Vol. 50, No. 1
Printed in U.S.A.
T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1968 by The Williams & Wilkins Co.
CREATINE PHOSPHOKINASE
A SPECTROPHOTOMETRIC METHOD WITH IMPROVED SENSITIVITY
JOSEPH W. HESS, M.D., KENNETH J. MTJRDOCK, B.S., AND GEORGE J. W. NATHO, B.S.
Department of Medicine, Wayne Stale University School of Medicine, Detroit, Michigan 48®07
A number of methods have been described
for measurement of creatine phosphokinase
(CPK) (creatine kinase, adenosine triphosphate (ATP)-creatine phosphotransferase)
activity. They include colorimetric10'12 and
fluorometric 2 ' 20 methods which measure
the amount of creatine or creatine phosphate
formed per unit time, a titrimetric method
which measures the rate of hydrogen ion
liberation as ATP is converted to adenosine
diphosphate (ADP), 1 ' 14 and spectrophotometric methods which require auxiliary
reactions to link the CPK reaction to oxidation of reduced nicotinamide adenine
dinucleotide (NADH) 22 or reduction of
nicotinamide adenine dinucleotide phosphate
(NADP+).18 An advantage of spectrophotometric methods is that the enzyme reaction
rate may be followed directly during the
period of incubation.
The modified procedure described in this
paper was developed initially to provide a
rapid, sensitive means for assaying tissue
CPK activity. It has also proved to be
useful for measuring serum CPK activity.
This method combines features of several
previously described spectrophotometric
methods 6 ' 15 . 18 . 21 with some additional modifications.
A T P + glucose ?
hexokinase
(2)
ADP + G-6-P
G-6-P + NADP -
G-6-PDH
6-phosphogluconic acid + NADPH 2
(3)
where G-6-P = glucose 6-phosphate and G-6PDH = glucose 6-phosphate dehydrogenase.
A Beckman DU spectrophotometer*
equipped with a Gilford Model 2000 multiple sample absorbance recording unitf
with a Heathkit Model EUW 20-A strip
chart recorder! was used to record absorbance. Cuvette compartment temperature was maintained at 30 ± 0.1 C. with
double thermospacers.
Enzymes and substrates were obtained
from Calbiochem (Los Angeles, Calif.) and
from Sigma Chemical Company (St. Louis,
Mo.).
Reagents
Buffer solutions.
Buffer I. Triethanolamine (TEA) 0.05 M,
pH 6.9.
Buffer II. TEA pH 7.S, 0.05 M (for dissolving creatine phosphate).
Other reagents. See Table 1.
MATERIALS AND METHODS
The basic method was first described by
Oliver.18 The "reverse" reaction of CPK
is coupled with two additional enzyme reactions to permit spectrophotometric measurement of the rate of reduction of NADP+
at 340 m/i.
CPK
ADP + creatine phosphate ,
(1)
ATP + creatine
Procedure
Reagents 1 through 6 were mixed in the
sequence given in Table 1. Microliter
pipettes were used for measurement of all
volumes of 0.1 ml. or less. The reaction
mixture was kept at 4 C. until immediately
prior to use when it was warmed to 30 C. in a
water bath. Reproducible results could be
obtained for at least 4 hr. after preparation.
After adjusting the spectrophotometer slit
Received August 24, 1967.
This study was supported in part by research
grants from the Michigan Heart Association, the
Muscular Dystrophy Association of America, and
the Detroit General Hospital Research Corporation.
* Beckman Instruments, Inc., Fullerton, Calif,
f Gilford Instrument Laboratories, Inc., Oberlin,
Ohio.
| Heath Company, Benton Harbor, Mich.
89
90
HESS ET
Vol. 50
AL.
TABLE 1
COMPOSITION OF REACTION M I X T U R E FOB C P K
Reagent
PROCEDURE
Stock Reagent Concentration
Aliquot
Final Cuvette
Concentration
ml.
1. Buffered glucosemagnesium solution, pl-l 6.9
TEA
Glucose
Magnesium acetate
2. Cysteine HCI-H-X)*- t
3. A D P - 3 H 2 0
Monosodiumf
4. N A D P - 4 H 2 0
Monosodiumf' f.
