Clinica Chimica Acta 282 (1999) 135–145

An inhibitor-free assay of acetylcholinesterase and

butyrylcholinesterase in the cerebrospinal fluid
a, b a
Harald H. Kluge *, Wolfram H. Kluge , Werner Hartmann
a
Department of Neurochemistry, Clinic of Neurology, Friedrich Schiller-University Jena,
Philosophenweg 3, D-07740 Jena, Germany
b
Clinic of Orthopaedics, ‘ Rudolf Elle’ Hospital Eisenberg, Friedrich Schiller-University Jena, Jena,
Germany

Received 25 August 1998; received in revised form 8 January 1999; accepted 12 January 1999

Abstract

An inhibitor-free assay for the simultaneous determination of acetylcholinesterase (AChE) and

butyrylcholinesterase (BChE) in the cerebrospinal fluid (CSF) is described. It is based on our
finding that the individual activity ratios of BChE on both its substrates acetylthiocholine (ACh)
and butyrylthiocholine (BCh) in the CSF and in the parallel serum are identical under conditions
of at least 5 mmol / l substrate concentration (Q BChE BChE
SE 5 Q CSF ). Considering that AChE only reacts
with ACh as substrate and occurs with negligible activities in the serum, the measured individual
activity ratio of BChE in the serum (Q BChE
SE ) and the total hydrolysis rate of ACh and BCh in the
CSF do allow a precise calculation of the AChE activity in the cerebrospinal fluid. The derivation
of the corresponding formula is demonstrated in detail. The inhibitor-free assay was compared
with procedures using cholinesterase inhibitors (BW284c51 for AChE and / or iso-OMPA for
BChE). Achieving widely identical results in particular between the procedure using the AChE
inhibitor and the inhibitor-free test, the latter has decisive advantages: (1) it avoids the use of
highly toxic inhibitors, (2) it minimizes the test volume needed, (3) it characterizes additionally
the status of the blood–CSF barrier by means of the BChE activity ratio in the CSF and in the
parallel serum.  1999 Elsevier Science B.V. All rights reserved.

Keywords: Acetylcholinesterase; Butyrylcholinesterase; Cerebrospinal fluid; Inhibitor-free test

*Corresponding author.

0009-8981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0009-8981( 99 )00021-2
136 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145

1. Introduction

Acetylcholinesterase (AChE, E.C. 3.1.1.7.) in the CSF is assayed to study

dysfunction of the central and peripheral nervous system (CNS). The enzyme is
assumed to be a biochemical marker for clinical diagnosis and prognosis [1–8].
Various methods to measure the enzymatic activity of AChE and butyryl-
cholinesterase (BChE, E.C. 3.1.1.8.) in the CSF have been employed. However
the results published are nonuniform. The discrepancies are due to varying
nosological as well as methodical approaches to the problem. Concerning the
latter, we have modified the colorimetric test according to Ellman et al. [9] to
create a specific assay for each enzyme avoiding the highly toxic inhibitors
iso-OMPA (anti-BChE) and BW284c51 (anti-AChE). The test scales down the
used CSF volume and offers additional information about the status of the
blood–CSF barrier.
Our concept is based on the following facts and conclusions:

1. The serum is dominated by BChE; AChE is present in traces. Because the

serum must be diluted to measure the BChE activity, AChE does not interfere
with the enzymatic tests.
2. BChE of the serum and of the CSF reacts with acetylcholine (ACh) and
butyrylcholine (BCh) in a well defined individual ratio of activity (Q SE and
Q CSF ). AChE only hydrolyses ACh.
3. If the individual ratio of BChE activity in the serum and in the CSF are
identical, it would be possible to calculate the AChE activity of the CSF by
means of the known serum ratio without the use of inhibitors.

Our studies indicate the correctness of this concept comparing analytical data
of tests using inhibitors with the results of the inhibitor free test. We describe the
advantages of the inhibitor-free assay.

