Determination of acetylcholinesterase and butyrylcholinesterase activity without dilution of biological samples

Article abstract of DOI:10.1515/chempap-2015-0117

Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known. The enzymes are important in the body and alteration of their activity has significant use in the diagnosis of poisoning, liver function, etc. Currently available methods for the determination of cholinesterases have some major drawbacks including various interferences and the inability to be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates were modified with indoxylacetate (100 μL of 10 mmol L^-1 solution) and used for cholinesterases assay after drying at 37 °C. The fact that indoxylacetate remains stable in dry state and serves simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 μL sample (solution of enzyme or plasma sample). The limit of detection is low enough to allow standard examination of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the standard Ellman's assay using plasma samples with BChE activity adjusted by carbofuran. The new method based on indoxylacetate seems promising for routine tests.

Full text of DOI:10.1515/chempap-2015-0117

Chemical Papers 69 (8) 1044–1049 (2015)
DOI: 10.1515/chempap-2015-0117
ORIGINAL PAPER
Determination of acetylcholinesterase and butyrylcholinesterase
activity without dilution of biological samples
Miroslav Pohanka*
Faculty of Military Health Sciences, University of Defense, Trebesska 1575, Hradec Kralove CZ-50001, Czech Republic
Received 18 January 2015; Revised 24 February 2015; Accepted 27 February 2015
Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known.
The enzymes are important in the body and alteration of their activity has significant use in the
diagnosis of poisoning, liver function, etc. Currently available methods for the determination of
cholinesterases have some major drawbacks including various interferences and the inability to
be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample
dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates
were modified with indoxylacetate (100 µL of 10 mmol L−1 solution) and used for cholinesterases
assay after drying at 37◦C. The fact that indoxylacetate remains stable in dry state and serves
simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit
of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 µL sample (solution
of enzyme or plasma sample). The limit of detection is low enough to allow standard examination
of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective
inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the
standard Ellman’s assay using plasma samples with BChE activity adjusted by carbofuran. The
new method based on indoxylacetate seems promising for routine tests.
c
ꢀ 2015 Institute of Chemistry, Slovak Academy of Sciences
Keywords: acetylcholinesterase, butyrylcholinesterase, cholinesterasemia, liver function test, in-
doxylacetate, Ellman’s assay
Introduction
& Lockridge, 2011; Lejus et al., 2006; Pohanka, 2015).
Both cholinesterases can serve as a biochemical
marker of some pathological processes. AChE is ac-
cessible in blood samples and immediate decrease
of its activity can be interpreted as poisoning with
some neurotoxic inhibitors such as nerve agents, some
drugs approved for e.g. Alzheimer disease, myasthe-
nia gravis, and/or pesticides such as carbofuran (Po-
hanka, 2012a; GhattyVenkataKrishna et al., 2013).
BChE has wider diagnostic applications than AChE
despite its lower physiological significance. Plasmatic
activity of BChE, cholinesterasemia can be used as
a liver function test because BChE is synthesized in
liver and secreted into blood (Iwasaki et al., 2007; Po-
hanka, 2013a). BChE assay can be applied for e.g.
acute hepatitis or liver cirrhosis diagnosis (Prellwitz
et al., 1976; Kemkes-Matthes et al., 1987). Beside the
Acetylcholinesterase (AChE; EC 3.1.1.7) and bu-
tyrylcholinesterase (BChE; EC 3.1.1.8) are the two
cholinesterases present in the human body. Despite
broad similarities between the two enzymes, their bi-
ological significance is different. AChE has particular
importance in the junction of nerves to affected cells,
where it terminates the neurotransmission by the neu-
rotransmitter, acetylcholine, hydrolysis and it can be
considered as a physiologically irretrievable enzyme
(Pohanka, 2011). Compared to AChE, BChE has no
major endogenous substrate hence its biological role is
hard to be understood. On the other hand, it does not
mean that BChE is completely useless. It is able to de-
grade some toxic compounds of natural origin as well
as drugs such as cocaine and succinylcholine (Duysen
*Corresponding author, e-mail: miroslav.pohanka@gmail.com
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1045
diagnostic purposes, cholinesterases can be used in the
construction of biosensors for neurotoxic compounds
assay.
and dimethyl sulfoxide were purchased from Penta-
Chemicals (Prague, Czech Republic). Deionized water
was prepared by an Aqua Osmotic system (Aquaos-
motic, Tisnov, Czech Republic).
