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DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS BY SUCCINYLDICHOLINE INHIBITION
Brit. J. Anaesth. (1973), 45,450
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS BY SUCONYLDICHOLINE INHIBITION J. KING AND DOROTHY GRTFFTN SUMMARY
METHODS AND MATERIALS The investigations of Kalow and Genest (1957) yielded the first means of detecting the inherited Cholinesterase activity was estimated by the method variants of serum cholinesterase, the possession of of Kalow and Lindsay (1955) and phenotype deterwhich predisposes to a sensitivity to the muscle mined simultaneously by dibucaine number (Kalow relaxant, succinyldicholine (suxamethonium, Scoline). and Genest, 1957), fluoride number (Harris and These workers differentiated what they termed the Whittaker, 1961) and chloride number (Whittaker, "usual" and "atypical" enzymes by means of dibu- 1968b) following the analytical scheme of King caine (cinchocaine, Nupercaine) inhibition of (1965) at 25°C. A Unicam SP8000 recording specbenzoylcholine hydrolysis. Harris and Wbittaker trophotometer was used, temperature control given (1961) using sodium fluoride as inhibitor not only by a Tecam water-bath. made a second differentiating parameter available Sera from six phenotypes, 100 normal homozybut through this were able to demonstrate the pre- gotes, E, u E a , 61 atypical heterozygotes, E, a E / , t sence of a further variant, the fluoride-resistant 25 fluoride-resistant heterozygotes, Ei u E, f , 20 isoenzyme. By means of high sodium chloride con- atypical homozygotes, E," E, a , 3 fluoride-resistant centration Whittaker (1968b) provided a further homozygotes, E / E / , and 12 atypical/fluoridemeans of segregation, indicating further variants and resistant heterozygotes, E, 1 E, f were studied. most useful since the chloride number (percentage inhibition) is the reverse of dibucaine and fluoride RESULTS in that the usual enzyme is inhibited least and the At a benzoylcholine concentration of 50 nM, which succinyldicholine sensitive variants, most. is optimal for the usual enzyme at 25 °C, the sucIn later studies, Kalow and Davies (1958) showed cinyldicholine concentration in the reaction mixture that many esterase inhibitors, including succinyldiwas varied from 5 (j.M to 100 mM. The inhibition choline, reacted differently with the usual and curves obtained for the six commonly-recognized atypical enzymes. Goedde, Held and Altland (1968) phenotypes are shown in figure 1 and these follow investigated the hydrolysis of succinyldicholine and the typical sigmoid pattern. The curves for the usual succinylmonocholine by human cholinesterase and and the atypical homozygotes are almost identical also the inhibition of benzoylcholine hydrolysis by with those obtained by Goedde, Held and Altland these compounds. With a different substrate, o-nitro(1968). From these studies maximal differentiation phenylbutyrate, McComb, LaMotta and Wetstone of the phenotypes was obtained at a 1 mM con(1965) used succinyldicholine to differentiate the centration of succinyldicholine in the reaction three usual/atypical phenotypes as did Garry (1971) mixture. employing butyrylthiocholine as substrate. In both these latter studies a clear differentiation was J. KING, B.SC, M.I.BIOL., L.R.I.C, F.I.MX.T., Department obtained correlating with dibucaine inhibition but of Biochemistry; DOROTHY GRIFFIN, University Department of Pathological Biochemistry; Royal Infirmary, slightly less effectively. Glasgow.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
The inhibition by succinyldicholine (suxamethonium, Scoline) of the hydrolysis of benzoylcholine by serum cholinesterases has been studied. An analytical scheme for the differentiation of the genetically-determined cholinesterase variants is described and the ranges of percentage inhibition (Scoline number) encountered for each phenotype are presented. Correlation between succinyldicholine, dibucaine, fluoride and chloride inhibitions in 221 individuals of 6 recognized phenotypes are illustrated and it is suggested that these groupings are not homogeneous.
