Cholinesterase

CHOLINESTERASE
REF 1119005
1 x 55 mL
CONTENTS
R1. Reagent 2 x 25 mL
CHOLINESTERASE
TOTAL AND INHIBITED
Enzymatic colorimetric method
KINETIC
For in vitro diagnostic use only
PRINCIPLE
SAMPLES
Cholinesterase (CHE) catalyzes the hydrolysis of butyrylthiocholine

substrate forming butyrate and thiocholine. The latter reduces 5,5mercaptobis-2-nitrobenzoic acid (DMNB) to 5-mercapto-2nitrobenzoate (5-MNBA), a colored compound.
The reaction is monitored kinetically at 405 nm by the rate of
formation of the yellow color produced, proportional to the activity
of CHE in the sample1.
Serum, EDTA or heparinized plasma. Moderate hemolysis does not

interfere.
Cholinesterase in serum or plasma is stable for several weeks
whether the specimen is stored at room temperature or under
refrigeration, and for 3 months at 20C.
Butyrylthiocholine + H2O
CHE
Butyrate + Thiocholine
pH 7.7
Tiocholine + DMNB
Oxidized thiocholine + 5-MNBA + H
Dibucain inhibition can be estimated by performing concurrent

assays in which dibucaine is present in the substrate mixture.
Percent inhibition is evaluated by comparison of activity in the
inhibited system with that in the uninhibited system. The resulting
dibucaine number allows the classification and identity of the
homozygous and heterozygous variants.
REAGENT COMPOSITION
R1
Buffer/Chromogen. Phosphate buffer 50 mmol/L pH 7.7,

DMNB 0.25 mmol/L. Powder.
R2
Substrate. Butyrylthiocholine iodide 7 mmol/L. Freeze-dried.
R3
Dibucaine. Dibucaine Clorhidrate 2.6 mmol/L.
STORAGE AND STABILITY

Store at 2-8C.
All the kit compounds are stable until the expiry date stated on the
label. Do not use reagents over the expiration date.
Store the vials tightly closed, protected from light and prevented
contaminations during the use.
Discard If appear signs of deterioration:
- Presence of particles and turbidity.
- Discard reconstitud reagents R1 and R2 if present an Blank
absorbance (A) at 405 nm > 0.700 in 1cm cuvette against
distilled water.
REAGENT PREPARATION
INTERFERENCES
Lipemia (intralipid),20 g/L does not interfere.

Bilirubin, 40 mg/dL does not interfere.
Hemoglobin, 16 g/L does not interfere.
Other drugs and substances may interfere4-5.
MATERIALS REQUIRED
Photometer or spectrophotometer with a thermostatted cell
compartment set at 25/30/37C, capable to read at 405 nm.
Stopwatch, strip-chart recorder or printer.
Cuvettes with 1-cm pathlength.
Pipettes to measure reagent and samples.
PROCEDURE
1. Preincubate working reagents and samples to reaction temperature
(see NOTES).
2. Set the photometer to 0 absorbance with distilled water.
3. Pipette into labelled cuvettes:
Temperature
25/30C
37C
Treatment
Without
inhibitor
With
inhibitor
Buffer/Chromogen
1.5 mL
1.5 mL
Inhibitor reagent
1.5 mL
1.5 mL
10 L
10 L
10 L
10 L
50 L
50 L
50 L
50 L
Sample
Sample dil 1:2
with saline
Substrate
Without
inhibitor
With
inhibitor
Working reagents.
1.
Buffer/Chromogen. Add 25 mL of distilled water into a vial of

R1. Cap. Shake. Stand for 15 min. before use. Stable for 6
weeks at 2-8C.
2.
Substrate. Add 2.0 mL of distilled water into a vial of R2. Mix.

Stable for 6 weeks at 2-8C. Excess substrate may be frozen
once.
3.
Inhibitor reagent. Mix 9 volumes of Buffer/Chromogen with 1

volumen of R3.
QUALITY SYSTEM CERTIFIED
ISO 9001 ISO 13485
4. Mix gently by inversion. Insert cuvette into the cell holder, start
stopwatch and record the initial absorbance.
5. Repeat the absorbance readings exactly after 30, 60 and 90
seconds.
6. Calculate the difference between absorbances.
7. Calculate the mean of the results to obtain the average change
in absorbance per second (A/30 sec).
LINEAR CHEMICALS S.L. Joaquim Costa 18 2 planta. 08390 Montgat, Barcelona, SPAIN
Telf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es
CALCULATIONS
Total cholinesterase
U/L = A/ 1 min x 23111 (37C)
U/L = A/ 30sec x 46222 (37C)
U/L = A/ 30sec x 23111 (25/30C)
Samples with A exceeding 0.250 at 405 nm should be diluted 1:10
with saline and assayed again. Multiply the results by 10.
If results are to be expressed as SI units apply:
U/L x 16.67 = nkat/L
Inhibited cholinesterase
To express the dibucaine number apply:
1
Percent inhibition =
U/mL with inhibitor

