Microsoft word - 178-13 composed
RESEARCH OPINIONS IN ANIMAL & VETERINARY SCIENCES
Comparison of an Alzheimer disease drug ability to bind acetylcholinesterase
using both electrochemical and spectrophotometric assays
Miroslav Pohanka1,2*, Vojtech Adam3,4 and Rene Kizek3,4
1Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-500 01 Hradec Kralove, Czech
Republic, European Union; 2Karel English College in Brno, Sujanovo namesti 356/1, 60200 Brno, Czech Republic
3Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno,
Czech Republic, European Union; 4Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel
University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, European Union
Acetylcholinesterase (AChE) is an enzyme playing important role in termination of neurotransmission within
cholinergic system. The enzyme is targeted by many compounds including nerve agents used for chemical warfare,
natural toxins and drugs for Alzheimer disease. Thousands of new inhibitors are prepared for pharmaceutical
purposes over the year and necessity to characterize them is given for this reason. In this work, standard
spectrophotometric test is compared to electrochemical test in order to assay AChE activity and measure its
inhibition. The both test were performed for characterization of an Alzheimer disease drug galantamine. The
spectrophotometric test was based on standard Ellman´s reaction. Electrochemical test used enzymatic hydrolysis to
acetylthiocholine with consequent electrochemical oxidation. In the experiments, we proved that electrochemical
test was able to reach better limit of detection for galantamine than the standard spectrophotometric test. Calculated
median inhibitory concentration was same for electrochemical and spectrophotometric tests. Interference in the
assay was tested as well. We proved that electrochemical test is reliable and more sensitive than the standard
spectrophotometric test. The electrochemical test would be preferred when median inhibitory concentration is the
acetylcholinesterase; Alzheimer disease; myasthenia gravis; huperzine; galantamine; donepezil; biosensor
To cite this article:
Pohanka M, V Adam and R Kizek, 2014. Comparison of an Alzheimer disease drug ability to
bind acetylcholinesterase using both electrochemical and spectrophotometric assays. Res. Opin. Anim. Vet. Sci.,
galantamine, huperzine), and drugs for myasthenia
gravis (neostigmine, pyridostigmine) can be exampled
Enzyme acetylcholinesterase (AChE; E.C. 184.108.40.206)
as inhibitors of AChE (Holzgrabe et al., 2007; Pohanka,
is involved in the regulation of cholinergic nerves
2011, 2012b; da Silva et al., 2011; de los Rios, 2012).
where it terminates neurotransmission by hydrolysis of
Necessity to analyze newly prepared drugs is given
neurotransmitter acetylcholine (Wessler and in pharmacological research. In the drug selection, Kirkpatrick, 2008; Pohanka, 2012a). The enzyme has a
AChE activity is analyzed in the presence of the tested
wide pharmacological and toxicological significance.
compound and the most potent inhibitors are given to
Many compounds including nerve agents used as
the next stages of preclinical tests. Currently, use of
chemical warfare (sarin, soman, tabun, VX), secondary
acetylthiocholine as a substrate and 5,5´-dithiobis-(2-
metabolites such as caffeine or aflatoxins, some drugs
nitrobenzoic acid) (further abbreviated as DTNB) as a
for Alzheimer disease (donepezil, rivastigmine, chromogen are used for spectrophotometric assay of*Corresponding author:
Miroslav Pohanka, Faculty of Military Health Sciences, University of Defence, Trebesska
1575, CZ-500 01 Hradec Kralove, Czech Republic, European Union. E-Mail: email@example.com; Tel: +420-973-251-519; Fax: +420-495-518-094.
Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207.
