CLIN. CIEM. 23/7,1329-1332(1977)
Creatine Kinase Isoenzyme Activity in Human Placenta and in
Serum of Women in Labor
Henry M. Laboda,’ and Venera J. Britton
Creatine kinase (EC 2.7.3.2) isoenzymes in extracts of
Materials and Methods
human placenta and in serum from nonpregnant women
Tissue and Serum Preparation
and women inlabor were separated on columnscontaining
Human heart and brain tissue were obtained at autopsy and
diethylaminoethyl-cellulose
and assayed. The distribution
human placenta specimens were obtained immediately
after
oftheisoenzymesin placenta (n = 10) was 80% BB (200
± 66 U/g (wet weight),19% MM (49 ± 30 U/g), and 1%
MB (2.6 ± 1.7 U/g). The geomefric mean for the serum BB
activity of the nonpregnant women (n = 50) was 0.6 ± 1.5
U/liter, as compared to 3.0 ± 1.4 U/liter for patients in
labor who had normal deliveries (n = 92). The arithmetic
mean for serum BB activity of labor patients with induced
labor (n = 20), premature labor (n = 7), cesariansection
(n = 6), or hypertension and pre-eclampsia (n = 6) did not
differ significantly from the arithmetic mean BB activity for
serum of labor patients with normal deliveries. However,
the arithmetic mean serum BB activity of patients with
stillbirths (n = 7) was significantly smaller than the arithmetic mean for normal labor patients.
Keyphrases
effect on serum creatine kinase activity
(and isoenzymes) of pregnancy and its complications,
fetal
status
Addftlonal
Activities
of creatine kinase (CK; EC 2.7.3.2) isoenzymes
have been measured after separation by anion-exchange
col-
umn chromatography.
2) and DEAE-cellulose
Although both DEAE-Sephadex (1,
(3, 4) have been used to separate CK
isoenzymes, we found that Nealon and Henderson’s (3, 4)
DEAE-cellulose
method gave better separation of the CK
isoenzymes,especiallyof the BB form. We subsequently used
this method to separate CK isoenzymes in human placenta
and in serum of women in labor.
Human placenta reportedly possesses CK activity of about
130 U/g (5). Tsung (6), using DEAE-Sephadex chromatography to separate placental creatine kinase isoenzymes, found
46-63% of the enzyme in the BB form, 6-22% in the MB form,
and 15-48% in the MM form. Because the BB form appears
to be the predominant
isoenzyme in the placenta, it was of
interest to compare values for serum BB in controls with those
for patientsduring labor.Ifserum CK BB isoenzyme varies
during pregnancy, it might be possibleto use such assays to
help monitor the status of the fetus during pregnancy. We
report here the measurement
of serum CK isoenzymes from
labor patients as compared to a nonpregnant
control group.
Department
of Medical Technology, College of Allied Health
Professions, Temple University,
Philadelphia,
Pa. 19140.
Current
address: Department
of Biochemistry,
Hahnemann
Medical College, Philadelphia,
Pa. 19102.
Received Jan. 12, 1977; accepted April 18, 1977.
samples were prepared as follows.We homogenized a given number of grams (wet weight) of tissue with
an equal number of milliliters of 50 mrnol/liter tnis(hydroxymethyl)methylamine
hydrochloride buffer (pH 7.5) containing, per liter, 500 mmol of sucrose, 2.5 mmol of magnesium
delivery.Enzyme
chloride, and 4 mmol of acetyl cysteine. The homogenate was
centrifuged
(100 000 X g, 60 mm, 4 #{176}C),
and portions of the
supernates from heart and brain were added to pooled human
serum to produce a serum control containing
all three CK
isoenzymes. This control was used in subsequent chromato-
graphic experiments. The placenta extracts were analyzed for
CK isoenzymes within 24 h.
specimens were obtained by venipuncture
from aphealthy,
nonpregnant
women within
the childbearing ages of 14 to 40 years, and from pregnant women who
were in labor. The blood specimens were centrifuged, the sera
removed, and 20 ,l of 2-mercaptoethanol
was added to about
2 ml of each serum, which then was stored at 4 #{176}C
and analyzed within 72 h.
