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Pesticide Poisoning, Succinylcholine-Induced Apnea, and Pseudocholinesterase
Laboratory Medicine Pesticide Poisoning, Succinylcholine-Induced Apnea, and Pseudocholinesterase Acetylcholinesterase is found in erythrocytes and in many other tissues, such as the lung, spleen, nerve endings, and gray matter of the brain. By hydrolyzing acetylcholine released from nerve endings, it regulates nervous transmission be tween preganglionic fibers and autonomic gan glia, postganglionic cholinergic nerves and invol untary muscle, and motor nerves and voluntary muscle and in some structures of the central ner vous system. Pseudocholinesterase is in the blood plasma, liver, pancreas, heart, and white matter of the brain. The biologic function of pseudocholines terase is unknown. It is synthesized by the liver. The serum pseudocholinesterase activity is de creased in patients with liver disease. Assay of pseudocholinesterase is useful in the diagnosis of pesticide poisoning and in the assessment of pa tients with prolonged apnea after administration of succinylcholine during anesthesia. The assay is also used to monitor liver function after liver transplantation. 1 Pesticide Poisoning.—Both acetylcholines terase and pseudocholinesterase are inhibited by organophosphates and carbamates. Workers in agriculture or organic chemical industries can be exposed by inhalation or by direct contact. Acute organophosphate poisoning causes blurred vi sion, dizziness, weakness, disorientation, head ache, nausea, vomiting, and muscle and abdomi nal cramps. Chronic subclinical exposure may result in sensory and motor peripheral neu ropathy, manifested by generalized weakness and ataxia. In acute poisoning, the pseudocholinester ase and acetylcholinesterase activities are 30 to 50% of normal by the time symptoms appear. 2,3 The normal ranges for pseudocholinesterase and acetylcholinesterase activity, however, are wide. Address reprint requests to Dr. M. F. Burritt, Section of Clini cal Chemistry, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 61:750-755, 1986
Therefore, patients may lose half their cholinesterase activity and still have values within the normal range. Consequently, baseline values for cholinesterase activities should be determined in all workers who have a high risk of exposure to organophosphates or carbamates. A decline of 30 to 50% from baseline cholinesterase activities indicates toxicity even if the activity is in the normal range. For monitoring exposure, assay of erythrocyte acetylcholinesterase is preferred to assay of serum pseudocholinesterase because the erythrocyte cholinesterase activity better reflects cholinesterase levels in nerve tissue. 3 If no baseline values are available and a low-normal cholinesterase activity is detected, organophosphate poisoning can be diagnosed retrospectively if serial measurements show an increase in pseudocholinesterase or acetylcholin esterase after the presumed exposure. The serum pseudocholinesterase will return to a baseline pla teau activity in 4 to 6 weeks, and the erythrocyte cholinesterase will return to a baseline plateau in 5 to 7 weeks. 2,3 Assessment of mild toxicity and recovery cannot be based on only one or two mea surements because of the normal biologic vari ability of pseudocholinesterase and erythrocyte cholinesterase activities—±20% and ±10%, re spectively.2,3 The measurements of cholinesterase activity after exposure are also used to determine when the patient can return to work. A low value for pseudocholinesterase activity does not always indicate pesticide poisoning. Low pseudocholinesterase activities are also present in patients with acute hepatitis, cirrhosis, advanced carcinoma, pregnancy, myocardial infarction, pul monary embolism, or acute infections or those who have undergone surgical procedures. 4 In addition, a low pseudocholinesterase activity may reflect a variant enzyme. The degree of inhibition of pseudocholinesterase by dibucaine distin guishes a low pseudocholinesterase activity due to genetic enzymopathy from that due to pesticide poisoning (see subsequent discussion). The effect of carbamates on pseudocholinester ase or acetylcholinesterase is more difficult to demonstrate. Unlike organophosphates, carba mates bind reversibly to enzymes. Therefore, the
750