AChE activity (Pohanka 2013c). The reaction is called as Ellman´s reaction in honour to team of doctor Ellman who firstly suggested the application (Ellman et al., 1961; Gorun et al., 1978; George and Abernethy, 1983; Runion et al., 1984). Though the assay is simple, sensitive and readily to use in the both laboratory and field conditions, there are some drawbacks. Beside instability of DTNB in water conditions, interference of nucleophilic substances including thiol, oxime and the other chemical groups can
be expected (Pohanka, 2012b, 2013b; Pohanka et al.,
Fig. 1: Principle of assay based on Ellman´s reaction. The
first step is catalyzed by acetylcholinesterase
electrochemical evaluation of AChE activity assay is
proposed as an alternative to the standard spectrophot-
ometric assay. It is believed that the new test can avoid the drawbacks typical for Ellman´s reaction. For the experiment purposes, standard drug galantamine, compound with proper chemical name (4aS
)- 5,6,9,10,11,12- hexahydro- 3-methoxy- 11-methyl- 4aH
-  benzofuro[3a,3,2-ef
]  benzazepin- 6-ol, for Alzheimer disease was chosen as compound inhibiting AChE and being measured by the standard test.
Materials and Methods
Fig. 2: Principle of electrochemical assay of acetylcholineste-
rase (AChE) activity.
The spectrophotometric assay was based on the
from BVT Technologies (Brno, Czech Republic). The
aforementioned application of acetylthiocholine chloride
sensors were sized 25.4×7.3×0.6 mm and they consisted
and DTNB as a chromogenic substance. Principle of the
from platinum working (dot shaped with diameter 1 mm),
assay is depicted in Figure 3. For the assay purposes,
silver covered with silver chloride reference, and platinum
lyophilized human recombinant AChE expressed in
auxiliary electrodes. Reagents used for the electro-
HEK293 cells with activity above 1,500 U per milligram
chemical assay were the same like the used in
of protein was purchased from Sigma Aldrich (Saint
Louis, Missouri, USA). AChE was solved in phosphate
Prior to the assay, sensor was linked with PalmSens
buffered saline (PBS) and activity was adjusted up 0.5
device (Palm Sens BV, Houten, Netherlands), inserted
U/µl for 1 mmol/l acetylthiocholine chloride. Reaction
into a disposable plastic cuvette with maximal volume 2
was performed using standard disposable PS cuvettes.
ml and operated by a computer using PsLite 1.8
The following reagents were subsequently given into the
(PalmSens BV) software. Into the cuvette, the following
cuvette: 100 µl of the tested solution in PBS, 200 µl of 0.4
reagents were added: 100 µl of the tested solution in PBS,
mg/ml DTNB in PBS, 590 µl of PBS and the solution was
790 µl of PBS and the solution was shaken and let to
shaken and let to incubate for 10 minutes. After that, 100
incubate for 10 minutes. After that, 100 µl of 10 mmol/l
µl of 10 mmol/l acetylthiocholine chloride was given into
acetylthiocholine chloride was injected. The mixture was
the cuvette, shaken and absorbance was measured at 412
shaken and current was measured chronoampero-
nm immediately after splitting and then after two minutes.
metrically after two minutes. Applied potential 640 mV
Mixture with acetylthiocholine replaced by pure PBS was
was given in order to receive oxidation of the released
used as a blank. Control measurement with sample
thiocholine. The potential was chosen using experiences
replaced by PBS was done as well. Galantamine was used
from the former experiment (Pohanka et al., 2013). In
as a standard compound inhibiting AChE. The all
control measurement, sample was replaced by PBS.
aforementioned reagents and standards used for the assay
purpose were achieved from Sigma-Aldrich.
Data processing and statistics
All samples were assayed in pentaplicate and the
both mean and standard deviation were calculated for
Electrochemical assay was done in similar conditions
each group of measurements. From the experimental data,
like the spectrophotometric one. Scheme of the assay is
percent of inhibition I was calculated using following
depicted as figure 4. Screen printed sensors were purchased
Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207.
Where SC is a signal received by either spectro-
photometric (absorbance) or electrochemical (current) assay for the control measurement. SI is a signal found for assay of the tested compound in given concentration.