2. Material and methods

Acetylthiocholine iodide (ACh), butyrylthiocholine iodide (BCh), AChE from

electric eel and from bovine erythrocytes, purified BChE from human serum,
BW284c51 (anti-AChE), iso-OMPA (anti-BChE), DTNB [5-59-dithio-bis-(2-
nitrobenzoic acid)], and eserine salicylate were obtained from Sigma-Aldrich.
Lumbar CSF samples were used for the investigations. To calculate the
individual ratio of the BChE activity on the substrates ACh and BCh, a serum
sample was drawn from the same person. To have available a sufficient CSF
volume for reliable tests, identical portions of CSF and serum samples from
three or four probands were pooled in the same relation.
 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145 137

2.1. Enzyme assays

AChE and BChE were determined spectrophotometrically using the approved

technique of Ellman et al. [9]. We standardized the test conditions as follows:
The reaction was performed at 378C in a total volume of 1.3 ml and stopped
after 20 min linear reaction time with 0.5 ml of 0.1 mmol / l eserine salicylate;
0.1 ml of CSF (cell-free) and 0.1 ml of the corresponding diluted serum were
used. The protein concentration of the serum had been adjusted to that of the
CSF by diluting it 100- to 200-fold with physiological NaCl. Thereby the total
protein concentration of both fluids tested extends up to 750 mg / l (see also
Table 3). The final concentration amounted to 5 mmol / l for the substrates ACh
and BCh, and to 1 mmol / l for DTNB. Final concentrations of 5 mmol / l
anti-AChE (procedure II) and of 0.5–5 mmol / l anti-BChE (procedure III) were
used for parallel inhibitor tests.
The 59-thio-2-nitrobenzoate produced enzymatically was measured at 412 nm.
Enzyme activities (nmol / ml 3 min) were calculated by multiplication of the
extinction difference (DE) during 20 min of linear enzymatic hydrolysis with the
factors F 5 65 for non-diluted CSF samples, F 5 6500 and F 5 13 000 for sera
diluted down to 1:100 and 1:200, respectively. These factors result from the
volumes of the reactants, the final volume of 1.8 ml, the dilution factor of the
sera, the reaction time of 20 min, and the micromolar extinction coefficient of
0.01388. The following formulae were employed to calculate the activities (see
Appendix) from the:

2.1.1. Inhibitor-free assay ( procedure I)

BChE of the serum on substrate ACh:
ACh
BChE SE 5 DE SE 3F (F from 6500 to 13 000)

BChE of the serum on substrate BCh:

BCh
BChE SE 5 DE SE 3F (F from 6500 to 13 000)

Individual ratio of serum BChE on substrates ACh and BCh:

Q BChE ACh BCh

SE 5 DE SE /DE SE

BChE of CSF on substrate BCh:

BCh
BChE CSF 5 DE CSF 3F (F 5 65)

AChE of CSF on substrate ACh:

AChE CSF 5 f DE ACh BChE

CSF 2 Q SE 3 DEBChE BCh
CSF g 3 F (F 5 65)
138 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145

2.1.2. Tests using inhibitors

AChE of CSF on substrate ACh with inhibitor anti-AChE (procedure II):

AChE CSF 5 f DE ACh ACh

CSF 2 DEBChE(anti-AChE) CSF g 3 F (F 5 65)

AChE of CSF on substrate ACh with inhibitor anti-BChE (procedure III):

AChE CSF 5 DEAChE(anti-BChE) ACh

CSF 3 F (F 5 65)

Results are given as means6S.D. for sample groups, or as means of the

individual differences 6S.E.M.. For statistical analysis, the t-test for significance
of differences between paired samples was used to calculate P values (SPSS for
Windows, version 6.0.1).

3. Results

3.1. Time and substrate kinetics

Under optimal final concentrations of the substrates (see below) the time
kinetics of AChE and BChE were linear over at least 20 min. Therefore, this
incubation time was used generally.
To study the substrate kinetics, certain experimental conditions had to be
taken into consideration: In the serum, the dependence of the BChE activity on
the substrate concentration could be tested without anti-AChE, because the
traces of AChE in the diluted sample do not interfere with the ACh hydrolysis
by BChE. In the CSF, however, the traditional difference method using anti-
AChE as an inhibitor must be applied to separate the BChE activity on ACh
from that of AChE on this substrate. An alteration of the BChE activity on ACh
and BCh (using 5 mmol / l final concentration of each substrate) by anti-AChE
had to be excluded in a preceding test series. Anti-AChE in a final concentration
of 5 mmol / l inhibits the AChE completely. The effect of this concentration was
tested on BChE. As shown in Table 1, the BChE activity ratio Q BChE was not
altered significantly by this inhibitor. Therefore, it is justified to compare
directly the BChE substrate kinetics of the CSF in the presence of 5 mmol / l
anti-AChE with that of the parallel serum in the absence of this inhibitor.
On the basis of this finding the substrate dependence of AChE and BChE was
tested in pools of equivalent CSF and serum samples from 20 probands. As seen
in Fig. 1, substrate saturation for AChE in the CSF was already established at a
final concentration of about 2 mmol / l ACh (Fig. 1a). The BChE of both fluids
did not show classical saturation kinetics on the two substrates. However, the
 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145 139

