Serum Creatine Phosphokinase: Evaluation of a

Commercial Spectrophotometric Method

JosephW. Hess, Roderick P. MacDonald, George J. W. Nafho,

and Kenneth J. Murdock

The clinical and laboratory applications of an assay method for serum creatine
phosphokinase (CPK) utilizing a reagent capsule were investigated. Continuous
recording of the change in absorbance at 340 mu, during the assay period gave a
slightly sigmoid curve. The time interval between serum addition and the beginning
of the linear portion of the absorbancecurve varied with the level of enzyme activity.
With samplesof low activity, up to 8 mm. elapsed before the linear phase began.
The rate of change in absorbanceincreased in a linear fashion with respect to in-
creasing CPK concentration, up to a level of about 370 U. When serum sampleswith
activity above this level were diluted and reassayed, and the data appropriately
corrected for dilution, significantly increased activity could be demonstrated in the
diluted samples.
Adenylate kinase from hemolyzed erythrocytes may interfere with measurement
of CPK activity in this assaysystem.This does not present a problem if samples with
visible hemolysisare excluded.
The range of normal human serum CPK values recommended for this method is
0-40 U./L. at 30#{176}.
Abnormal values in patients with injuries or diseasesof skeletal
muscle were as high as 55,000 U. After acute myocardial infarction, elevated serum
CPK levels were detected within 4 hr. after the onset of chest pain. In some of
these patients CPK activity did not return to normal for a week or more.
Properly used, this method provides a reliable and convenient assay procedure for
serum CPK activity.

lvi EASUREMENT of serum creatine phosphokinase (CPK, creatine

kinase, ATP-creatine phosphotransferase) activity is gaining increas-
ing acceptance as a laboratory procedure of value in the diagnosis of
disorders affecting skeletal and cardiac muscle and in family studies
of pseudohypertrophic muscular dystrophy (1-12). Non-muscular tis-

From the Department of Medicine, Wayne State University School of Medicine, Detroit,
Mich. 48207, and the Department of Laboratories, Harper Hospital, Detroit, Mich. 48201.
Supported in part by research grants from The Michigan Heart Association, Muscular
Dystrophy Associations of America, and Detroit General Hospital Research Corporation.
Received for publication Jan. 3, 1967; accepted for publication May 29, 1967.

994
Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 995

sues other than brain (9, 13-15) do not contain high levels of CPK;
therefore, this enzyme is more tissue-specific than widely distributed
enzymes such as the transaminases and dehydrogenases. Tile relatively
greater tissuespecificityof CPK makes itsmeasurement more valuable
clinicallythan that of the transaminases and dehydrogenases, the ones
commonly used to confirm the presence of myocardial and skeletal
muscle injury.
Several methods have been proposed for estimating serum CPK
activity. Each method gives different unit values, depending upon (1)
whether the “forward” or “reverse” action is used; (2) whether a
sulfhydryl compound is present in the reaction mixture; (3) the tem-
perature at which the enzyme activity is determined; (4) the technic
used to measure the rate of product formation of the primary reaction;
and (5) miscellaneous variables such as substrate and cofactor con-
centration, and pH.
We have evaluated a recently developed commercial reagemit capsule
method* for measuring creatine phosphokinase activity. This method
is based upon the spectrophotometric CPK assay procedure described
by Oliver (13).
Materials and Methods
In this procedure the “reverse” reaction of CPK is coupled to tile
indicator reaction as follows:
CPK
(1) Creaiine phosphate + ADP _______ Creatine + ATP

Hexokina8e
(2) ATP + Glucose ADP + Glucose-6-phosphate

G-6-PDH
(3) G-6-P + NADP+ ‘6-Phosphogluconate + NADPLI + H

The reduction of NADP+ per unit time is measured spectrophoto-

metrically at 340 m. The final concentration of substrates, emizymes,
and cofactors are as follows: Creatine phosphate, 1.7 mM; ADP, 2 mM;
NAPD, 4 mM; hexokinase, 1 U.t/3 ml.; U-6-P1)H, 1 U.t/3 nil.; glu-
cose, 10 mM; Mg SO4, 5 mM; cysteine, 4 mM; and tris buffer, 0.07 M, pH
7.0. Oliver reported that AMP, when present in concentrations five- to
tenfold greater than ADP, inhibits the adenylate kinase (myokinase)
activity usually encountered in muscle homogenates (13). The supplier
has included AMP 20 mM in the reagent mixture, thus theoretically
eliminating the necessity for a separate reagent blank.
The reagents were supplied dry in capsules, individually sealed in
*Avaimable as CPK Calsuls from Calbiocliem, Los Angeles, Calif.
tOne unit will catalyze the turnover of 1 gmole of substrate per minute at 300.
 a
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Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 997

