Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene.

The silent phenotype of human butyrylcholinesterase (BChE), present in most human populations in frequencies of approximately 1/100,000, is characterized by the complete absence of BChE activity or by activity <10% of the average levels of the usual phenotype. Heterogeneity in this phenotype has been well established at the phenotypic level, but only a few silent BCHE alleles have been characterized at the DNA level. Twelve silent alleles of the human butyrylcholinesterase gene (BCHE) have been identified in 17 apparently unrelated patients who were selected by their increased sensitivity to the muscle relaxant succinylcholine. All of these alleles are characterized by single nucleotide substitutions or deletions leading to distinct changes in the structure of the BChE enzyme molecule. Nine of the nucleotide substitutions result in the replacement of single amino acid residues. Three of these variants, BCHE*33C, BCHE*198G, and BCHE*201T, produce normal amounts of immunoreactive but enzymatically inactive BChE protein in the plasma. The other six amino acid substitutions, encoded by BCHE*37S, BCHE*125F, BCHE*170E, BCHE*471R, and BCHE*518L, seem to cause reduced expression of BChE protein, and their role in determining the silent phenotype was confirmed by expression in cell culture. The other four silent alleles, BCHE*271STOP, BCHE*500STOP, BCHE*FS6, and BCHE*I2E3-8G, encode BChES truncated at their C-terminus because of premature stop codons caused by nucleotide substitutions, a frame shift, or altered splicing. The large number of different silent BCHE alleles found within a relatively small number of patients shows that the heterogeneity of the silent BChE phenotype is high. The characterization of silent BChE variants will be useful in the study of the structure/function relationship for this and other closely related enzymes.

Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase.

The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier we reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work we have identified two different point mutations associated with the fluoride-resistant phenotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members.

DNA mutation associated with the human butyrylcholinesterase K-variant and its linkage to the atypical variant mutation and other polymorphic sites.

Genomic DNA from two families exhibiting the K-variant phenotype of serum butyrylcholinesterase was amplified by PCR and sequenced to determine the molecular basis of this variant. The K-variant phenotype was found to be associated with a DNA transition from guanine to adenine at nucleotide 1615, which caused an amino acid change from alanine 539 to threonine (GCA----ACA; Ala539----Thr). There was a 30% reduction of serum butyrylcholinesterase activity associated with this mutation. Amplification and sequencing of DNA from a random sample of 47 unrelated people gave a frequency of .128 for the K-variant allele. Thus, 1 person in 63 should be homozygous for the K-variant, making the K-variant the most common butyrylcholinesterase variant. The K-variant mutation was also found to be present in 17 (89%) of 19 butyrylcholinesterase genes containing the point mutation which causes the atypical phenotype of butyrylcholinesterase (GAT----GGT; Asp70----Gly). The presence of the K-variant in the same molecule as the atypical variant does not contribute to the qualitative change in the atypical enzyme, but it most likely accounts for the approximately one-third reduction in Vmax of butyrylcholinesterase activity in atypical serum. Two additional point mutations located in noncoding regions of the gene were also observed to be in linkage disequilibrium with the K-variant mutation. As many as four different point mutations have been identified within a single butyrylcholinesterase gene. Inhibition tests of the enzyme in plasma are usually used to distinguish the K-variant from the usual enzyme when the former is present with the heterozygous atypical variant (AK phenotype vs. UA phenotype). Inhibition tests were performed on plasma enzyme from the four possible genotypic combinations of the heterozygous atypical mutation with or without the K-variant mutation on either allele; we found that the AK phenotype was caused by three genotypes (A/K, AK/K, and U/A) and that the UA phenotype was caused by two genotypes (U/A and U/AK).

Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----GGAG).

A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.

Changes in resistance to mouth opening induced by depolarizing and non-depolarizing neuromuscular relaxants.

Mouth opening was measured in 43 children anaesthetized with isoflurane and paralysed with vecuronium or suxamethonium. Measurements of mouth opening were made for up to 10 min after loss of the adductor pollicis twitch and cessation of muscle fasciculations. In 22 patients receiving suxamethonium, a significant (P less than 0.001) reduction in mean mouth opening occurred in the 60 s after loss of twitch and cessation of fasciculations. Mouth opening reductions could last for up to 10 min after the loss of twitch, beyond the return of the twitch. One patient experienced "masseter spasm"; he did not develop malignant hyperpyrexia during 2.5 h of isoflurane anaesthesia. Patients receiving vecuronium showed a significant (P less than 0.0006) increase in mouth opening. In 20 subjects, mouth opening was generated with a small (1.67 N) and a larger (4.32 N) force. Proportionally equal reductions in mouth opening were obtained with either force after suxamethonium administration. Relatively equal increases with either force followed vecuronium administration. Isolated masseter spasm is not pathognomonic for malignant hyperpyrexia. If the diagnosis of malignant hyperpyrexia is contemplated, signs of hypermetabolism, such as increases in end-tidal carbon dioxide concentration during constant minute ventilation, should be sought.