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS
451
100-•
90
80-
_6 S
iv
z
50--
IE
40+
t 'V • - ^
c 2 3 4 -log Molar SiKCinyldKholirw
FIG.
1. Inhibition of cholinesterase variants by succinyldicholine.
4>
S
30
I20
Ei" Ei»
f
Ei» Ei'
10-
Eif Ei'
E U E°
Ei« Ei» E ^ Ei' ............
Ei» Ei»
FIG.
2. Percentage inhibitions of cholinesterase phenotypes by mM succinyldicholine.
By employing a 4 mM aqueous solution of suc- tion in conjunction with dibucaine, fluoride and cinyldicholine (397 mg succinyldicholine chloride chloride inhibition respectively. The best segregadihydrate/litre) in the assay scheme of King (1965) tion is obtained with a combination of Scoline and the percentage inhibition (Scoline number) was fluoride numbers. The segregation obtained with determined in sera obtained from patients who had dibucaine and fluoride inhibitions on the same exhibited an abnormal response to die relaxant, from population is shown in figure 6. A point in the their relatives, and from normal individuals. The centre of a symbol in these diagrams indicates two results obtained from this survey over a period of sera with identical numbers. two years are illustrated in figure 2 and demonstrate Preincubation of dilute serum in the presence of that a differentiation of phenotypes is obtained at 1 mM succinyldicholine for periods up to 60 minutes least as good as that obtained by the use of dibucaine did not alter the percentage inhibition significantly. inhibition. In figure 2, the closed drcles for die This is not surprising as Kalow (1959a) showed that usual phenotype each represent three sera, the open the normal and atypical enzymes at this concentracircles, one specimen. Three anomalous values tion of succinyldicholine hydrolysed about 80 (jjnol obtained from serum samples of two sisters were and less than 5 [xmol/min/1. respectively at 30°C. assigned to the atypical homozygote group where This is equivalent to hydrolysis of 96 nmol and they are shown as open drcles. less than 6 nmol per hour by 20 JAI of serum which Figures 3, 4 and 5 illustrate die phenotype from figure 1 would not result in any significant differentiation obtained by succinyldicholine inhibi- change in inhibition. The figures given by Goedde,
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
"5
60'
452
BRITISH JOURNAL OF ANAESTHESIA
10090 •
90-
W
80'
80'
E'Ef
f. 70' •
70o
I
ojj
60
60-
•D
z
SO40
"o 30
30 20 •
%"•
20
10
Y0
20
30
?0
50
60
70
80
Dibucoine Numkxr
FIG.
10-
•+•
3. Distribution of Scoline and dibucaine numbers.
•+• •+•+• •+•+20 30 40 50 60 70 Chloride Number FIG. 5. Distribution of Scoline and chloride numbers.
10
90- •
-*-
90'
80' -
80-
70 •
70-
60'
H I
E°Ef
"o o
50'
" • E°Ef
40-
"> 40-
a
30-
30-
V.
20
20-
1010-
-+•
10
-•20
30
•+-
40
•+-
SO
•+•
60
•+•
70
Fluoride Number FIG.