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effect of carbamates is reduced by dilution during the assay. Furthermore, as carbamates bind to the assay substrate, enzyme inhibition lessens. Some degradation of carbamate may also occur before or during the assay. Thus, cholinesterase activity may be spuriously normal or only mildly reduced after severe carbamate exposure. 2 Succinylcholine-Induced Apnea.—Succinylcholine is a muscle relaxant commonly used dur ing anesthesia. It competes with acetylcholine for binding to receptors at the neuromuscular junc tion. It is hydrolyzed by plasma pseudocholines terase. (The ester type of local anesthetics, such as procaine, are also hydrolyzed by plasma pseudo cholinesterase.) The effect of succinylcholine per sists until a large proportion of the dose has been removed from the plasma. 5 When pseudocholines terase activity is normal, the action of succinyl choline is short-lived, and spontaneous res pirations return within 2 to 10 minutes after administration of the drug. In approximately 1 of 2,500 patients given succinylcholine, however, clinically significant apnea occurs and persists for minutes to several hours. 6 In a series of 225 patients who had prolonged apnea after receiving succinylcholine, 66% had an inherited abnormal pseudocholinesterase variant. 7 Another 6% had an acquired deficiency of pseudocholinesterase due to causes such as liver disease, chronic debili tating disease, or carcinoma. In 15% of the pa tients, the serum pseudocholinesterase activity was normal, and the prolonged apnea was due to other causes such as an overdose of succinylcho line, hyperventilation, or central or peripheral respiratory depression. In 13% of the patients, no reason for the prolonged apnea was established. Thus, the detection of pseudocholinesterase var iants is necessary in the evaluation of succinylcholine-induced apnea. The Genetics of Pseudocholinesterase.— Pseudocholinesterase is encoded at two autosomal gene loci, designated Ei and E2.5 Several molecu lar variants of pseudocholinesterase are due to alleles for the Ei locus. The Ei u allele codes for the common enzyme with normal activity, and the "wild type" normal homozygote may be desig nated ΕιΈι υ . The Ei a , E / , and E, s alleles code for variant forms of the enzyme. The Ei a Ei a enzyme has weak activity. The E / allele codes for vari ants that are less sensitive than the common ΕΓΕι" enzyme to fluoride inhibition. In most cases, enzymes determined by the E / allele are
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less sensitive than the common Ei u Ei u enzyme to dibucaine inhibition as well.5 The Ei s allele codes for absent activity, and the homozygote for Ei s has no serum pseudocholinesterase activity. Thus, Ei s homozygotes are especially at risk for pro longed succinylcholine-induced apnea, as in clas sic mendelian recessive inheritance. The variants most commonly encountered in association with prolonged succinylcholineinduced apnea are those that reflect genotypes Ei a Ei a and ΕΓΕΛ 7 Persons whose pseudocholines terase reflects genotype Ε 1 Έ / and ΕΓΕι δ are usually not sensitive to succinylcholine. 5,8 Fur thermore, because the genotype ETEi a is rela tively common, many cases of prolonged succinyl choline-induced apnea are associated with this genotype, despite the fact that most persons with this genotype are not overly sensitive to succinyl choline. 7 Those homozygous for Ei a or Ei s or who have the genotype Ei a Ei s are almost always overly sensitive to succinylcholine. 5,8 When a person with prolonged succinylcholineinduced apnea has one of the variant enzymes, the total serum pseudocholinesterase activity is usually low. Prolonged apnea may occur, how ever, in those persons with the Ei u Ei a and Ei a Ei a enzyme variants despite normal serum pseudo cholinesterase activity. 7 Dibucaine Inhibition.—The pseudocholines terase of Ei a Ei a genotype is more resistant to inhibition by dibucaine than is Ε 1 Έ Λ Except for variants of genotypes Ε Γ Ε Λ and rarely Ε Γ Ε / , the other abnormal enzyme variants have reduced sensitivity to dibucaine inhibition. 6,9 Therefore, the degree of dibucaine inhibition can be used to detect variant enzymes. The activity of serum pseudocholinesterase is assayed with butyrylthiocholine as the substrate in the presence and ab sence of dibucaine. Succinylcholine is not used as the substrate in the assay of pseudocholinester ase because of the technical difficulties. A "dibu caine number" can then be calculated from the following formula: 10 / 1 _ activity with dibucaine \ in„ \ activity without dibucaine / The dibucaine number represents the percentage reduction in cholinesterase activity due to addi tion of dibucaine inhibitor. Typical values for dibucaine inhibition are shown in Table l. 6 Exact genotyping often necessitates the use of an additional enzyme inhibitor (such as urea)
