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Methods for serum cholinesterase assay and classification of genetic variants
87
Clinica Chimica Acta, 183 (1989) 87-90 Elsevier
CCA 04479
Short Communication
Methods for serum cholinesterase assay and classification of genetic variants Mauro Panteghini 1’ Laboratorio Analisi Chimico-Cliniche, Spedah Civili, Brescia (Italy) (Received 24 August 1988; revision received 15 December 1988; accepted 31 December 1988)
Key words: Serum cholinesterase; Acetylcholinesterase
Introduction
Two cholinesterases are found in man: the one in red cells, nerve endings, lung, spleen, and grey matter of the brain is commonly called acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, EC 3.1.1.7), the other in serum, liver, pancreas, heart, and white matter, is called serum cholinesterase (CHE, acylcholine acylhydrolase, EC 3.1.1.8). Only the serum enzyme is currently clinically relevant, and used as a test of hepato-cellular function, to confirm acute organophosphate insecticide poisoning, and to detect increased sensitivity to the muscle relaxant succinylcholine (suxamethonium). Sensitivity to succinylcholine
The depolarizing neuromuscular blocking agent succinylcholine is usually hydrolyzed in plasma in a few minutes by CHE. This rapid hydrolysis is responsible for this muscle relaxant’s conveniently short action, but some patients cannot hydrolyze succinylcholine rapidly and so a ‘normal’ dose of succinylcholine (1 mg/kg i.v.) may cause apnea for several hours following cessation of anesthesia. In other words, CHE from these individuals have a lower affinity for the choline ester substrates than has the enzyme from normals. The enzyme variants in subjects with increased succinylcholine sensitivity have lower total activity and resist inhibition by the local anesthetic dibucaine or fluoride, or both. Four main allelic genes at the locus El are the determinants for the principal clinical variants of CHE: the normal gene (U = usual), the atypical gene (A = atypical), the fluoride-resistant gene (F = fluoride-resistant), and the silent gene (S = silent). The first is greatly inhibited by dibucaine and fluoride, while the second is resistant to such inhibition. The variant associated with the F gene is Correspondence to: Dr. M. Panteghini, lo Laboratorio Analisi Chirnico-Cliniche, SpedaIi Civili, 25123 Brescia, Italy.
OOOS-8981/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
88
resistant to inhibition by fluoride but not to dibucaine. Finally, the gene may also not express itself (S gene}. Additional relatively rare genes (J and K) have been described recently [l]. Assays of CHE activity and methods for CHE phenotyping Kalow and Genest first defined the standard test distinguishing the common CHE variants, using benzoylcholine as substrate [2]. They further noted that 10e5 M dibucaine inhibits the ‘usual’ enzyme by about 80% but the ‘atypical’ enzyme only by about 20%. Percentual inhibition by dibucaine under standard conditions was termed the Dibucaine Number. However, this classic method creates several problems. First, the serum sample must be diluted. Second, although the absorption of the substrate (benzoylcholine) and its hydrolysis product differs most at 235 nm, because of high absorbancy of serum at this wavelength, measurement should be made at 240 nm on the steep portion of the substrate’s absorption curve, with a large decrease in sensitivity. Third, this method is unsuitable for mechanization. Thus, for long, the routinely used assays were calorimetric, such as the procedure of Ellman et al. [3], which measures the sulphydryl derivative of choline liberated by the CHE from acylthiocholines, in conjunction with differential inhibitors to dist~g~sh the variants. Acetyl-, butyryl-, and propionylt~~ho~ne have been used as substrates. Although propionylthiocholine