foil containers with a desiccant. The contents of one capsule were

dissolved in 2.90 ml. distilled water in a cuvet (I-cm. light path). The
spectrophotometer was adjusted to give zero absorballce for distilled
water at 340 m. After the reagent solution was warmed to 30#{176}, 0.1 ml.
serum was added and the solution thoroughly mixed. The rate of change
in absorbance was determined as described below.
In this study, Beckman PU spectrophotometers attached to Gilford
Model 2000 multiple-sample absorbance recording units with Honeywell
or Heathkit strip chart recorders were used to follow the enzyme
activity graphically. Temperature was maintained at 30 ± 0.10 with
double thermospacers. The linear portion of the absorbance curve (see
below) was used to calculate the rate of change in absorbance ( A)
during a 5-mm. assay period.
Enzyme units for this assay are defined according to the general
recommendations of the International Union of Biochemistry (TUB):
One unit is that amount of CPK activitywhich resultsin the reduction
of 1 pmole of NA]JP per minute under the specifiedconditions (16).
One liter has been chosen as the unit volume. The increase in ab-
sorbance per 5 mm. is multiplied by 965 to convert A per 5 mm. to
units per liter.The constant was derived from the following formula:
10,000 (dilution factor) X 3 ml assay vol. - 965
6.22 X 5 (mm.) -

Results and Discussion

We have found that this CPK method gives reproducible results and
is technically simple to use. #{149}Similar
experience has been reported by
Rosalki (17). However, there are certain details which deserve atten-
tion when this method is used.
Characteristics of Reaction
This method is designed for use with any ultraviolet spectrophotom-
eter; however, a continuous recording of the change in absorbance
gives a sigmoid curve which may not be fullyappreciated ifthe reaction
curve is not monitored by means of a continuous recording. The ab-
sorbance curve may be divided into three phases: Phase A, an initial
lag period during which the reaction begins; Phase B, a period of
linear activity; and Phase C, a period of decremental slowing of ac-
tivity as conditions become suboptimal (Fig. 1).
The duration of Phase A is variable and is partially dependent on
the level of enzyme activity (Table 1).
In Table 1, serum from a patient with muscular dystrophy was as-
sayed for CPK activity at various dilutions to illustrate the influence
998 HESS ET AL. Clinical Chemistry

Table 1. EFFECT OF ENZY ME CONCENT RATI ON ON RESULT S OF CPK As SAY

U./L. Duration (mm.)

Corrected for
Serum concentration A/S mm. Uncorrected dilution Phase A Phase B

Undiluted 0.594 570 570 4.0 4

1:2 0.419 405 810 55 7
1:3 0.377 362 1086 7.5 6
1:3.5 0.348 334 1170 7.5 6
1:4 0.269 258 1032 7.5 10
1:8 0.141 135 1080 8.0 12

of the level of activity on the time of onset and duration of the linear
portion (Phase B) of the absorbance curve (far right column). The
effect of dilution of samples with high activity is shown in the center
columns.
In general, samples with high activity begin Phase B earlier than
samples with low activity. Six minutes after addition of serum only
19 of 31 samples with a 5-mm. i A of less than 0.350 had begun their
limmear phase. However, at 8 mm. all samples were in Phase B. Once
limmear, the reaction remained so for about 0.45 absorbance units. Thus,
the duration of the linear phase was dependent upon the rate of activity
as shown by the slope of the curve. For Phase B to last 5 mm. or more,
the slope should be no greater thami about 0.08 absorbance units per
minute.
Another consideration was the range over which the rate of change
in absorbance was linear with respect to the concentration of CPK. As
shown in Fig. 2, the linear relationship ends at a 5-mm. A of about
0.375 (370 1IJ./L.). The effect of diluting a serum sample with high
activity is shown imi Column 4, Table 1.
1mmview of the characteristics described above, if an absorbance
recording unit is not used, we recommend that an 8-mm. period, instead
of the 6-mimi. recommended in the supplier’s package insert, be allowed
to elapse between addition of serum and recording of the initial reading
for . A. Furthermore, samples with A/S miii. greater thami 0.350
absorbance units should be diluted and reassayed.
1mmperformimig enzyme assays some workers prefer noting elapsed
time per given absorbaimee interval rather than measuring absorbance
change per time interval. in our experience, measurement of absorbance
change per time interval with this procedure is the more convenient of
the two methods because serum from some normal human subjects has
extremely low CPK activity amid the time for performing the assay
would be unnecessarily prolonged if one waited for a predetermined
absorbance change to occur. There may be some special applications
Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 999