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Table 1.—Typical Values for Dibucaine Inhibition for Various Genotypes of Pseudocholinesterase Genotype Dibucaine inhibition (%) Ε,Έ," 78-86 Ε,Έ," 53-72 Ε,Έ," 13-27
because the dibucaine numbers of some of the enzyme variants overlap. A dibucaine number of less than 30 indicates that the enzyme variant is either Ei a Ei a or Ei a Ei s and implies a high risk for succinylcholine-induced apnea. 6 Screening for Variants.—Because succinyl choline-induced apnea is rare, the preoperative screening of all patients is unwarranted. When unexplained prolonged succinylcholine-induced apnea does occur, however, the serum pseudocho linesterase activity and the degree of dibucaine inhibition should be measured. Furthermore, if a pseudocholinesterase variant is demonstrated, close relatives of the patient should be screened for this variant so that they can be informed about the potential risk of prolonged succinyl choline-induced apnea. Thomas C. Nelson, M.D. Resident in Pathology* Mary F. Burritt, Ph.D. Section of Clinical Chemistry, Department of Laboratory Medicine
REFERENCES
1. Evans DB, Lehmann H: Pseudocholinesterase activity in liver transplantation. Lancet 1:1040-1044,1971 2. Ducatman AM, Moyer TP: The role in the clinical labora tory in the evaluation of the polygenated-polycyclic tox ins: DDT, PCBs, dibenzodioxins and dibenzofurans, chlordecone (Kepone), and hexachlorophene. In Thera peutic Drug Monitoring Continuing Education and Qual ity Control Program, Washington, DC, American Asso ciation for Clinical Chemistry, January 1983 3. Midtling JE, Barnett PG, Coye MJ, Velasco AR, Romero P, Clements CL, O'Malley MA, Tobin MW, Rose TG, Monosson IH: Clinical management of field worker organophosphate poisoning. West J Med 142:514-518, 1985 4. Moss DW, Henderson AR, Kachmar JF: Enzymes. In Textbook of Clinical Chemistry. Edited by NW Tietz. Philadelphia, WB Saunders Company, 1986, pp 746-751 5. Brown SS, Kalow W, Pilz W, Whittaker M, Woronick CL: The plasma cholinesterases: a new perspective. Adv Clin Chem 22:1-123, 1981
*Mayo Graduate School of Medicine, Rochester, Minnesota.
Mayo Clin Proc, September 1986, Vol 61
6. Viby-Mogensen J, Hanel HK: A Danish Cholinesterase Research Unit. Acta Anaesth Scand 21:405-412,1977 7. Viby-Mogensen J, Hanel HK: Prolonged apnoea after suxamethonium: an analysis of the first 225 cases re ported to the Danish Cholinesterase Research Unit. Acta Anaesth Scand 22:371-380, 1978 8. Whittaker M: Genetic aspects of succinylcholine sensitiv ity. Anesthesiology 32:143-150,1970 9. Schoeffler P, Viallard JL, Monteillard C, Canis M, Gutnecht JL, Haberer JP: Neonatal respiratory distress related to congenital pseudocholinesterase abnormality. Ann Fr Anesth Reanim 3:225-227, 1984 10. Arnold WP: Dibucaine numbers. Clin Chem News, Au gust 1983, No. 14
Serum /32-Microglobulin /52-Microglobulin (/32-M) is a small protein (molecu lar weight, 11,800) that was isolated from the urine of patients with proteinuria. It is normally present on the surface of all nucleated cells, and it forms the light chain of the human leukocyte antigen (HLA). The small size of /32-M enables it to pass through the glomerular membrane, but normally it is almost completely reabsorbed by the proximal renal tubules. An increased serum concentration of ß2-M is characteristic of several benign condi tions, such as chronic inflammation, liver disease, renal dysfunction, and some acute viral infec tions. In the nephrotic syndrome and other dis orders of renal tubular function, the /32-M concen tration is low in serum but increased in urine. The serum /32-M concentration may be increased in patients with several types of malignant le sions, especially myeloma, lymphoma, and leuke mia of B-lymphocyte lineage. In our study, the value of serum /32-M as an indicator of prognosis was assessed in 100 patients with multiple my eloma. Compared with other variables such as serum creatinine, staging, morphologic subtype, and plasma cell labeling index, serum /?2-M showed the best correlation with survival. The median survival was 12 months for those patients with myeloma who had high serum ßz-M concen trations (greater than 4 //g/ml) (4 mg/liter) and 43 months for those who had normal or low values (less than 4 /yg/ml) (4 mg/liter). The cerebrospinal fluid concentration of /32-M may also reflect the presence of a malignant lesion. The /32-M concentration in the cerebrospiAddress reprint requests to Dr. J. A. Katzmann, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN 55905.