4. Distribution of Scoline and fluoride numbers.
10
20
?0
•+•
40
•+-
50
•+-
60
-+70
Fluoride Number FIG. 6. Distribution of dibucaine and fluoride numbers.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
J <0 +
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS Held and Altland (1968) for spontaneous hydrolysis of succinyldicholine are of the same order as that for the atypical enzyme. DISCUSSION
Certainly among the "silent gene" homozygotes heterogeneity has been demonstrated (Goedde and Altland, 1968; Rubinstein et aL, 1970) and either explicitly or implicitly the presence of subtypes has been suggested by chloride inhibition (Whittaker, 1968b; King and Dixon, 1970), by the effect of n-butyl alcohol (Whittaker, 1968a) or by substrate affinities (Irwin and Hein, 1966). The three sera of controversial phenotype can readily be explained on either premise but the concept that there may be numerous subtypes of cholinesterase gains support from the three anomalous results from two sisters who were classified as atypical homozygotes despite the unusually high inhibition values. This, in turn, could suggest that there is not a single entity at present classed as the "usual", "atypical" or '^fluoride-resistant" enzyme but that each of these designations embraces a heterogenous population. There may therefore well be a continuous range of cholinesterases within that which is termed a phenotype, and depending not only upon the enzymic properties of the molecular species, mainly turnover number, but also on the relative stabilities of the different variants in the circulation. ACKNOWLEDGEMENTS
We are grateful to our many anaesthetist colleagues in the West of Scotland Region who have referred cases of "Scoline sensitivity". Garry, P. J. (1971). Serum cholinesterase variants: examination of several differential inhibitors, salts and buffers used to measure enzyme activity. Clin. Cherru, 17, 183. Goedde, H. W , and Altland, K. (1968). Evidence for different "silent genes" in human serum pseudocholinesterase polymorphism. Ann. N.Y. Acad. Sci., 151, 540. Held, K. R., and Altland, K. (1968). Hydrolysis of succinyldicholine and succinylmonocholine in human serum. Mol. Pharmacol, 4, 274. Harris, H., and Whittaker, M. (1961). Differential inhibition of human serum cholinesterase with fluoride: recognition of two new phenotypes. Nature (Lond.), 191, 496. Irwin, R. L., and Hein, M. M. (1966). The substrate specificity of atypical cholinesterase in relation to phenotypes. Biochem. Pharmacol., 15, 145. Kalow, W. (1959a). The distribution, destruction and elimination of muscle relaxants. Anesthesiology, 20, 505. (1959b). Cholinesterase types; in Ciba Foundation Symposium on Biochemistry of Human Genetics. London : Churchill. Davies, R. O. (1958). The activity of various esterase inhibitors towards atypical human strum cholinesterase. Biochem. Pharmacol., 1, 183. Genest, K. (1957). A method for the detection of atypical forms of human serum cholinesterase: determination of dibucaine numbers. Canad. J. Biochem., 35, 339.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
The technical advantages of the use of succinyldicholine in differentiating the serum cholinesterase variants are limited. Less skill is required in preparing the reagent. For a litre of solution an error of a few milligrams in the required 397 mg of succinyldicholine chloride dihydrate will not alter the results, whereas with dibucaine and sodium fluoride 13.7 mg and 8.4 mg respectively must be accurately weighed. There is doubtless an advantage in interpreting results with succinyldicholine inhibition since the difference between the extremes of the usual phenotype and atypical homozygote are greater than that obtained with dibucaine. Figure 3 indicates, however, that there is a close correlation between die two inhibitor numbers. It is only a comparison of figures 4 and 6 which shows any divergence of interpretation. Three of the specimens classified as E,* E,' by Scoline and fluoride number combination (figure 4) would probably be classed as E, u E, a by dibucaine and fluoride numbers (fig. 6). It is not possible by family studies to decide this matter although in one case involved in an apnoeic episode it may be possible at a later date, if there are any children. The point may appear trivial but it does introduce a wider concept. In our hands the assay of cholinesterase activity has an error of ±2.4% and replicate estimates of dibucaine numbers around 20 and 80 have maximum variations of + 3 and ± 1 respectively. The scatter of inhibitor constants for any phenotype cannot therefore be explained by a lack of precision in the assay procedure and the ranges of inhibitor numbers illustrated are real and not a result of experimental error. It should be emphasized that for the values reported here in which a result was doubtful or of a controversial nature it was confirmed by repeated estimations. Kalow (1959b) considered two possible causes for the wide range of dibucaine inhibitions encountered in atypical heterozygotes: that is, either there were numerous subtypes of cholinesterase with differing kinetic properties, or the amount of enzyme due to each gene was separately controlled. He concluded that the latter was the case and that heterozygous sera contained a mixture of the two enzymes in various proportions, and further that these proportions were genetically determined.
