An inhibitor-free assay of acetylcholinesterase and butyrylcholinesterase in the cerebrospinal fluid

Kluge, H.; Kluge, Wolfram H.; Hartmann, W.

doi:10.1016/s0009-8981(99)00021-2

Cited by 9 publications

(8 citation statements)

References 9 publications

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“…Until recently we have been using the inhibitor-free test, which permits for simultaneous analysis of both cholinesterases in the CSF [ 18 ]. The inhibitor-free method is based on the finding that the ratio of individual activities of BChE on its both substrates ACh and BCh in the CSF and in the serum obtained at the same time are identical with substrate concentrations of at least 5 mmol/l.…”

Section: Discussionmentioning

confidence: 99%

“…It does not appear advisable to judge whether the bupivacaine-inhibition test or the inhibitor-free test is more precise when one considers the minor differences between test results. We could previously show that the inhibitor-free procedure offers widely identical results in comparison to the difference method using BW284c51 as AChE-inhibitor which proved to be a trustworthy assay [ 9 , 11 , 12 , 15 , 18 ]. We could also show that the assay utilizing iso-OMPA as inhibitor for BChE exclusively [ 14 ] is unreliable if applied without correction of methodical errors [ 18 ].…”

Section: Discussionmentioning

confidence: 99%

“…Therefore the majority of colorimetric test procedures published to date are using relatively specific (highly toxic) inhibitors to distinguish the two different cholinesterases in the test system. We introduced an inhibitor-free test for AChE and BChE in the CSF [ 18 ]. During our recent studies we performed in vitro inhibition tests on AChE and BChE using various pharmacological agents [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

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Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase

Kluge

Bauer

et al. 2001

BMC Biochem

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Background Most test systems for acetylcholinesterase activity (E.C.3.1.1.7.) are using toxic inhibitors (BW284c51 and iso-OMPA) to distinguish the enzyme from butyrylcholinesterase (E.C.3.1.1.8.) which occurs simultaneously in the cerebrospinal fluid. Applying Ellman's colorimetric method, we were looking for a non-toxic inhibitor to restrain butyrylcholinesterase activity. Based on results of previous in vitro studies bupivacaine emerged to be a suitable inhibitor. Results Pharmacokinetic investigations with purified cholinesterases have shown maximum inhibition of butyrylcholinesterase activity and minimal interference with acetylcholinesterase activity at bupivacaine final concentrations between 0.1 and 0.5 mmol/l. Based on detailed analysis of pharmacokinetic data we developed three equations representing enzyme inhibition at bupivacaine concentrations of 0.1, 0.2 and 0.5 mmol/l. These equations allow us to calculate the acetylcholinesterase activity in solutions containing both cholinesterases utilizing the extinction differences measured spectrophotometrically in samples with and without bupivacaine. The accuracy of the bupivacaine-inhibition test could be confirmed by investigations on solutions of both purified cholinesterases and on samples of human cerebrospinal fluid. If butyrylcholinesterase activity has to be assessed simultaneously an independent test using butyrylthiocholine iodide as substrate (final concentration 5 mmol/l) has to be conducted. Conclusions The bupivacaine-inhibition test is a reliable method using spectrophotometrical techniques to measure acetylcholinesterase activity in cerebrospinal fluid. It avoids the use of toxic inhibitors for differentiation of acetylcholinesterase from butyrylcholinesterase in fluids containing both enzymes. Our investigations suggest that bupivacaine concentrations of 0.1, 0.2 or 0.5 mmol/l can be applied with the same effect using 1 mmol/l acetylthiocholine iodide as substrate.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase

Kluge

Bauer

et al. 2001

BMC Biochem

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“…We could previously show that the inhibitor-free procedure offers widely identical results in comparison to the difference method using BW284c51 as AChE-inhibitor which proved to be a trustworthy assay [9,11,12,15,18]. We could also show that the assay utilizing iso-OMPA as inhibitor for BChE exclusively [14] is unreliable if applied without correction of methodical errors [18]. So far no substance has been found which completely inhibits BChE without also affecting AChE.…”

Section: Discussionmentioning

confidence: 99%

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Kluge

Bauer

et al. 2001

BMC Biochem

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Background: Most test systems for acetylcholinesterase activity (E.C.3.1.1.7.) are using toxic inhibitors (BW284c51 and iso-OMPA) to distinguish the enzyme from butyrylcholinesterase (E.C.3.1.1.8.) which occurs simultaneously in the cerebrospinal fluid. Applying Ellman's colorimetric method, we were looking for a non-toxic inhibitor to restrain butyrylcholinesterase activity. Based on results of previous in vitro studies bupivacaine emerged to be a suitable inhibitor.

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“…The current determination for AchE or Ach is based on the determination of the rate of hydrolysis of the substrate. The techniques most commonly used for the determination are spectrophotometry [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], high-performance liquid chromatography (HPLC), gas chromatography (GC) using a flame ionization detection, GC with mass spectrometry (GC-MS) [23][24][25][26] and amperometric [27][28][29][30][31][32][33][34][35][36] and potentiometric sensors [27,[37][38][39][40][41][42][43][44]. Most of the potentiometric AchE-based Ach sensors involve pH detection.…”

Section: Introductionmentioning

confidence: 99%

Electrometric assay for the determination of acetylcholine using a sensitive sensor based on carbon paste

Barsoum

Watson

Mahdi

et al. 2004

Journal of Electroanalytical Chemistry

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An inhibitor-free assay of acetylcholinesterase and butyrylcholinesterase in the cerebrospinal fluid

Cited by 9 publications

References 9 publications

Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase

Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase

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Electrometric assay for the determination of acetylcholine using a sensitive sensor based on carbon paste

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