Differing effect of agonist and antagonist muscle relaxants on cat jaw muscles.

Mouth closure and an increased resistance to mouth opening follow succinylcholine administration in humans. To elucidate the effects of succinylcholine on masticatory muscle function, mouth opening in the cat, produced by a constant test force, was measured during steady state halothane anesthesia. After baseline measurements, either succinylcholine (0.3 mg.kg-1 of body weight) or vecuronium (0.1 mg.kg-1 of body weight) was infused intravenously, and mouth opening measurements were repeated for up to 30 min. Concomitantly, muscle relaxant effect was quantified by measurement of the neurally-evoked tibialis anterior muscle response. All animals given succinylcholine displayed active jaw closure, which was followed by an increased resistance to mouth opening. This increased resistance was present after cessation of fasciculations and during complete twitch suppression. It lasted beyond the time at which the limb muscle twitch had fully recovered. Vecuronium administration was associated with a decreased resistance to mouth opening without a closing action. The initial jaw closure and the subsequently increased resistance to mouth opening after succinylcholine administration during halothane anesthesia in the cat are comparable with mouth opening changes after succinylcholine administration during inhalation anesthesia in humans. The cat may serve as an animal model for study of the mechanisms involved in responses of jaw muscles to succinylcholine with use of techniques inappropriate in humans.

Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.

Differing interactions between hexamethonium and tubocurarine, pancuronium or alcuronium at the neuromuscular junction.

The action of hexamethonium has been investigated in the rat phrenic nerve-hemidiaphragm preparation, alone and in combination with the neuromuscular blocking agents tubocurarine, pancuronium and alcuronium. Hexamethonium alone in concentrations between 3.55 x 10(-3) and 7.1 x 10(-3) mol litre-1 produced neuromuscular blockade in a dose-dependent manner. Low concentrations of hexamethonium antagonized the neuromuscular blocking effect of all three neuromuscular blocking drugs, less with tubocurarine than with the other two. Increasing the concentration of hexamethonium produced potentiation of the neuromuscular blocking effect, this being greater with tubocurarine than with either pancuronium or alcuronium. The cholinesterase activity in rat diaphragm homogenates was inhibited by hexamethonium. This inhibition was only significant at concentrations greater than those which resulted in antagonism and cannot, therefore, explain the observed antagonism. The mechanism of these observations is discussed with respect to the known behaviour of a combination of antagonists acting within a receptor system.

Increased masticatory muscle stiffness during limb muscle flaccidity associated with succinylcholine administration.

The resistance of mouth opening to a constant force of 1.7 N was measured in 44 pediatric subjects anesthetized with enflurane and paralyzed with succinylcholine or vecuronium. Measurements were made during a deep level of anesthesia before relaxant administration, immediately after the loss of the adductor pollicis muscle twitch and 45 s later. In 22 patients receiving succinylcholine, there was a significant reduction in mean mouth opening (from 16.9 +/- 2.8 to 12.6 +/- 4.3 to 13.0 +/- 4.3 mm; P less than 0.0005) and an increase in jaw stiffness (from 102.3 +/- 21.9 to 154.5 +/- 77.4 to 150.5 +/- 77.0 Nm/degree; P less than 0.02) immediately after disappearance of the evoked thenar muscle twitch, as well as 45 s later. In six patients receiving succinylcholine, measurements were continued at 1 min intervals; mouth opening reduction and jaw stiffness increase lasted up to 10 min and extended beyond the return of visible twitch. One patient had a reduction of mouth opening from 20 to less than 1 mm; his corresponding jaw stiffness changed from 83.4 to 3335.4 Nm/degree. This patient, considered by us to have masseter spasm, required several attempts at tracheal intubation due to an increased resistance to mouth opening, as did one other patient. Patients receiving vecuronium showed a significant (P less than 0.02) increase of mouth opening 45 s following loss of twitch (from 19.8 +/- 3.6 to 20.9 +/- 4.1 mm; jaw stiffness changed from 87.0 +/- 15.3 to 83.0 +/- 17.2 Nm/degree). Anesthesia and surgery proceeded normally; in most patients, in excess of 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of vecuronium and atracurium in an in vitro nerve-muscle preparation.