453
BRITISH JOURNAL OF ANAESTHESIA
454
cents-vingt et un individus appartenant a six phenotypes reconnus et il est suggeri que ces groupements ne sont pas homogenes.
Kalow, W., and Lindsay, H. A. (1955). A comparison of optical and manometric methods for the assay of human serum cholinesterase. Canad. J. Biochem., 33, 568. King, J. (1965). Practical Clinical Enzymology. London: Van Nostrand. Dixon, R. I. (1970). A source of error in the determination of inhibitor constants of serum cholinesterase. Brit. J. Anaesth., 42, 698. McComb, R. B., LaMotta, R. V. and Wetstone, H. J. (1965). Procedure for detecting atypical serum cholinesterase using o-nitrophenylbutyrate as substrate. Clin. Chem., 11, 645. Rubinstein, H. M., Dietz, A. A., Hodges, L. K., Lubrano, T., and Czebotar, V. (1970). Silent cholinesterase gene: variations in the properties of serum enzyme in apparent homozygotes. J. Clin. Invest., 49, 479. Whittaker, M. (1968a). Differential inhibition of human serum cholinesterase with n-butyl alcohol: recognition of new phenotypes. Acta. genet. (Basel), 18, 335. (1968b). The pseudocholinesterase variants: differentiation by means of sodium chloride. Acta. genet. (Basel), 18, 556.
Es wurden Untersuchungen angestellt fiber die Hemmung der Hydrolyse von Benzoylcholin durch Succinyldicholin (Suxamethonium, Scholine) mittels Serumcholinesterasc wurde untersucht. Es wird ein analytisches Schema fur die Differenzierung der genetisch festgelegten Cholinesterasevarianten beschrieben. Die VerhSltnisse der prozentuellen Hemmung (Scholinezahl) welche jedem Phanotyp entsprechen werden dargelegt. Die Korrelation der Hemmung bei Sucdnyldichohxi, Dibucaine, Fluorid und Chlorid wird an 221 einzelnen Fallen fur sechs festgestellte Phanotypen aufgezeichnet. Es wird angenommen, dafi diese Gruppen jedoch nicht homogen sind.
DIFFERENCIATION DES VARIANTES DE LA CHOLINESTERASE SERIQUE A L'AIDE DTTNE INHIBITION PAR LA SUCCINYL-DICHOLINE
DEFERENCIACION DE LAS VARIANTES DE COLINESTERASA EN EL SUERO POR INHIBICION DE LA SUCCINILCOLINA
DIFFERENZIERUNG VON VARIANTEN DER SERUM-CHOLINESTERASE DURCH HEMMUNG MIT SUCCINYLDICHOLIN ZUSAMMENFASSUNG
RESDMEN
Ha sido estudiada la inhibition por la succinilcoUna (suxametonio, Scoline) de la hidrolisis de benzoilcolina por las colinesterasas sericas. Se describe un esquema analitico para la diferenciaci6n de las variantes de colinesterasa de determinacion gendtica y se presentan las escalas de inhibicion porcentual (Numero Scoline) encontradas para cada tipo. Se ilustra sobre la correlaci6n entre la inhibici6n de la succinilcoUna, dibucaina, fluoruros y cloruros, en doscientos veintiuno individuos de seis fenotipos reconocidos, sugiriendose que estos grupos no son homogeneos.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
SOMMAIRE
L'inhibition par la succinyl-dicholine (suxamethonium, ScoUne) de l'hydrolyse de la benzoylcholine par les cholinesterases siriques, a tx& itudiee. Un schema analytique en vue d'une differenciation des variantes de la chohnesterase, genetiquement determinees, est decrit et il est fait itat des marges d'inhibition en pourcentage (Nombre de ScoUne) recontrees pour chaque phenotype. Une illustration des correlations existant entre les inhibitions exercees par la succinyl-dicholine, la dibucaine, les fluorures et les chlorures a et6 donnee a partir de deux-
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS BY SUCONYLDICHOLINE INHIBITION J. KING AND DOROTHY GRTFFTN SUMMARY
