Butyrylcholinesterase Phenotype Research Articles

Timing of blood sampling for butyrylcholinesterase phenotyping in patients with prolonged neuromuscular block after mivacurium or suxamethonium.

Variants of butyrylcholinesterase are frequently associated with prolonged response to suxamethonium or mivacurium. Butyrylcholinesterase (BChE) can be characterized by phenotyping and determination of genotype. Inappropriate timing of blood sampling might interfere with phenotyping methods. However, guidelines regarding delay between exposure to anaesthesia and testing are not clearly defined. In this study, the BChE activity and phenotype in an early (T1) and late (T2) phase were compared and the phenotype/genotype correlation was assessed. Patients with a prolonged paralysis after mivacurium or suxamethonium were selected after ethical committee approval and written consent. BChE activity was based on butyrylthiocholine hydrolysis rate and phenotyping on differential inhibition of BChE activity with dibucaine and fluoride. DNA sequencing allowed genotypic characterization. We included the results of 20 patients with prolonged neuromuscular block (NMB) induced by mivacurium or suxamethonium. In these patients, BChE activity was different at T1 and T2 (2120 [1506-2733] UL-1 and 4055 [2810-5301] UL-1 , respectively; P=0.0014; values are mean [95% CI]). When phenotyping was possible, phenotyping at T1 and T2 yielded identical results. Phenotyping failed to identify one new variant (p.Tyr146Cys) and the K variant in 14 of 16 patients. Anaesthesia interfered with BChE activity, but not with phenotyping. Phenotyping can be performed on blood drawn during or immediately after recovery of mivacurium or suxamethonium to screen for clinically relevant variants of BChE. However, accurate diagnosis of BChE deficiency needs further confirmation by determination of genotype.

Characterization of four BCHE mutations associated with prolonged effect of suxamethonium.

Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a "silent" BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower kcat value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (Ω-loop) can explain the modification of the kinetic parameter of the mutated protein.

Activity and polymorphisms of butyrylcholinesterase in a Polish population

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents.In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3′UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type).In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5′UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type.To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

The allele frequency of T920C mutation in butyrylcholinesterase gene is high in an Indian population

The genetic variants of butyrylcholinesterase (BChE) are the cause of concern in individuals experiencing prolonged apnea on administration of muscle relaxants. In an Indian community called Vysya, a variant (L307P; T920C) was associated with imperceptible plasma BChE. Since BChE has several pharmacological significances in humans, the identification of its variants having altered activity is very important. Previous studies for identifying the mutants in a population were based only on its functional attributes (phenotypic characters) such as esterase activity, dibucaine number and fluoride number. Generally phenotyping method is not considered as the accurate methodology till date though it might be used as a primary screening tool. Molecular biology provides a better technique in identifying these variants. Our aim was to screen this particular community living in South India for the heterozygosity of T920C mutation by phenotypic and genotypic analysis and to find the reliability between the two methods. We analysed 266 individuals for the heterozygosity of T920C. Based on BChE phenotypes, we found that 95% of the individuals are heterozygous. Real-time PCR based genotyping revealed that 96% of individuals are heterozygous. The allele frequency for the mutant allele C was 0.52 which confirmed that the genetic pool of this allele is much higher in Vysya. Also, we observed that genotyping correlates 97% with the phenotype in our study. Further, both phenotype and genotype of age matched other ethnic group do not show any preponderance to this mutation authenticating the vulnerability of Vysyas to this mutation is dominant.

Butyrylcholinesterase (BChE) activity is associated with the risk of preeclampsia: influence on lipid and lipoprotein metabolism and oxidative stress

Objective: To determine the butyrylcholinesterase (BChE) activity and phenotypes in preeclampsia and its possible association with lipid and lipoprotein metabolism and oxidative stress in preeclamptic women.Methods: In a case–control study, 101 pregnant women with normal pregnancy and 198 women with preeclampsia from Western Iran were studied. The serum BChE activity and phenotypes were measured using spectrophotometric method. The apolipoprotein E (APOE) genotypes were identified using PCR-RFLP. The serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined by HPLC and commercial kits, respectively.Results: The BChE activity and the frequency of non-usual BChE phenotype in preeclamptic women were significantly lower and higher, respectively compared to controls. There was a higher BChE activity in the presence of APOE ε3ε4 compared to ε3ε3 genotype in preeclamptic women. In addition, there were significant positive correlations between BChE activity and the levels of low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, total cholesterol (TC) and TAC. However, there was a negative but significant correlation between BChE activity and MDA level.Conclusions: Our study for the first time indicated that BChE activity might be involved in the pathogenesis of preeclampsia through influence on lipid and lipoprotein metabolism and oxidative stress.

