Cargando…

Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kluge, Wolfram H, Kluge, Harald H, Bauer, Heike I, Pietsch, Stefan, Anders, Jens, Venbrocks, Rudolf A
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2001
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC64563/
https://www.ncbi.nlm.nih.gov/pubmed/11801199
http://dx.doi.org/10.1186/1471-2091-2-17
_version_ 1782120134520864768
author Kluge, Wolfram H
Kluge, Harald H
Bauer, Heike I
Pietsch, Stefan
Anders, Jens
Venbrocks, Rudolf A
author_facet Kluge, Wolfram H
Kluge, Harald H
Bauer, Heike I
Pietsch, Stefan
Anders, Jens
Venbrocks, Rudolf A
author_sort Kluge, Wolfram H
collection PubMed
description 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.
format Text
id pubmed-64563
institution National Center for Biotechnology Information
language English
publishDate 2001
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-645632002-01-23 Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase Kluge, Wolfram H Kluge, Harald H Bauer, Heike I Pietsch, Stefan Anders, Jens Venbrocks, Rudolf A BMC Biochem Research Article 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. BioMed Central 2001-12-21 /pmc/articles/PMC64563/ /pubmed/11801199 http://dx.doi.org/10.1186/1471-2091-2-17 Text en Copyright © 2001 Kluge et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
spellingShingle Research Article
Kluge, Wolfram H
Kluge, Harald H
Bauer, Heike I
Pietsch, Stefan
Anders, Jens
Venbrocks, Rudolf A
Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title_full Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title_fullStr Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title_full_unstemmed Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title_short Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
title_sort acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC64563/
https://www.ncbi.nlm.nih.gov/pubmed/11801199
http://dx.doi.org/10.1186/1471-2091-2-17
work_keys_str_mv AT klugewolframh acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase
AT klugeharaldh acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase
AT bauerheikei acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase
AT pietschstefan acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase
AT andersjens acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase
AT venbrocksrudolfa acetylcholinesteraseassayforcerebrospinalfluidusingbupivacainetoinhibitbutyrylcholinesterase