5. G-6-PD
6. Hoxokinase
Total volume
7. Creatine phosphate disodium saltf
9.40
7.5 Gm. ( 6 . 6 m l . ) / l .
4.0 G m . / l .
2.4 G m . / l .
110 mg./ml. of Buffer I
33 mg./ml. of Buffer I
0.20
0.20
0.05 M
0.02 M
0.01 M
0.01 M
0.001 M
42 mg./ml. of Buffer I
0.20
0.0008 M
0.01
0.01
10.02
0.5 U./ml.
1 U./ml.
500 units§/ml. in 2.4 M (NH4)2SO-4
1300 units§/ml. in 2.4 M (NH 4 ) 2 S0 4
69 mg./ml. of Buffer If
0.020 M
* Readjust pH to 6.9 by adding 1 or 2 drops of T E A following addition of cysteine to buffered glucose
magnesium solution.
f Stored in freezer.
f We have tried one batch of the lithium salt of A D P and found t h a t the C P K activity measured
with this reagent mixture was about 50% of t h a t obtained when the sodium salt was used. We therefore recommend t h a t the lithium salt not be used without preliminary experiments to compare results
with those obtained with the sodium salt.
§ One unit, 1 /iM s u b s t r a t e converted per min. 25 C.
width to near 0 absorbance, using distilled
water as a blank, the steps outlined in
Table 2 were followed. The method may be
used with 1- or 3-ml. cuvettes, although
we have routinely used the smaller size for
reasons of economy. Sample aliquots may
be 0.1 or 0.01 of the final cuvette volume.
For serum analyses we used 10-jul. samples,
and for tissue homogenates we used 100-jul.
samples in total assay volumes of 1.0 ml.
The reasons for these choices are detailed
below. Unless otherwise specified, all of
the serum assays described in this paper
were performed with 10-jul. samples.
A reference serum with moderately elevated CPK activity was included in each
day's analyses as a check on reproducibility.
Reagents and technic were re-evaluated
whenever variation greater than 10% from
previous results was noted.
the formation of 1 /zM of NADPH 2 per
min.5 One liter was used as the standard
unit volume for serum assay. To correct the
absorbance data to units, AAi/5 min.(blank
activity) was subtracted from AA 2 /5 min.
to give the net AA/5 min. The formula for
conversion of net AA/5 min. to units
is as follows:
Enzyme units
Net AA/5 min.
X conversion factor = units per 1.
One unit of CPK activity is defined as
that, amount of activity which will lead to
Net AA/5 min. X dilution factor
(10 or 100) X 1000 (converts ml. tol.)
6.22 (/xM extinction coefficient
forNADP) X 5 min.
= jiM NADPH 2 formed per min.
per I. of sample
The constants in the formula may be reduced
to a single factor for converting net AA/5
min. to units per liter:
The appropriate conversion factors are given
C H E A T I N E P H O S P H O KINASE
TABLE 2
PROCEDURE SEQUENCE AND CONVERSION FACTORS*
l-ml. Cuvettes
1.
2.
3.
4.
5.
0.90
Reaction mixture (Table 1)
0.01
Sample
Record AA f o r 5 m i n . (blank ictivity or A/ti)
0.10
Add creatine phosphatef
Record AA for 5 min. (A.42)
1.01
Total volume
3200
Conversion factor
3-ml. Cuvettes
ml.
ml.
0.80 ml.
0.10 ml.
2.70 ml.
0.03 ml.
2.40 ml.
0.30 ml.
ml.
0.10 ml.
0.30 ml.
0.30 ml.
ml.
1.00 ml.
320
3.03 ml.
3200
3.00 ml.
320
* T h e table shows the steps in the procedure after preparation and temperature equilibration of the
reaction mixture. Modifications permitting use of 1- and 3-ml. cuvettes and sample volumes 0.01 and
0.10 of the final volume are shown. For application of conversion factors, see section on enzyme units
under "Materials and M e t h o d s " in the text.
f There may be a lag period of 1 to 2 min. between addition of s u b s t r a t e and the development of a
constant reaction rate. If an absorbance recorder is not used, allow 3 min. to elapse between s u b s t r a t e
addition and the initial absorbance reading for AA2 . Separate cuvettes for simultaneous measurement
of blank and of C P K activity may be prepared if desired.
on the bottom line of Table 2. For tissue
assay the formula must be altered to take
into account the appropriate tissue dilution
factors.
R E S U L T S AND
DISCUSSION
A ssay Conditions
Buffers and pH. CPK activity was compared in reaction mixtures buffered with
TEA or tris-maleate buffers over a pH
range of 5.0 to 8.0. Fifteen to 20% more activity was consistently observed in the TEAbuffered reactions at all points between pH
5.5 and S.0. Maximal CPK activity with
TEA buffer was observed at pH 5.8, but
buffer capacity was unsatisfactory below
pH G.5. The standard selected for this
method was pH 6.9 because it was well
within the range of adequate buffer capacity
and enzyme activity was S0% of maximum.
Substrate, enzyme, and co-factor concentrations. The enzyme activity with various
concentrations of creatine phosphate (CP)
was determined and the results are plotted
in Figure 1. The Michaelis constant (Km) for
this reaction calculated from a LineweaverBurke plot was 2,5 X 10~3 M. This Km
is close to the values which may be calculated from the report of Conn and Anido2
for their fluorometric method (3.S X 10 -3 M)
and Nielsen and Ludvigsen's published
*
8
Creoline
12
16
20
21
28
32
36
Phosphole Concentration {mmoles)
F I G . 1. Enzyme activity versus final s u b s t r a t e
concentration. Conditions were the same as in
Table 2 except for variations in creatine phosphate
concentration.
modification15 of Oliver's method18 (4.0 X
10- 3 M). A final CP concentration of 0.020 M
was selected as the standard concentration
for this procedure.
Additional studies showed that the concentrations of ADP, glucose, NADP, hexokinase, and G-6-PDH shown in Table 1
were also optimal for this system.
Effect of sulfhydryl (/roups. Beginning with
Ennor and Rosenberg4 a number of workers
have observed an activating effect of
sulfhydryl (SH) reagents on CPK activity. 10 ' "• 1 4 ' 1 7 ' 1 S Cysteine was chosen for the
present method. The effect of various concentrations of cysteine on the reaction rate
of the present system is shown in Table 3.
92
HESS ET AL.
TABLE 3
EFFECT
OF V A R I O U S CONCENTRATIONS OF
C Y S T E I N E ON C P K
ACTIVITY*
Final Cysteine Concentration
Net 4/1/5 Min.
M/l.
0.071
0.2G2
0.316
0.342
0.338
0.320
0
0.001
0.005
0.010
0.020
0.040
* I n this experiment, the routine procedure was
followed except for variation of cysteine concentration. The enzyme source was serum from a patient receiving intramuscular injections of penicillin every 4 hr. (100 pi. serum sample).
TABLE 4
COMPARISON OF E F F E C T OF V A R I O U S S I I R E A G E N T S
ON SERUM C P K
Compound
None
Cysteine
Mercaptoethanol
Glutathione
Dithiothreitol
Dithiothreitol
ACTIVITY
Final
Concentration
CPK Activity*
M/l.
unils/l.
0.01
0.01
0.01
0.01
0.005
35
170
160
100
167
177
* One unit is denned as 1 /xM of s u b s t r a t e converted per minute a t 25 C.
In contrast to the 5-fold increase in serum,
the CPK activity of rat skeletal muscle
homogenates was increased approximately
2-fold by 0.01 M cysteine.
To compare the effect of various SH
reagents on the present assay system, an
experiment was performed using a single
serum sample without and with 0.01 M
concentrations of cysteine, mercaptoethanol,
glutathione, and dithiothreitol (DTT). Since
D T T has two SH groups per molecule, a
concentration of 0.005 M was also used.
All of the SH compounds tested enhanced
the CPK activity approximately 5-fold
(Table 4).
Effect of adenosine monophosphate (AMP).
Myokinase (adenylate kinase) acts upon
ADP in this assay system to form ATP
and AMP. Thus, myokinase from erythro-
Vol. 50
cytes, muscle, or other sources may form
ATP and activate the auxiliary enzyme
systems to give the "blank" activity seen
prior to addition of creatine phosphate.
Oliver reported that AMP 10-fold in excess
of ADP would inhibit myokinase activity. 18
We were interested to learn whether AMP
would also have an effect on CPK as measured in this system. The results, part of
which are illustrated in Table 5, show that,
whereas AMP did indeed reduce blank
activity to negligible levels, it also reduced
CPK activity up to 34%. AMP was therefore excluded from the routine assay procedure.
Range of linear response. The range over
which AA had a linear relation to the concentration of CPK was estimated by plotting
the AA/5 min. versus the proportion of
serum present in 100- and 10-^1. aliquots
of serial water dilutions of serum samples
with high CPK activity. The results are
shown in Figure 2. The AA/5 min. increased
linearly with increasing concentrations of
enzyme to 1.40 absorbance units per 5 min.
when 10-jul. samples were used, and to 0.40
absorbance unit when 100-/*1. samples were
used. Thus, the smaller sample volume was
associated with a more extensive range of
linearity.