Mice BALB/c (Velaz, Unetice, Czech Republic)
were used as the source of plasma samples. A group of
12 female mice were used for the purpose. The mice
Disparate protocols for the determination of cholin-
esterases activity have been presented. In past, sim-
ple spectrophotometric tests based on 5,5ꢀ-dithiobis-
(2-nitrobenzoic) acid as the chromogenic reagent and
acetyl- (or butyryl-)thiocholine as the substrate (Ell-
man et al., 1961; George & Abernethy, 1983; Gorun
et al., 1978), potentiometric tests with pH titration
(Bazire et al., 2011), and electrochemical sensors and
biosensors (Khaled et al., 2010; Pohanka, 2014) have
been employed. The mentioned testes are based on an
addition of reagents to the sample followed by mea-
surements of the enzyme activity. In case of spec-
trophotometric tests, the addition of a chromogenic
substance and a substrate are necessary. Methods such
as pH titration can be applied directly with added
substrate. On the other hand, the shift of pH or the
necessity to add a neutralizing buffer are evident dis-
advantages of these methods.
All the described methods have a major drawback:
dilution of samples by the used reagents. It can be a
problem when cholinesterase activity in e.g. blood or
serum is to be measured to diagnose poisoning by a
reversible inhibitor. No or only limited decrease of the
cholinesterase activity can be determined by the stan-
dard methods while the contrary is true. The neces-
sity to use chromogenic compounds and substrates of
cholinesterase resulting in spontaneous reactions pro-
viding colored products is another disadvantage of the
standard methods. In the present paper, a new method
suitable for the determination of cholinesterases activ-
ity in biological samples is established and validated.
The new method is expected to be reliable and avail-
able enough to be applicable in diagnostic tests in-
cluding diagnosis of overdosing by e.g. Alzheimer dis-
ease drugs huperzine, donepezil, and galantamine. The
presented method is designed to be performed by a
simple addition of the sample with no necessity of its
pretreatment or addition of other reagents.
weighted (19
1) g and they were two months old
on the day of euthanasia. Prior to euthanasia, the
mice were kept in standard conditions recommended
for small rodents (temperature of (22 2)◦C, humid-
ity of (50 10) %, light/dark period each of 12 h, no
limitations in access to chow and water). The experi-
ment was approved as well as supervised by the ethical
committee (Faculty of Military Health Sciences, Uni-
versity of Defence, Hradec Kralove, Czech Republic).
The mice were sacrificed under carbon dioxide
anesthesia by cutting the jugular vein. Blood was
collected into tubes with lithium heparin (Dialab,
Prague, Czech Republic) and centrifuged at 1000g for
5 min. Finally, plasma was isolated by a micropipette.
Spectrophotometry was performed for AChE as
well as for BChE solutions in phosphate buffered
saline with pH 7.4. Standard disposable PS cuvettes
with the optical length of 0.01 m and spectrophotome-
ter adjusted to the wavelength of 412 nm were used.
The cuvettes were filled with the following reagents:
i) 400 L of 1 mmol L−1 5,5ꢀ-dithiobis-(2-nitroben-
zoic) acid;
ii) 100 L of enzyme solution or plasma;
iii) 400 L of phosphate buffered saline with pH
7.4;
iv) 100 L of 10 mmol L−1 butyrylthiocholine chlo-
ride in case of the BChE assay or 10 mmol L−1 acetyl-
choline chloride for the AChE assay.
After the final substance addition, the absorbance
was measured immediately and then after 5 min. En-
zyme activity was calculated using the extinction co-
efficient ε = 14.150 L mol−1 cm−1 for pH 7.4 (Eyer et
al., 2003).
Indoxylacetate was dissolved in ethanol or dime-
thyl sulfoxide up to the concentration of 10 mmol L−1
and 100 L of the solution were injected per one well
of a 96 well-microplate. The plate was left to dry for at
least 12 h. In the total, 100 L of the sample were ap-
plied per one well and the absorbance at 670 nm was
measured immediately and then after 3 h. The wave-
length was chosen as optimal in compliance with the
referred works (Villatte et al., 2001; Pohanka, 2012b;
de Melo et al., 2006). The principle of the indoxylac-
etate based reaction is depicted in Fig. 1.