METHODS AND MATERIALS The investigations of Kalow and Genest (1957) yielded the first means of detecting the inherited Cholinesterase activity was estimated by the method variants of serum cholinesterase, the possession of of Kalow and Lindsay (1955) and phenotype deterwhich predisposes to a sensitivity to the muscle mined simultaneously by dibucaine number (Kalow relaxant, succinyldicholine (suxamethonium, Scoline). and Genest, 1957), fluoride number (Harris and These workers differentiated what they termed the Whittaker, 1961) and chloride number (Whittaker, "usual" and "atypical" enzymes by means of dibu- 1968b) following the analytical scheme of King caine (cinchocaine, Nupercaine) inhibition of (1965) at 25°C. A Unicam SP8000 recording specbenzoylcholine hydrolysis. Harris and Wbittaker trophotometer was used, temperature control given (1961) using sodium fluoride as inhibitor not only by a Tecam water-bath. made a second differentiating parameter available Sera from six phenotypes, 100 normal homozybut through this were able to demonstrate the pre- gotes, E, u E a , 61 atypical heterozygotes, E, a E / , t sence of a further variant, the fluoride-resistant 25 fluoride-resistant heterozygotes, Ei u E, f , 20 isoenzyme. By means of high sodium chloride con- atypical homozygotes, E," E, a , 3 fluoride-resistant centration Whittaker (1968b) provided a further homozygotes, E / E / , and 12 atypical/fluoridemeans of segregation, indicating further variants and resistant heterozygotes, E, 1 E, f were studied. most useful since the chloride number (percentage inhibition) is the reverse of dibucaine and fluoride RESULTS in that the usual enzyme is inhibited least and the At a benzoylcholine concentration of 50 nM, which succinyldicholine sensitive variants, most. is optimal for the usual enzyme at 25 °C, the sucIn later studies, Kalow and Davies (1958) showed cinyldicholine concentration in the reaction mixture that many esterase inhibitors, including succinyldiwas varied from 5 (j.M to 100 mM. The inhibition choline, reacted differently with the usual and curves obtained for the six commonly-recognized atypical enzymes. Goedde, Held and Altland (1968) phenotypes are shown in figure 1 and these follow investigated the hydrolysis of succinyldicholine and the typical sigmoid pattern. The curves for the usual succinylmonocholine by human cholinesterase and and the atypical homozygotes are almost identical also the inhibition of benzoylcholine hydrolysis by with those obtained by Goedde, Held and Altland these compounds. With a different substrate, o-nitro(1968). From these studies maximal differentiation phenylbutyrate, McComb, LaMotta and Wetstone of the phenotypes was obtained at a 1 mM con(1965) used succinyldicholine to differentiate the centration of succinyldicholine in the reaction three usual/atypical phenotypes as did Garry (1971) mixture. employing butyrylthiocholine as substrate. In both these latter studies a clear differentiation was J. KING, B.SC, M.I.BIOL., L.R.I.C, F.I.MX.T., Department obtained correlating with dibucaine inhibition but of Biochemistry; DOROTHY GRIFFIN, University Department of Pathological Biochemistry; Royal Infirmary, slightly less effectively. Glasgow.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
The inhibition by succinyldicholine (suxamethonium, Scoline) of the hydrolysis of benzoylcholine by serum cholinesterases has been studied. An analytical scheme for the differentiation of the genetically-determined cholinesterase variants is described and the ranges of percentage inhibition (Scoline number) encountered for each phenotype are presented. Correlation between succinyldicholine, dibucaine, fluoride and chloride inhibitions in 221 individuals of 6 recognized phenotypes are illustrated and it is suggested that these groupings are not homogeneous.
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS
451
100-•
90
80-
_6 S
iv
z
50--
IE
40+
t 'V • - ^
c 2 3 4 -log Molar SiKCinyldKholirw
FIG.