U/mL without inhibitor
x 100
Cholinesterase levels in serum has been used as a test for the liver
function, as an indicator of possible insecticide poisoning and for
the detection of patients with atypical forms of the enzyme. As a
measure of hepatic disease function it a appears to add little to
more commonly used laboratory measurements. However, CHE is
a sensitive parameter to poisoning by inhalation or contact with
organic phosphorus compounds that inhibit cholinesterase activity.
Among them are many organic insecticides, such as Parathion,
Sarin and tetraethylpyrophosphate4.
The genetic control of serum CHE activity has some practical
importance. Two forms of serum CHE have been recognized. One
has been called normal and the other atypical. Individuals
homozygous for the atypical gene can be distinguished ready
from the homozygous normal. The homozygous abnormal has
very low CHE levels and the abnormal CHE is not inhibited by
dibucaine. The homozygous normal has much higher levels of
serum cholinesterase inhibitable by dibucaine, while the
heterozygous has intermediate levels and response to the
inhibitors. This fact has clinical importance in regard to the
administration of muscle relaxants (succinylcholine). Homozygous
abnormals may develop prolonged apnea after they receive
succinylcholine.
NOTES
REFERENCE VALUES
Total cholinesterase3
1.
Serum, plasma
2.
Children
Males and females > (40 years)
3.5-8.5 KU/L (58.3-141.7 ktal/L)
Females, (16-39 years)

2.8-7.4 KU/L (46.7-123.3 ktal/L)
Nonpregnant, not taking contraceptives
Females, (18-41 years)
Pregnant or taking contraceptives
3.
2.4- 6.0 KU/L (40.0-100.0 ktal/L)
Buffer/Chromogen and Substrate can be mixed

proportionally in tests or analysers using the serum as starter.
The mixture is stable for 2 hours at 15-25C.
This method may be used with different instruments. Any
application to an instrument should be validated to
demonstrate
that
results
meets
the
performance
characteristics of the method. It is recommended to validate
periodically the instrument. Contact to the distributor for any
question on the application method.
Clinical diagnosis should not be made on findings of a single
test result, but should integrate both clinical and laboratory
data.
Inhibited cholinesterase
Dibucaine number
ANALYTICAL PERFORMANCE
Percent inhibition
Normal homozygous
70-90
Heterozygous subjects
35-75
Atypical homozygous
0-20
QUALITY CONTROL
To ensure adequate quality control (QC), each run should include a
set of controls (normal and abnormal) with assayed values handled
as unknowns.
REF
REF
1980005 HUMAN MULTISERA NORMAL

Borderline level of cholinesterase. Assayed.
1985005 HUMAN MULTISERA ABNORMAL
Elevated level of alkaline cholinesterase. Assayed.
If the values are found outside of the defined range, check the
instrument, reagents and procedure.
Each laboratory should establish its own Quality Control scheme
and corrective actions if controls do not meet the acceptable
tolerances.
CLINICAL SIGNIFICANCE
The cholinesterase of the serum (CHE) has been referred as
pseudocholinesterase
to
distinguish
it
from
the
true
acetylcholinesterase (AcCHE) of the red cells and nerve tissue.
QUALITY SYSTEM CERTIFIED
ISO 9001 ISO 13485
- Detection Limit : 244 U/L

- Linearity : Up to 10000 U/L
- Precision:
U/L
Within-run
Between-run
Mean
5.11
8.01
5.11
8.01
SD
0.04
0.106
0.091
0.227
CV%
0.79
1.36
1.78
2.84
10
10
10
10
- Sensitivity : 0.08 mA / U/L cholinesterase.

- Correlation: This assay (y) was compared with a similar commercial
method (x). The results were:
N =50 r= 0.995 y = 0.987 x 0.005
The analytical performances have been generated using on automatic
instrument. Results may vary depending on the instrument.
REFERENCES
1. Knedel, M. y Bttger. Klin. Wschr. 45 : 325 (1967).
2. Poppe, W.A. and Tritsler. J. Clin: Chem. Biochem. 21 : 381 (1983).
3. Clinical Chemistry. Kaplan, L.A. y Pesce, A.S. The C.V. Mosby
Co. 3rd Ed. (1996).AACC
4. Tietz. N.W. Clinical Guide to Laboratory Tests, 3th Edition.
W.B. Saunders Co. Philadelphia, PA.1995.
5. Young DS. Effects of drugs on clinical laboratory tests, 5th ed
Press, 2000.
LINEAR CHEMICALS S.L. Joaquim Costa 18 2 planta. 08390 Montgat, Barcelona, SPAIN
Telf. (+34) 934 694 990 Fax. (+34) 934 693 435. website www.linear.es
B1119-3/0901
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CHOLINESTERASE

For in vitro diagnostic use only

Cholinesterase (CHE) catalyzes the hydrolysis of butyrylthiocholine

Serum, EDTA or heparinized plasma. Moderate hemolysis does not

Oxidized thiocholine + 5-MNBA + H

Dibucain inhibition can be estimated by performing concurrent

Buffer/Chromogen. Phosphate buffer 50 mmol/L pH 7.7,

Substrate. Butyrylthiocholine iodide 7 mmol/L. Freeze-dried.

Dibucaine. Dibucaine Clorhidrate 2.6 mmol/L.

STORAGE AND STABILITY

Lipemia (intralipid),20 g/L does not interfere.

Buffer/Chromogen. Add 25 mL of distilled water into a vial of

Substrate. Add 2.0 mL of distilled water into a vial of R2. Mix.

Inhibitor reagent. Mix 9 volumes of Buffer/Chromogen with 1

QUALITY SYSTEM CERTIFIED

ISO 9001 ISO 13485

U/mL with inhibitor

3.5-8.5 KU/L (58.3-141.7 ktal/L)

Females, (16-39 years)

Buffer/Chromogen and Substrate can be mixed

1980005 HUMAN MULTISERA NORMAL

QUALITY SYSTEM CERTIFIED

ISO 9001 ISO 13485

- Detection Limit : 244 U/L

- Sensitivity : 0.08 mA / U/L cholinesterase.

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