Document ID: EPA-HQ-OPP-2004-0405-0033
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-02-08T05:00Z

NHEERL­
NTD­
CMTB­
SP/
98­
001­
001
February
10,
1998
Page
1
Procedural
Section
1.0
Scope
and
Application
1.1
The
radiometric
method
is
used
to
measure
cholinesterase
activity.
The
assay
employs
minimal
dilution
and
short
run
times.

1.2
The
method
is
the
most
accurate
means
for
determining
cholinesterase
activity
in
tissues
from
animals
that
have
been
treated
with
reversible
inhibitors.

1.3
The
assay
should
be
used
for
cholinesterase
determinations
in
tissues
containing
hemoglobin,
e.
g.,
erythrocytes
or
high
glutathione
levels,
e.
g.,
liver.

2.0
Prerequisites
2.1
Equipment
and
Supplies
Water
bath
Analytical
balance
Vortexers
Stopwatch/
Timer
Pipets
Scintillation
counter
7
ml
scintillation
vials
Ice
Laboratory
glassware
All
equipment
and
supplies
are
considered
standard
laboratory
devices
or
items
and
do
not
need
to
meet
critical
specifications.

2.2
Chemicals
0.2M
Sodium
Phosphate,
monobasic
(
Sigma,
S­
9638):
27.6
g
NaH2PO4
in1000
ml
ddH2O
0.2M
Sodium
Phosphate,
dibasic
(
Sigma,
S­
9763):
28.4
g
Na2HPO4
in1000
ml
ddH2O
0.1M
Sodium
Phosphate
buffer,
pH
8.0:
380
ml
0.2M
Na2HPO4
(
dibasic)
20
ml
0.2M
NaH2PO4
(
monobasic),
adjust
pH
to
8.0,
double
volume
by
adding
ddH2O
and
refrigerate
0.1M
Sodium
Phosphate
buffer,
pH
8.0
+
1%
Triton
(
Sigma,
T­
6878)
Acetylcholine
Iodide
(
Acetyl­
3H)
(
New
England
Nuclear,
NET­
113)
24mM
Acetylcholine
Iodide
(
Sigma,
A­
7000):
65
mg
Acetylcholine
Iodide
in10
ml
0.05M
KPO4
buffer
&
freeze
1M
KH2PO4,
monobasic
(
Sigma,
P­
5379):
136.1
g
KH2PO4
in1000
ml
ddH2O
NHEERL­
NTD­
CMTB­
SP/
98­
001­
001
February
10,
1998
Page
2
1M
K2HPO4,
dibasic
(
Sigma,
P­
5504):
228.3
g
K2HPO4
in1000
ml
ddH20
1M
Potassium
Phosphate
buffer,
pH
7.0:
39
ml
1M
KH2PO4
(
monobasic)
+
61
ml
1M
K2HPO4
(
dibasic),
adjust
pH
to
7.0
and
refrigerate
0.05M
Potassium
Phosphate
buffer,
pH
7.0:
Dilute
10
ml
of
1M
stock
with
190
ml
ddH2O
Stop
Solution:
23.6
g
Chloroacetic
acid
(
1M)
(
Sigma,
C­
0266)
5
g
Sodium
hydroxide
(
0.5M)
(
Fisher,
S320­
500)
29.2
g
Sodium
chloride
(
2M)
(
Sigma,
S­
9625)
Add
ddH2O
to
a
total
volume
of
250
ml
Scintillation
cocktail:
5
g
2,5­
Diphenyloxazole
(
Sigma,
D­
4630)
0.3
g
1,4­
Bis[
2­(
5­
Phenyloxazolyl)]
benzene
(
Sigma,
P­
3754)
100
ml
Isoamyl
alcohol
(
Sigma,
I­
3643)
Add
Toluene
(
Burdick
&
Jackson,
347­
4)
to
a
total
volume
of
1000
ml
Ultima
Gold
(
Packard,
6013324)

2.3
Biological
Samples
should
be
stored
at
­
80

C
in
an
alarm
equipped
freezer
Tissues
will
be
received
already
appropriately
diluted
and
homogenized
in
0.1M
Sodium
Phosphate
buffer,
containing
1%
Triton
Keep
samples
on
ice
during
preparation
2.4
Personnel
Training
Technical
personnel
should
receive
radiation
safety
training
and
be
instructed
and
deemed
capable
by
the
Principle
Investigator
in
performing
the
method
prior
to
initiating
the
procedure
alone.
In
many
cases,
the
assay
requires
the
skill
of
two
technicians
when
short
run
times
are
employed.