Therefore, patients may lose half their cholinesterase activity and still have values within the normal range. Consequently, baseline values for cholinesterase activities should be determined in all workers who have a high risk of exposure to organophosphates or carbamates. A decline of 30 to 50% from baseline cholinesterase activities indicates toxicity even if the activity is in the normal range. For monitoring exposure, assay of erythrocyte acetylcholinesterase is preferred to assay of serum pseudocholinesterase because the erythrocyte cholinesterase activity better reflects cholinesterase levels in nerve tissue. 3 If no baseline values are available and a low-normal cholinesterase activity is detected, organophosphate poisoning can be diagnosed retrospectively if serial measurements show an increase in pseudocholinesterase or acetylcholin esterase after the presumed exposure. The serum pseudocholinesterase will return to a baseline pla teau activity in 4 to 6 weeks, and the erythrocyte cholinesterase will return to a baseline plateau in 5 to 7 weeks. 2,3 Assessment of mild toxicity and recovery cannot be based on only one or two mea surements because of the normal biologic vari ability of pseudocholinesterase and erythrocyte cholinesterase activities—±20% and ±10%, re spectively.2,3 The measurements of cholinesterase activity after exposure are also used to determine when the patient can return to work. A low value for pseudocholinesterase activity does not always indicate pesticide poisoning. Low pseudocholinesterase activities are also present in patients with acute hepatitis, cirrhosis, advanced carcinoma, pregnancy, myocardial infarction, pul monary embolism, or acute infections or those who have undergone surgical procedures. 4 In addition, a low pseudocholinesterase activity may reflect a variant enzyme. The degree of inhibition of pseudocholinesterase by dibucaine distin guishes a low pseudocholinesterase activity due to genetic enzymopathy from that due to pesticide poisoning (see subsequent discussion). The effect of carbamates on pseudocholinester ase or acetylcholinesterase is more difficult to demonstrate. Unlike organophosphates, carba mates bind reversibly to enzymes. Therefore, the
750
Mayo Clin Proc, September 1986, Vol 61
effect of carbamates is reduced by dilution during the assay. Furthermore, as carbamates bind to the assay substrate, enzyme inhibition lessens. Some degradation of carbamate may also occur before or during the assay. Thus, cholinesterase activity may be spuriously normal or only mildly reduced after severe carbamate exposure. 2 Succinylcholine-Induced Apnea.—Succinylcholine is a muscle relaxant commonly used dur ing anesthesia. It competes with acetylcholine for binding to receptors at the neuromuscular junc tion. It is hydrolyzed by plasma pseudocholines terase. (The ester type of local anesthetics, such as procaine, are also hydrolyzed by plasma pseudo cholinesterase.) The effect of succinylcholine per sists until a large proportion of the dose has been removed from the plasma. 5 When pseudocholines terase activity is normal, the action of succinyl choline is short-lived, and spontaneous res pirations return within 2 to 10 minutes after administration of the drug. In approximately 1 of 2,500 patients given succinylcholine, however, clinically significant apnea occurs and persists for minutes to several hours. 6 In a series of 225 patients who had prolonged apnea after receiving succinylcholine, 66% had an inherited abnormal pseudocholinesterase variant. 7 Another 6% had an acquired deficiency of pseudocholinesterase due to causes such as liver disease, chronic debili tating disease, or carcinoma. In 15% of the pa tients, the serum pseudocholinesterase activity was normal, and the prolonged apnea was due to other causes such as an overdose of succinylcho line, hyperventilation, or central or peripheral respiratory depression. In 13% of the patients, no reason for the prolonged apnea was established. Thus, the detection of pseudocholinesterase var iants is necessary in the evaluation of succinylcholine-induced apnea. The Genetics of Pseudocholinesterase.— Pseudocholinesterase is encoded at two autosomal gene loci, designated Ei and E2.5 Several molecu lar variants of pseudocholinesterase are due to alleles for the Ei locus. The Ei u allele codes for the common enzyme with normal activity, and the "wild type" normal homozygote may be desig nated ΕιΈι υ . The Ei a , E / , and E, s alleles code for variant forms of the enzyme. The Ei a Ei a enzyme has weak activity. The E / allele codes for vari ants that are less sensitive than the common ΕΓΕι" enzyme to fluoride inhibition. In most cases, enzymes determined by the E / allele are
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less sensitive than the common Ei u Ei u enzyme to dibucaine inhibition as well.5 The Ei s allele codes for absent activity, and the homozygote for Ei s has no serum pseudocholinesterase activity. Thus, Ei s homozygotes are especially at risk for pro longed succinylcholine-induced apnea, as in clas sic mendelian recessive inheritance. The variants most commonly encountered in association with prolonged succinylcholineinduced apnea are those that reflect genotypes Ei a Ei a and ΕΓΕΛ 7 Persons whose pseudocholines terase reflects genotype Ε 1 Έ / and ΕΓΕι δ are usually not sensitive to succinylcholine. 5,8 Fur thermore, because the genotype ETEi a is rela tively common, many cases of prolonged succinyl choline-induced apnea are associated with this genotype, despite the fact that most persons with this genotype are not overly sensitive to succinyl choline. 7 Those homozygous for Ei a or Ei s or who have the genotype Ei a Ei s are almost always overly sensitive to succinylcholine. 5,8 When a person with prolonged succinylcholineinduced apnea has one of the variant enzymes, the total serum pseudocholinesterase activity is usually low. Prolonged apnea may occur, how ever, in those persons with the Ei u Ei a and Ei a Ei a enzyme variants despite normal serum pseudo cholinesterase activity. 7 Dibucaine Inhibition.—The pseudocholines terase of Ei a Ei a genotype is more resistant to inhibition by dibucaine than is Ε 1 Έ Λ Except for variants of genotypes Ε Γ Ε Λ and rarely Ε Γ Ε / , the other abnormal enzyme variants have reduced sensitivity to dibucaine inhibition. 6,9 Therefore, the degree of dibucaine inhibition can be used to detect variant enzymes. The activity of serum pseudocholinesterase is assayed with butyrylthiocholine as the substrate in the presence and ab sence of dibucaine. Succinylcholine is not used as the substrate in the assay of pseudocholinester ase because of the technical difficulties. A "dibu caine number" can then be calculated from the following formula: 10 / 1 _ activity with dibucaine \ in„ \ activity without dibucaine / The dibucaine number represents the percentage reduction in cholinesterase activity due to addi tion of dibucaine inhibitor. Typical values for dibucaine inhibition are shown in Table l. 6 Exact genotyping often necessitates the use of an additional enzyme inhibitor (such as urea)
752
LABORATORY MEDICINE
Table 1.—Typical Values for Dibucaine Inhibition for Various Genotypes of Pseudocholinesterase Genotype Dibucaine inhibition (%) Ε,Έ," 78-86 Ε,Έ," 53-72 Ε,Έ," 13-27
because the dibucaine numbers of some of the enzyme variants overlap. A dibucaine number of less than 30 indicates that the enzyme variant is either Ei a Ei a or Ei a Ei s and implies a high risk for succinylcholine-induced apnea. 6 Screening for Variants.—Because succinyl choline-induced apnea is rare, the preoperative screening of all patients is unwarranted. When unexplained prolonged succinylcholine-induced apnea does occur, however, the serum pseudocho linesterase activity and the degree of dibucaine inhibition should be measured. Furthermore, if a pseudocholinesterase variant is demonstrated, close relatives of the patient should be screened for this variant so that they can be informed about the potential risk of prolonged succinyl choline-induced apnea. Thomas C. Nelson, M.D. Resident in Pathology* Mary F. Burritt, Ph.D. Section of Clinical Chemistry, Department of Laboratory Medicine