All measurements were made in five replicates and
the mean as well as the standard deviation were cal-
culated. The saturation curve was constructed by
non-linear curve fitting using the Michaelis–Menten
equation and the data were processed in Origin 9.1
(OriginLab Corporation, Northampton, MA, USA).
Enzyme activity was expressed in U where 1 U re-
Experimental
In the experiment, human recombinant AChE
(from HEK 293 cells, obtained as lyophilized pow-
der ≥ 1500 U per mg of protein) and human re-
combinant BChE (expressed in goat, obtained as
lyophilized powder with the enzyme activity ≥ 500
U per mg of protein) were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Phosphate buffered
saline pH 7.4 and carbofuran in analytical purity
were bought from Sigma–Aldrich as well. Acetylthio-
choline chloride, butyrylthiocholine chloride, 5,5ꢀ-di-
thiobis-(2-nitrobenzoic) acid, indoxylacetate, bovine
serum albumin, ascorbic acid, donepezil, and tetraiso-
propyl pyrophosphoramide (iso-OMPA) were received
from Litolab (Chudobin, Czech Republic). Ethanol
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M. Pohanka/Chemical Papers 69 (8) 1044–1049 (2015)
Fig. 1. Principle of the reaction based on the indoxylacetate hydrolysis.
sponds to 1 mol of acetylthiocholine (butyrylthio-
choline in BChE case) converted per 1 min in the
given solution. Signal vs. noise equal to three crite-
rion (S/N = 3) was applied as the limit of detection
calculation.
Results and discussion
The microplates were prepared as described in the
Experimental. A solution of indoxylacetate in ethanol
and/or dimethyl sulfoxide was used. Microplates were
dried in either laboratory conditions or in an incu-
batory box adjusted to 37◦C. The experiment was
aimed at the selection of an optimal protocol to get
a thin and homogenous layer of indoxylacetate. Dis-
persion of the obtained values was assessed. The use
of ethanol as a solvent and drying in an incubatory
box provided the best results. While the combina-
tion of ethanol and drying at 37◦C provided optical
density of wells in quite a narrow interval of values:
0.033 0.006, lower temperature resulted in a broader
Fig. 2. Determination of the Michaelis constant for indoxylac-
etate as the substrate and AChE ( ) or BChE ( )
•
as the enzymes. Non-linear curve fitting (Michaelis–
Menten kinetics) was applied in order to determine
the constant. Error bars indicate standard deviation for
n = 5.
interval: 0.034
0.011. Results obtained when us-
ing dimethyl sulfoxide were worse compared to those
when using ethanol: 0.037 0.014 for the higher and
ited by the access to substrate but it is directed by
the enzyme activity. Higher indoxylacetate concentra-
tions are not reasonable because the layer on the well
surface is too strong and typically not optically ho-
mogenous.
Calibration for standard commercial AChE and
BChE when using the prepared microplates can be
seen in Fig. 3. Limits of detection for the tested
cholinesterases were calculated from the calibration
plots and they are valid for a sample with the volume
of 100 L. BChE was assayed with the limit of detec-
tion of 0.71 U (or 12 nkat) per 100 L of the sample.
AChE was assayed with a limit of detection higher
compared to that of BChE (2.8 U; 47 nkat). The ob-
tained limits of detection were low enough to enable
the determination of cholinesterases in biological sam-
ples. In one example, cholinesterasemia was reported
0.035
0.017 for the lower temperature. The find-
ings correspond with visual observation when the wells
treated with the ethanol solution and dried at 37◦C
appeared to have the finest homogeneity. This process-
ing of the wells was chosen for the following assays.
