Dibucaine numbers

Neuromuscular blockade produced by succinylcholine and mivacurium can be prolonged in patients with a genetically abnormal variant of plasma cholinesterase. The dibucaine number is a measure of the ability of a patient to metabolize succinylcholine and can be used to identify at-risk patients. Under standardized test conditions, dibucaine inhibits the normal enzyme by 80% and the abnormal enzyme by only 20%. Many genetic variants of plasma cholinesterase have been identified, although the dibucaine-related variants are the most important. Given the rarity of these genetic variants, plasma cholinesterase testing is not a routine clinical procedure. 

W. Kalow, K. Genest, A Method for the Detection of Atypical Forms of Serum Cholinesterase. Determination of Dibucaine Numbers , Can. J. Biochem., 35, 339-346 (1957). 

Kalow, W. and Staron, N. On distribution and inheritance of atypical forms of human serum cholinesterase, as indicated by dibucaine numbers. Can J Biochem Physiol 1957, 35 1305-1320. 

Kalow and Genest, using benzoylchohne as a classic substrate, demonstrated the qualitative difference in CHEs. Based on the differences as sensitivity to inhibition by the local anesthetic dibucaine, they developed a simple test to classify the type of CHE as usual, intermediate, or atypical. With lO mol/L dibucaine ( dibucaine number ), the usual CHE is inhibited by 80%, but atypical CHE is inhibited by only 20%. Subjects heterozygous for the normal and atypical gene show about 60% inhibition of CHE. To differentiate other genotypes, sodium fluoride can be used as CHE inhibitor. 

Relative activity Dibucaine number Fluoride number ... 

Nine of the remaining patients had dibucaine numbers (measured by the choline oxidase procedure) characteristic of the EyEy genotype and the sera of six had no activity with succinylcholine (Section 3.2). These 15 patients had cholinesterases whose properties appear to be quite different from those of any other variants that have been reported, with one possible exception. The six patients whose serum exhibited no activity with succinylcholine but did show the usual level of activity with benzoylcholine, represent a probable new phenotype. The nine patients who had DN values characteristic of the usual phenotype when using benzoylcholine as substrate but DN values characteristic of the atypical enzyme when using succinylcholine, must also be classified as representing... 

It is sometimes recommended that if it is necessary to administer succinylcholine to a patient who is known to be overly susceptible, very small doses be used, Lee-Son et al. (L21) reported an interesting case in which the drug was administered on a number of occasions to a patient who had a dibucaine number (Section 2.2) of 23, and who may have been homozygous for the atypical gene, or heterozygous for the atypical... 

Fig. 3. Pattern of elution of tetramer from 10 ml of the same AU-heterozygous serum as was used in the experiment of Fig. 2, with a longer affinity chromatography column (1 x 30 cm). Assignment of the tetrameric composition of each fraction was based upon the dibu-caine number and total activity, and the calculated dibucaine numbers for each possible tetramer. The homologous tetramers at the beginning and end, A4 and U4, had dibucaine numbers of 15 and 78, respectively. (After La Du and Choi, L2.)...

Dibucaine number Phenotype Genotypes Number found Number expected Ratio— found/ expected... 

Differentiation based on dibucaine inhibition. The most commonly used agent for differentiating serum cholinesterase variants is dibucaine, which acts as an inhibitor of the enzyme (Sections 2.2 and 6.2). Its use for the differentiation of cholinesterase variants was first introduced by Kalow and Genest (Kll) in 1957, and it has been and continues to be extensively used for this purpose. The assay is performed in two cuvettes, one with substrate alone and one with substrate plus dibucaine. Both cuvettes contain M/15 phosphate buffer (Sorensen) at pH 7.4 and 5 X 10 mol/liter benzoylcholine chloride. One of the cuvettes contains 1 X 10 mol/liter dibucaine. The initial velocity at 26°C is measured in both cuvettes by determining the rate of change of absorbance at 240 nm. The dibucaine number, DN, is the percentage of inhibition by dibucaine, and is calculated as DN = 100 (v - t> )/o, where V is the uninhibited velocity and v is the velocity measured in the presence of the inhibitor. DN values obtained by this method for the most widely studied cholinesterase variants are presented in Table 1 (see Section 2.1). 