1. Inhibition of cholinesterase variants by succinyldicholine.
4>
S
30
I20
Ei" Ei»
f
Ei» Ei'
10-
Eif Ei'
E U E°
Ei« Ei» E ^ Ei' ............
Ei» Ei»
FIG.
2. Percentage inhibitions of cholinesterase phenotypes by mM succinyldicholine.
By employing a 4 mM aqueous solution of suc- tion in conjunction with dibucaine, fluoride and cinyldicholine (397 mg succinyldicholine chloride chloride inhibition respectively. The best segregadihydrate/litre) in the assay scheme of King (1965) tion is obtained with a combination of Scoline and the percentage inhibition (Scoline number) was fluoride numbers. The segregation obtained with determined in sera obtained from patients who had dibucaine and fluoride inhibitions on the same exhibited an abnormal response to die relaxant, from population is shown in figure 6. A point in the their relatives, and from normal individuals. The centre of a symbol in these diagrams indicates two results obtained from this survey over a period of sera with identical numbers. two years are illustrated in figure 2 and demonstrate Preincubation of dilute serum in the presence of that a differentiation of phenotypes is obtained at 1 mM succinyldicholine for periods up to 60 minutes least as good as that obtained by the use of dibucaine did not alter the percentage inhibition significantly. inhibition. In figure 2, the closed drcles for die This is not surprising as Kalow (1959a) showed that usual phenotype each represent three sera, the open the normal and atypical enzymes at this concentracircles, one specimen. Three anomalous values tion of succinyldicholine hydrolysed about 80 (jjnol obtained from serum samples of two sisters were and less than 5 [xmol/min/1. respectively at 30°C. assigned to the atypical homozygote group where This is equivalent to hydrolysis of 96 nmol and they are shown as open drcles. less than 6 nmol per hour by 20 JAI of serum which Figures 3, 4 and 5 illustrate die phenotype from figure 1 would not result in any significant differentiation obtained by succinyldicholine inhibi- change in inhibition. The figures given by Goedde,
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
"5
60'
452
BRITISH JOURNAL OF ANAESTHESIA
10090 •
90-
W
80'
80'
E'Ef
f. 70' •
70o
I
ojj
60
60-
•D
z
SO40
"o 30
30 20 •
%"•
20
10
Y0
20
30
?0
50
60
70
80
Dibucoine Numkxr
FIG.
10-
•+•
3. Distribution of Scoline and dibucaine numbers.
•+• •+•+• •+•+20 30 40 50 60 70 Chloride Number FIG. 5. Distribution of Scoline and chloride numbers.
10
90- •
-*-
90'
80' -
80-
70 •
70-
60'
H I
E°Ef
"o o
50'
" • E°Ef
40-
"> 40-
a
30-
30-
V.
20
20-
1010-
-+•
10
-•20
30
•+-
40
•+-
SO
•+•
60
•+•
70
Fluoride Number FIG.