was recommended in a proposed ‘selected’ method [4], some investigator has found that this fails to match the reproducibility of using butyrylthiocholine, a widely used substrate [5]. The method is gaining favour for monitoring enzymic activity to assess an in~~du~s susceptibility to suxamethonium sensitivity 161.In the original work of Dietz et al. [4] the diagnostic efficiency was about 98%; using this as screening method, a CHE activity greater than 2.5 SD below mean activity for ‘usual’ enzyme makes the existence of an abnormal phenotype unlike 161. In 1984, Panteghini and Bonora f7] developed a reliable routine assay amenable to mechanization, using benzoylcholine as substrate and the enzymatic assay of Okabe et al. [8]. In this procedure the choline released from benzoylcholine is monitored at 500 nm by choline-oxidase coupled with a peroxidase detection system 191. This approach overcomes the difficulties of monitoring be~oylcho~e at 240 nm, is more precise (between-day analysis gives CV between 3.5 and 5.6%), and matches the obtained by dibucaine inhibition results with those obtained by the
TABLE I Reference ranges for selected cholinesterase phenotypes using the method in ref. [7]. Phenotype
No. of cases
Cholinesterase activity (U/l)
Dibucaine no. (W)
uu UA AA
370 215 24
2 117 (1754-3 883) 1340 (190-l 920) 625 (370-1600)
80 (76-84) 66 (45-73) 26 (17-35)
89
method of Kalow (Table I). Inclusion of other inhibitors (e.g. fluoride or Ro 02-0683) may resolve the rarer variants [lo]. The alternative use of p-hydroxybenzoylcholine as substrate, in which NADPH is consumed via ~-hy~oxybe~oate hydroxylase, has been evaluated in the detection of genetic CHE variants Ill]. Precision of this method is very satisfactory (between-day CV < 2.7%), but the curves of inhibition of ‘usual’ and ‘atypical’ CHE forms by various concentrations of dibucaine show that its dis~~~nation of ‘atypical’ phenot~es is below the benzoylcholine method [ 111. The latter allows convenient determination of CHE phenotypes with inhibitors, but this substrate is not recommended for definitive CHE assays to predict suc~nylcho~ne sensitivity [6]. Recently, succinylcholine and its analogue succinyldit~~ho~ne have been proposed as substrates to assess succinylcholine sensitivity by measurement of total enzymic activity. Succinyldithiocholine is used in the Ellman reaction [12], whereas with succinylcholine the liberated choline is assayed by coupled reactions involving choline-oxidase and peroxidase [13,14]. Both procedures appear to be precise (between-day CV between 1.3 and 3.7%) and amenable to mechanization, permitting routine use in any laboratory. Preliminary results show that the phenotypes UU, UA, and AA can usually be separated by these procedures,
1
2
3
4
5
6
7
8
0
10
Propionyldithiocholinr activity lkU/l) Fig. 1. Comparison between serum cholinesterase activity obtained using the method in ref. 12 (y-axis) and the method in ref. 4, assumed as reference procedure (x-axis). Triangle: ‘usual’ (UU) enzyme (n = 46); circle: ‘ heterozygous’ (UA) enzyme (n = 56); square: ‘atypical’ (AA) enzyme (n = 7). Circled symbols indicate patients treated with succinylchotine and who developed apnea; dashed lines indicate 2.5 SD below the mean activity for ‘usual’ enzyme, tentativeiy chosen as dividing lines between the cholinesterase activities of sensitive and nonsensitive individuals.
90
with a good correlation with the propionylthiocholine method (Fig. l), but it is difficult to define activities at which succinylcholine sensitivity is substantially increased [15]. Still, these methods may increase diagnostic discrimination and even predict duration of succinylcholine-induced apnea. Clearly clinical validation remains as the crucial step of method development. Acknowledgements
Partially supported by Poli Industria Sclavo S.p.A., Siena, Italy.