That simple reagent dilution was not the
cause of the difference in ranges noted in
Figure 2 was shown by demonstrating that
10% dilution of the reaction mixture with
water did not change the results obtained
with a 10-jul. sample of serum. This observation suggested that serum inhibitors (chloride and other ions,13 and perhaps other
factors) or counteracting enzyme reactions
may have been present to a significant degree when serum comprised 10 % of the total
assay volume. Further evidence of an inhibitory effect of serum factors was provided
by a comparison of duplicate determinations
of CPK activity on a series of serum samples using 100- and 10-/*1. aliquots of the
same serum (Table 6). These sera all had
CPK activity which gave AA/5 min. of
less than 0.400 unit with 100-jul. samples.
Lower values were consistently observed
with the 100-/ul. samples. Furthermore,
there was not a consistent mathematical
relationship between the values obtained
July 1968
93
CREA TINE PH OSPHOKJ NA SE
TABLE 5
E F F E C T OF AMP
ON SEBUM C P K
ACTIVITY*
A/1/5 Min.
Serum
Sample Volume
Before CP
Net A/1/5 Min.
Inhibition
Afte r CP
+ AMP
-AMP
+AMP
-AMP
+AMP
0.054
0.044
0.011
0.007
0.014
0.000
0.004
0.000
0.000
0.000
0.508
0.476
0.206
0.075
0.069
0.390
0.352
0.131
0.045
0.041
%
riA
B
C
D
E
im
100
10
10
10
0.454
0.432
0.195
0.068
0.055
0.390
0.34S
0.131
0.045
0.041
14
20
33
34
25
* Means of duplicate analyses using the routine procedure with and without AMP, 0.01 M. N o t e
inhibition of blank activity (before CP) as well as the inhibition of C P K activity by AMP.
TABLE 6
COMPARISON OF R E S U L T S USING 10- AND 100-^1.
SICUUM SAMPLES
CPK Activity
10,il.
IOOMI.
units/l.
30
40
%
50
SERUM
IN
60
70
80
9C
SAMPLE
F I G . 2. Enzyme activity plotted versus serum
concentration in serial water dilutions of sera with
high C P K activity to illustrate influence of sample size on range of linear response. Different sera
were used for tlie 10- and 100-MI- sample curves.
with 10-yul. samples and those obtained with
100-^1. samples. This was not the case
when assaying dilute muscle homogenates
where the number of unknown variables in
the sample could be more adequately controlled. More is said about this problem
below.
Precision of the method. Analysis of the
precision of the method was performed on a
series of 40 duplicate determinations on
serum samples with CPK activity greater
than 100 units. These assays were performed
over a period of more than 1 year. The formula
S.D.
•
/
N
1
2
3
4
5
6
7
8
9
10
45
71
58
109
29
64
80
83
48
35
22
47
30
44
10
20
42
22
16
13
d, is the percentage of difference of each
sample from the mean of the duplicates,
and N is the number of sets of duplicates.
Per cent difference rather than difference
in units was used for d because of the wide
range over which serum CPK values vary.
According to this calculation, the standard
deviation was 2.7%.
Influence of sample pipettes on results. We
were aware that the sensitivity of this assay
system would accentuate small differences
in sample volumes. Microliter pipettes from
two different suppliers were on hand in our
laboratory, although we have used one type
of pipette* for nearly all of our work. We
as described by Henry and Dryer 7 was
u s e d , w h e r e S . D . is t h e s t a n d a r d d e v i a t i o n ,
* Scientific Industries, Inc., Springfield, Mass.
94
H E S S ET
compared data from six consecutive assays
(separate 10-JKI. pipettes were used for each
sample) with each brand of pipette using a
single serum sample. The results showed a
mean value of 1070 units (range 1040 to
1117) when the serum samples were measured with Accupettesf and 1210 units
(range 116S to 1242) when samples were
measured with Speedipets,* a 13% difference between mean values (p < 0.05).
These results illustrate that one is not safe
in assuming volume consistency between
the microliter pipettes of various manufacturers, and that precision may be affected
if sample volume is not carefully controlled.
Enzyme stability. Total CPK activity of
serum as measured by this method was
stable for periods of 1 week or more when
sera were stored in the refrigerator at 4 C.
or frozen at —IS C. The presence of a
sulfhydryl reagent is of prime importance.17
Experience with a commercial method which
also includes cysteine has given similar
results.9
Application of Method
Normal human serum CPK activity. Sera
from 120 adult subjects (laboratory personnel and blood bank donors) were analyzed to ascertain the range of normal
values. The subjects were screened for
history of active heart or skeletal muscle
disease, trauma or strenuous exercise within
the preceding 72 hr., or recent intramuscular injections. Additional sera from 28
healthy children, age 13 and younger, who
were attending the Pediatrics Clinic, were
analyzed to obtain data on a younger age
group. Data from male and female adults
and children were analyzed separately.
Normal equivalent deviate (N.E.D.) plots
of data on both males and females gave a
linear distribution of values when units
were plotted versus N.E.D. The mean and
the range for each group were taken directly
from the N.E.D. versus units plot as described by Henry and Dryer. 7 There was an
appreciable difference in the means and the
normal ranges of adult males and females.
Inasmuch as the data on male and female
f Scientific Products, E v a n s t o n , 111.
Vol. 50
AL.
TABLE 7
NORMAL HUMAN SIOKUM C P K
ACTIVITV
CPK Activity
Group
No.
Mean
Age
Age
Range
Mean
y-
80
Male
adults
40
Female
adults
17
Male
children
11
Female
children
28
Children
both
sexes
* % . , Observed
Limits
Range
units/1
33
18-59
71
0-150
10-192
38
18-70
48
0-110
10-110
7.4
1-13
74
10-110
8.3
1-13
69
13-99
7.9
1-13
74
2-148
10-110
children showed essentially the same distribution, these data were pooled for determination of the range of normal values.
Data on the various groups are shown in
Table 7. Only one observed value (adult
male, CPK activity 192 units) fell outside
of the range of normal as defined by the
N.E.D. plot.
Serum CPK activity was plotted versus
age for each group. Except for slightly
higher values in 1- and 2-year-old male and
female children, there was no consistent
correlation between age and level of CPK
activity.
Muscle homogenate analyses. The present
method has been used for measuring CPK
activity of muscle homogenates or of fractions thereof. Very dilute homogenates were
required to bring the activity within the
linear portion of the assay system. For
normal rat muscle, 1:5,000 dilution (w/v)
was necessary for a 10-M1- sample and
1:50,000 dilution for a 100-/il. sample.
(We used 0.03 M phosphate-buffered 0.025
M sucrose, pH 7.4, for homogenate preparation and dilution.) There was little or
no blank activity at these dilutions and, in
contrast to serum, the results obtained
with 10- and 100-/^1. samples were quite
comparable when corrected for dilution
differences. The larger (100-/ul.) pipettes
may be preferable for work with some types
of tissue homogenates because they are
July
I96S
CHEATINE PHOSPHOKINASE
95
TABLE 8
COMPARISON
OF C P K
ACTIVITY
USING
VARIOUS
COMMERCIALLY
AVAILAIILE M E T H O D S
ANU THE
AUTHORS' METHOD
Method
Boehringer and Soehne*
Boehringer and Soehne
Sigma 40, UVJ
Sigma 060
Sigma 520
Calbiochem, Calsul§
Authors (noncommercial)
Original Author
Tanzer and Gilvarg 22
Tanzer and Gilvarg
Tanzer and Gilvarg
K u b y etal.12
Hughes 1 0
Oliver 18
Oliver
Direction of Reaction
Forward
Forward
Forward
Forward
Reverse
Reverse
Reverse
SH Reagent
No
Yesf
Yes
Yes
Yes
Yes
Yes
Unit/I. 30 C.
34
245
330
245
312
1190
3200
* Boehringer unci Soehne G m b H , Mannheim, Germany.
t Cysteine 0.01 M, final concentration, added to Boehringer & Soehne reaction mixture. The sample
used for this comparison was a 1'.5 water dilution of fresh serum from a patient with Duchenne type
muscular dystrophy. The unit values have been corrected for dilution. All analyses were completed
within a period of 0 hr. and all results are expressed in the units defined in the text.
X Sigma Chemical Company, St. Louis, Mo.
§ Calbiochem, Los Angeles, Calif.
less likely to become plugged with tissue
particles.
CPK activity in the range of 3000 to
4000 units per Cm. wet weight has been
measured in fresh, normal, young rat psoas
muscle with this procedure. This figure compares with 430 units per Gm. wet weight
from. Oliver's data on rat muscle with his
original method,18 and S00 units per Gm. wet
weight from the data of Tanzer and
Gilvarg.22
Comparison -with other methods. In order to
provide a basis for comparing the results
obtained by the method here described with
those of several commercially available
reagent kit methods, CPK activity of a
single serum sample was assayed using the
different procedures. The results, converted
to micromoles of substrate per minute per
liter, are shown in Table S.