4. Distribution of Scoline and fluoride numbers.
10
20
?0
•+•
40
•+-
50
•+-
60
-+70
Fluoride Number FIG. 6. Distribution of dibucaine and fluoride numbers.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
J <0 +
DIFFERENTIATION OF SERUM CHOLINESTERASE VARIANTS Held and Altland (1968) for spontaneous hydrolysis of succinyldicholine are of the same order as that for the atypical enzyme. DISCUSSION
Certainly among the "silent gene" homozygotes heterogeneity has been demonstrated (Goedde and Altland, 1968; Rubinstein et aL, 1970) and either explicitly or implicitly the presence of subtypes has been suggested by chloride inhibition (Whittaker, 1968b; King and Dixon, 1970), by the effect of n-butyl alcohol (Whittaker, 1968a) or by substrate affinities (Irwin and Hein, 1966). The three sera of controversial phenotype can readily be explained on either premise but the concept that there may be numerous subtypes of cholinesterase gains support from the three anomalous results from two sisters who were classified as atypical homozygotes despite the unusually high inhibition values. This, in turn, could suggest that there is not a single entity at present classed as the "usual", "atypical" or '^fluoride-resistant" enzyme but that each of these designations embraces a heterogenous population. There may therefore well be a continuous range of cholinesterases within that which is termed a phenotype, and depending not only upon the enzymic properties of the molecular species, mainly turnover number, but also on the relative stabilities of the different variants in the circulation. ACKNOWLEDGEMENTS
We are grateful to our many anaesthetist colleagues in the West of Scotland Region who have referred cases of "Scoline sensitivity". Garry, P. J. (1971). Serum cholinesterase variants: examination of several differential inhibitors, salts and buffers used to measure enzyme activity. Clin. Cherru, 17, 183. Goedde, H. W , and Altland, K. (1968). Evidence for different "silent genes" in human serum pseudocholinesterase polymorphism. Ann. N.Y. Acad. Sci., 151, 540. Held, K. R., and Altland, K. (1968). Hydrolysis of succinyldicholine and succinylmonocholine in human serum. Mol. Pharmacol, 4, 274. Harris, H., and Whittaker, M. (1961). Differential inhibition of human serum cholinesterase with fluoride: recognition of two new phenotypes. Nature (Lond.), 191, 496. Irwin, R. L., and Hein, M. M. (1966). The substrate specificity of atypical cholinesterase in relation to phenotypes. Biochem. Pharmacol., 15, 145. Kalow, W. (1959a). The distribution, destruction and elimination of muscle relaxants. Anesthesiology, 20, 505. (1959b). Cholinesterase types; in Ciba Foundation Symposium on Biochemistry of Human Genetics. London : Churchill. Davies, R. O. (1958). The activity of various esterase inhibitors towards atypical human strum cholinesterase. Biochem. Pharmacol., 1, 183. Genest, K. (1957). A method for the detection of atypical forms of human serum cholinesterase: determination of dibucaine numbers. Canad. J. Biochem., 35, 339.
Downloaded from http://bja.oxfordjournals.org/ at Fudan University on May 2, 2015
The technical advantages of the use of succinyldicholine in differentiating the serum cholinesterase variants are limited. Less skill is required in preparing the reagent. For a litre of solution an error of a few milligrams in the required 397 mg of succinyldicholine chloride dihydrate will not alter the results, whereas with dibucaine and sodium fluoride 13.7 mg and 8.4 mg respectively must be accurately weighed. There is doubtless an advantage in interpreting results with succinyldicholine inhibition since the difference between the extremes of the usual phenotype and atypical homozygote are greater than that obtained with dibucaine. Figure 3 indicates, however, that there is a close correlation between die two inhibitor numbers. It is only a comparison of figures 4 and 6 which shows any divergence of interpretation. Three of the specimens classified as E,* E,' by Scoline and fluoride number combination (figure 4) would probably be classed as E, u E, a by dibucaine and fluoride numbers (fig. 6). It is not possible by family studies to decide this matter although in one case involved in an apnoeic episode it may be possible at a later date, if there are any children. The point may appear trivial but it does introduce a wider concept. In our hands the assay of cholinesterase activity has an error of ±2.4% and replicate estimates of dibucaine numbers around 20 and 80 have maximum variations of + 3 and ± 1 respectively. The scatter of inhibitor constants for any phenotype cannot therefore be explained by a lack of precision in the assay procedure and the ranges of inhibitor numbers illustrated are real and not a result of experimental error. It should be emphasized that for the values reported here in which a result was doubtful or of a controversial nature it was confirmed by repeated estimations. Kalow (1959b) considered two possible causes for the wide range of dibucaine inhibitions encountered in atypical heterozygotes: that is, either there were numerous subtypes of cholinesterase with differing kinetic properties, or the amount of enzyme due to each gene was separately controlled. He concluded that the latter was the case and that heterozygous sera contained a mixture of the two enzymes in various proportions, and further that these proportions were genetically determined.