3.0
Special
Considerations
3.1
Avoid
tissue
volumes
and
lengthy
run
times
which
may
use
up
the
substrate
(
keep
disintegrations
per
minute
[
DPM]

60,000).

3.2
If
tissues
are
treated
with
a
reversible
inhibitor,
short
run
times
and
minimal
dilutions
should
be
used
to
avoid
reactivation.
Historical
data
from
our
laboratory
suggests
the
following
guidelines:
Plasma­
undiluted,
20

l
for
3
minutes
Erythrocytes­(
1:
10)
dilution,
40

l
for
10
minutes
Brain­(
1:
50)
dilution,
20

l
for
10
minutes
Heart­(
1:
10)
dilution,
40

l
for
5
minutes
4.0
Procedure
NHEERL­
NTD­
CMTB­
SP/
98­
001­
001
February
10,
1998
Page
3
1.
Set
water
bath
temperature
to
26

C­
temperature
verified
with
mercury
thermometer
2.
Prepare
substrate
as
follows:
a.
500

l
of
24mM
Acetylcholine
Iodide
stock
solution
b.
1.5
ml
0.05M
Potassium
Phosphate
buffer,
pH
7.0
c.
20

l
3H­
Acetylcholine
Iodide
d.
Vortex.
Transfer
20

l
to
a
7
ml
scintillation
vial
and
add
5
ml
Ultima
Gold.
Vortex.
Count
on
Easy
Count
program
on
scintillation
counter.
Need
a
minimum
of
220,000
DPM.
3.
Perform
a
trial
run
to
determine
the
appropriate
amount
of
tissue
and
length
of
the
run
time
to
be
used
for
the
assay
by:
a.
Pipet
several
varying
amounts
of
tissue,
in
duplicate,
into
7
ml
vials.
Record
in
notebook.
b.
Add
0.05M
Potassium
Phosphate
buffer,
pH
7.0,
so
that
the
tissue
volume
+
buffer
volume=
80

l
c.
Pipet
80

l
of
0.05
Potassium
Phosphate
buffer,
pH
7.0
in
a
separate
7
ml
vial
to
run
a
blank
d.
Place
vials
into
the
26

C
water
bath
e.
Add
20

l
of
3H­
Acetylcholine
Iodide
substrate
to
each
vial;
vortex
f.
When
a
set
run
time
has
expired,
add
100

l
of
stop
solution
to
each
vial;
vortex
g.
Add
5
ml
of
scintillation
cocktail
to
each
vial
h.
Cap
and
invert
vials
before
placing
on
scintillation
counter
to
count
NOTE:
Lowest
value
should
be
at
least
2­
3
times
the
blank
and
the
highest
value
should
be

60,000
DPM.
If
counts
are
too
high,
reduce
the
sample
volume
and/
or
the
length
of
the
run
time.
If
counts
are
too
low,
first
try
increasing
the
sample
volume;
then,
increase
the
run
time
if
necessary.
4.
Prepare
substrate/
buffer
solution
according
to
the
following
formula:
(
100­
a)/
20=
X
X*
b=
Y
Y­
b=
Z
Where:
a=

l
of
sample
used
for
assay
b=
ml
of
substrate
originally
prepared
Y=
final
volume
of
substrate/
buffer
solution
in
ml
Z=
ml
of
0.05M
Potassium
Phosphate
buffer,
pH
7.0
to
be
added
to
the
substrate
that
was
originally
prepared
Example:
2
ml
of
substrate
has
been
prepared
and
it
has
been
determined
from
the
pre­
run
that
20

