Manual colorimetric methods for pseudocholinesterase and red cell (true) cholinesterase.

exist for cholinesterase assay, but each has drawbacks (1, 2). This paper presents an improved version of Garry and Routh's modification (3) of Ellman's method, as well as variations of this method for the assay of " true " cholinesterase in red cells and for the determination of the dibucaine number. The basic reactions are: Milwaukee, Wis. 53233), 2.0 mg/mI in 0.2 molar potassium phosphate buffer, pH 8.0. This solution is stable 1 week at 2-8#{176}C. Enzyme inhibitor. Quinidine sulfate, 0.03 g/ 100 ml, in water. Red cell cholinesterase inhibitor. Caffeine, 2 g/ 100 ml, in water. mg/ml in 0.2 molar potassium phosphate buffer at pH 8.0. This solution is stable for one week, stored frozen. Color reagent. DTNB3 Prepare buffer-color-substrate reagent by adding 1.0 ml of substrate to 5.0 ml of color reagent. Into each of two test tubes, labeled " Unknown " and " Blank, " pipet 0.6 ml of the combined buffer-color-substrate reagent. Incubate the tubes at 37#{176}C and add 100 Ml of a 10-fold dilution of unhemolyzed serum to the Unknown tube. Alternatively, 10 Mlof undiluted serum may be used. After a 10-mm incubation, the enzyme reaction is inhibited by adding 10 ml of quinidine sulfate. The Unknown tube is read in a 19-mm cuvet at 450 nm against the Blank set for 0 absorbance. Units are read from the standard curve. This report describes both an improved version of Garry and Routh's color-imetric assay for serum pseudocholinesterase [CLIN. CHEM. 11, 91 (1965)] and a variation of this method for use in measuring cholinesterase in red cells. These procedures require either 0.1 ml of serum (diluted 10-fold) cr 0.1 ml of packed, heparinized erythrocytes (diluted 100.fold). The reaction involves the hydrolysis of acetylthiocholine to acetic acid and thiocholine, and the quantitation of the thiocholine with 5,5'-dithiobis-2-nitrobenzoic acid. In the procedure, the sample of diluted serum or cells is added to the combined buffer-color-substrate reagent at 37#{176}C. After incubation for 10 mm, quinidine sulfate is added to retard the reaction and the color is read at 450 nm. Blanks are required with each red cell sample, but not for sera. The results correlate well with those obtained by Michel's standard pH assay, Kalow's benzoylchol me uv-rate reaction, Caraway's colorimetric procedure, and ElIman's colorimetric rate reaction. A method for determination of dibucaine-inhibited serum pseudocholinesterase is also described.