Determination of Serum and Urine Amylase
with Use of Procion Brilliant Red M-2BS Amylopectin
Sylvan M. Sax, Anna B. Bridgwater, and John J. Moore
We describe a sensitive, precise assay of serum or urine amylase activity,
with use of a new substrate, Procion Brilliant Red M-2BS-Amylopectin.
After 0.2 ml of serum or urine is incubated with substrate for 10 mm at 37#{176}C,
ethylene glycol monomethyl ether is added to precipitate proteins and
larger substrate particles. Clarity and chromogenicity of the final solution
are not sensitive to small changes in temperature or concentrations of the
various reagents. No interferences necessitating preparation of specimen
blanks have been encountered. Human salivary amylase, assayed by a
reference saccharogenic method, is used for calibration. When read against
the resulting curve, normal sera and urines give activities comparable to
those obtained with the reference method. Human pancreatic extract,
sera and urines from pancreatitis patients, and macroamylasemia serum
show higher activities by the proposed method.
Additional Keyphrases
normal
values
.
Pi-ocio-n Brilliant
Dye U-nits
#{149}
Both dyed amyloses
(1, 2) and arnvlopectins
(1, 3) are used in the measurement
of a-amylase
(cc-i ,4-glucan
4-glucanohydrolase,
EC 3.2.1.1) activity.
Because
of their insolubility,
the former
must
be used at high concentrations
to give
adequate
sensitivity
in the heterogenous
incubation mixtures
(1, 2, 4-6). A soluble dyed amylopectin,
highly sensitive
for measuring
amylase,
has been described
by Babson
et al. (3). Their
substrate,
Reactone
Red
2B-Amylopectin,
is
precipitated
with an aqueous methanolic
solution
of tannic acid after incubation.
We propose a new substrate,
made by treating
amylopectin
with the dichlorotriazene
dye, Procion
Brilliant
Red M-2BS. The properties
of this substrate and its use in a sensitive
amylase
assay,
applicable
to serum or urine, are described.
An
improved
precipitating
agent is used in the procedure. Finally,
a calibration
by use of human
salivary amylase is proposed.
From the Department
of Pathology,
The Western
Pennsylvania
Hospital,
4800 Friendship
Ave., Pittsburgh,
Pa. 15224.
Received Nov. 23, 1970; accepted Jan. 19, 1971.
Red ]lI-2BS-Amylopectin
substrate
macroam ylase-mia, pancreatitis
diagnosis of
Materials and Methods
Reagents
Procion Brilliant Red M-2B8-Amylopectin.1
Amylopectin
(“Ramaliii
G,” Stein Hall and Co., New
York, N.Y. 10016) is treated with Procion Brilliant
Red M-2BS (ICI America,
Inc., Stamford,
Conn.
06904) in the same proportions
and under experimental
conditions
described
by Dudman
and
Bishop (7) for the dye-labeling
of a variety of polysaccharides.
Buffered
diluent.
Heat to dissolve
8.662 g of
Na2HPO4, 5.31 g of KH2PO4, 10 g of NaCl, 1.35 g
of methyl
p-hydroxybenzoate,
and 0.27 g of
propyl
p-hydroxybenzoate
in 900 ml of water.
Adjust pH to 6.9 at 25#{176}C
with NaOH. Dilute to
1 liter with water, and mix. This solution is stable
for at least three months at 4#{176}C.
Substrate.
Add 3 g of Procion
Brilliant
Red
1
This
material
may
46 Worthington
Dr.,
trade name “Lyosine
be
obtained
Maryland
lied.”
from
Heights,
Reliable
Mo.
63042,
Reagents,
under
the
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 311
.7
.6
.5
C.,
.2
0
Original
720
Concentration
760
800
(mi/I)
840
880
of Ethylene Glycol Monomethyi Ethyl
Fig. 1. Effect of varying the ethylene glycol monomethyl
ether concentration on final absorbance readings of
undigested substrate (
), partially digested sub.
strate (
), and exhaustively digested substrate
add 0.2 ml of water to RB and X. Mix and place in
37#{176}C
water bath for at least 3 mm.