Atracurium and vecuronium were compared when given alone and in combination in the in vitro rat phrenic nerve-hemidiaphragm preparation stimulated via the phrenic nerve. The slopes of the log dose response curves of atracurium and vecuronium were parallel; their ED50s were 1.12 +/- 0.0035.10(-5)M and 5.89 +/- 0.16.10(-6)M, respectively. The combination's log dose response curves were significantly shifted to the left when compared with those of either relaxant alone; an increased potency is displayed by the combination. These observations indicate nondepolarizing muscle relaxant synergy for the combination of equal proportions of vecuronium and atracurium. The synergistic interaction of vecuronium and atracurium in this in vitro-model is not dependent on pharmacokinetic factors such as uptake, distribution, and biodegradation as are present in the in vivo animal models and in humans. Synergy of vecuronium and atracurium in vitro is a new finding and is consistent with hypotheses of multiple receptor sites and different modes of action of the "competitive" neuromuscular blocking agents. This degree of synergy, seen in the in vitro animal data, if extrapolatable to humans, is probably of little clinical significance.

The neuromuscular blocking effect of trimetaphan alone and in combination with different non-depolarizing muscle relaxants in the rat.

The effect of trimetaphan alone and in combination with pancuronium, tubocurarine or metocurine (dimethyl tubocurarine) has been examined on the rat phrenic nerve diaphragm preparation. Trimetaphan alone produced neuromuscular blockade in an all-or-none fashion once a concentration of between 2.39 X 10(-4) and 3.00 X 10(-4) M had been exceeded. Concentrations of trimetaphan below this threshold produced a dose-dependent potentiation of all three non-depolarizing relaxants studied. This potentiation was equal for tubocurarine and metocurine, but less for pancuronium.

The effects of succinylcholine on mouth opening.

Mouth opening and the resistance to opening developed by the muscles of mastication were measured in 63 children anesthetized with halothane and relaxed with succinylcholine, pancuronium, or vecuronium. Measurement of mouth opening, induced by a constant test force, was made when each patient was deeply anesthetized, as judged by clinical parameters. Succinylcholine, vecuronium, or pancuronium was then administered. The mouth opening measurement was repeated immediately after the loss of limb muscle twitch response and 45 s following the loss of twitch response. For the 24 patients receiving succinylcholine, there was a significant reduction in mean mouth opening (P less than 0.0001) and a significant increase in jaw stiffness (P less than 0.0001) immediately after limb relaxation. Forty-five seconds after full limb relaxation was attained, the mean mouth opening was still reduced (P less than 0.0001) and the mean jaw stiffness was still increased (P less than 0.0003) in the succinylcholine group. Patients receiving either vecuronium or pancuronium did not show a significant change of mouth opening or jaw stiffness following limb relaxation. Three patients, who received succinylcholine, required several attempts at tracheal intubation due to increased resistance to mouth opening. Anesthesia and surgery proceeded in all patients. None of the patients developed malignant hyperthermia. In view of the fact that a reduction in mouth opening was a constant finding when succinylcholine was administered during halothane anesthesia, the assumption that isolated "masseter spasm" or jaw stiffness heralds malignant hyperthermia should be reconsidered.

The effects of calcium-blocking agents on sympathetic responses to acute haemorrhagic shock in dogs.

The effects of three calcium channel-blocking agents, verapamil, nifedipine and diltiazem, given intravenously, have been studied on the cardiovascular and sympathetic responses to acute haemorrhagic shock in anaesthetized dogs. Following acute haemorrhage, cardiac output, mean pulmonary artery pressure, mean right atrial pressure, and pulmonary capillary wedge pressure all fell, and adrenaline, noradrenaline and plasma renin activity rose. In the presence of each calcium antagonist the fall in cardiac output, mean pulmonary artery pressure, central venous pressure and pulmonary capillary wedge pressure was similar, and the rise in catecholamine levels unaffected. The rise in plasma renin activity following diltiazem 0.02 mg kg-1 or 0.1 mg kg-1 or nifedipine 0.01 mg kg-1 was similar to values in a control group, whereas in those receiving verapamil 0.15 mg kg-1 or 0.6 mg kg-1, or nifedipine 0.05 mg kg-1, the rise was greater.

Cyanosis and cardiovascular depression in a neonate: complications of halothane anesthesia or phenylephrine eyedrops?

A 3-week-old patient underwent cataract extraction under halothane anesthesia. After induction of anesthesia and instillation of phenylephrine hydrochloride eyedrops, the patient experienced cyanosis and cardiovascular depression. Her condition improved over the next 10 minutes. The possible contributions of halothane and phenylephrine to this response are discussed.

Treatment of acute deficits of moyamoya disease with verapamil.

A 7 year old girl with moyamoya disease presented with progressive right arm and leg weakness and muteness. Intravenous administration of verapamil was followed by immediate and lasting improvement in motor function and return of some expressive language. A second infusion of verapamil during angiography produced an increased opacification of collateral vessels in the left basal ganglia, suggesting drug-induced vasodilation and reversal of ischaemia as a mechanism for the observed clinical improvement. Calcium-channel blockers may be useful in the acute management of selected patients with moyamoya disease.