Luminescence 2001;16:299304 DOI: 10.1002/bio.

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Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay

Boni Yavo1, Iguatemy L. Brunetti2, Luiz M. da Fonseca2, Luiz H. Catalani3 and Ana Campa1*
1 2

Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, CP 66.083, 05389970 Sao Paulo, Brazil Faculdade de Ciencias Farmaceuticas, Universidade Estadual Paulista, CP 502, 14801902 Araraquara, Brazil 3 Instituto de Qumica, Universidade de Sao Paulo, CP 26.077, 05599970 Sao Paulo, Brazil Received 1 September 2000; revised 10 March 2001; accepted 14 March 2001

ABSTRACT: In a previous study, we showed that puried commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)H2O2 system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPBserumHRPH2O2 system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at 4C. Addition of aliesterase inhibitors, such as uoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specic substrate benzoylcholine causes a delay in both O2 uptake and light emission, while the specic acetylcholinesterase substrate, acetyl-b-methylcholine, had practically no effect. Puried butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The nding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specic chemiluminescent assay for this enzyme. Copyright # 2001 John Wiley & Sons, Ltd. KEYWORDS: esterase; butyrylcholinesterase; pseudocholinesterase; horseradish peroxidase; chemiluminescence

INTRODUCTION
In a previous study, we developed a new analytical assay for esterase based upon the chemiluminescent oxidation of enols catalysed by horseradish peroxidase (HRP) (1). The enzyme-catalysed hydrolysis of 2-methyl-1-propenylbenzoate (MPB) produces 2-methyl-1-propenol, the enol form of isobutyraldehyde (IBAL), which is oxidized by HRPH2O2O2, leading to a presumed dioxetane intermediate (2), whose cleavage yields light emission.

kidney, duodenum, brain and liver, the last contributing to the esterase activity found in serum. Hence, in principle, altered plasma esterase activity can be used as a biochemical marker of hepatic diseases (4, 5). Another source of esterase activity in serum is paraoxonase. This activity is physically associated with high density lipoprotein (HDL) and has been implicated in the detoxication of organophosphates and possibly in the prevention of low density lipoprotein (LDL) peroxidation (6, 7).

The analytical potential of this reaction has subsequently been extended by demonstrating that the esterase present in monocytes, but not granulocytes, hydrolyses MPB and by the application of this nding in the distinction between monocytic leukemias (3). In mammals, high esterase activities are found in the
*Correspondence to: A. Campa, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, CEP 05508-900, Sao Paulo, Brazil. Email: anacampa@usp.br Contract/grant sponsor: Fundacao de Amparo a Pesquisa do Estado de ` Sao Paulo (FAPESP), Brazil. Contract/grant sponsor: Conselho Nacional de resenvolvimento Cientico e Tecnolo gico (CNPq), Brazil. Copyright 2001 John Wiley & Sons, Ltd.

The aim of this study was to verify the existence of esterase activity capable of hydrolysing MPB in serum. An activity specically related to butyrylcholinesterase (or pseudocholinesterase) is shown to hydrolyse MPB and trigger light emission when coupled with the HRP H2O2 reaction.

MATERIALS AND METHODS

HRP type I (E.C. 1.11.1.7), bovine erythrocyte acetylcholinesterase type XII-S (E.C. 3.1.1.7), human serum

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Figure 1. Oxygen uptake (A) and light emission (B) of the MPBserumHRPH2O2 system (&). Controls without MPB (!), serum (~), HRP (*) and H2O2 (^) are also shown.

butyrylcholinesterase (E.C.3.1.1.8), eserine, sodium uoride, trichlorfon, parathion, chloroquine, acetyl-b methylcholine and benzoylcholine were from Sigma. Hydrogen peroxide was from Aldrich. Isobutyraldehyde (Merck) was freshly distilled over KOH. The synthesis and identication of 2-methyl-1-propenyl benzoate has been described in Yavo et al. (1). Serum was obtained from healthy fasted persons and used without any further procedures. Although sera were used in all experiments, a possible interference of EDTA was checked. Serum can be replaced by plasma without any interference to the results. Chemiluminescence was followed in a BioOrbit Model 1251 Luminometer (intensity in mV and 1 mL nal volume) or in an EG&G Berthold Model LB96V (intensity in RLU/s and 0.3 mL of nal volume). Oxygen consumption was measured with a Yellow Springs Model 5300 oxygen monitor (3 mL of nal volume). Unless otherwise stated, the assay mixture contained MPB (1 mmol/L)/serum (50 mL/mL)/HRP (2.77 units/ mL)/H2O2 (74 mmol/L) in 0.05 mol/L phosphate buffer, pH 7.4, at 37C. The action of butyrylcholinesterase and acetylcholinesterase in substitution of serum was also tested.

RESULTS AND DISCUSSION

The MPB-serum-HRP-H2O2 system consumes oxygen and emits light (Fig. 1). These data indicate the presence of an esterase activity in serum capable of hydrolysing MPB to the enol form of isobutyraldehyde, which is then
Copyright 2001 John Wiley & Sons, Ltd.