REFERENCES
1. Evans DB, Lehmann H: Pseudocholinesterase activity in liver transplantation. Lancet 1:1040-1044,1971 2. Ducatman AM, Moyer TP: The role in the clinical labora tory in the evaluation of the polygenated-polycyclic tox ins: DDT, PCBs, dibenzodioxins and dibenzofurans, chlordecone (Kepone), and hexachlorophene. In Thera peutic Drug Monitoring Continuing Education and Qual ity Control Program, Washington, DC, American Asso ciation for Clinical Chemistry, January 1983 3. Midtling JE, Barnett PG, Coye MJ, Velasco AR, Romero P, Clements CL, O'Malley MA, Tobin MW, Rose TG, Monosson IH: Clinical management of field worker organophosphate poisoning. West J Med 142:514-518, 1985 4. Moss DW, Henderson AR, Kachmar JF: Enzymes. In Textbook of Clinical Chemistry. Edited by NW Tietz. Philadelphia, WB Saunders Company, 1986, pp 746-751 5. Brown SS, Kalow W, Pilz W, Whittaker M, Woronick CL: The plasma cholinesterases: a new perspective. Adv Clin Chem 22:1-123, 1981
*Mayo Graduate School of Medicine, Rochester, Minnesota.
Mayo Clin Proc, September 1986, Vol 61
6. Viby-Mogensen J, Hanel HK: A Danish Cholinesterase Research Unit. Acta Anaesth Scand 21:405-412,1977 7. Viby-Mogensen J, Hanel HK: Prolonged apnoea after suxamethonium: an analysis of the first 225 cases re ported to the Danish Cholinesterase Research Unit. Acta Anaesth Scand 22:371-380, 1978 8. Whittaker M: Genetic aspects of succinylcholine sensitiv ity. Anesthesiology 32:143-150,1970 9. Schoeffler P, Viallard JL, Monteillard C, Canis M, Gutnecht JL, Haberer JP: Neonatal respiratory distress related to congenital pseudocholinesterase abnormality. Ann Fr Anesth Reanim 3:225-227, 1984 10. Arnold WP: Dibucaine numbers. Clin Chem News, Au gust 1983, No. 14
Serum /32-Microglobulin /52-Microglobulin (/32-M) is a small protein (molecu lar weight, 11,800) that was isolated from the urine of patients with proteinuria. It is normally present on the surface of all nucleated cells, and it forms the light chain of the human leukocyte antigen (HLA). The small size of /32-M enables it to pass through the glomerular membrane, but normally it is almost completely reabsorbed by the proximal renal tubules. An increased serum concentration of ß2-M is characteristic of several benign condi tions, such as chronic inflammation, liver disease, renal dysfunction, and some acute viral infec tions. In the nephrotic syndrome and other dis orders of renal tubular function, the /32-M concen tration is low in serum but increased in urine. The serum /32-M concentration may be increased in patients with several types of malignant le sions, especially myeloma, lymphoma, and leuke mia of B-lymphocyte lineage. In our study, the value of serum /32-M as an indicator of prognosis was assessed in 100 patients with multiple my eloma. Compared with other variables such as serum creatinine, staging, morphologic subtype, and plasma cell labeling index, serum /?2-M showed the best correlation with survival. The median survival was 12 months for those patients with myeloma who had high serum ßz-M concen trations (greater than 4 //g/ml) (4 mg/liter) and 43 months for those who had normal or low values (less than 4 /yg/ml) (4 mg/liter). The cerebrospinal fluid concentration of /32-M may also reflect the presence of a malignant lesion. The /32-M concentration in the cerebrospiAddress reprint requests to Dr. J. A. Katzmann, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN 55905.