At timed intervals,
pipet 0.2 ml of the albumin
solution
into RB, 0.2 ml of serum into X, and
0.2 ml of urine into U. Mix after each addition and
return to 37#{176}C
water bath.
After incubating
for exactly
10 mm, forcibly
blow 5 ml of EGME into each tube, shake, and place
outside of water bath.
Add 0.2 ml of the zinc sulfate solution
to all
tubes, shake vigorously,
and centrifuge
for 5 mm
at about 700 g.
Read absorbance
of supernatant
fluids at 517
nm, setting RB to zero. If activity
is over 1200
Dye Units, repeat
the entire test on a fourfold
dilution of specimen with the albumin solution.
(- - -)
Calibration
M-2BS-Amylopectin
slowly,
with
magnetic
stirring,
to 100 ml of buffered
cliluent. Heat to
85#{176}C
with stirring to disperse uniformly.
Remove
heat and stir magnetically
for an additional
10 mm.
Filter through
a sintered-glass
funnel of coarse
porosity.
This reagent is stable for about a month
at 4#{176}C.
Albumin solution.Dissolve 3 g of bovine albumin
(“Fraction
V,” Pentex Div., Miles Laboratories,
Inc., Kankakee,
Ill. 60901) and 25 mg of NaN3 in
50 ml of NaC1 solution
(8.5 g/liter).
This solution
is stable for at least a month at 4#{176}C.
EGME.
Mix 400 ml of water
and sufficient
ethylene glycol monomethyl
ether to make 2 liters in
a volumetric
flask. Cool; add 4 drops of “Antifoam
Q Compound”
(Dow Corning
Corp.,
Midland,
Mich. 48641).
Dilute
to volume
with ethylene
glycol monomethyl
ether and mix. This solution,
stored in a brown bottle, is stable.
Zinc sulfate solution.
One hundred
grams of
ZnSO4
71120 per liter of aqueous solution.
Reference enzyme solutions.
Pool saliva specimens obtained
from at least five persons. Dilute
10-fold with NaC1 solution (9 g/liter),
and centrifuge. The supernatant
fluid is used in preparing
both calibration
solutions
and control serum. In
the former, the supernatant
fluid is mixed with a
diluent containing
8.5 g of NaCl per liter and 60 g
of bovine albumin per liter; the resulting dilutions
should be used on the day of preparation
(see
Calibration).
The control
serum is prepared
by
adding
sufficient
supernatant
fluid to pooled
human serum to give an activity of approximately
400 Somogyi
saccharogenic
units; it is stored in
small aliquots in the freezer.
Chromogeriic Procedure
1 ml of the substrate
solution
into
tubes, labeled RB (reagent
blank),
X
(unknown
or control
serum),
and U (unknown
urine). Add 0.2 ml of the albumin solution to U;
Measure
centrifuge
312 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
Prepare
a sufficient
number
of the calibration
solutions,
with activities
ranging
up to approximately
1200 Somogyi
saccharogenic
units,
to
obtain a curve with a minimum of eight calibration
points. Analyze each dilution several times by the
chromogenic
procedure
and by a suitable reference
method (8, 9). Plot averages of absorbance
readings
obtained
with the former method against Somogyi
units contained
in the calibration
solutions.
With
human saliva as the enzyme source, the following
equation pertains:
1 Dye
Unit
=
1 Somogyi
saccharogenic
unit
Other Procedures
Spectral
absorption
curves were obtained
with
a Model 220 spectrophotometer;
other measurements were made on a Model 300 spectrophotometer (both from Gilford
Instrument
Labs, Inc.,
Oberlin, Ohio 44074).
The reference amylase method used in this study
is Somogyi’s
1960 modification
of his original
saccharogenic
procedure
(8).
Results and Discussion
Method Development
Procion
numerous
Brilliant
Red M-2BS was selected from
mono- and dichlorotriazene
dyes tested,
because
of its high color intensity
per unit weight
and the sensitivity
of the corresponding
dyelabeled amylopectin
to human pancreatic
amylase.