of the assay method, however, where a narrow range of activity may

be encountered and where measurement of elapsed time per given
absorbance change would be convenient.

Effect of Diluent
In a study with another method, in which the sample volume consti-
tuted one-third of the final volume, we found that saline diluent

.600

C .500
E
I’)
S.-

‘ci

.200

tOO

9
II .025 .050 .075 .100
SERUM (ml)
Fig. 2. Serial dilution of serum with high CPK activity. Note change in relationship of
enzyme activity to enzyme concentration above A/5 nun, of 0.3 75.

inhibited CPK activity (9). Noda et al. had previously reported inhibi-
tion of CPK activity by chloride (18). Comparative studies using the
present method have shown that serum samples diluted with distilled
water and with saline give essentiallyidentical results.The lack of
effect of saline dilueimt in this system can probably be attributed to a
30-fold dilution of the sample.

Effect of Hemolysis
Hemolyzed serum with hemoglobin levels above 50 mg./100 ml. (defi-
nitely visible hemolysis) tended to have lower CPK activity than the
same serum without hemolysis in the Tanzer-Gilvarg assay system (9).
We were therefore interested in observing the effect of hemolysis in
the present method. Distilled water and serum dilutions of a hypotonic
hemolysate were studied.
Packed fresh erythrocytes were washed twice in 20 vol. 0.85% NaCl;
2 ml. were then mixed with 6 ml. distilled water and allowed to stand
for 1 hr. at room temperature. After centrifugation (2000 g for 15 mm.)
1000 HESS ET AL. Clinical Chemistry

the supernatant was removed and used in the experiment. The hemo-
globin concentration was 4.6 gm./100 ml. Distilled water was used for
dilution of the hemolysate. Representative data obtained from this
series of experiments are shown in Table 2. The hemoglobin concentra-
tions in Column 2, Table 2, were calculated with the dilution factor.
At a hemoglobin concentration of approximately 70 mg./100 ml. the
enzyme activity of hemolysate diluted with water was equivalent to
9 U. Lower dilutions had progressively higher levels of activity. A
number of studies has shown that human erythrocytes do not have ap-
preciable CPK activity. Additional experiments with our own modifica-
tion of Oliver’s method, in which we could control the composition of
the reagent mixture, led us to the conclusion that the interfering enzyme
was erythrocyte adenylate kinase (myokinase).
This conclusion was based upon the following observations and theo-
retical considerations: (1) The reaction rate was linear for several
minutes, suggesting that the interfering factor was an enzyme and not
a substrate such as ATP. (2) All substrates and enzymes of the CPK
reagent mixture except creatine phosphate had to be present in order
for the reaction to proceed. Since the CPK and adenylate kinase (AK)
reactions may be interchanged as the first reaction of the three-stage
Oliver procedure, our observation suggested that the APP present in
the CPK reagent mixture was serving as substrate for AK. (3) We
obtained enzyme activity similar to that seen with hemolysate when
10 l. of a 1:1000 dilution of commercially available AK (Boehringer
and Soehne) was added to the reagent mixture. (4) The Embden-
Meyerhof pathway was excluded as a source for ATP formation in the
system, because time reaction was not stimulated by addition of 10 mM
fructose 1-6 diphosphate nor inhibited by the addition of arsenate. (5)
AMP (0.01 M) complete suppressed the blank activity of very dilute
solutions of hemolysate and partially inhibited the activity of more
concentrated hemolysates. As mentioned above, AMP is a known in-
hibitor of adenylate kinase. (6) Erythrocytes are known to have com-
paratively high levels of adenylate kinase activity (19).