Concordance of Butyrylcholinesterase Phenotype With Genotype

Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.

Determination of butyrylcholinesterase (BChE) phenotypes to predict the risk of prolonged apnea in persons receiving succinylcholine in the healthy population of western Iran

Objective: The best known clinical application of serum BChE assay is to predict abnormally prolonged apnea following the application of the muscle relaxant succinylcholine. The aim of the present study was to assess the frequency of BChE phenotypes and to predict the risk of apnea for those receiving succinylcholine among the residents in western Iran. Methods: We examined the frequency of nine BChE phenotypes in 1548 volunteers including 816 males and 732 females with the mean age of 35 ± 15 years from an apparently healthy group living in western Iran. The frequencies of BChE phenotypes were determined using BChE activity measurements and by inhibition with dibucaine, fluoride, and the compound Ro2-0683 (Hoffman-La-Roche). Results: The reference range for serum total BChE activity was 4600–14000 U/L (using butyrylthiocholine iodide as substrate). The mean value obtained for men (9030 U/L) was significantly ( p < 0.05) higher than that for women (8550 U/L). The frequencies of four alleles U, A, F, S were calculated to be 0.9826, 0.0165, 0.008 and 0.001, respectively. The frequency of phenotypes of BChE was as follows: normal phenotype (UU) 95.5%, moderate sensitive to succinylcholine including UA,US,UF phenotypes was 3.9% and hypersensitive to succinylcholine (AA, AF, AS, FF, SS) was 0.58%. Conclusion: This study indicates that the population of western Iran has a medium frequency of succinylcholine-sensitive individuals compared to other populations. We suggest that determination of BChE activity and phenotype by the micro automated method is well suited to pre-operative screening and detection of at-risk of prolonged apnea in persons receiving succinylcholine in the healthy population of western Iran.

Novel mutation and multiple mutations found in the human butyrylcholinesterase gene

Background: Mutations in human butyrylcholinesterase (BChE) are linked to low BChE activity and abnormal response to muscle relaxants. Methods: Twenty Chinese patients with hepatic disease and low cholinesterase activity, and one Japanese patient and her mother were tested for BChE activity and BChE phenotype. The butyrylcholinesterase (BCHE gene) was amplified by polymerase chain reaction (PCR) and sequenced. Mutant BChE was expressed in 293 cells. Results: A novel mutation was found in one Chinese patient at nucleotide 943, where A was changed to T (943 A→T), causing substitution of threonine 315 by serine (T315S). The T315S mutant had half of the normal BChE activity. One Japanese patient with low BChE activity had three nucleotide substitutions, 355 C→T, 988 T→A, and 1615 G→A. The amino acid substitutions were Q119stop, L330I, and A539T, respectively. The single mutant L330I had low BChE activity, but the double mutant L330I/A539T had normal activity. Conclusions: The L330I and the novel T315S mutation caused a decreased BChE activity. The T315S mutation is one of the first BChE mutations reported in the Chinese population. Multiple mutations in BChE may interact with each other in an intramolecular manner.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17–20, 24–31 and 34–53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.

Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses

Yael Loewenstein, Averell Gnatt, Lewis F. Neville, Haim Zakut and Hermona Soreq: Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1993, 17(6): 905–926. 1. 1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. 4. Thus, it was found that asp70→gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425→Pro with gly70 gives rise to the “atypical” butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.

Aspartate-70 to glycine substitution confers resistance to naturally occurring and synthetic anionic-site ligands on inovo produced human butyrylcholinesterase

The "atypical" allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nucleotide substitution conferring a change of aspartate-70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the "atypical" BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine-425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of the resultant recombinant BuChE variants. The presence of the Gly-70 mutation alone was found to render the enzyme resistant to 100 microM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the "normal," Asp-70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly-70 BuChE failed to be reactivated by 10 mM of the cholinesterase-specific oxime pyridine 2-aldoxime methiodide (2-PAM); a concentration restoring about 50% of activity in the "normal" Asp-70 recombinant enzyme. The Pro-425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly-70 mutation.(ABSTRACT TRUNCATED AT 250 WORDS)