Software Origin 8 (OriginLab Corporation,
Northampton, MA, USA) was used for data processing
and statistics evaluation. Limit of detection was calculated
from confidence interval (95 %) in calibration plot. Results and Discussion
Galantamine was chosen as a representative Fig. 3: Spectrophotometric assay of galantamine. Error bars
compound used for Alzheimer disease therapy. It is well
indicate standard deviation for n=5. The data were
tolerated by patients and it has good efficacy in the
fitted (full line) and confidence interval for
therapy (Bond et al., 2012). It was discovered by
probability level 0.05
Mashkovsky and Kruglikova-Lvova as a secondary
metabolite in Caucasian snowdrops Galanthus
sp. in early 1950s (Heinrich and Teoh, 2004). Currently, galantamine is sold over the world under trade names RazadyneTM, RazadyneTMER, ReminylTMER, and Reminyl®, and it remains as one of the most relevant compounds used for the therapy.
Calibration for galantamine was done by the both
spectrophotometry and electrochemistry in concentration range 10-9 – 10-2 mol/l (final concentration in the cuvette). PBS was used for control measurement. Calibration is depicted as figure 1 for the spectrophotometry and figure 2 for the electrochemistry. When searched limit of detection, electrochemical assay was able to detect as low as 5×10-9 mol/l of galantamine. Comparing to it,
spectrophotometric assay have limit of detection 2×10-9
mol/l for galantamine. When comparing the two limits of
Fig. 4: Electrochemical assay of galantamine. Description is
detection, the electrochemical method was able to detect
the same as in figure 1.
3.7 times lower amount of galantamine. However, the
lower limit of detection does not privilege any method if
not immobilized for the assay purposes. Decision not to
accuracy to estimate median inhibitory concentration is
use immobilization was initiated by decision to establish
method close to standard spectrophotometric test.
For the above mentioned reason, the both methods
Immobilization of AChE would lead to preparation of a
were fitted by Boltzmann function and median inhibitory
biosensor and further improvement to limits of detection.
concentration was calculated. The median inhibitory
However, comparison of such experimental data and data
concentration for galantamine was 2.19×10-6 mol/l when
from spectrophotometric test would be complicated.
used spectrophotometry and 2.09×10-6 mol/l when used
Limits of detection known from literature about AChE
electrochemistry. The data are insignificant one to each
based biosensors are slightly lower than the here
other when used analysis of variance and probability level
described device. In an example, AChE based
0.05. The two methods were correlated one to each other.
electrochemical biosensor using Co phtalocyanine and
Coefficient of determination R2 was 0.997. Slope for the
carbon electrodes was used for assay of organophosphate
correlation was 1.04. Considering the calculated median
pesticides with limit of detection 2 ppb for malaoxon
inhibitory concentration, we can claim that the (Ivanov et al., 2011). In another paper, biosensor electrochemical assay is reliable and very close in the
containing AChE immobilized on carbon nanotubes was
used to detect chosen pesticides and nerve agents (Liu and
The reached limits of detection are quite good for
Lin, 2006). Limit of detect for organophosphate paraoxon
characterization of drugs. On the other hand, AChE was
Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207.
Table 1: Interference in the assays in percent of control
The Ministry of Education, Youth and Sports of the
Czech Republic is gratefully acknowledged for project
LH11023. Financial support from CEITEC CZ.1.05/
1.1.00/02.0068 is highly acknowledged as well.
Interference was measured using acetone oxime,
cysteine, and ethanol. The interference is resumed in table
1. The compounds were given into the cuvette up to final
Bond, M., Rogers, G., Peters, J., Anderson, R., Hoyle, M.,
concentration 0.1 mmol/l for acetone oxime and cysteine.