Table 2. EFFECT 0 F HEMOLYSIS 0 N RATE OF NADP REDUCTIO N (CALSUL CPK METHOD)

Hemolysate

Dilution Hb. cone. (gm./100 ml.) A/mm.

1:2 2.30 0.175

1:4 1.15 0.147
1:16 0.29 0.035
1:64 0.07 0.009
1:128 0.04 0.002
Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 1001

The data in the right-hand column of Table 2 suggest that time effect
of hemolysis on blank activity in the method under investigation was
negligible below a hemoglobin concentration of 40-50 mg./100 ml. This
is approximately the hemoglobin level at which hemolysis becomes
visually detectable in serum samples. The data suggest that if care is
taken to exclude all hemolyzed serum samples, interference from eryth-
rocyte adenylate kinase need not become a problem in the clinical
laboratory.

Influence of Temperature
The difference in CPK activity determined on time same serum sample
at 25.0#{176},
27.0#{176},
and 30.0#{176}is shown in Table 3. The temperature of the
reagent solutions in the cuvet compartment was carefully monitored
and accurately maintaimmed at the teniperature specified (± 0.1#{176})
throughout the assay period. The formula for calculating per cent in-
crease in activity is as follows:
Units at higher temperature - Units at lower temperature_ 100
Units at lower temperature

The data show a 7-8% change in activity per centigrade degree.

We have found that approximately 15 mm. is required for solutions
in cuvets at 25.0#{176}
to equilibrate to 30.00 after being placed in the cuvet
compartment of the Beckman PU with double thermospacers. This lag
period is due to the relatively poor heat conduction in this system. The
period for temperature equilibration may be shortened by pre-warming
the distilled water, cuvets, and cuvet holder to 30#{176}.

Storage
Serum samples frozen at -18#{176}
for 1 or more weeks showed only
slight changes in enzyme activity. Data on the effect of storage are
shown in Table 4.

Table 3. EFFECT OF TEMPERATURE ON ENZYME ACTIVITY (MEANs OF DUPLICATE ASSAYS)

Sample Cuvet temperature C) CPK activity (U/LI Increase per decree

1 30.0 169
7.7%
27.0 137
7.1%
25.0 120
2 30.0 123
7.5%
27.0 102
7.3%
25.0 89
1002 HESS fT AL. Clinical Chemistry

Table 4. EFFECT OF STORAGE AT -18#{176}

ON CPK ACTIVITY IN SERUM

CPK activity (U. L.)

Diagnosis Days frozen Fresh serum Thawed serum Change (%)

Myocardial infarction 8 134 133 -0.7

Myocardial infarction 6 269 276 +2.6
Myocardial infarction 6 144 146 + 1 .4
Myocardial infarction 7 500 515 +3.0
Polymyositis 10 280 250 -10.7
Alcoholic myopathy 12 113 115 +1.5
Alcoholic myopathy 7 126 110 -12.7
Alcoholic myopathy 20 7700 7890 +2.5
Muscle trauma 13 226 210 -4.4

Reagent Standardization and Stability

A single serum sample was assayed 5 times on the same day, with
different shipments of two different lots of reagent capsules (Table 5).
All assays were performed within 1 hr. on Apr. 19, 1966. The most
recent shipments gave the highest values, with close agreement between
duplicate and triplicate determinations.
Reproducibilityof Assay
In order to provide an indication of the precision of the method, the
variation of 18 sets of duplicate serum CPK determinations performed
over a period of several days was analyzed, with a modification of the
procedure outlined by Henry and Dryer (20). The calculations were
based on the difference between duplicate samples (A and B) ex-
pressed as
A B x 100 = d. -

A + B
2
/ !d ‘\
The mean variation where N = the number of sets of
duplicates, was 5.6%. The stamidard deviation(s) obtained by

S
VN
was 2.4%, thus giving 95% linmits of ± 5%.

Table 5. COMPARISON OF CPK ACTIVITY OBTAINED WITH DIFFERENT REAGENT SHIPMENTS

AND SINGLE SERUM SAMPLE

Date Received Lot No. U./L./min.

Dec. 20, 1963 57080 178

Dec. 20, 1965 67106 180
Feb. 21, 1966 67106 161, 162
Mar. 14, 1966 67106 190
Apr. 11, 1966 57106 192, 194, 202
Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 1003

Frozen aliquots of 0.3-0.5 ml. of a “reference” serum sample with

moderately high CPK activity has provided a satisfactory means for
periodic checking of reproducibility.