Table 1
Effect of anti-AChE (5 mmol / l final concentration) on the activity ratio of BChE (Q BChE ) from
different sources. Grouped ratios as means6S.D., pairs differences between activity ratios as
means6S.E.M.. Upper and lower limits in parentheses
Activity ratios (Q BChE ) Pairs differences
Non-inhibited Anti-AChE inhibited
Purified BChE 0.4860.02 0.4860.02 0.00560.003
(N58) (0.45–0.51) (0.45–0.51) n.s.
BChE of serum 0.5460.02 0.5360.03 0.01160.011
(N522) (0.50–0.57) (0.47–0.57) n.s.
Purified BChE added to serum 0.5560.03 0.5460.02 0.01460.010
(N522) (0.51–0.57) (0.49–0.56) n.s.

curves became nearly parallel at substrate concentrations greater than 3 mmol / l

in the CSF as well as in the corresponding serum (Fig. 1a,b). The activity ratios
(Q BChE ) reached almost equal figures in both fluids (Fig. 1c). To confirm the
latter finding, the Q BChE of 25 CSF and corresponding serum pools were
analysed in a different series, each pool containing equivalent samples from four
probands. Table 2 displays the results. Using the t-test for paired samples, the
differences between Q BChE BChE
CSF and Q SE were not significant. The BChE activity
ratio on ACh and BCh in the CSF is identical to the BChE activity ratio on ACh
and BCh in the parallel serum if the final substrate concentration is at least 4
mmol / l. To standardize the test system, 5 mmol / l final concentrations of both
substrates were used generally.
Earlier investigations with purified BChE suggested a possible non-specific
rise of its activity ratio on ACh and BCh if isolated serum or CSF proteins were
added. To simulate the protein ranges of ventricular CSF, lumbar CSF and
diluted serum (between 100 and 750 mg / l) BChE activity ratios of the purified
and serum enzyme were compared in the presence of increasing albumin and
serum protein concentrations. As seen in Table 3, the activity ratio of purified
BChE increased slightly with the addition of albumin alone, but considerably
with addition of mixed serum proteins. The stimulating effect on the hydrolysis
of ACh is obviously greater than on the hydrolysis of BCh. And yet, because
this activation had already grown to an optimum at a protein concentration of
about 100 mg / l, an interference of varying serum or CSF protein concentrations
in the test procedures can be excluded.
The observations mentioned so far are essential to derive the fundamental
equation for the inhibitor-free assay on the AChE activity in the CSF (see
Section 2 and Appendix) and to discuss the tests on the BChE activity in the
CSF and in the serum.
140 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145

Fig. 1. Substrate kinetics of AChE in the CSF and BChE in the CSF and in the serum. (a) AChE
on ACh and BChE on ACh and BCh in the CSF (according to procedure II). (b) BChE on ACh
and BCh in the corresponding serum (according to procedure I). (c) BChE activity ratios (Q BChE
SE
BChE
and Q CSF ) on substrates ACh and BCh in the CSF and in the serum (calculated from a and b).
 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145 141

Table 2
BChE activity ratios (Q BChE ) on ACh and BCh in the CSF (with inhibitor anti-AChE) and in the
corresponding serum (non-inhibited). Grouped ratios as means6S.D., pairs differences as
means6S.E.M., upper and lower limits in parentheses
Q BChE BCHE
CSF (A) Q SE (B) Pairs differences (A2B)
0.5660.02 0.5560.01 0.00560.003 (n.s.)
(0.52–0.57) (0.52–0.56) (from 20.02 to 0.03)