Chimica S.p.A., Milano, Italy, and by
References 1 Evans RT. Cholinesterase. phenotyping: clinical aspects and laboratory applications. CRC Crit Rev Clin Lab Sci 1985;23:35-64. 2 Kalow W, Genest K. A method for the detection of atypical forms of human serum cholinesterase. Determination of dibucaine numbers. Can J Biochem Physiol 1957;35:339-346. 3 Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95. 4 Dietz AA, Rubinstein HM, Lubrano T. Calorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholine-ditbiobis(nitrobenzoic acid) procedure. Clin Chem 1973;19:1309-1313. 5 Whittaker M, Britten JJ, Dawson PJG. Comparison of a commercially available assay system with two reference methods for the determination of plasma cholinesterase variants. Clin Chem 1983;29:1746-1751. 6 Evans RT, Wroe J. Is serum cholinesterase activity a predictor of succinyl choline sensitivity? An assessment of four methods. Clin Chem 1978;24:1762-1766. 7 Panteghini M, Bonora R. Evaluation of a new continuous calorimetric method for determination of serum pseudocholinesterase catalytic activity and its application to a centrifugal fast analyser. J Clin Chem Clin Biochem 1984;22:671-676. 8 Okabe H, Sagesaka K, Nakajima N, Noma A. New enzymatic assay of cholinesterase activity. Clin Chim Acta 1977;80:87-94. 9 Artiss JD, McGowan MW, Strandbergh DR, Zak B. A procedure for the kinetic calorimetric determination of serum cholinesterase activity. Clin Chim Acta 1982;124:141-148. 10 Evans RT, Wardell J. On the identification and frequency of the J and K cholinesterase phenotypes in a Caucasian population. J Med Genet 1984;21:99-102. 11 Panteghini M, Bonora R, Pagani F. Evaluation of a new method for cholinesterase determination. Clin Biochem 1986;19:161-165. 12 Panteghini M, Bonora R, Pagani F. An alternative approach to the prevention of succinyldicholineinduced apnoea. J Clin Chem Clin Biochem 1988;26:85-90. 13 Abemethy MH, George PM, Melton VE. A new succinylcholine-based assay of plasma cholinesterase. Clin Chem 1984;30:192-195. 14 George PM, Joyce SL, Abemethy MH. Screening for plasma cholinesterase deficiency: an automated succinylcholine based assay. Clin Biochem 1988;21:159-162. 15 Faye S, Evans RT. Is succinyldicholme the substrate of choice for the measurement of cholinesterase activity? Clin Chem 1986;32:1477-1480.
Clinica Chimica Acta, 183 (1989) 87-90 Elsevier
CCA 04479
Short Communication
Methods for serum cholinesterase assay and classification of genetic variants Mauro Panteghini 1’ Laboratorio Analisi Chimico-Cliniche, Spedah Civili, Brescia (Italy) (Received 24 August 1988; revision received 15 December 1988; accepted 31 December 1988)
Key words: Serum cholinesterase; Acetylcholinesterase
Introduction
Two cholinesterases are found in man: the one in red cells, nerve endings, lung, spleen, and grey matter of the brain is commonly called acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, EC 3.1.1.7), the other in serum, liver, pancreas, heart, and white matter, is called serum cholinesterase (CHE, acylcholine acylhydrolase, EC 3.1.1.8). Only the serum enzyme is currently clinically relevant, and used as a test of hepato-cellular function, to confirm acute organophosphate insecticide poisoning, and to detect increased sensitivity to the muscle relaxant succinylcholine (suxamethonium). Sensitivity to succinylcholine
The depolarizing neuromuscular blocking agent succinylcholine is usually hydrolyzed in plasma in a few minutes by CHE. This rapid hydrolysis is responsible for this muscle relaxant’s conveniently short action, but some patients cannot hydrolyze succinylcholine rapidly and so a ‘normal’ dose of succinylcholine (1 mg/kg i.v.) may cause apnea for several hours following cessation of anesthesia. In other words, CHE from these individuals have a lower affinity for the choline ester substrates than has the enzyme from normals. The enzyme variants in subjects with increased succinylcholine sensitivity have lower total activity and resist inhibition by the local anesthetic dibucaine or fluoride, or both. Four main allelic genes at the locus El are the determinants for the principal clinical variants of CHE: the normal gene (U = usual), the atypical gene (A = atypical), the fluoride-resistant gene (F = fluoride-resistant), and the silent gene (S = silent). The first is greatly inhibited by dibucaine and fluoride, while the second is resistant to such inhibition. The variant associated with the F gene is Correspondence to: Dr. M. Panteghini, lo Laboratorio Analisi Chirnico-Cliniche, SpedaIi Civili, 25123 Brescia, Italy.
OOOS-8981/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
88
resistant to inhibition by fluoride but not to dibucaine. Finally, the gene may also not express itself (S gene}. Additional relatively rare genes (J and K) have been described recently [l]. Assays of CHE activity and methods for CHE phenotyping Kalow and Genest first defined the standard test distinguishing the common CHE variants, using benzoylcholine as substrate [2]. They further noted that 10e5 M dibucaine inhibits the ‘usual’ enzyme by about 80% but the ‘atypical’ enzyme only by about 20%. Percentual inhibition by dibucaine under standard conditions was termed the Dibucaine Number. However, this classic method creates several problems. First, the serum sample must be diluted. Second, although the absorption of the substrate (benzoylcholine) and its hydrolysis product differs most at 235 nm, because of high absorbancy of serum at this wavelength, measurement should be made at 240 nm on the steep portion of the substrate’s absorption curve, with a large decrease in sensitivity. Third, this method is unsuitable for mechanization. Thus, for long, the routinely used assays were calorimetric, such as the procedure of Ellman et al. [3], which measures the sulphydryl derivative of choline liberated by the CHE from acylthiocholines, in conjunction with differential inhibitors to dist~g~sh the variants. Acetyl-, butyryl-, and propionylt~~ho~ne have been used as substrates. Although propionylthiocholine was recommended in a proposed ‘selected’ method [4], some investigator has found that this fails to match the reproducibility of using butyrylthiocholine, a widely used substrate [5]. The method is gaining favour for monitoring enzymic activity to assess an in~~du~s susceptibility to suxamethonium sensitivity 161.In the original work of Dietz et al. [4] the diagnostic efficiency was about 98%; using this as screening method, a CHE activity greater than 2.5 SD below mean activity for ‘usual’ enzyme makes the existence of an abnormal phenotype unlike 161. In 1984, Panteghini and Bonora f7] developed a reliable routine assay amenable to mechanization, using benzoylcholine as substrate and the enzymatic assay of Okabe et al. [8]. In this procedure the choline released from benzoylcholine is monitored at 500 nm by choline-oxidase coupled with a peroxidase detection system 191. This approach overcomes the difficulties of monitoring be~oylcho~e at 240 nm, is more precise (between-day analysis gives CV between 3.5 and 5.6%), and matches the obtained by dibucaine inhibition results with those obtained by the
TABLE I Reference ranges for selected cholinesterase phenotypes using the method in ref. [7]. Phenotype
No. of cases
Cholinesterase activity (U/l)
Dibucaine no. (W)
uu UA AA
370 215 24
2 117 (1754-3 883) 1340 (190-l 920) 625 (370-1600)
80 (76-84) 66 (45-73) 26 (17-35)
89
method of Kalow (Table I). Inclusion of other inhibitors (e.g. fluoride or Ro 02-0683) may resolve the rarer variants [lo]. The alternative use of p-hydroxybenzoylcholine as substrate, in which NADPH is consumed via ~-hy~oxybe~oate hydroxylase, has been evaluated in the detection of genetic CHE variants Ill]. Precision of this method is very satisfactory (between-day CV < 2.7%), but the curves of inhibition of ‘usual’ and ‘atypical’ CHE forms by various concentrations of dibucaine show that its dis~~~nation of ‘atypical’ phenot~es is below the benzoylcholine method [ 111. The latter allows convenient determination of CHE phenotypes with inhibitors, but this substrate is not recommended for definitive CHE assays to predict suc~nylcho~ne sensitivity [6]. Recently, succinylcholine and its analogue succinyldit~~ho~ne have been proposed as substrates to assess succinylcholine sensitivity by measurement of total enzymic activity. Succinyldithiocholine is used in the Ellman reaction [12], whereas with succinylcholine the liberated choline is assayed by coupled reactions involving choline-oxidase and peroxidase [13,14]. Both procedures appear to be precise (between-day CV between 1.3 and 3.7%) and amenable to mechanization, permitting routine use in any laboratory. Preliminary results show that the phenotypes UU, UA, and AA can usually be separated by these procedures,
1
2
3
4
5
6
7
8
0
10
Propionyldithiocholinr activity lkU/l) Fig. 1. Comparison between serum cholinesterase activity obtained using the method in ref. 12 (y-axis) and the method in ref. 4, assumed as reference procedure (x-axis). Triangle: ‘usual’ (UU) enzyme (n = 46); circle: ‘ heterozygous’ (UA) enzyme (n = 56); square: ‘atypical’ (AA) enzyme (n = 7). Circled symbols indicate patients treated with succinylchotine and who developed apnea; dashed lines indicate 2.5 SD below the mean activity for ‘usual’ enzyme, tentativeiy chosen as dividing lines between the cholinesterase activities of sensitive and nonsensitive individuals.