Clinical Application
Normal Values
The data on serum CPK activity of normal subjects were analyzed
according to age and sex (Table 6). Frequency distribution was also
plotted and there was no significant skewing. Previous studies where a
sulfhydryl compound has been included in the reaction mixture have
shown slightly higher serum CPK values in males than in females
(21-23). Our data also show a slightly higher mean value for adult
males than for females but the range of values observed in samples
from the 2 groups was identical. Normal children below 6 years of age
have been reported to have higher values than older children and adults
(21). With the present method we did not find a clinically significant
difference in serum CPK values between children and adults. On the
basis of the above data, we recommend that a single normal range of
0-40 U./L. be applied to adults and children of both sexes when this
method is used at 30#{176}.

Abnormal Values
Table 7 shows the range of abnormal values which may be en-
countered. A significant finding which deserves emphasis because it is
often overlooked is that multiple intramuscular injections may give
rise to appreciable elevations of serum CPK activity. The highest value
shown here was obtained from a child who was receiving intramuscular
aqueous penicillin every 4 hr. for pneumonia. Generally, under these
circumstances children will show greater elevations than adults,
probably because of their smaller extracellular fluid volumes.
An example of the pattern of serum CPK elevation following acute
myocardial infarction confirmed by electrocardiography is shown in
Fig. 3. Data on serum glutamic oxaloacetic transaminase (GOT) and
lactic dehydrogenase (LDH) are included for comparative purposes.

Table 6. NORMAL SERUM CPK \ALITES (CALSUL METHOD, 30#{176})

.Vo. of Observed
Subjects sample., range .1! can ± S.D.

Adult males (18 yr. and older) 31 8-36 21 7-36

Adult females (18 yr. and older) 40 8-36 18 3-33
Both sexes (6-17 yr.) 15 6-36 16 0-39
Both sexes (0-6 yr.) 11 0-33 15 0-35
All 97 0-36 22 5-39
1004 HESS fT AL. Clinical Chemistry

Table 7. REPRESENTATIVE SERUM CPK VALUES ASSOCIATED WITH VARIOUS

CLINICAL CONDITIONS

CPK (U. L.)

No. of
Diagnosis patients Range Mean

Pseudohypertropic muscular dystrophy 12 97-1350 729

1\Iyocardial infarction 20 77-1400 382
Polymyositis 2 380-816 598
Acute myoglobinuria 2 1100-55000
Intramuscular penicillin injections
Adults, every 4 hr. 4 36-170 106
Adults, every 12 hr. 2 10-38 24
Children under 12, every 4 hr. 5 86-207 151
Children under 12, every 12 hr. 4 85-320 129
Auto accidents, children, ages 2-10 6 58-470 192
Gunshot wounds, adults 3 301-437 309
Active liver disease 8 2-32 16

0. B. 74 M
500- Anteroseptal Ml
C’,
I-
#{149}
CPK (Calsul)
z o GOT (Reitman-Frankel)
400 #{149}
LDH (MacDonald-
Simpson)
I-
0
300
0
C 2100
C
200 1750
C-) l400
1050 D
100 _7 -‘ =
(VU
40 3501
I I I I I I I I I I I
I’ 2 4 6 8 10 12
Chest DAYS
Pain
Fig. 3. Comparison of serum CPK, GOT, and LDH values in patient with acute myocardial
infarction.

We have found that the serum CPK activity after myocardial infarc-
tion may be elevated within 4-6 hr. after the onset of chest pain and
may remain elevated from 2 to 7 or 8 days. In general, we have observed
longer periods of elevation with this method than when the Tanzer-
Gilvarg procedure was used (9).
If carefully followed, this method is reliable and provides a relatively
simple means for estimating serum CPK activity.
Vol. 13, No. II, 1967 CREATINE PHOSPHOKINASE 1005

References
1. Ebashi, S., Toyokura, V., Momoi, H., and Sugata, H., High creatine phosphokinase ac-
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(1959).
2. Okinaka, S., Kumagal, H., Ebashi, S., Sugata, H., Momoi, H., Toyokura, V., and Fujie, V.,
Serum creatine phosphokinase activity in progressive muscular dystrophy and neuro-
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