Miners, A., Moxham, T., Davis, S., Thokala, P.,
Ethanol was added up to concentration 10% (v
Wailoo, A., Jeffreys, M. and Hyde, C. 2012. The
purpose of this part of experiment, volume of applied PBS
effectiveness and cost-effectiveness of donepezil,
described above was decreased for 100 µl and the tested
galantamine, rivastigmine and memantine for the
compounds were applied just in volume 100 µl.
treatment of Alzheimer's disease (review of
As seen in the table, cysteine was oxidized by applied
Technology Appraisal No. 111): a systematic review
voltage and caused interference more than 100 % when
and economic model. Health Technology Assessment
applied final concentration 0.1 mmol/l in the electro-
chemical assay. The same interference was found in
da Silva, V.B., de Andrade, P., Kawano, D.F., Morais,
spectrophotometric assay. The fact that free thiol can
P.A.B., de Almeida, J.R., Carvalho, I., Taft, C.A. and
interfere is not surprising, because it is well known for
da Silva, C. 2011. In silico design and search for acetylcholinesterase inhibitors in Alzheimer's disease
Ellman´s reaction (Prokofieva et al., 2012). It is
with a suitable pharmacokinetic profile and low
frequently used for the both electrochemical and
toxicity. Future Medicinal Chemistry
, 3: 947-960.
spectrophotometric assay of thiol containing antioxidants
de los Rios, C. 2012. Cholinesterase inhibitors: a patent
(Pohanka, 2012c; Tipple and Rogers, 2012; Squellerio et
review (2007-2011). Expert Opinion on Therapeutic
al., 2012). Interference of an oxime group containing
compound is not surprising as well. This kind of
Ellman, G.L., Courtney, K.D., Andres, V. and
compounds causes oximolysis. The oximolysis leads to
Featherstone, R.M. 1961. A new and rapid
spontaneous reaction with DTNB (Worek et al., 2012;
colorimetric determination of acetylcholinesterase
Pohanka, 2012a, 2013a). Electrochemical assay is not
activity. Biochemical Pharmacology
, 7: 88-95.
sensitive to the interference because oxime is not redox
Fekonja, O., Zorec-Karlovsek, M., El Kharbili, M.,
active during the measurement. Ethanol influences the
Fournier, D. and Stojan, J. 2007. Inhibition and
assay in course of AChE inhibition. The inhibitory effect
protection of cholinesterases by methanol and
is not specific and it can be anticipated for other organic
ethanol. Journal of Enzyme Inhibition and Medicinal
solvents as well (Turdean and Turdean, 2008; Pietsch et
al., 2009). Ethanol is a typical example of such solvents
George, P.M. and Abernethy, M.H. 1983. Improved
(Fekonja et al., 2007). When considered the mentioned
Ellman procedure for erythrocyte cholinesterase.
results. We can claim that the main advantage of the
electrochemical assay lays in ability to avoid interference
Gorun, V., Proinov, I., Baltescu, V., Balaban, G. and
by some nucleophilic substances. On the other hand,
Barzu, O. 1978. Modified Ellman procedure for assay
compounds inhibiting AChE and free thiol containing
of cholinesterases in crude enzymatic preparations.
compounds will cause the same interference in the
, 86: 324-326.
spectrophotometrical like in the electrochemical assay.
Heinrich, M. and Teoh, H.L. 2004. Galanthamine from
snowdrop-the development of a modern drug against
Alzheimer's disease from local Caucasian knowledge.
Electrochemical assay was introduced as a tool for a
Journal of Ethnopharmacology
, 92: 147-162.
fast and reliable testing of drugs inhibiting enzyme AChE.
Holzgrabe, U., Kapkova, P., Alptuzun, V., Scheiber, J.
The new assay was found to be more sensitive than the
and Kugelmann, E. 2007. Targeting acetylcholine-
traditional spectrophotometric test. Moreover, the
sterase to treat neurodegeneration. Expert Opinion on
electrochemical assay is less sensitive to interference
, 11: 161-179.
caused by nucleophilic substances. On the other hand,
Ivanov, A.N., Younusov, R.R., Evtugyn, G.A., Arduini,
electrochemical assay is not advantageous if there is a
demand to calculate enzyme activity in katals.
Acetylcholinesterase biosensor based on single-
Electrochemical assay should be privileged above the
walled carbon nanotubes-Co phtalocyanine for
spectrophotometric test when median inhibitory
organophosphorus pesticides detection. Talanta
Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207.