The Procion reactive dyes are patented
products,
and the manufacturer
has disclosed
only the
following
information
concerning
Brilliant
Red
M-2BS:
the molecular
weight is about 700, the
molecule
contains
an azo chromophore
with a
dichlorotriazene
reactive
group, and the commercial product has a purity of approximately
65%.
When heated to 85#{176}C
in water, Pro cion Brilliant
C.,
C
0
0
.0
4
0
20
30
40
Minutes of Incubation
Fig. 2. Increase of absorbance with incubation time,
with human serum (270 Somogyi saccharogenic units) as
the enzyme source
Red M-2BS-Amylopectin
forms an opaque
colloidal dispersion,
which can be reprecipitated
by
adding methanol to a final concentration
of 500 ml/
liter. We attempted
to use methanolic
tannic acid
(3) to precipitate
larger substrate
particles
after
incubation
with serum or urine. The following
difficulties
were encountered:
(a) supernatant
fluids were not consistently
clear; (b) calibration
curves departed
sharply
from linearity;
(c) the
solubility
of intermediate-sized
split
products
sharply depended
on temperature
and the concentrations
of phosphate,
methanol,
tannic acid, and
protein;
(d) the batch-to-batch
variability
in the
appearance
and properties
of tannic acid could not
be overcome
by various purification
procedures;
and (e) the reagent
had a short shelf-life. These
difficulties were successfully
surmounted
by development
of a two-reagent
precipitation
technique.
The added procedural
step could not be omitted;
without
zinc sulfate,
supernatant
fluids are not
perfectly
clear. However,
that
disadvantage
is
minor in view of the consistent
clarity and reproducible absorbance
readings
obtained.
Some protein also is apparently
required
for clear supernatant fluids. Therefore,
albumin has been included
with the reagent
blank and urine specimens.
Supernatant
fluids absorb maximally
at 517 nm.
The wavelength
of maximum
absorbance
does not
depend on the extent of hydrolysis
of substrate.
Concentrations
of constituents
of the incubation
mixture may affect final absorbance
by influencing
either the rate of enzyme activity or the solubility
of substrate
or split products
in the precipitants.
Concentrations
of precipitating
reagents
can only
affect the final solubilities.
We measured
the effect
of various concentrations
of each substance
used
in the proposed procedure
on the final absorbance
readings
of the reagent
blank
(representing
undigested
substrate),
control
serum
(representing
partially
hydrolyzed
substrate),
and on digested
substrate
(in which the substrate
was exhaustively
hydrolyzed
by human salivary amylase).
A ± 10%
variation
in NaC1, Procion Brilliant
Red M-2BSAmylopectin,
ZnSO4, albumin,
or phosphate
concentration
from that specified in the proposed procedure had no notable effect. A 20% increase in
phosphate
slightly increases the absorbance
of the
digested
substrate.
Various
concentrations
of
ethylene
glycol monomethyl
ether have a considerable
effect on the solubility
of intermediatesized split products,
hut little on either unreacted
or extensively
degraded
substrate
(Figure 1). The
colors of the reagent
blank and of the digested
substrate
both increase with increasing p11, making
it difficult
to determine
the optimum
pH for
enzyme activity.
However,
the peak is broad and
shows little variation
between pH 6.8 and 7.1.
Plots of absorbance
vs. incubation
time and vs.
enzyme concentration
are shown in Figures 2 and
3. Substitution
of human
pancreatic
extract
for
human saliva as the enzyme source does not significantly
alter the shape of the latter curve.
To study possible interferences
and to establish
the need for running
specimen
blanks,
a large
number
of such blanks were prepared
from sera
and urine specimens of hospital patients.
Although
most sera were ordinary
in appearance,
some were
icteric, hemolyzed,
or lipemic,
and some urines
were unusually
dark, bloody,
turbid,
or icteric.
Some bilirubin does appear to be extracted
by the
precipitating
solvent.