3.2. Comparison of the inhibitor-free test with procedures using anti-AChE

or anti-BChE

To check the validity of the inhibitor-free (I) assay in comparison with the
traditional difference procedures using anti-AChE (II) or anti-BChE (III), the
AChE and BChE activities of 25 pooled CSF samples were tested in parallel
with all three methods. The activities extended from 3 to 27 nmol / ml3min for
AChE and from 8 to 22 nmol / ml3min for BChE, thus including the normal
range (about 10–20 and 8–15 nmol / ml3min for AChE and BChE, respective-
ly). The results (Table 4) are presented as percental deviations from the activity
measured with the inhibitor-free assay. For further assessment the measured
activities were divided into subgroups.
The percental differences between the inhibitor-free test (I) and the procedure
using anti-AChE (II) were insignificant concerning AChE as well as BChE. The
deviations between the subgroups were nearly identical for both enzymes.
Comparing the procedure using anti-BChE (III) with the inhibitor-free test,
about 8% lower AChE activity and about 5% lower BChE activity were
determined. These differences were significant, the deviations being larger in the
subgroup with the lower activities. The drawback of the method employing
anti-BChE became already obvious in a pilot study: whereas purified BChE was
inhibited completely by 0.5 mmol / l final concentration of anti-BChE, the serum
enzyme often showed a residual activity even with 5 mmol / l final inhibitor

Table 3
Activity ratios of BChE from different sources (Q BChE ) on ACh and BCh in dependence upon the
protein concentration
Enzyme source Range of protein Q BChE
concentration
Purified BChE – (N521) 0.4860.02
Purified BChE in the ,100 mg / l (N515) 0.4860.02
presence of albumin .100 mg / l (N527) 0.5160.02
Endogeneous BChE from diluted sera 100–750 mg / l (N57) 0.5660.02
Purified BChE added to endogeneous
BChE from diluted sera 100–750 mg / l (N57) 0.5560.03
142 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145

Table 4
Test procedures I–III on AChE and BChE activity in the CSF: percental deviations between the
inhibitor-free method (procedure I; activity5100%) and assays using anti-AChE (procedure II) or
anti-BChE (procedure III) as inhibitors
Activity tested with procedure I Percent deviation from procedure I (100%) to
(total and partial range)
(nmol / ml3min) Procedure II Procedure III
(mean6S.E.M.) (mean6S.E.M.)
AChE
3–27 21.060.51 28.561.43
(total, N525) (n.s.) (P,0.001)
3.0–15.0 21.661.45 210.362.54
(N58) (n.s.) (P, 0.01)
15.1–27.0 20.760.36 26.961.83
(N517) (n.s.) (P,0.001)
BChE
8–22 1.260.81 24.960.91
(total, N525) (n.s.) (P,0.001)
8.0–15.0 1.361.15 26.161.31
(N513) (n.s.) (P,0.001)
15.1–22.0 1.260.87 23.661.18
(N512) (n.s.) (P,0.01)

concentration. We could also confirm that a concentration of over 1 mmol / l

anti-BChE inhibits AChE substantially.

4. Discussion

Discussing the conflicting data concerning the alteration of the CSF AChE in
neuropsychiatric disorders, Davis and Goodnick mentioned among other con-
cerns the controversy around assay procedures used to measure the AChE
activity [10]. We do agree with this comment and extend it with regard to
alterations of the BChE activity. Because the BChE does not only derive from
neuronal tissues but to a much larger extent from the blood the ratio of its
activity between the CSF and the corresponding serum can be used as an
additional parameter for the status of the blood–CSF barrier.
When AChE and BChE are occurring simultaneously in body fluids or tissues,
assay procedures hitherto published made use of the specific inhibitors anti-
AChE and anti-BChE to separate the two different cholinesterases. The reason
being their overlapping substrate specificity: AChE only hydrolyses ACh but
BChE breaks down ACh as well as BCh.
Practically the use of anti-AChE would be adequate if one calculated the
 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145 143