l
of
sample
will
be
assayed
(
100­
20

l)/
20=
4
4*
2
ml=
8
ml
Therefore,
6
ml
of
0.05M
Potassium
Phosphate
buffer,
pH
7.0,
will
be
added
to
the
2
ml
of
substrate
already
prepared
for
a
total
of
8
ml.
Rationale:
By
diluting
the
substrate
with
the
buffer
in
this
manner,
the
technician
NHEERL­
NTD­
CMTB­
SP/
98­
001­
001
February
10,
1998
Page
4
can
eliminate
a
pipetting
step
that
can
save
time
and
improve
assay
accuracy.
5.
Pipet
the
appropriate
amount
of
tissue,
in
duplicate,
into
7
ml
vials
6.
Pipet
the
same
amount
of
0.05M
Potassium
Phosphate
buffer,
pH
7.0
into
2
vials
for
substrate
blanks
7.
Place
all
vials
in
the
26

C
water
bath
8.
Add
the
appropriate
volume
of
substrate/
buffer
solution
to
each
vial
(
tissue
volume
+
substrate/
buffer
solution
volume=
100

l);
vortex
9.
After
the
appropriate
run
time
has
elapsed,
stop
the
reaction
by
adding
100

l
of
solution;
vortex
10.
Add
5
ml
of
scintillation
cocktail
to
each
vial,
cap
and
invert
11.
Prepare
2
total
count
vials
by
placing
the
same
volume
of
substrate/
buffer
solution
that
was
used
in
the
assay
into
(
2)
7
ml
vials
and
adding
5
ml
of
Ultima
Gold
12.
Count
on
the
scintillation
counter
within
24
hours
Quality
Control
Section
1.0
All
samples
are
run
in
duplicate.

1.1
The
assay
uses
the
analysis
of
blanks.

1.2
Calculations:
a.
Data
should
be
expressed
in
nmol/
minute
b.
Scintillation
counter
reports
results
in
DPM
c.
Correct
the
averaged
DPM
value
for
dilution,

l
of
tissue
and
run
time
used
for
the
assay
d.
Complete
the
conversion
by
taking
the
corrected
average
DPM
value/(
total
counts/
specific
activity
constant
of
the
radioisotope),
where
specific
activity
constant
is
determined
by
the
following
calculation:
Take
the
specific
activity
expressed
in
Ci/
mmol
from
the
product
sheet
that
accompanies
the
radioisotope,
convert
to
mCi/

mol
(/
1000),
convert
to

Ci/
nmol
(/
1000),
multiply
the
number
by
2,200,000
DPM
(
DPMs=
1

Ci)
to
get
DPM/
nmol,
and
complete
conversion
to
DPM/
pmol
(/
1000)

Example:
40

l
of
erythrocytes
diluted
at
(
1:
10)
were
run
for
10
minutes.
The
mean
DPM
value
was
16582.92.
Total
counts
for
the
assay
were
101026.5
DPM.
The
specific
activity
constant
of
the
radioisotope
was
121.8
DPM/
nmol.
Mean
DPM
DPM/
min(/
10)
dilution
correction(*
10)
nmol/
min(
TC/
SA)
nmol/
min/
ml(/
40*
1000)
16582.92
1658.29
16582.92
19.99
499.82
1.3
Warnings
3H­
Acetylcholine
Iodide
is
a
radioisotope.
Laboratory
personnel
should
wear
the
appropriate
personal
protective
equipment.
Avoid
spills,
contamination
and
discard
waste
in
NHEERL­
NTD­
CMTB­
SP/
98­
001­
001
February
10,
1998
Page
5
accordance
with
the
US
EPA
Radiation
Safety
Manual.

1.4
Record
keeping
Requirements
All
sample
inventories,
correspondence,
etc.
for
a
study
are
kept
in
a
labeled
hanging
file
folder
in
the
laboratory's
file
cabinet.
As
assays
are
run,
notations
on
procedure,
including
any
problems,
are
documented
in
the
technician's
laboratory
notebook.
Resulting
data
is
organized
in
Excel
(
Microsoft
Office
`
97)
files.
Hard
copies
are
printed
and
placed
in
the
study
file.

References
1.
Johnson,
CD
and
Russell,
RL.
A
rapid,
simple
radiometric
assay
for
cholinesterase
suitable
for
multiple
determinations.
Anal.
Biochem.,
1975,
64,
229­
238.