However,
a commercial
control
serum,
containing
20.8 mg of bilirubin
(chiefly unconjugated)
per 100 ml, gave a blank
2.4
2.2
2.0
1.8
.6
‘C
.4
0
.0
1.2
I.0
.8
.6
.4
.2
0
200
Enzyme Concentration
600
000
1400
in Somogyi Sacchorogenic
Units
Fig. 3. Plot of absorbance vs. enzyme concentration.
with human saliva as the enzyme source
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 313
Table 1. Relative Activities of Different Enzyme Sources, as Assayed by Proposed Procedure and
by Reference Saccharogenic Method
Enzyme
source
Normal human serum
Pooled human serum
Human pancreatitis serum’
Human macroamylasemia serumd
Human
urine, low activity
Human urine, elevated activity
Human saliva’
“Versatol-E” reference serum
Human pancreas, postmortem’
Range of
activities,
Dye Units
35-165
N”
D/S6 ratio.
mean and SD
25
0.99
5
85-99
±
0.11
0.938
0.26
D=1.275S+9.2
0.981
0.09
D=1.084S-12.1
0.985
0.04
±
±
193-1340
12
1.30
857
1
1.59
52-307
15
332-551
6
1.25 ± 0.14
9
2
1.05 ± 0.04
0.67
1
1.49
‘Number of individual specimens or individual
Ratio of Dye Units to Somogyi units.
batches of pooled specimens
Problems of Calibration
It is desirable
to express
enzyme
activities
in
terms
of cmoles
of substrate
transformed
per
minute (10). Where the substrate
and products are
not easily defined in terms of molecular weight and
structure,
it is permissible
to regard the active
groups measured
as representing
an equal number
of molecules
of product-e.g.,
reducing
groups
from starch or titratable
carhoxyl
groups from
triglycerides.
Difficulties
in implementing
the
I.U.B. recommendations
(10) with this and other
chromogenic
amylase
methods
are enumerated
below:
Whereas,
in saccharogenic
methods,
there is
a 1 : 1 correspondence
between
molecules
of substrate transformed
and reducing groups appearing,
the relationship
is more complex in chromogenic
methods.
A single enzymatic
cleavage may result
in the appearance
of 0, 1, or >1 dye residues,
depending
upon the solubilities
of the fragments
and the number
of dye moieties
per solubilized
fragment.
This
consideration
applies
to dyelabeled amylose as well as amylopectin.
#{149}
The ultimate solubility of a given fragment is
affected
by more or less arbitrary
experimental
conditions
unrelated
to enzyme activity.
#{149}
The molecular
weight of the dye marker is
unknown.
#{149}
The dichlorotriazene
dye, as supplied by the
manufacturer,
contains
a considerable
amount
of
impurities.
These may vary from batch to batch,
and could affect the chromogenic
properties
of the
substrate.
314 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
For
r
analyzed.
‘Chronic or acute pancreatitis
indicatedas physician’sdiagnosison patient’schart.
d Macroamylasemia
demonstrated by chromatography
on Sephadex 200.
‘Dilutionsof primaryextractsprepared in physiological saline to give activities in a measurable
value of only 8 Dye Units. Since no other significant
blanks have been encountered
with serum or urine
to date, the preparation
of specimen blanks appears
to be unnecessary.
Correlation
coefficients.
equation
D=1.044S-3.9
1.02 ±
1.00
Regression
these
reasons,
range.
we have
chosen
to report
activities
in Dye Units,
yielding about the same
results (at least with normal sera) as those reported
in the familiar
Somogyi
units. Table 1 shows the
ratio of results by the proposed
method
in Dye
Units to results by the reference method in Somogyi
units
(“D/S
ratio”)
for a number
of enzyme
sources. Fortuitously,
the ratios of human saliva
and human pooled serum do not differ significantly
at the 95% limits of confidence
(t = 1.7). Therefore, human saliva is recommended
for preparing
calibration
curves. It should be noted that the
commercial
control
serum,2 which gives a D/S
ratio of unity by the method of Babson et al. (3),
gives a much lower ratio by the proposed method;
that product is therefore unsuitable
for calibrating
the latter procedure.