BChE activity only by its hydrolysis rate with BCh. The AChE activity would
be equivalent to the difference between the non-inhibited and the inhibited
hydrolysis rate of ACh (corresponding to procedure II in this paper).
On the other hand, the exclusive use of the inhibitor anti-BChE would be
sufficient too, because the hydrolysis rate of ACh measured directly represents
the AChE activity (corresponding to procedure III in this paper). However, as
shown in Table 4 using this inhibitor, the activities differed considerably from
those obtained by the difference method using anti-AChE. This discrepancy is
caused by the frequently incomplete inhibition effect of anti-BChE on BChE
even in millimolar final concentrations. In addition anti-BChE can inhibit the
AChE. Therefore, this method is unreliable.
Considering these aspects and to avoid the highly toxic inhibitors, we did find
a reliable method to measure accurately the AChE as well as the BChE activity
in the CSF as a fluid containing both enzymes side by side.
The decisive point in developing the inhibitor-free assay was to define the
component of the BChE activity on ACh against the total ACh hydrolysis rate in
the CSF. This was guaranteed by affirming the identity of the individual BChE
activity ratios in the serum and the CSF under conditions of substrate final
concentrations of more than 4 mmol / l (Fig. 1c and Table 2). Given this identity
it is sufficient to determine the BChE activity only in the serum where the AChE
inhibitor is not necessary. Multiplying this CSF-equivalent intraindividual serum
ratio by the hydrolysis rate of BCh in the CSF, the product equals the BChE
activity on ACh. Consequently, the difference between the total and this BChE
corresponding ACh hydrolysis rate represents the AChE activity of the CSF:

AChE CSF 5 f DE ACh BChE

CSF 2 Q SE 3 DEBChE BCh
CSF g 3 F

For the exact derivation of the formula see Appendix.

To standardize the test conditions it appeared safe to define the final substrate
concentration at 5 mmol / l.
According to this formula, the inhibitor-free procedure on AChE and BChE
only demands the measure of the hydrolysis rates of ACh and BCh in the serum
and in the CSF. These hydrolysis rates do not depend on the total protein
concentrations of the CSF or the diluted serum sample within the methodical
detection limits (Table 1).
As shown in Table 4, the results (procedure I) widely equalled those of the
difference method using anti-AChE as inhibitor (procedure II) and, consequently
differed from those of the method using anti-BChE (procedure III). The
deviations between the procedures I and II did not exceed 2% thus being
negligible. Though there exist trustworthy methods with both procedures, the
former has important advantages: (1) it avoids the use of highly toxic
substances, (2) it minimizes the volume of CSF needed (no inhibitor-related
144 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145

extra tests), (3) it simultaneously determines the BChE activity of the serum,
thus (4) allowing conclusions concerning the status of the blood–CSF barrier by
the ratio of BChE SE to BChE CSF in comparison to that of albumin. The test
requires minimal material effort and can be performed at any routine laboratory.

Acknowledgements

The authors wish to thank Gudrun Venth and Ines Herrmann for their precise
efforts in the assays performed.

Appendix

To derive the fundamental Eq. (A.6) for the calculation of the AChE activity
in the CSF using the inhibitor-free assay the following conditions had to be
included stepwise:
The BChE activity ratio Q BChE
SE in the diluted serum is calculated from the
quotient of the extinction differences according to the activities on the substrates
ACh and BCh:
Q BChE ACh BCh
SE 5 DE SE /DE SE (A.1)
BChE activity rate in the CSF and the serum of the same proband is identical:
Q BChE BChE
SE 5 Q CSF (A.2)
Because the extinction difference measured in the CSF on ACh is equal to the
sum of AChE and BChE activities on this substrate,
DE ACh ACh ACh
CSF 5 DEBChE CSF 1 DEAChE CSF (A.3)
the extinction difference based on the BChE activity can be derived from Eqs.
(A.2) and (A.3):

DEBChE ACh ACh ACh BChE

CSF 5 DE CSF 2 DEAChE CSF 5 Q SE
BCh
3 DEBChE CSF (A.4)
The extinction difference based on the AChE activity can be isolated from Eq.
(A.4)
DEAChE ACh ACh BChE BCh
CSF 5 DE CSF 2 Q SE 3 DEBChE CSF (5)
From Eq. (A.5) the AChE activity can be calculated by multiplication with the
factor F (see Section 2):

AChE CSF 5 f DE ACh BChE

CSF 2 Q SE 3 DEBChE BCh
CSF g 3 F (A.6)
 H.H. Kluge et al. / Clinica Chimica Acta 282 (1999) 135 – 145 145

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