Two other pairs of tubes are prepared to determine the percentage of inhibition by dibucaine (concentration in reaction mixture 0.03 mmol/liter) and by sodium fluoride (concentration in reaction mixture 4 mmol/liter). In each case one out of the pair of tubes serves as the blank. The inhibitor is added to all tubes with the substrate. The remainder of the assay procedure is identical to that of the uninhibited reaction. The percentages of inhibition by dibucaine (dibucaine number) and by fluoride (fluoride number) are calculated as described in Section 6.3.2.2. 

The relationship of BuChE activity and succinylcholine can be somewhat different, however. One author25 reports on an individual whose BuChE activity was 3-fold higher than normal. His dibucaine number was normal, and he was found to be relatively resistant to succinylcholine. His sister and daughter also had high BuChE activities. The author of this report suggests that this abnormality is autosomal dominant and that it represents another genetic abnormality of BuChE. 

A patient anaesthetised with fluroxene and nitrous oxide demonstrated 100% blockade with suxamethonium (succinylcholine) and tubocurar-ine. About 50 minutes later when twitch height had fully returned and tidal volume was 400 mL, she was given lidocaine 50 mg intravenously for premature ventricular contractions. She immediately stopped breathing and the twitch disappeared. About 45 minutes later the tidal volume was 450 mL. Later it was found that the patient had a dibucaine number (a measure of cholinesterase activity) of 23%. ... 

Another report describes a man whose recovery from neuromuscular blockade with suxamethonium was very prolonged. He had attempted suicide approximately 2 weeks earlier with dimpylate (diazinon), a household insecticide. His pseudocholinesterase was found to be 2.5 units/L (normal values 7 to 19) and his dibucaine number (a measurement of cholinesterase activity) was too low to be measured. ... 

Palleschi G, Lavagnini MG, Moscone D, Pilloton R, D Ottavio D, Evangelisti ME. 1990. Determination of serum cholinesterase activity and dibucaine numbers by an amperometric choline sensor. Biosensors and Bioelectronics 5 27-35. 

Pseudocholinesterase (EC 3.1.1.8). Enzyme present in serum but shows atypical kinetics Condition first discovered with introduction of suxamethonium (suc-cinyl dicholine) as muscle relaxant in electroconvulsion therapy. This drug is normally rapidly hydrolysed by pseudocholinesterase, and its effects last only a few minutes Afiected subjects (1 in 2,000 Europeans) develop prolonged muscular paralysis and apnea (up to 2 hours) after normal drug dose. Condition screened for by measuring inhibition of serum cholinesterase by dibucaine percent inhibition is called dibucaine number (80% for normal enzyme, 20% for atypical enzyme at lO M dibucaine). Dibucaine nmnbers of about 62 % also occur in 4 % of Europeans, who possess about equal amounts of normal and atypical forms... 

Normal plasma cholinesterase (pseudocholinesterase) is 80% inhibited by the anaesthetic dibucaine, i.e. it has a dibucaine number of 80. In individuals with suxamethonium sensitivity, the cholinesterase differs from the normal form and is less susceptible to dibucaine inhibition, i.e. it has lower dibucaine numbers. This enzyme behaviour is used in phenotyping members of an affected family, since heterozygotes have dibucaine numbers intermediate between those of normals and homozygotes. 

The most common mutant is the so-called dibucaine-resistant enzyme variant. It is characterized principally by its reduced afiSnity for a series of substrates and inhibitors as compared with the normal pseudocholinesterase. Although at high concentrations succinyldicholine can also be converted by the dibucaine-resistant enzyme variant, the absence of enzymatic hydrolysis under pharmacologic conditions is adequately explained by the large difference in the Michaelis constants of succinyldicholine with the normal enzyme with the dibucaine-resistant variant Usually the variant is identified by the reduced inhibition of its reaction with the substrate benzoylcholine produced by the local anaesthetic dibucaine (cinchocaine). The percentage of inhibition by dibucaine under standard conditions, defined as the dibucaine number (DN). is about 20 for the dibucaine-resistant variant and app oximately 80 for the ordinary enzyme. Serum activity from heterozygotes having both enzymes is inhibited about 40-70%. 

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