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A simple staining method to detect serum cholinesterase activity
Clinica Chimica Ada, Elsevier
164 (1987) 241,242
241
CCA 03765
Letter
to the editor
A simple staining method to detect serum cholinesterase activity * Dear Editor, Serum cholinesterase (ChE; EC 3.1.1.8) isoenzyme patterns have been actively analysed for genetic variants [l-5]. Here we report a rapid method for the precise and reproducible staining of ChE in serum. Our staining method was as follows: PTCI (propionylthiocholine iodide, Sigma Chemical Co., St. Louis, MO, USA.) was hydrolysed by ChE, and liberated thiocholine directly coupled to MTT (thiazolyl blue (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide, Sigma) producing a reduced form MTT (formazan). Since the products are insoluble in water, they precipitate from the staining solution and the reaction proceeded in an irreversible direction. ChE was detected by the formazan form from a sequence of reactions coupled to ChE activity. We used electrophoresis on a cellulose acetate membrane (Cellogel, Chemetron Co., Ltd., Milan, Italy) in order to select our staining conditions for ChE. We applied 2 pl of human serum to the Cellogel membrane, carried out electrophoresis for one hour at 1 mA/cm and stained the electrophoresed membrane for 10 min at 37OC. After we decoloured the stained membrane using 2% acetic acid, we found the integral value of absorbance of the stained band with a Cliniscan reflectance densitometer (Helena Laboratory, Japan) at 570 nm. The staining solution contained PTCI, MTT 1 mg/ml in a 25 mmol/l sodium potassium phosphate buffer, pH 7.6 [6,7]. We stained human serum sample containing normal ChE activity with solution containing from 2.5 mmol/l to 20 mmol/l (2.5, 5.0, 10.0, 20.0) of PTCI. Finally, we selected 10 mmol/l of PTCI as the substrate concentration. When PTCI was removed from the staining solution, no band was stained. By using the staining solution selected above, we obtained the following results. Linearity was well maintained. The precision (within-run and between-run; n = 10 each) were all within 6%. Analytical recovery was satisfactory by adding two known amounts of lyophilised human ChE (Sigma) to a human serum sample. Our staining method for ChE activity analysis is precise, accurate, simple to use and equal the previous method [l-5] in the other aspects. After electrophoresis using 4-30% polyacrylamide gradient gel (PAA 4/30, Pharmacia Fine Chemicals Co., Uppsala, Sweden) as a supporting media [8], the gel can be stained by our
* This work was supported Japan
in part a grant in aid of Scientific
Research
from the Ministry
(No. 59570998).
0009-8981/87/$03.50
0 1987 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Education,
242
(+I
Fig. C,, C,( was
t-1
1. Polyacrylamide gradient gel electrophoresis. Two human sera were electrophoresed, and C,, C,, and C, which were detected in most of population, and C, which were detected only in people with +) variant by Harris et al [l], were stained by our ChE staining method. Control without Cs band shown in lane 1 and C, variant was shown in lane 2.
method (Fig. 1). Our method may be used to analyse ChE isoenzymes to detect genetic variants and various diseases. Masato Maekawa +*, Kayoko Sudo b and Takashi Kanno a a Department of Laboratory Medicine, Hamnmatsu University, School of Medicine, Hamamatsu City, and ’ Department of L.uboratoty Medicine, Jikei University, School of Medicine. Tokyo (Japan)
References 1 Harris H, Hopkinson DA, Robson EB. Two-dimensional electrophoresis of pseudocholinesterase components in normal human serum. Nature (London) 1962;196:1296-1298. 2 Bemsohn J, Barron KD, Hess A. Cholinesterases in serum as demonstrated by starch gel electrophoresis. Proc Sot Exp Biol Med 1961;108:71-73. 3 Juul P. Human plasma cholinesterase isoenzymes. CIin Chim Acta 1968;19:205-213. 4 Yoshida A, Motulsky AC. A pseudocholinesterase variant (E Cynthiana) associated with elevated plasma enzyme activity. Am J Hum Genet 1969;21:486-498. 5 LaMotta RV, Woronick CL. Molecular heterogeneity of human serum cholinesterase. Clin Chem 1971;17:135-144. 6 Dietz AA, Rubinstein HM, Lubrano T, Carry PJ, Medera-Orsini F, Strever BC. Calorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholine-dithiobis (nitrobenzoic acid) procedure. In: Cooper CR, and King JS, eds. Selected methods for the small clinical chemistry laboratory, Vol. 8. Washington, DC: American Association for Clinical Chemistry, 1977;41-46. 7 Maekawa M, Sudo K, Kanno T. Immunochemical approach to the question of inactive subunits in cases of lactate dehydrogenase B subunit deficiency. CIin Chem 1986;32:116-119. 8 Maekawa M, Sudo K, Kanno T. Characteristics of the complex between alkaline phosphatase and immunoglobulin A in human serum. Clin Chim Acta 1985;150:185-195.
* Correspondence to: M. Maekawa, Department School of Medicine, Handa-cho 3600, Hamamatsu
of Laboratory Medicine, City, 431-31, Japan.