oxidized by HRPH2O2. The exclusion of MPB, serum, HRP or H2O2 caused a marked reduction in oxygen uptake and completely abolished light emission. The dependence of the integrated light emission on substrate, HRP and H2O2 concentrations, at a xed volume of serum, is shown in Fig. 2 (AC). It is important to note that, in the range of HRP and MPB concentrations assayed, modications were observed in light intensity, but not in light emission kinetics. In contrast to what was observed for these two parameters, variations in the H2O2 concentration clearly affect both the kinetics and integrated light emission (Fig. 2C). Hydrogen peroxide is needed for the conversion of native HRP to the active compound I. This compound abstracts an electron from the enol to form HRP-compound II and a radical that reacts with oxygen, giving rise to the chemiluminescent reaction. Compound II can also accept an electron from the substrate or other source to regenerate the native enzyme (2). Consequently, H2O2 is continuously consumed during the reaction and the shorter duration of the chemiluminescent reaction at lower H2O2 concentration reects this consumption. How different volumes of serum affect integrated light emission is shown in Fig. 2D. A linearity between light emission and serum volume was seen only until 50 mL serum/mL of reaction. The serum-triggered emission from MPBHRPH2O2 is partially inhibited by incubating the serum at 56C for 1560 min and is completely abolished by serum heat inactivation for 4 h. Storage of serum at 20C (up to 2 weeks) or even at 4C (up to 4 days) retained most of the ability to trigger the emission. The relative stability of the
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Figure 2. Effect of substrate (A), HRP (B) and H2O2 (C) concentrations and volume of serum (D) on the integrated light emission of the MPBserumHRPH2O2 system.

serum enzyme involved here is consistent with that described for serum esterase (8, 9). In one case, serum was dialysed against 0.05 mol/L phosphate buffer, pH 7.4, at 4C (24 h, with three exchanges), using a 100,000 Da cut-off membrane. Dialysis of the serum resulted in approximately two-fold increase in the light emission relative to the original intensity, probably the result of reduced interference from low molecular weight quenchers. The type of esterase responsible for the activity found in serum was ascertained through the addition of specic inhibitors and competition with specic substrates. Chloroquine, NaF and the organophosphorous agent trichlorfon were considered as inhibitors of aliesterases
Copyright 2001 John Wiley & Sons, Ltd.

(10, 11). Of these, chloroquine inhibited oxygen consumption and the chemiluminescence of the IBALHRP H2O2 reaction. Since this reaction is a free model of the oxidation step of our system (2), chloroquine was disregarded. The effect of trichlorfon and NaF on the MPBserumHRPH2O2 system is shown in Fig. 3. The addition of each aliesterase inhibitor completely prevented light emission from the MPBserumHRPH2O2 system, but had no effect on the oxygen consumption or light emission of the model IBALHRPH2O2 reaction. The same behaviour was observed for eserine, a specic cholinesterase inhibitor (10, 12) (Fig. 4). The identity of the cholinesterase activity present in serum that hydrolyses MPB was further veried through
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Figure 3. Inhibition of the light emission produced by the MPBserumHRPH2O2 system by (A) trichlorfon (&, control; ^, 5.8; !, 11.6 and *, 93.4 mmol/L) and (B) sodium uoride (&, control; *, 2; and ~, 25 mmol/L).

the addition of specic substrates (13). The human butyrylcholinesterase-specic substrate, benzoylcholine, caused a marked delay in both light emission (Fig. 5A) and O2 uptake (Fig. 5B). These data clearly show that benzoylcholine competes with MPB as a substrate for serum esterase. Moreover, commercial serum butyrylcholinesterase triggered a very intense light emission in the MPBbutyrylcholinesteraseHRPH2O2 system (Fig. 6). On the other hand, the specic acetylcholinesterase substrate, acetyl b-methylcholine, had no signicant effect on either of these two parameters. Similarly, a commercial sample of puried acetylcholinesterase (0.070.98 U/mL) did not exhibit activity. Another expected esterase source in serum is paraoxonase. This enzyme is associated with high density lipoprotein (HDL) but is not present in the low density lipoprotein (LDL) fraction (6, 7). In the reaction studied here, the participation of lipoproteins as a source of serum esterase was not expected, since paraoxonase requires calcium for activity and dialysis of serum did not decrease light emission.

CONCLUSIONS
Figure 4. Inhibition of the light emission produced by the MPBserumHRPH2O2 system by eserine (&, control; *, 0.04; ^, 0.22; and !, 4.45 mmol/L).
Copyright 2001 John Wiley & Sons, Ltd.

It was ascertained that butyrylcholinesterase in serum can hydrolyse MPB, yielding a substrate for the HRPH2O2 catalysed chemiluminescent reaction. This suggests the
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Figure 5. Light emission kinetics (A) and oxygen consumption (B) of the MPBserumHRPH2O2 system in the absence and presence of benzoylcholine (&, control; *, 0.4; ~, 1.0; !, 2.0; and ^, 4.0 mmol/L).

use of this system as a specic chemiluminescent assay for butyrylcholinesterase. Apart from being a potentially useful biochemical marker for the evaluation of hepatic diseases (4, 5), organophosphorus poisoning (9) and veno-occlusive disease (14), additional applications of butyrylcholinesterase determination can be envisioned, given its role in lipoprotein metabolism, especially that associated with diabetes (1517). Butyrylcholinesterase activity is also related to a poor prognosis index in cancer patients (18). Esterase activity also seems to be a useful marker in cerebrospinal uid for meningitis (19) and in amniotic uid for prenatal diagnosis (20, 21). The chemiluminescent determination of butyrylcholinesterase in other biological uids besides serum should benet from the expected lower interference from proteins in these media. Acknowledgements The authors are indebted to the Fundacao de Amparo a ` Pesquisa do Estado de Sao Paulo (FAPESP), Sao Paulo, Brazil, and to the Conselho Nacional de Desenvolvimento Cientco e Tecnolo gico (CNPq), Braslia, Brazil for nancial support and to Professor Frank Quina (Universidade de Sao Paulo) for reviewing this manuscript.
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Figure 6. Light emission kinetics of the MPBbutyrylcholinesteraseHRPH2O2 system (&, 0.8; *, 1.6; ~, 3.2 U butyrilcholinesterase/mL).
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