453
BRITISH JOURNAL OF ANAESTHESIA
454
cents-vingt et un individus appartenant a six phenotypes reconnus et il est suggeri que ces groupements ne sont pas homogenes.
Kalow, W., and Lindsay, H. A. (1955). A comparison of optical and manometric methods for the assay of human serum cholinesterase. Canad. J. Biochem., 33, 568. King, J. (1965). Practical Clinical Enzymology. London: Van Nostrand. Dixon, R. I. (1970). A source of error in the determination of inhibitor constants of serum cholinesterase. Brit. J. Anaesth., 42, 698. McComb, R. B., LaMotta, R. V. and Wetstone, H. J. (1965). Procedure for detecting atypical serum cholinesterase using o-nitrophenylbutyrate as substrate. Clin. Chem., 11, 645. Rubinstein, H. M., Dietz, A. A., Hodges, L. K., Lubrano, T., and Czebotar, V. (1970). Silent cholinesterase gene: variations in the properties of serum enzyme in apparent homozygotes. J. Clin. Invest., 49, 479. Whittaker, M. (1968a). Differential inhibition of human serum cholinesterase with n-butyl alcohol: recognition of new phenotypes. Acta. genet. (Basel), 18, 335. (1968b). The pseudocholinesterase variants: differentiation by means of sodium chloride. Acta. genet. (Basel), 18, 556.
Es wurden Untersuchungen angestellt fiber die Hemmung der Hydrolyse von Benzoylcholin durch Succinyldicholin (Suxamethonium, Scholine) mittels Serumcholinesterasc wurde untersucht. Es wird ein analytisches Schema fur die Differenzierung der genetisch festgelegten Cholinesterasevarianten beschrieben. Die VerhSltnisse der prozentuellen Hemmung (Scholinezahl) welche jedem Phanotyp entsprechen werden dargelegt. Die Korrelation der Hemmung bei Sucdnyldichohxi, Dibucaine, Fluorid und Chlorid wird an 221 einzelnen Fallen fur sechs festgestellte Phanotypen aufgezeichnet. Es wird angenommen, dafi diese Gruppen jedoch nicht homogen sind.
DIFFERENCIATION DES VARIANTES DE LA CHOLINESTERASE SERIQUE A L'AIDE DTTNE INHIBITION PAR LA SUCCINYL-DICHOLINE
DEFERENCIACION DE LAS VARIANTES DE COLINESTERASA EN EL SUERO POR INHIBICION DE LA SUCCINILCOLINA
DIFFERENZIERUNG VON VARIANTEN DER SERUM-CHOLINESTERASE DURCH HEMMUNG MIT SUCCINYLDICHOLIN ZUSAMMENFASSUNG
RESDMEN
Ha sido estudiada la inhibition por la succinilcoUna (suxametonio, Scoline) de la hidrolisis de benzoilcolina por las colinesterasas sericas. Se describe un esquema analitico para la diferenciaci6n de las variantes de colinesterasa de determinacion gendtica y se presentan las escalas de inhibicion porcentual (Numero Scoline) encontradas para cada tipo. Se ilustra sobre la correlaci6n entre la inhibici6n de la succinilcoUna, dibucaina, fluoruros y cloruros, en doscientos veintiuno individuos de seis fenotipos reconocidos, sugiriendose que estos grupos no son homogeneos.
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SOMMAIRE
L'inhibition par la succinyl-dicholine (suxamethonium, ScoUne) de l'hydrolyse de la benzoylcholine par les cholinesterases siriques, a tx& itudiee. Un schema analytique en vue d'une differenciation des variantes de la chohnesterase, genetiquement determinees, est decrit et il est fait itat des marges d'inhibition en pourcentage (Nombre de ScoUne) recontrees pour chaque phenotype. Une illustration des correlations existant entre les inhibitions exercees par la succinyl-dicholine, la dibucaine, les fluorures et les chlorures a et6 donnee a partir de deux-