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CLIN. CHEM. 19/9,994-997 (1973)
Evaluation of Direct and Indirect Coupled Enzyme Assay
Systems for Measurement of Creatine Kinase Activity
E. C. Dinovo,1 D. S. Miyada, and R. M. Nakamura
assay
of creatine
kinase by the method
of
and Cohen with the Technicon “SMA
12/60”
is compared
with two indirect coupled-enzyme
assay
methods.
The direct method was linear to greater
than 2000 U/liter,
whereas
the indirect
methods
Direct
Siegel
were linear to 250 and 1500 U/liter,
with use of seri-
al dilutions of a creatine kinase preparation.
Increasing the activity of the auxiliary enzymes, hexokinase
and glucose-6-phosphate
dehydrogenase,
in one of
the coupled enzyme assay methods increased its linear range from 250 to 700 U/liter.
The observed
findings are explained on the basis of the fundamental differences
between
direct and indirect coupledenzyme assay systems and, within the latter system,
the effect of the activitiesof auxiliary enzymes.
Additional Keyphrases: apparent
with dilution
A utoAnalyzer
activity
in crease in CK specific
kit methods of enzyme assay
#{149}
We have
observed
significant
discrepancies
in
serum CK2 activities,
depending
upon whether
the
assays were performed
by the Siegel and Cohen procedure (1) on the Technicon
“SMA 12/60” or by the
coupled-enzyme
assay systems
of the Calbiochem
or
Worthington
companies.
When
sera with elevated
CK activity
were diluted
and re-assayed
by the Calbiochem
reagent system, the specific activity
was observed
to increase
significantly.
This unexplained
dilution
effect was noted previously
by other investigators (2-6). The increases
in specific activity
varied
unpredictably
with different
sera, but increases
of
100-200% have been common.
Such increases
in specific activity
are difficult
to reconcile
with current
theories
of enzyme
action;
nevertheless,
they have
been reported
and apparently
accepted.
We compare
here results of the direct and indirect
coupled enzyme assay methods
for the determination
of CK and give an explanation
for the cause of the
dilution effect.
From the Department
of Pathology,
Orange
County
Medical
Center,
Orange,
Calif. 92668; and The University
of California,
Irvine, Calif.
1 Postdoctoral
Fellow in Clinical Chemistry.
2Nonstandard
abbreviations
used:
CK, creatine
kinase
(EC
2.7.3.2.);
HK, hexokinase
(EC 2.7.1.1) GPD, glucose-6-phosphate
dehydrogenase
(EC 1.1.1.49).
Received Aug. 28, 1972; accepted June 4, 1973.
994
CLINICAL
CHEMISTRY,
Vol. 19, No. 9, 1973
Material and Methods
“Multienzyme
III” (Lot No. 3043120002Al;
Hyland,
Division
of Travenol
Laboratories,
Inc., Costa Mesa,
Calif. 92626) was used as the source of CK. It was
reconstituted
according
to manufacturer’s
instructions and then serially diluted
with sodium
chloride
solution
(9 g/liter).
The serially
diluted
specimens
were assayed by three methods:
A: “SMA
12/60”
(Technicon
Instruments
Corp.,
Tarrytown,
N. Y. 10591). The direct assay for creatine by the Siegel and Cohen (1) modification
of the
Hughes (7) colorimetric
assay for creatmne.
B: “Statzyme
CPK”
(Lot OLA and 11B; Worthington
Biochemical
Corp.,
Freehold,
N. J. 07728).
Coupled
enzymatic
assay system
of Oliver
(8) as
modified by Rosalki (9).
C. “CPK
kit” (Lot No. 21050; Calbiochem,
La
Jolla, Calif. 92037). Coupled
enzymatic
assay system
of Oliver (8) as modified by Rosalki (9).
Rate measurements
by Methods
B and C were
made at 37 #{176}C
on the Gilford 300-N equipped
with
Thermo-Cuvette
3017 and Data Lister 4008. In these
studies,
1.0 ml of Worthington
or Calbiochem
CK
substrate
solution
was heated to 37 #{176}C
in a temperature-controlled
heating
block,
20 l of the diluted
Multienzyme
was added,
stirred,
and pumped
into
the 37 #{176}C
temperature-controlled
cuvet, and the absorbance
at 340 nm recorded
for at least 10 mm. All
three systems
contained
sulfhydryl
activators
and
the three series of assays were usually run within one
or two days.
To test for auxiliary
enzyme insufficiency,
the Calbiochem
substrate
was fortified
with added HK and
GPD. The amounts
of auxiliary
enzymes
were increased
by adding
10 pl of solution
No. 5 from the
Boehringer
CK kit No. 15926-containing,
per milliliter, 1 mg of HK and 1 mg of GPD-to
each test
tube before the serially diluted specimens
were added.
Results
Results
obtained
by the direct method
and those
obtained
by the indirect
coupled-enzyme
assay systems were compared
by determination
of specific activity (activity
per unit weight of the enzyme)
at different dilutions
of the enzyme.
The results of assay-
Table 1. Effect of Dilution on CK Activity as Measured by Methods A, B, and C
DIlutions
Observed activity
(U/liter)
of diluted
solutions
Calculated activity
for undiluted sample
1
Coupled-enzyme
method C
Coupled-enzyme
method B
Direct method A
1220
Coupled-enzyme
method C
Coupled-enzyme
method B
Direct method A
1220
1/2
1/4
1/8
830
550
330
180
820
400
200
1560
1/16
1/32
90
100
2020
1060
530
260
130
1660
2200
2640
2880
2880
3120
3280
3200
3200
3200
4040
4240
4240
4160
4160
The observed results were obtained directly from assays; whereas, the results for the original undiluted sample were calculated
lated activity = observed X (I/dilution).
Method A: SMA 12/60.
Method B: Statzyme CPK Kit (Worthington).
Method C: CPK Kit (Calbiochem).
by this formula:
calcu-
Table 2. Effect of Auxiliary Enzyme
Concentrations on CK Activity as
Measured by Method C
Dilutions
r
LU
I-.
-J
>I>
C,
1/10
1/5
1/2
240
376, 396
609
252
488,498
913
Activity (U/liter)
without added auxiliary
enzymes
Activity(U/liter)
with added auxiliary
enzymes
4
Experimental details are found in the ‘Methods’ section.
Dilutions of Hyland Multienzyme Ill were assayed using Method C with
and without added HK and GPD.
a Calbiochem CPK Kit.
0
0.1
0.2
RELATIVE
0.3
04
ENZYME
0.5
0.6
CONCENTRATION
Fig. 1. Enzyme activityvs. relativeCK concentration by
Methods A, B and C. Activityin U/liter,as measured by
theirrespective methods
ing the same serial dilutions
of an elevated
CK control by the three different
methods
are presented
in
Figure
1 and Table 1. The linearity
of the methods
is compared
in Figure 1. The direct method
(Method A) showed little change in specific activity,
varying from 4040 to 4240 U/liter over a sixteen-fold
range
of dilution.
Linearity
of CK activity
was maintained
to 2020 U/liter.
The reported
high of 2020 U/liter
observed with the SMA 12/60 was obtained
by reading “O.D.”
(absorbance)
on the meter and converting
to U/liter.
Method
B of the coupled-enzyme
assay systems
showed
little
change
in specific
activity,
varying
from 3120 to 3280 U/liter
over a 16-fold change
in
dilution.
We estimate
that linearity
was maintained
to about 1500 U/liter.
On the other hand, the specific activity
with Method
C varied from 1220 with the
undiluted
control to 2880 U/liter
with a 32-fold dilution. Specific
activity
increased
almost 2.5-fold with
the 32-fold change in dilution.
Method
C was linear
to about 250 U/liter.
Note that even in the linear
portions
of Figure 1, the activities
of the three methods are not equal. For example,
even though
the results for the 16-fold dilution
are in the linear range
for all three methods,
values of 280, 200, and 180 U/
liter are given by methods
A, B, and C, respectively.
The effect of auxiliary
enzymes
on CK activity
as
measured
by Method
C is demonstrated
in Table 2.
A considerably
increased
activity
of CK is seen when
the substrate
solution
of Method
C is fortified
by
added HK and GPD. Although
the observed
increase
is small at low CK concentrations
in or near the linear range of the assay (252 vs. 240 U/liter),
at moderately
elevated
concentrations
of CK outside
the
linear range of the method
there was almost a 50%
increase in activity (913 vs. 609 U/liter).
Discussion
Coupled
enzyme
assays
are deceptively
and one should be aware of their limitations
to avoid some of their pitfalls.
All three
used here make use of one or more of the
reactions:
CLINICALCHEMISTRY,
simple,
if one is
methods
following
Vol.19,No.9, 1973
995
1.
Primary
Creatine
reaction:
phosphate
ADP
+
creatine
2. Auxiliary
ATP
+
ADP
reaction:
Hexokina8e
ATP + glucose
p
glucose-6-phosphate
3.
+
Indicator
reaction:
Glucose-6-phosphate
NADP
+
or
NAD
6-phosphogluconate
+
NADPH
or
NADH
+
H
The direct method
of Siegel and Cohen (1) measures the rate of the primary
reaction.
The coupledenzyme
assay method
of Oliver (8), as modified
by
Rosalki
(9), in which reagent
systems
manufactured
by Calbiochem
and Worthington
were used, involve
all three reactions.
Their rates are measured
as a decrease in absorbance
of the reaction
mixture
at 340
nm as either NADP
or NAD
is reduced to NADPH
or NADH, respectively.
The direct
method
gave results
for rate that are
linearly
related
to enzyme
dilution
(Figure
1). The
reaction
rate, therefore,
was zero order over the dilution range investigated,
which was greater than 2000
U/liter.
Dilutions
with saline from twofold to 32-fold
had no effect other than to decrease
enzyme
activity
in proportion
to the dilution.
Furthermore,
other factors that might
conceivably
cause deviations
from
linearity,
such as back reactions
and substrate
insufficiency,
were without
apparent
effect within
the
limits of this system.
On the other hand,
the indirect
coupled-enzyme
systems,
Methods
B and C, gave results that deviated from linearity
to different
extents
(Figure
1).
Method
B was linear to about 1500 U/liter;
whereas,
Method
C was linear to only 250 U/liter.
Their differences did not end there. Method
B yielded significantly
higher
activities
for the same
specimens,
amounting
to 12 and 99% at a 32-fold and twofold
dilution,
respectively
(Table 1).
Basically,
there are two areas for discussion:
the
differences
observed
between
the direct and indirect
systems
and the differences
observed
between
the
two indirect
assay systems.
The significance
of these
findings
are explained
by basic principles
of clinical
enzymology.
In the indirect
coupled-enzyme
assay
systems
we used
(see equations),
the measured
change in absorbance
is two reactions
removed
from
the primary
reaction,
and therefore
measures
CK activity indirectly.
This change would be a direct measure of CK activity
only if the velocity
of auxiliary
enzymes
approached
infinity
and the reactions
were
irreversible.
In this situation,
as soon as a CK turnover occurred,
the products
would be converted
immediately
by the auxiliary
enzymes
and a molecule
996
CLINICAL
CHEMISTRY,
Vol. 19. No. 9, 1973
of NAD+
or NADP+
(depending
upon the system
used) would be immediately
converted
to NADH or
NADPH,
respectively,
by the indicator
enzyme.
The
steady
state concentration
of ATP and glucose-6phosphate
would be infinitesimally
low, and no lag
period
would be observed.
This situation
is rarely
approached
in the laboratory.
Normally,
a finite
time interval
occurs between
turnover
of CK and the
appearance
of NADH.
Time
is required
for the
build-up
of the steady-state
concentrations
of the intermediate
products,
thus decreasing
the observed
NADH
rate with respect
to the actual
rate of CK
turnover.
In addition,
enzymatic
reactions
are generally reversible
and its effect would be to further
decrease the observed
rate. Therefore,
the measured
NADH rate is always less than the true CK rate in
coupled
enzyme
systems.
The amplification
of this
difference
creates the disparity
observed
between
the
direct and the coupled enzyme assay systems.
Confirmatory
evidence
is provided
by Rosalki and
Tarlow (10, 11), who assayed
CK by a direct fluorometric method,
measured
the reaction
product,
creatine,
by reaction
with ninhydrin,
and compared
these results with those obtained
by the Oliver-Rosalki method
(9). The results
by the fluorometric
method
were higher
by 30 and 100% with CK in
human serum and skeletal muscle, respectively.
