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Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase.

Kluge WH, Kluge HH, Bauer HI, Pietsch S, Anders J, Venbrocks RA - BMC Biochem. (2001)

Bottom Line: Based on results of previous in vitro studies bupivacaine emerged to be a suitable inhibitor.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic of Orthopedics, Rudolf Elle Hospital Eisenberg, Friedrich-Schiller-University Jena, Germany. wlfrmklg@aol.com

ABSTRACT

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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Activity of purified AChE and BChE (in %; hairline at 5% and 100%) in relation to bupivacaine concentration (1.0–5.0 × 10-4 mol/l).
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Figure 3: Activity of purified AChE and BChE (in %; hairline at 5% and 100%) in relation to bupivacaine concentration (1.0–5.0 × 10-4 mol/l).

Mentions: The influence of bupivacaine on the activity of both cholinesterases is demonstrated in Figure 2. AChE activity and BChE activity were determined in separate analyses. BChE inhibition above 95% of its activity was not achieved even at bupivacaine concentration of 1 mmol/l. The remaining BChE activity of 5% must therefore be added to all BChE activities measured under the influence of bupivacaine. Using drug concentrations which have minimum influence on AChE activity but cause maximum inhibition of BChE we selected three bupivacaine concentrations (0.1; 0.2 and 0.5 mmol/l) for further investigation (Figure 3).


Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase.

Kluge WH, Kluge HH, Bauer HI, Pietsch S, Anders J, Venbrocks RA - BMC Biochem. (2001)

Activity of purified AChE and BChE (in %; hairline at 5% and 100%) in relation to bupivacaine concentration (1.0–5.0 × 10-4 mol/l).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64563&req=5

Figure 3: Activity of purified AChE and BChE (in %; hairline at 5% and 100%) in relation to bupivacaine concentration (1.0–5.0 × 10-4 mol/l).
Mentions: The influence of bupivacaine on the activity of both cholinesterases is demonstrated in Figure 2. AChE activity and BChE activity were determined in separate analyses. BChE inhibition above 95% of its activity was not achieved even at bupivacaine concentration of 1 mmol/l. The remaining BChE activity of 5% must therefore be added to all BChE activities measured under the influence of bupivacaine. Using drug concentrations which have minimum influence on AChE activity but cause maximum inhibition of BChE we selected three bupivacaine concentrations (0.1; 0.2 and 0.5 mmol/l) for further investigation (Figure 3).

Bottom Line: Based on results of previous in vitro studies bupivacaine emerged to be a suitable inhibitor.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic of Orthopedics, Rudolf Elle Hospital Eisenberg, Friedrich-Schiller-University Jena, Germany. wlfrmklg@aol.com

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus