different esterases. However, we find only four esterases in human plasma, and one of these, AChE, is present in negligible amounts. This leaves only BChE, PON1, and albumin to carry out ester hydrolysis.
Our analysis assumes that any esterase present in significant quantity would have been stained with alpha or beta-naphthylacetate. This assumption is verified
Acknowledgement
Supported by U.S. Army Medical Research and Materiel Command Grants DAMD17-01-1-0776 and DAMD17-01-2-0036 (to O.L.), UNMC Eppley Cancer Center Support Grant P30CA36727, U.S. Army Research, Development & Engineering Command Grant W911SR-04-C-0019 (to O.L.), French Office of the Defense Cooperation Attache Grant DGA/DSP/STTC PEA 03CO001 (to P.M.), and Indian funding agency, AICTE, New Delhi Grant F. No. 8019/RDII/BOR/R&D 226/2001 (to R.B.). MS was funded by the UNMC College of Medicine Dean's
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