Concentration of indoxylacetate underwent opti-
mization and the saturation curve was constructed
from the experimental data (Fig. 2). Indoxylacate
in the given concentration was applied on plates in
the amount of 100 L per one well and left to dry
at 37◦C for 12 h. The Michaelis constant, Km, was
equal to (1.36
0.17) mmol L−1 for BChE and
(4.36 0.53) mmol L−1 for AChE. In the following as-
says, the indoxylacetate concentration of 10 mmol L−1
was used as an optimum because the assay is not lim-
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(Colovi´c et al., 2013; Darreh-Shori & Soininen, 2010;
Sochocka et al., 2008). Compared to donepezil, iso-
OMPA is a specific inhibitor of BChE but not AChE
(Pohanka, 2013b; Harel et al., 1992). The inhibitors
were added to the indoxylacetate solution up to the
concentration of 1 mmol L−1 and left to dry. As seen
in Fig. 4, the use of inhibitors initiated the decrease
of the AChE activity in case of donepezil application
and of the BChE activity in case of iso-OMPA appli-
cation. Activities obtained in measurements No. 2 and
3 corresponded well with those obtained for the mix-
ture in measurement No. 1 (Fig. 4). Donepezil had no
effect on the assayed BChE activity and iso-OMPA
had no effect on the AChE activity assay (data not
shown). Ascorbic acid, glutathione, and albumin had
no potential to interfere in the assay. It is an advan-
tage of the assay because both glutathione and al-
bumin are quite strong interferents in cholinesterase
determination when using the Ellman’s assay (Gius-
tarini et al., 2013; Pohanka, 2012b; Prokofieva et al.,
2012). Indoxylacetate is not chemically reactive with
such compounds; hence, an assay based on indoxy-
lacetate is not sensitive to the interference. From this
point of view, it should be emphasized that indoxylac-
etate is quite stable and less reactive when compared
to 5,5ꢀ-dithiobis-(2-nitrobenzoic) acid applied in the
Ellman’s assay.
Fig. 3. Calibration for AChE ( ) and BChE ( ) solutions us-
•
ing microplates with indoxylacetate. Activity expressed
for a sample of 100 L. Error bars indicate the standard
deviation for n = 5.
The method was validated using plasma samples
from the BALB/c mice. For the experiment purposes
it was necessary to achieve samples with gradually in-
creasing enzyme activity. The obtained plasma sam-
ples were mixed together and then divided into five
groups: in the first, no treatment was applied; in the
other four, carbofuran was added up to the concen-
tration of 1 mol L−1, 5 mol L−1, 25 mol L−1
,
and 125 mol L−1, respectively. BChE activity in the
plasma samples was determined using the calibration
presented in Fig. 3 and the results are depicted in
Fig. 5. It is clearly visible that the method based on
indoxylacetate well correlates with the standard Ell-
man’s assay as confirmed by the quite high coefficient
of determination of 0.976.
Fig. 4. Interference testing: 1 – 60 U of AChE and 60 U of
BChE for sample of 100 L; 2 – 60 U of AChE and 60
U of BChE with an addition of donepezil, 1 mmol L−1
;
3 – 60 U of AChE and 60 U of BChE with addition
of iso-OMPA, 1 mmol L−1; 4 – 6 U of BChE; 5 –
1 mmol L−1 of ascorbic acid; 6 – 1 mmol L−1 of re-
duced glutathione; 7 – 1 mg mL−1 of albumin. Error
bars indicate standard deviation for n = 5.
Conclusions
to be approximately 8 U mL−1 (Rastogi et al., 2008)
or 18–67 U mL−1 in another source (Guemei et al.,
2001).
The method described here seems to be promis-
ing for a routine assay of both AChE and BChE.
The use of inhibitors such as donepezil and/or iso-
OMPA can further improve the assay efficiency and
specificity. The possibility to employ an assay with-
out sample dilution is the major advantage. The ab-
sence of glutathione or albumin interference is an-
other advantage of the presented assay. The assay
can be easily used for the determination of poison-
ing by a reversible inhibitor of cholinesterases or for
in vitro characterization of drugs. Overall simplic-
ity and low cost of the assay support its applica-
tion.
Interferences were also examined. Experimental
data from the interference tests are summarized in
Fig. 4. In compliance with the expectation from the
calibration plot, the obtained signal is the highest
when AChE and BChE are both presented in the
sample. From this point of view, cholinesterases can
be considered as interferents of each other. In order
to distinguish the activity of AChE and BChE, spe-
cific inhibitors can be used. Donepezil has been cho-
sen as a compound inhibiting AChE but not BChE
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M. Pohanka/Chemical Papers 69 (8) 1044–1049 (2015)
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