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Four methods for measuring serum cholinesterase activity have been applied to sera of normal individuals and of patients shown to be sensitive to short-acting muscle relaxants of the succinyldicholine type. They have been assessed according to their ability to differentiate between sensitive and insensitive individuals on the basis of enzyme activity measurements alone. The method described, based upon that of Dietz et al. [Clin. Chem. 19, 1309 (1973)], in which propionylthiocholine is used as substrate, is best for this purpose, being capable of identifying over 90% of affected individuals with no false positives. Acetylcholine and butyrylthiocholine are slightly inferior substrates in this respect, and benzoylcholine gives little useful information.

For assaying plasma cholinesterase (EC 3.1.1.8) activity and phenotyping by means of dibucaine inhibition, we have compared a commercially available kit, in which butyrylthiocholine is used as substrate, with two reference methods, one using benzoylcholine and the other propionylthiocholine. With 50 different samples of three of the most common genetic variants, we could clearly differentiate the variants with benzoylcholine and dibucaine, whereas there was some overlap of the E1uE1u and E1uE1a phenotypes with the other two substrates at 30 degrees C. The phenotypes were better differentiated at 25 degrees C, and in our hands the use of butyrylthiocholine was preferable to propionylthiocholine for phenotyping with dibucaine. The affinity of the usual and atypical homozygotes for fluoride with butyrylthiocholine gave an inverted response to the affinity of these variants for the anion with benzoylcholine. We suggest that this may be explained by the role of the chromogen or its products in the assay procedure with the thiocholine substrate.

A simple ultraviolet method has been developed for determining serum or plasma cholinesterase activity. The cholinesterase acts on acetyl- or butyrylthiocholine to release thiocholine, which reacts with 2,2'- or 4,4'-dithiodipyridine, showing absorbance at 324 or 343 nm, respectively. The activity can be measured kinetically and also after incubation for 3 to 5 min at 20° to 37°C. A volume of 10 µl of serum or plasma is sufficient for the determination. This method is well suited for clinical investigation as well as experimental studies.

A simplified manual and automated method is described for measuring serum cholinesterase activity and identifying the homozygous "usual," heterozygous, and homozygous "atypical" forms of the enzyme. With butyrylthiocholine as substrate at pH 7.4, the activity is determined in two 0.05 mol/liter buffer systems, Tris and phosphate. The "usual" enzyme has the same activity in both buffer systems. The phosphate buffer inhibits the heterozygous enzyme about 13% and the "atypical" enzyme about 43% relative to its activity in Tris buffer. The range of normal serum cholinesterase values (measurements on plasma from 1842 preschool children) is 2.10 to 5.25 U (mean, 3.67 U). In this population, 3.28% of the Caucasians, but only 0.29% of the Negroes, were heterozygous for the trait.

We describe a useful technique to identify "fluoride-resistant" serum cholinesterase variants (E1uE1f and E1fE1f) by using the method of Garry to detect "atypical" variants and to measure serum cholinesterase activity [Clin. Chem. 17, 192 (1971)]. With butyrylthiocholine as substrate, sodium fluoride is used as a differential inhibitor in both Tris and phosphate buffer systems (50 mmol/liter, pH 7.4, 25°C). Fluoride inhibition values are plotted on two-dimensional graph paper, Tris plus sodium fluoride vs. phosphate plus sodium fluoride. Inhibition values for the "fluoride-resistant" and "atypical" variants are located in specific quadrants, which permits unequivocal identification of each variant. Examination of 836 Caucasian preschool children revealed a gene frequency of 0.0066 for the "fluoride" variant. No "fluoride-resistant" variants were found in 168 Negro preschool children examined.

Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

Four methods for measuring serum cholinesterase activity have been applied to sera of normal individuals and of patients shown to be sensitive to short-acting muscle relaxants of the succinyldicholine type. They have been assessed according to their ability to differentiate between sensitive and insensitive individuals on the basis of enzyme activity measurements alone. The method described, based upon that of Dietz et al. [Clin. Chem. 19, 1309 (1973)], in which propionylthiocholine is used as substrate, is best for this purpose, being capable of identifying over 90% of affected individuals with no false positives. Acetylcholine and butyrylthiocholine are slightly inferior substrates in this respect, and benzoylcholine gives little useful information.

The technical and clinical usefulness of three substrates for serum cholinesterase were compared. The diagnostic value of acetylthiocholine was equal to that of butyrylthiocholine and superior to that of o-nitrophenylbutyrate. Technically, butyrylthiocholine is superior to the other two substrates chiefly because of its greater affinity for serum cholinesterase. A kinetic method using o-nitrophenylbutyrate is described that is capable of carrying out 60 tests per hour with good results.