Blood
parently
Electrophoresis
on Agarose Gel
Sample volumes of 1 to 2 zl were applied
(Corning-AC!,
on agarose films
Palo Alto, Calif. 94306) and electrophoresed
at 4 #{176}C
in sodium
barbital
buffer
(50 mmol/liter,
pH 8.0)for
20 mm at 170 V. After electrophoresis the films were layered
with CK substrate (Corning-AC!),2 incubated at 37 #{176}C
for 20
mm, and dried at 100-140 #{176}C
for10 to 20 mm. CK activity in
the electropherograms
was detected
under
ultraviolet
light
at 360 nm.
lsoenzyme
Separation
on DEAE-Cellulose
CK isoenzymes were separated on DEAE-cellulose
as described by Nealon and Henderson
(3, 4). Dithiothreitol(5
mmol/liter)
was added to portions of the MM-, MB-, and
BB-eluting buffers before use. The DEAE-cellulose (Cellex
D, DEAE; Bio-Rad Laboratories, Richmond, Calif. 94804;
exchange capacity, 0.70 mEq/g) was equilibrated
in the
MM-eluting
buffer for 24 h. The cellulosewas then washed
twice with the MM-eluting
buffer and poured into a column
2
Containing
ADP, MgCI2, L-cysteine,
glucose, creatine
phosphate,
NAD+, hexokinase, and glucose-6-phosphate dehydrogenase, in unspecified amounts.
CLINICAL CHEMISTRY, Vol. 23, No. 7, 1977
1329
Table 1. Creatine Kinase Isoenzyme Activity of
Human Placenta8
Crsatlne
P.rc.nt
kin...
i.o.nzym.
SD
acty,U/ge’
MM
MB
47.8
2.6
BB
Total CK
29.6
ci
tot&
1.7
19
1
200.2
66.2
80
250.8
62.0
100
#{149}
CK Isoenzyme activIty per gram of human placenta,
average of data on 10
specimens.
b
Wet wt.
Table 2. ArIthmetic and Geometric Means of
Serum MB and BB Creatine Kinase Isoenzymes
for Nonpregnant Women and Women in Labor
with Normal Deliveries
Activity
MB
drained, 0.25 ml of specimen, with a CK activityof no more
than 600 U/liter, was added and allowed to enter the cellulose
bed. The CK isoenzymes were eluted from the cellulose as
described by Nealon and Henderson
(3,4). We evaluated the
reproducibility of the DEAE-cellulose separation
by applying
control sera with high, medium, and low total CK activities
onto 10 DEAE-cellulose
columns. (The control sera were
prepared as described and so contained all three CK isoen-
zymes.) Overall, the results confirm that column chromatography on DEAE-cellulose reproducibly separates CK isoenzymes. For example, the means and coefficients of variations
(CV) of the BB isoenzyme activities in the control sera with
high, medium, and low totalCK activity were, respectively:
8.4 ± 0.3U/liter,
CV = 3.6%; 5.0 ± 0.3 U/liter, CV = 6.0%; and
2.8 ± 0.3 U/liter, CV = 10.7%.
Arithmetic mean
SD
mean
activitywas measured by the following procedure, a
modification
of the method of Oliver (7) and Rosaiki (8). The
final assay mixture (including the enzyme sample) consisted
of a 50 mmol/liter tris(hydroxymethyl)methylamine-histidine
buffer, pH 6.7, containing, per liter, 1.0 mmol of ADP, 5.0
mmol of magnesium acetate, 1.6 mmol of NAD+, 20.0 mmol
of 2-mercaptoethanol, 3.7 mmol of AMP, 20.0 mmol of creatine phosphate, and, per milliliter, about 0.5 U each of hexokinase (EC 2.7.1.1, from yeast) and glucose-6-phosphate
dehydrogenase (EC 1.1.1.49, from Leuconostoc mesteraides).
The reaction mixtures were pre-incubated
with the enzyme
sample at 30 #{176}C
for5 to 7 mm before the change of absorbance
of NADH was measured at 340 nm with a Gilford Model
2400-40 recording spectrophotometer. All measurements were
made while the change in absorbance was linear with respect
to time and before more than 4% of the substrate had been
consumed.
Concentration
of lsoenzyme
Fractions
To investigate the purityof the CK isoenzymes eluted from
the DEAE-celiuiose
columns, we concentrated
the fractions
corresponding to the peaks for each isoenzyme for 1 to 2 h at
4 #{176}c
with a Model B-15 Minicon concentrator (Amicon Corp.,
Lexington, Mass. 02173). The concentrates (about 100-fold)
were then electrophoresed.