The differences
in results obtained
by the two coupled-enzyme
systems
(Methods
B and C) can be explained
on the basis of the concentration
differences
of the auxiliary
enzymes.
It will be recalled
that
Method
B yielded significantly
higher CK activities
on aliquots
from the same specimen
and possessed
a
longer linear range (Figure 1). In a closer look at the
reagent
composition
of these two systems,
the primary difference
was found to be in the activities
of
the auxiliary
enzymes,
HK and GPD. In Method
B,
2 and 4 U were used per assay, respectively,
of HK
and GPD; whereas
in Method
C, 1 U of each was
used per assay.
Table
2 shows that increasing
the
activity
of the auxiliary
enzyme
in the substrate
of
Method
C not only produced
a significant
increase
in
CK activity,
but also extended
the linear range of the
calibration
curve. Accordingly,
the use of relatively
low concentrations
of the auxiliary
enzymes
is implicated,
at least in part, for the differences
observed
between the two coupled-enzyme
systems.
As further
evidence,
4 and 2 U/assay
of HK and
GPD have been reported
to be optimal
(12). In another commercial
enzyme
system for the determination of CK-alleged
to be capable
of measuring
CK
activity
to 1500 U/liter
without
dilution
and in
which a modification
of the Oliver-Rosalki
procedure
is used-i
and 10 U, respectively,
of HK and GPD is
used per assay.
On the basis of the discrepancies
noted above we
believe the dilution
effect to be an artifact
created
by the overextension
of the linear range of coupledenzyme
systems
for measuring
CK activity.
This effect is usually
seen with the Oliver-Rosalki
method
when systems
are using insufficient
quantities
of the
auxiliary
enzymes.
In all reported
cases of the dilution effect observed
with CK, the enzyme
system of
Oliver and Rosalki
was the one used, with only one
exception.
In every case, where applicable,
the concentrations
of the auxiliary
enzymes,
HK and GPD,
were less than 1 U per assay. In the exception
noted,
Craig et a!. (4) reported
that the dilution
effect was
present
with coupled-enzyme
assays as well as with
the direct
colorimetric
method
of Hughes
(7). Our
results
by the direct method
of Siegel and Cohen, a
modification
of the Hughes
method,
show clearly
that there is no increase
in CK activity
upon dilution. Conceivably,
this lack of agreement
might be
because
they did not activate
CK with a thiol compound, inasmuch
as they make no mention
of its use.
The most conclusive
evidence
against the dilution
effect is its absence
with the direct method
over the
span of a 16-fold dilution
range. No increase
in CK
activity
was observed
with a serially
diluted
specimen having
a CK activity
exceeding
2000 U/liter.
With coupled
enzyme
systems,
evidence
is provided
that can be misconstrued
as a manifestation
of the
dilution
effect. Method
C showed a 74% increase
in
activity
for aliquots
of the same specimen
used in
the direct
methods.
Further
support
against
the
dilution
effect is provided
in Table 2, which shows
that by simply
increasing
the activity
of auxiliary
enzymes
it was possible,
simultaneously,
to increase
CK activity,
to lengthen
the linear
range
of the
assay, and to diminish
the dilution
effect. We conclude that the use of coupled-enzyme
systems,
particularly
those in which low concentrations
of the
auxiliary
enzymes,
HK and GPD are used, results in
an early deviation
from linearity.
The misuse of this
system by its application
to CK activities
above the
linear range creates the dilution effect.
We present
evidence
that illustrate
the basic differences
between
the direct and the indirect
coupledenzyme
assay methods
for the determination
of CK,
for the differences
noted
between
coupled-enzyme
assay methods,
and against
the persistent
dilution
effect of CK. Basically,
these results and conclusions
provide
another
case in support
contention
that the three-component
measurement
of enzyme
activity
aged; however,
if they are used,
indicator
(auxiliary)
enzymes
to
should be present (14).
We thank
Judy Smoke,
M.T.,
work was supported
in part by
of Bergmeyer’s
(13)
reaction
for the
should be discourthe proper ratio of
measured
enzymes
for technical
assistance.
USPHS
Training
Grant
This
GM
02049-03.
References
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kinase
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Vol. 19, No. 9, 1973
Clin.
997