A summary of normal human serum CPK
values obtained with the present method
and those reported for several previously
published methods is shown in Table 9.
The unit values from all studies have been
converted to the unit system described in
this paper. Table 9 illustrates the influence
of methodology on the level of activity
measured in apparently normal adults and
indicates some of the variables which influence results. Differences in methodology
may be accentuated when measuring CPK
activity of abnormal sera, as is evident
from the data presented in Table S.
' Isoenzyme analysis. The reaction mixture as described in Table 1 has been
modified and successfully used for identifying isoenzymes of CPK following electrophoresis. When the mixture was used
for this purpose, cysteine was omitted
(cysteine reduces the dye immediately)
and 1 ml. of phenazine methosulfate (1
mg. per ml.) and 6 mg. of dry nitroblue tetrazolium were added per 10 ml. of
reaction mixture. The reaction mixture was
divided into two parts and creatine phosphate was added to only one portion in
order to provide solutions for both blank
and CPK activity. Cellulose acetate strips
were moistened either in the "blank" or
"CPK" solutions and were laid over previously electrophoresed tissue extracts (cellulose acetate strips, barbital buffer 0.075 n,
pH S.6, 25 v/cm.) for 45 min. Color development was allowed to proceed in a moist
chamber at 37 C. for 1 hr. Blue bands appeared where NADPH 2 formation had occurred. CPK isoenzyme bands, three dark
and one faint, were identified from 1:10
homogenates of the left ventricular myocardium of a rat. Homogenates of the lateral
(lightest colored) portion of rat psoas gave
one dark and one faint band. When both red
and white psoas were homogenized, one
TABLE 9
Direction of
Reaction
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Reverse
Forward
Forward
Forward
Forward
Method
Spectrophotometry
Spectrophotometry
Spectrophotometry
Spectrophotometry
Fluorometrie
Fluorometric
Colorimetric
Colorimetric
Spectrophotometry
Spectrophotometric
Colorimetric
Colorimetric
Authors
Present study
Hess el al.9
Nielson andLudvigscm 1 5
Fleischer el al.s
Sax and Moore 2 0
Conn and Anido 2
Hughes 1 0
Dreyfus el al.3
Richterich el al.n
Hess el al.s
Okinaka el al."
N u t t a l and W e d i n "
6.9
7.0
6.75
7.0
6.8
6.8
7.4
7.4
9.0
9.0
9.0
9.0
pH
30
30
30
37.5
25
37
37
38
37
25
38
37
C.
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
SulfT e m p . hydryl
Reagent
COMPARISON OF N O R M A L A D U L T H U M A N SKRUM C P K
Range
11
0.3
33
95
0-0.8
0-35
5-39
substrale/min./
1. serum
22
ju.l/
Mean
97
No.
B o t h sexes combined
29
189
19
47
22
30
22
80
No.
9.5
17.6
0.8
6.5
29.8
27.2
73.8
37.8
71
tiM
Mean
Range
4.3-20.1
6-42
0-3.8
23-18
28-32
13-57
29-190
13-03
0-150
substrale/min./
1 serum
Males
Mean and R a n g e (95% Limits
ACTIVITY R E P O R T E D BY V A R I O U S A U T H O R S
19
116
21
50
18
31
28
40
No.
9.0
12.8
0.4
3.8
22.5
25.8
58.4
27.7
48
fx.\I
Mean
Range
3.5-23
6-25
0-3.3
0.9-16
21-24
13-52
31-108
6-49
0-110
substrate/min./
1. serum
Femal es
o
July 1968
CREATINE
PHOSPHOKINASE
dark and two faint bands usually appeared.
The soleus gave one dark and one faint band.
SUMMARY
A sensitive spectrophotometric method for
estimation of creatine phosphokinase activity of body fluids and tissue fractions is
described. The assay procedure uses the
reverse reaction (creatinine phosphate —>
creatine) with two auxiliary reactions at pH
6.9 in the presence of cysteine. A sample
volume 0.01 of the final assay volume is
preferred for serum assays. Sample volumes
0.10 or 0.01 of the final volume may be used
for assay of dilute muscle homogenates.
Normal human serum CPK activity was:
adult males, 0 to 150 units (mean 71);
adult females, 0 to 100 units (mean 58);
and children, 2 to 148 units (mean 74).
CPK activity of rat psoas muscle was 3000
to 4000 units per Gm. wet weight. The
reagent mixture may be easily adapted for
isoenzyme analyses.
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