Statistical
A Student’s
Analysis of the Data
t -testwas performed to compare the arithmetic
1330 CLINICALCHEMISTRY,Vol. 23, No. 7, 1977
GB
3.2
0.7
0.7
2.1
1.5
1.5
0.8
0.6
1.8
3.0
1.5
1.5
1.8
1.4
means of the data. However when an F test of the standard
deviations of the arithmetic meansshowed that the variances
differed significantly,
we used a Student’s t -test of the geometric means (9) to compensate for any skewness in the data.
The data from the nonpregnant females and normal deliveries
are illustrated
as frequency bar graphs to demonstrate
the
distributionand skewness of the CK isoenzyme activities.
Results
Ten placenta extracts were chromatographed
on DEAEcelluloseforanalysis of CK isoenzyme activity (Table 1). The
BB isoenzyme is the predominant
form of CK in the placenta,
with an average activityof 200 U/g, followedby MM (48 U/g)
and MB (2.6U/g).
We determined the CK isoenzyme activity in blood specimens from 50 apparently healthy nonpregnant women and
in blood specimens from 150 labor patients who were divided
into the following groups: normal delivery, induced labor,
premature labor, cesarean sections, hypertension
and preeclampsia, and stillbirths.
forthe nonpregnant
CK
MB
1.1
0.7
The results for the total CK-MM,
Enzyme Assay
GB
U/iltsr
SD
allowed to pass through the column. when the column had
(n=92)
(n50)
Geometric
with a 4-ml reservoir and a diffusion disc plug. The final dimensions of the cellulose column were 7 X 36 mm.
Before the sample was added to the column, two 1-mi aliquota of the MM-eluting buffer containingdithiothreitol were
Labor patI.nts
Nonprsgnant
-MB, and -BB activities
women and labor patients
with normal
deliveries are shown as histograms in Figure 1 (A-D). Table
2 compares the arithmetic
and geometric means of the MB
and BB activities
forthe nonpregnant women and those with
normal deliveries.
Table 3 compares the arithmetic
means for
the totalCK-MM,
-MB, and -BB activities
for the normal
deliveries and the other groups of labor patients.
Discussion
In our hands, the DEAE-cellulose
method of Nealon and
is highly reproducible and discretely
sepa-
Henderson (3, 4)
rates
the creatine
kinase
isoenzymes.
The results of our study show that the placenta possesses
an average total creatinekinase activityof 250 U/g (SD ± 62),
about twice that reported by Sammour et al. (5). The BB activity in the placenta averaged 80% of the total CK activity,
followed by 19% MM and 1% MB. These percentages
differ
significantly from those reported by Tsung (6): 46-69% BB,
6-22% MB, and 15-48% MM-values
based on results for only
two placenta specimens.
Our results indicate that normal serum BB activities range
from undetectable
to 2.0 U/liter, data that contrast to the
findings of Nealon and Henderson (4), who reported
essentially no serum BB activity in a group of normal laboratory
personnel, a difference that may be attributed
to the technique of analysis of CK activity.
In our study we found it
necessary to use relatively large sample volumes to detect BB
isoenzyme activity in the control group after fractionation
of
the serum samples. The significant
in
the serum of nonpregnant
BB isoenzyme
activity
women may be associated with
MM ISOENZYME
TOTAL CM
(MMMB#{149}
BB)
30-
o
Normal Non-Pregnant Femoles (nR5O)
#{149}
Labor Patients with Normal Deliveries (n-92)
o Normal Non-Pregnant Females(n.50)
#{149}
Labor Patients with Normal DeliverIes (n.92)
20
20-
U
U
C
U-
I
$0-
10-
I..
0
I
I
50
100
I
I
150
I
I
I
200
250
MM Activity
U/Liter
Total CK Activity U/Liter
MB ISOENZYME
BB
o Normal
Non-Pregnant
Females (n5O)
#{149}
Labor Patients with Normal Deliveries
(n=92)
30-
30
20
ISOENZYME
o
Normal Non-PregnantFemales (n.50)
#{149}
Labor PatientswithNormal Deliveries
(n 92)
20-
>‘
U
&
C.
SI
S1
I
Li.
10-
10-
III.
0-
.
-.--
0
3.0
9.0
MB Activity
U/Liter
0-I
0
Ii
ii.
