IN vitro experiments at room temperature with benzoylcholine as a substrate showed that 10–50 μm fluoride, the concentration usually observed with sevoflurane anesthesia, 1inhibits pseudocholinesterase (PChE) by 16–56%. 2PChE is the enzyme responsible for the metabolism of mivacurium. Mivacurium dose requirements to maintain neuromuscular blockade are decreased by sevoflurane. 3In addition to the well-known potentiation of nondepolarizing neuromuscular blocking agents by volatile anesthetics, this might be caused by an inhibition of PChE. The aim of this study was to investi-gate whether fluoride concentrations between 10 and 1,000 μm inhibit PChE at 37°C using butyrylthiocholine and mivacurium as substrates.
Materials and Methods
After approval by the local ethics committee (Faculty of Medicine, University of Regensburg, Regensburg, Germany) and with written, informed consent, serum samples were obtained from 10 healthy volunteers (6 men, 4 women; 24–39 yr of age). The subjects did not have diseases or take drugs that would interfere with PChE activity. The serum samples were spiked with 0 (fluoride-free control), 10, 50, 100, and 1,000 μm fluoride (NaF; Merck, Darmstadt, Germany), and PChE activity was measured by the standard procedure recommended by the German Society for Clinical Chemistry and the American Association for Clinical Chemistry, which use butyrylthiocholine (Bayer, Leverkusen, Germany) as substrate and a temperature of 37°C. 4The metabolism of mivacurium was measured at 37°C by adding 18 μm mivacurium 5(Mivacuriumchloride; GlaxoWellcome, Hamburg, Germany) to 4 ml of the fluoride-free control and the serum samples spiked with 10, 50, 100, and 1,000 μm fluoride. At timed intervals (1, 2, 3, 4, 6, 8, and 10 min), aliquots of 500 μl were withdrawn and put into vials containing 0.5 mg echothiophate (Wyeth-Ayerst, Pearl River, NY) to stop hydrolysis immediately. The concentrations of the three mivacurium isomers were determined by stereospecific high-performance liquid chromatography. 6An Agilent Technologies (Waldbronn, Germany) HP1090 high-pressure liquid chromatograph with an HP1046A programmable fluorescence detector was used. The detection limit was 0.009 μm for each of the isomers.
Data Analysis and Statistics
Because the clearance of the cis-cis isomer does not depend on PChE activity, 7only the half-lives of the cis-trans and trans-trans isomers were calculated with KINETICA 2000 (InnaPhase Corp., Philadelphia, PA). The dependence of the cis-trans and trans-trans isomer half-lives on PChE activity in the fluoride-free samples was evaluated by linear regression analysis. To compare PChE activity and cis-trans and trans-trans isomer half-lives between the four fluoride groups (10, 50, 100, and 1,000 μm) and the fluoride-free control group (0 μm), a one-way analysis of variance with a post hoc Dunnett test was performed. P < 0.05 was considered statistically significant. The SPSS for Windows software package was used (version 10.0; SPSS Inc., Chicago, IL). The post hoc calculated power was 90% using GPOWER (Franz Faul & Edgar Erdfelder, Bonn, Germany) for MS-DOS (Microsoft, Unterschleißheim, Germany).
In the fluoride-free samples, there was a strong negative correlation between PChE activity and the cis-trans isomer half-life (r =−0.84, P = 0.002) as well as between PChE activity and the trans-trans isomer half-life (r =−0.86, P = 0.001). The PChE activity in the four fluoride groups did not differ from the fluoride-free control group. The same applied to the cis-trans and the trans-trans isomer half-life up to 100 μm fluoride, whereas 1,000 μm led to a significant increase of both half-lives (table 1).
The main finding of our study was that fluoride concentrations up to 100 μm do not inhibit PChE at 37°C. These fluoride concentrations did not alter cis-trans and trans-trans half-lives of mivacurium (table 1).
Using butyrylthiocholine as the substrate, no inhibition of PChE was observed with fluoride concentrations as high as the toxic concentration of 1,000 μm (table 1). A study of the mechanism of PChE inhibition by fluoride suggests that the binding of fluoride to PChE reduces the space available for the substrate. 8This could explain why 1,000 μm fluoride affects the hydrolysis of the bulky mivacurium, with a molecular weight of 1,029, but not that of the smaller butyrylthiocholine, with a molecular weight of 190. With regard to the investigation by Kambam et al. , 21,000 μm fluoride led to a 95% inhibition of PChE. Their study was performed with benzoylcholine as the substrate and at room temperature. PChE activity heavily depends on temperature. In genotypically normal individuals, PChE activity at 37°C is twice as high as that measured at 25°C. 9Furthermore, lower temperatures promote the inhibition of PChE by fluoride. 8Although the PChE assay using benzoylcholine as the substrate and a temperature of 25°C is widely used, 10,11other studies dealing with PChE activity 3,7use the assay with butyrylthiocholine as the substrate, probably because it is recommended by international societies for clinical chemistry. 4In conclusion, fluoride inhibition of PChE seems to depend on the substrate and temperature used.
As expected from former studies, 5,7the half-lives of the cis-trans and trans-trans isomers in the fluoride-free samples depended on PChE activity. The mean in vitro half-lives were 0.61 min for the cis-trans isomer and 0.76 min for the trans-trans isomer (table 1). Referring to a personal communication, Lien et al. 7reported a longer in vitro half-life for the cis-trans isomer (1.30 vs. 0.83 min for the trans-trans isomer). The reason for the conflicting results is unclear. What could be in favor of our result is that the shorter in vitro half-life of the cis-trans isomer we found corresponds to the higher clearance of this isomer in vivo (106 vs. 63 ml · min−1· kg−1for the trans-trans isomer). 7To summarize, this in vitro study does not provide evidence that fluoride concentrations up to 100 μm, the concentration observed with sevoflurane anesthesia, inhibit PChE.
The authors thank Karl-J. Lackner, M.D., Associated Professor, Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany, for the measurements of PChE activity.