Evaluation
Examination
of Table 1 further
reveals that,
with normal urines, the proposed method might be
expected
to yield results that are comparable
to
those by the reference
procedure.
However,
the
proposed
method
discriminates
more effectively
than does the reference procedure
between normal
or abnormal
sera and urines. The enhanced
activities
observed
in clinically
proved
cases of
pancreatitis
and macroamylasemia
as well as the
excellent
correlation
between
results by the proposed and reference methods strongly suggest that
we are indeed
measuring
amylase
activity.
The
high values observed in cases of macroamylasemia
are in contrast with results by a different chromogenie method (4). In our hands, other chromogenic
“Versatol
N. J.
E,”
Warner-Chilcott
Laboratories,
Morris
Plains,
methods
greatly
have
from
unity
shown
D/S
for normal
ratios
urine
that
depart
specimens.
Normal ranges were estimated
from analyses of
specimens
from 30 female and 17 male student
nurses and new hospital employees.
There was no
significant
sex-attributable
difference
in means.
The combined
data, although
slightly skewed for
the higher
values,
more nearly
approximate
a
gaussian
(normal)
rather than a log normal distribution.
The mean and standard
deviation
are
87.5 ± 26.5 Dye Units.
Precision
was measured
by taking aliqiiots
of
serum and urine specimens
with both normal and
elevated
activities,
analyzing
them in separate
runs, and calculating
standard
deviations
of the
paired results.
Twenty
specimens
ranged
in activity from 37 to 154 Dye Units (av, 88; SD, 5.5;
cv, ± 6.3%).
Twenty
other
specimens
ranged
from 231 to 2700 Units (av, 620; SD, 34.2; cv,
±5.5%).
References
1. Rinderknecht,
H., Wilding,
method
for the determination
(1967).
P., and Haverback,
B. J., A new
of a-amylase.
Experientia
23, 805
2. Klein, B., Foreman,
J. A., and Searcy, H. L., The synthesis
and utilization
of Cibachron
Blue-Amylose:
A new chromogenic
substrate
for determination
of amylase
activity.
Anal. Biochem.
31, 412 (1969).
3. Babson, A. L., Tenitey,
amylase
(1970).
substrate
and
S. A., and
Megraw,
it.
procedure.
CLIN.
CHEM.
assay
4. Take, S., Berk, J. E., and Fridhandler,
new simplified
dye method
Chim. Ada 26, 533 (1969).
C.,
An
improved
indebted
to Dr. J. Edward
Berk and I)r. Louis
Fridhandler,
College
of Medicine,
University
of California
at
Irvine,
for characterizing
the macroamylasemia
specimen
by
column chromatography.
We thank Michole Ryan, The Western
Pennsylvania
Hospital,
for preparing
the figures.
T.,
amylase
activity.
on a
Clin.
Ratliff,
C. H., and
assay
using
a new
Searcy,
H. L., New
of serum
amylase
W. F., and Bishop,
C. T., Electrophoresis
on cellulose
acetate.
Can. J. Chem.
8. Somogyi,
i\L., Modifications
of two
amylase.
CLIN. CHEM. 6, 23 (1960).
We are much
amylase
39
Amer. J. Clin. Palhol. 53, 627 (1970).
6. Klein,
B., Foreman,
J. A., and
genie substrate
for determination
CLIN. CHEM. 16, 32 (1970).
7. Dudman,
polysaccharides
(1968).
16,
L., Observations
for assaying
5. Hall, F. F., Culp, T. W., Hayakawa,
Hightower,
N.
starch derivative.
E., New
methods
chromoactivity.
of dyed
46, 3079
for the assay
of
9. Amador,
E., and Wacker,
W. E. C., Enzymatic
methods
used
for diagnosis.
In Methods of Biochemical
Analysis
13; Glick, D.,
Ed. Interscience
Publishers,
New York, N.Y., 1965, p 334.
10. King,
units-an
E. J.,
attempt
and Campbell,
at international
D.
M., International
enzyme
agreement.
Clin. Chim. Acta
6, 301 (1961).
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 315