3.0
6.0
BB ActivityU/Uter
9.0
Fig.1.A-D. Histograms for total creatine kinase, and CK isoenzyme MM, MB, and BB activities in serum ofnonpregnant
women
and labor patients with normal deliveries
menses, because the uterus has been shown to contain a significant percentage of the BB isoenzyme (10). However, information on the menstrual status was not available for the
control serum samples used in this study.
Student’s t-test of the geometric means of the data for
nonpregnant women and normal deliveries (P <0.025) indicated that the serum MB and BB activities are greater in the
latter. The apparent increase in MB activities in the serum
of women with normal deliveries is somewhat difficult to assess,sincethe samples we used in the calibration and precision
study contained higher MB activity than did the serum
samples. However, the coefficient of variation for BB at similar activities was only 10.7%; thus the differences we saw in
the MB isoenzyme activities appear to be significant.
CLINICALCHEMISTRY,Vol. 23, No. 7, 1977 1331
Table 3. Arithmetic Mean and Standard DeviatIons for Activities of Serum CK Isoenzymes of Labor
Patients, by Category
induced
Normal
deliveries
n92
Av
SD
labor
n20
Av
Cesarean
Prematur#{149}
labor
(n7
SD
SD
Av
and
pr.-eciampsia
sections
n6
Av
Hypertension
SD
Stillbirths
n7
Av
SD
18.2
1.5
1.2
20.0
42.5
1.3
2.1
45.8
n6
SD
Av
U/liter
MM
MB
BB
Total
42.6
2.1
3.2
47.9
29.4
1.5
1.5
30.1
38.4
1.3
2.7
42.2
23.9
1.0
1.2
25.3
40.9
2.4
3.4
46.7
24.6
1.5
1.0
24.3
We compared the arithmetic
means for CK isoenzyme activities in normal deliveries to those for the other delivery
groups by use of Student’s t -test. None of the groups except
the stillbirth group differed significantly from the normal
delivery group in total CK-MM, -MB, or -BB activities. The
stillbirth group had significantly (P <0.025) lower mean MB
and BB activities
than did the normal-delivery group.
From our data we can make two observations.
First, serum BB isoenzyme activity is greater in labor patients than in nonpregnant
women. We think the most probable source of the additional
serum BB isoenzyme is the placenta, although the uterus has not been ruled out. The increased serum BB activity may be due to stress imposed upon
the placenta and uterus during labor or it may be a conse-
quence of normal tissue turnover in pregnancy.
The second observation
is that serum BB activity of the
group with the stillbirths
is apparently
lower than for the
normal-delivery
group. This implies that serum BB activity
decreases when the fetus dies.
This study was a preliminary
investigation
of the serum CK
distribution
in labor patients, and additional data are required. Such studies should include serum BB activities
throughout
normal pregnancy and in pregnancies in which
difficulties are encountered by eitherfetusor mother, and the
BB activities in amniotic fluid.
49.1
2.2
2.9
54.2
34.6
1.1
1.4
36.2
Biochemistry,
39.4
1.9
3.4
44.6
for their cooperation
and assistance.
We are especially
grateful to Vern L. Schramm, Ph.D., Department
of Biochemistry,
for his advice and assistance.
References
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(1974).
2. Mercer, D., and Varat, M. A., Detection of cardiac-specific creatine
kinase isoenzyme in sera with normal or slightly increased
atine kinase activity. Clin. Chem. 21, 1088 (1975).
392 (1975).
4. Nealon, D. A., and Henderson, A. R., Measurement of brain-specific creatine kinase isoenzyme activity in serum. Clin. Chem. 21, 1663
(1975).
5.
Sammour, M. B., Fattah, M. M. A., Ibrahim, F. K., and Ramadan,
M. A., Creatine phosphokinase
activity in maternal cord blood and
placenta of normal pregnancy and in EPH-gestosis.
Biochem.
Med.
11,205(1974).
6.
Tsung, S. H., Creatine kinase isoenzyme patterns
obtained at surgery. Clin. Chem. 22, 173 (1976).
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CLINICAL
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phokinase
This work was in partial completion of requirements for the Master
degree in Medical Technology under the Allied Health
Profession Advanced Traineeship Program, DHEW 1A02AH0041301. We thank Frances L. Hutchison, M.D., Department of Obstetrics
total cre-
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in serum by ion-exchange column chromatography
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method
and myokinase.
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24.1
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1.3
25.8
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phos-
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Armitage, P., Statistical
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