90
with a good correlation with the propionylthiocholine method (Fig. l), but it is difficult to define activities at which succinylcholine sensitivity is substantially increased [15]. Still, these methods may increase diagnostic discrimination and even predict duration of succinylcholine-induced apnea. Clearly clinical validation remains as the crucial step of method development. Acknowledgements
Partially supported by Poli Industria Sclavo S.p.A., Siena, Italy.
Chimica S.p.A., Milano, Italy, and by
References 1 Evans RT. Cholinesterase. phenotyping: clinical aspects and laboratory applications. CRC Crit Rev Clin Lab Sci 1985;23:35-64. 2 Kalow W, Genest K. A method for the detection of atypical forms of human serum cholinesterase. Determination of dibucaine numbers. Can J Biochem Physiol 1957;35:339-346. 3 Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95. 4 Dietz AA, Rubinstein HM, Lubrano T. Calorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholine-ditbiobis(nitrobenzoic acid) procedure. Clin Chem 1973;19:1309-1313. 5 Whittaker M, Britten JJ, Dawson PJG. Comparison of a commercially available assay system with two reference methods for the determination of plasma cholinesterase variants. Clin Chem 1983;29:1746-1751. 6 Evans RT, Wroe J. Is serum cholinesterase activity a predictor of succinyl choline sensitivity? An assessment of four methods. Clin Chem 1978;24:1762-1766. 7 Panteghini M, Bonora R. Evaluation of a new continuous calorimetric method for determination of serum pseudocholinesterase catalytic activity and its application to a centrifugal fast analyser. J Clin Chem Clin Biochem 1984;22:671-676. 8 Okabe H, Sagesaka K, Nakajima N, Noma A. New enzymatic assay of cholinesterase activity. Clin Chim Acta 1977;80:87-94. 9 Artiss JD, McGowan MW, Strandbergh DR, Zak B. A procedure for the kinetic calorimetric determination of serum cholinesterase activity. Clin Chim Acta 1982;124:141-148. 10 Evans RT, Wardell J. On the identification and frequency of the J and K cholinesterase phenotypes in a Caucasian population. J Med Genet 1984;21:99-102. 11 Panteghini M, Bonora R, Pagani F. Evaluation of a new method for cholinesterase determination. Clin Biochem 1986;19:161-165. 12 Panteghini M, Bonora R, Pagani F. An alternative approach to the prevention of succinyldicholineinduced apnoea. J Clin Chem Clin Biochem 1988;26:85-90. 13 Abemethy MH, George PM, Melton VE. A new succinylcholine-based assay of plasma cholinesterase. Clin Chem 1984;30:192-195. 14 George PM, Joyce SL, Abemethy MH. Screening for plasma cholinesterase deficiency: an automated succinylcholine based assay. Clin Biochem 1988;21:159-162. 15 Faye S, Evans RT. Is succinyldicholme the substrate of choice for the measurement of cholinesterase activity? Clin Chem 1986;32:1477-1480.