Hamamatsu
University,
164 (1987) 241,242
241
CCA 03765
Letter
to the editor
A simple staining method to detect serum cholinesterase activity * Dear Editor, Serum cholinesterase (ChE; EC 3.1.1.8) isoenzyme patterns have been actively analysed for genetic variants [l-5]. Here we report a rapid method for the precise and reproducible staining of ChE in serum. Our staining method was as follows: PTCI (propionylthiocholine iodide, Sigma Chemical Co., St. Louis, MO, USA.) was hydrolysed by ChE, and liberated thiocholine directly coupled to MTT (thiazolyl blue (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide, Sigma) producing a reduced form MTT (formazan). Since the products are insoluble in water, they precipitate from the staining solution and the reaction proceeded in an irreversible direction. ChE was detected by the formazan form from a sequence of reactions coupled to ChE activity. We used electrophoresis on a cellulose acetate membrane (Cellogel, Chemetron Co., Ltd., Milan, Italy) in order to select our staining conditions for ChE. We applied 2 pl of human serum to the Cellogel membrane, carried out electrophoresis for one hour at 1 mA/cm and stained the electrophoresed membrane for 10 min at 37OC. After we decoloured the stained membrane using 2% acetic acid, we found the integral value of absorbance of the stained band with a Cliniscan reflectance densitometer (Helena Laboratory, Japan) at 570 nm. The staining solution contained PTCI, MTT 1 mg/ml in a 25 mmol/l sodium potassium phosphate buffer, pH 7.6 [6,7]. We stained human serum sample containing normal ChE activity with solution containing from 2.5 mmol/l to 20 mmol/l (2.5, 5.0, 10.0, 20.0) of PTCI. Finally, we selected 10 mmol/l of PTCI as the substrate concentration. When PTCI was removed from the staining solution, no band was stained. By using the staining solution selected above, we obtained the following results. Linearity was well maintained. The precision (within-run and between-run; n = 10 each) were all within 6%. Analytical recovery was satisfactory by adding two known amounts of lyophilised human ChE (Sigma) to a human serum sample. Our staining method for ChE activity analysis is precise, accurate, simple to use and equal the previous method [l-5] in the other aspects. After electrophoresis using 4-30% polyacrylamide gradient gel (PAA 4/30, Pharmacia Fine Chemicals Co., Uppsala, Sweden) as a supporting media [8], the gel can be stained by our
* This work was supported Japan
in part a grant in aid of Scientific
Research
from the Ministry
(No. 59570998).
0009-8981/87/$03.50
0 1987 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Education,
242
(+I
Fig. C,, C,( was
t-1
1. Polyacrylamide gradient gel electrophoresis. Two human sera were electrophoresed, and C,, C,, and C, which were detected in most of population, and C, which were detected only in people with +) variant by Harris et al [l], were stained by our ChE staining method. Control without Cs band shown in lane 1 and C, variant was shown in lane 2.
method (Fig. 1). Our method may be used to analyse ChE isoenzymes to detect genetic variants and various diseases. Masato Maekawa +*, Kayoko Sudo b and Takashi Kanno a a Department of Laboratory Medicine, Hamnmatsu University, School of Medicine, Hamamatsu City, and ’ Department of L.uboratoty Medicine, Jikei University, School of Medicine. Tokyo (Japan)
References 1 Harris H, Hopkinson DA, Robson EB. Two-dimensional electrophoresis of pseudocholinesterase components in normal human serum. Nature (London) 1962;196:1296-1298. 2 Bemsohn J, Barron KD, Hess A. Cholinesterases in serum as demonstrated by starch gel electrophoresis. Proc Sot Exp Biol Med 1961;108:71-73. 3 Juul P. Human plasma cholinesterase isoenzymes. CIin Chim Acta 1968;19:205-213. 4 Yoshida A, Motulsky AC. A pseudocholinesterase variant (E Cynthiana) associated with elevated plasma enzyme activity. Am J Hum Genet 1969;21:486-498. 5 LaMotta RV, Woronick CL. Molecular heterogeneity of human serum cholinesterase. Clin Chem 1971;17:135-144. 6 Dietz AA, Rubinstein HM, Lubrano T, Carry PJ, Medera-Orsini F, Strever BC. Calorimetric determination of serum cholinesterase and its genetic variants by the propionylthiocholine-dithiobis (nitrobenzoic acid) procedure. In: Cooper CR, and King JS, eds. Selected methods for the small clinical chemistry laboratory, Vol. 8. Washington, DC: American Association for Clinical Chemistry, 1977;41-46. 7 Maekawa M, Sudo K, Kanno T. Immunochemical approach to the question of inactive subunits in cases of lactate dehydrogenase B subunit deficiency. CIin Chem 1986;32:116-119. 8 Maekawa M, Sudo K, Kanno T. Characteristics of the complex between alkaline phosphatase and immunoglobulin A in human serum. Clin Chim Acta 1985;150:185-195.
* Correspondence to: M. Maekawa, Department School of Medicine, Handa-cho 3600, Hamamatsu
of Laboratory Medicine, City, 431-31, Japan.
Hamamatsu
University,