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Luminescence 2001;16:299–304 DOI: 10.1002/bio.

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ORIGINAL RESEARCH

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

Ï Ï Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, CP 66.083, 05389±970 Sao Paulo, Brazil Ä Ä Ï Ï Faculdade de Ciencias Farmaceuticas, Universidade Estadual Paulista, CP 502, 14801±902 Araraquara, Brazil 3 Instituto de Quõmica, Universidade de Sao Paulo, CP 26.077, 05599±970 Sao Paulo, Brazil Â Ä Ä Received 1 September 2000; revised 10 March 2001; accepted 14 March 2001

ABSTRACT: In a previous study, we showed that purified 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 MPB–serum–HRP–H2O2 system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at 4°C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O2 uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-b-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific 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 HRP–H2O2–O2, 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 detoxification 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 finding in the distinction between monocytic leukemias (3). In mammals, high esterase activities are found in the
*Correspondence to: A. Campa, Faculdade de Ciencias Farmaceuticas, ˇ ˇ ˜ ˜ 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 Cientifico 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 specifically 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 MPB–serum–HRP–H2O2 system (&). Controls without MPB (!), serum (~), HRP (*) and H2O2 (^) are also shown.

butyrylcholinesterase (E.C.3.1.1.8), eserine, sodium fluoride, 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 identification 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 final volume) or in an EG&G Berthold Model LB96V (intensity in RLU/s and 0.3 mL of final volume). Oxygen consumption was measured with a Yellow Springs Model 5300 oxygen monitor (3 mL of final 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 37°C. 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 HRP–H2O2. 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 fixed volume of serum, is shown in Fig. 2 (A–C). It is important to note that, in the range of HRP and MPB concentrations assayed, modifications 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 reflects 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 MPB–HRP–H2O2 is partially inhibited by incubating the serum at 56°C for 15–60 min and is completely abolished by serum heat inactivation for 4 h. Storage of serum at À20°C (up to 2 weeks) or even at 4°C (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 MPB–serum–HRP–H2O2 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 4°C (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 specific inhibitors and competition with specific 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 IBAL–HRP– 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 MPB–serum–HRP–H2O2 system is shown in Fig. 3. The addition of each aliesterase inhibitor completely prevented light emission from the MPB–serum–HRP–H2O2 system, but had no effect on the oxygen consumption or light emission of the model IBAL–HRP–H2O2 reaction. The same behaviour was observed for eserine, a specific cholinesterase inhibitor (10, 12) (Fig. 4). The identity of the cholinesterase activity present in serum that hydrolyses MPB was further verified through
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Figure 3. Inhibition of the light emission produced by the MPB–serum–HRP–H2O2 system by (A) trichlorfon (&, control; ^, 5.8; !, 11.6 and *, 93.4 mmol/L) and (B) sodium fluoride (&, control; *, 2; and ~, 25 mmol/L).

the addition of specific substrates (13). The human butyrylcholinesterase-specific 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 MPB–butyrylcholinesterase–HRP–H2O2 system (Fig. 6). On the other hand, the specific acetylcholinesterase substrate, acetyl b-methylcholine, had no significant effect on either of these two parameters. Similarly, a commercial sample of purified acetylcholinesterase (0.07–0.98 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 MPB–serum–HRP–H2O2 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 HRP–H2O2 catalysed chemiluminescent reaction. This suggests the
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Figure 5. Light emission kinetics (A) and oxygen consumption (B) of the MPB–serum–HRP–H2O2 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 specific 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 (15–17). 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 fluid for meningitis (19) and in amniotic fluid for prenatal diagnosis (20, 21). The chemiluminescent determination of butyrylcholinesterase in other biological fluids besides serum should benefit 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 Cientıfico e Tecnolo ´ ´gico (CNPq), Brasılia, Brazil ´ for financial 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 MPB–butyrylcholinesterase–HRP–H2O2 system (&, 0.8; *, 1.6; ~, 3.2 U butyrilcholinesterase/mL).
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B. Yavo et al. 12. Boyer PD. Structure and control. In The Enzymes, vol 1, Boyer PD (ed.). Academic Press: New York, 1970; 124–127. 13. Sawbney AK, Lott JA. Acetylholinesterase. In Lott JA, Wolf PL (eds). Clinical Enzymology, 2nd ed Rich and Associates: New York, 1986; 1–26. 14. Kami M, Mori S, Tanikawa S et al. Risk factors for hepatic venoocclusive disease after bone marrow transplantation: retrospective analysis of 137 cases at a single institution. Bone Marrow Transpl. 1997; 20: 397–402. 15. Kutty KM, Payne RH. Serum pseudocholinesterase and very-lowdensity lipoprotein metabolism. J. Clin. Lab. Anal. 1994; 8: 247– 250. 16. Abbot CA, Mackness MI, Kumar S et al. Relationship between serum butyrylcholinesterase activity, hypertriglyceridemia and insulin sensitivity in diabetes mellitus. Clin. Sci. 1993; 85: 77–81. 17. Annapurna V, Senciall I, Davis AJ, Kutty KM. Relationship between serum pseudocholinesterase and triglycerides in experimentally induced diabetes mellitus in rats. Diabetologia 1991; 34: 320–324. 18. Maltoni M, Pirovano M, Nanni O et al. Biological indices predictive of survival in 519 Italian terminally ill cancer patients. Italian Multicenter Study Group on palliative care. J. Pain Symptom. Managem. 1997; 13: 1–9. 19. Popovic L, Skerk V, Batinica S, Zupancic B. Cholinesterase in plasma and cerebrospinal fluid in patients with viral meningitis. Lijec. Vjesn. 1996; 118: 108–109. 20. Elejalde BR, Peck G, de Elejalde MM. Determination of cholinesterase and acetylcholinesterase in amniotic fluid. Uses in prenatal diagnosis and quality control. Clin. Genet. 1986; 29: 196– 203. 21. Wald NJ, Barlow RD, Cuckle HS, Turnbull AC, Goldfine C, Haddow JE. Ratio of amniotic fluid acetylcholinesterase to pseudocholinesterase as an antenatal diagnostic test for exomphalos and gastroschisis. Br. J. Obstet. Gynaecol. 1984; 91: 882–884.

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Luminescence 2001;16:299–304