Liu, G. and Lin, Y. 2006. Biosensor based on self-
J., Masarik, M., Eckschlager, T. and Kizek, R. 2012.
assembling acetylcholinesterase on carbon nanotubes
Voltammetry Assay for Assessment of Oxidative
for flow injection/amperometric detection of organo-
Stress linked Pathologies in Brain Tumor suffered
phosphate pesticides and nerve agents. Analytical
Childhood Patients. International Journal of
, 7: 11978-11992.
Pietsch, M., Christian, L., Inhester, T., Petzold, S. and
Prokofieva, D.S., Jenkins, R.O. and Goncharov, N.V.
Gutschow, M. 2009. Kinetics of inhibition of
2012. Microplate biochemical determination of
acetylcholinesterase in the presence of acetonitrile.
Russian VX: Influence of admixtures and avoidance
of false negative results. Analytical Biochemistry
Pohanka, M. 2011. Cholinesterases, a target of
pharmacology and toxicology. Biomedical Papers
Runion, T.H., Smith, C.L. and Stoll, R.E. 1984.
Modification of Ellman technique for measuring
Pohanka, M. 2012c. Acetylcholinesterase based dipsticks
erythrocyte cholinesterase using a centrifichem-500
with indoxylacetate as a substrate for assay of
analyzer. Clinical Chemistry
, 30: 1060-1060
organophosphates and carbamates. Analytical Letters
Squellerio, I., Caruso, D., Porro, B., Veglia, F., Tremoli,
E. and Cavalca, V. 2012. Direct glutathione
Pohanka, M. 2012b. Acetylcholinesterase inhibitors: a
quantification in human blood by LC-MS/MS:
patent review (2008-present). Expert Opinion on
comparison with HPLC with electrochemical
, 22: 871-886.
detection. Journal of Pharmaceutical and Biomedical
Pohanka, M. 2012a. Alpha7 nicotinic acetylcholine
receptor is a target in pharmacology and toxicology.
Tipple, T.E. and Rogers, L.K. 2012. Methods for the
International Journal of Molecular Sciences
determination of plasma or tissue glutathione levels.
Methods in Molecular Biology
, 889: 315-324.
Pohanka, M. 2013a. Cholinesterases in biorecognition and
Turdean, G.L. and Turdean, M.S. 2008. Synergetic effect
biosensor construction, a review. Analytical Letters
of organic solvents and paraoxon on the immobilized
acetylcholinesterase. Pesticides in Biochemistry and
Pohanka, M. 2013b. Spectrophotomeric assay of aflatoxin
B1 using acetylcholinesterase immobilized on standard
Wessler, I. and Kirkpatrick, C.J. 2008. Acetylcholine
microplates. Analytical Letters
, 46: 1306-1315.
beyond neurons: the non-neuronal cholinergic system
Pohanka, M., Fusek, J., Adam, V. and Kizek, R. 2013.
in humans. British Journal of Pharmacology
Carbofuran assay using gelatin based biosensor with
acetylcholinesterase as a recognition element. Worek, F., Eyer, P. and Thiermann, H. 2012. International Journal of Electrochemical Science
Determination of acetylcholinesterase activity by the
Ellman assay: A versatile tool for in vitro research on
Pohanka, M., Hynek, D., Kracmarova, A., Kruseova, J.,
medical countermeasures against organophosphate
Ruttkay-Nedecky, B., Sochor, J., Adam, V., Hubalek,
poisoning. Drug Testing and Analysis
, 4: 282-291.
Translocations: Migration and Social Change An Inter-Disciplinary Open Access E-Journal ISSN Number: 2009-0420 ___________________________________________________________________ The views and experiences of members of new communities in Ireland: perspectives on mental health and well-being. School of Nursing, Dublin City University, Glasnevin, Dublin 9, Ireland ________________
2010 Step Therapy Criteria Effective January 1, 2010 Step Therapy Group Description Step Therapy Criteria Requires member has filled at least a 60-day supply of a step 1 drug (i.e. long-acting beta agonist, Spiriva or inhaled corticosteroid) in the last 120 days before Advair will be covered Requires member must have a documented 60-day trial of either Antabuse or naltrexone before