CLIN. CHEM. 24/11, 2054-2057
(1978)
JIP©I
Creatine Kinase lsoenzyme of High Relative Molecular Mass in
Serum of a Cancer Patient
Hojyo Yuu, Yasushi Takagi, Osamu Senju, Jun-ichiro Hosoya, Kunihide Gomi, and Toru lshii
We describe an atypical serum creatine kinase isoenzyme
in the serum of a woman with cancer of the left breast. This
isoenzyme migrated toward the cathode, closely following
the MM isoenzyme on agarose gel electrophoresis.
Its
relative molecular
mass was estimated to be about
325 000, fourfold that of normal creatine kinase. It is more
heat-stable and is inhibited more by urea than the normal
MM isoenzyme. lsoenzyme monomer B activity was observed to be 20 U/liter in the serum, as measured with use
of an antibody against the M monomer. On anion-exchange
column analysis, creatine kinase activity was observed
only in the MM fraction, in spite of the fact that B activity
was observed in the patient’s serum. Results of the immunological investigation make it unlikely that the atypical
isoenzyme is linked to immunoglobulin or beta-lipoprotein.
It may have been present as the result of modification of
normal creatine kinase by the therapeutic radiation the
patient was receiving.
Creatine kinase (CK;’ EC 2.7.3.2) is separable into three
different components by several methods (1-5). Each isoenzyme is a dimer composed of two subunits (B or M), each
having a relative molecular mass of 40 000 (1). The brain
isoenzyme (CK-BB) consists of two identical B subunits, the
muscle isoenzyme (CK-MM) contains two identical M subunits, and the intermediate isoenzyme (CK-MB) is a hybrid
containing both subunits. On electrophoresis on agarose gel,
CK-BB, CK-MB, and CK-MM migrate closest to the positions
of prealbumin, a2-globulin, and -y-globulin, respectively.
In recent years, many investigators have discussed diagnostic and clinical evaluation of CK isoenzymes in acute
myocardial infarction (AMI). We have also examined CK
isoenzymes in various pathologic conditions including AMI
(6).
We describe here an atypical serum CK isoenzyme of high
relative molecular mass, which migrates cathodically on
agarose gel electrophoresis. Such an isoenzjme
has never been
Department
of Clinical Pathology, Showa University, School of
Medicine,Shinagawa-ku,Tokyo 142,Japan.
1 Nonstandard
abbreviations used: CK, creatine kinase (EC 2.7.3.2;
ATP:creatine
N-phosphotransferase);
CK-BB, brain isoenzyme of
CK; CK-MM, skeletal muscle isoenzyme of CK; CK-MB, musclebrain hybrid isoenzymeof CK; m-CK, CK in the mitochondria.
Received July 7, 1978; accepted Aug. 25, 1978.
2054
CLINICAL CHEMISTRY,
Vol. 24, No. 11, 1978
reported
before,
and we refer to it in this report
as “macro-
CK.”
Case History
The patient, a 52-year-old woman, underwent radical
mastectomy after diagnosis of cancer of the left breast in
March 1977; tele-cobalt
therapy
(6000 R, or 1.55 Ci/kg) was
administered
over the left supraclavicular
fossa and the mediastinum during five months.
In July, metastasis
of the
cancer to the skin of the left anterior chest region was found,
so the patient received irradiation over the anterior thoracic
wall, as well as local injection with Picibanil, a streptococcal
preparation, and telecobalt-therapy with 1800 R over the left
axillary fossa. At the end of September, remarkable retention
of pericardial fluid and jaundice was noted, and a grade I
atrio-ventricular block was found on electrocardiography.
The patient died on October 12. No autopsy was performed.
The main laboratory findings (normal range of enzyme activity in parentheses) at the end of September were as follows:
total protein
56 g/liter, albumin/globulin
ratio 0.8, IgG 15
g/liter, IgA 2.46 g/liter, 1gM 2.07 g/Iiter, aspartate
aminotransferase
200 U/liter (5-35 U/liter),
alanine aminotransferase 34 U/liter (3-30 U/liter), lactate dehydrogenase 4896
U/liter (50-400 U/liter), CK 93 U/liter (10-60 U/liter), ‘yglutamyl transferase 528 U/liter (0-30 U/liter), leucine aminopeptidase 605 U/liter (27-142 U/liter), alkaline phosphatase
19.3 U (0.5-3.5 U, Bessey-Lowry), acid phosphatase 0.79 U
(0.1-0.5
U, Bessey-Lowry).
Materials and Methods
Serum and pericardial fluid from the patient were used for
analysis. Total CK activity was determined at 37 #{176}C
by the
method of Rosalki (7) with “CPK Stat-Pack” reagents (Calbiochem, Los Angeles, Calif.). An electrophoretic method for
CK isoenzymes was carried out by a modification (6,8) of the
fluorescence technique of Somer and Konttinen (2). Electrophoretic patterns were scanned with a Densitron FR-i
(Jookoo Sangyo Co., Tokyo, Japan). CK isoenzymes were also
separated
on a mini-column of DEAE-Sepharose CL-6B
(Pharmacia Fine Chemicals AB, Uppsala, Sweden) as previously reported (5, 6, 9). Our elution procedure was similar to
that described by Nealon and Henderson (4), but the serum
was diluted with tris(hydroxymethyl)aminomethane
buffer
(50 mmol/liter, pH 7.0) before it was applied to the column.
Thin-layer
gel filtration on Sephadex G-200 superfine
(A)
(B)
Fig. 2. Thin-layer gel filtration patterns of serum creatine kinase
on Sephadex G-200, superfine
Thin-layer gel filtration was developed for 2 h with phosphate buffer (67
mmol/liter,pH 7.4). Position of fractions was observed by ultraviolet fluorescence
asdescribedin the text. (1) and(3), the patient’sserum; (2) normal serum. Arrows
(a)
(b)
.1
indicate the serum protein fractions. A: Albumin,& 19G.M 1gM.The patient’s
serum cK shows fluorescent emission, not only between the albumin and lgG
fractions, but also the lgG and 1gMfractions
E
I
C
-
C
0
Fig. 1. Agarose gel electrophoretic patterns of serum creatine
kinase isoenzymes (A) and change of fluorometric scan of
electropherograms (B)
The cathode isattheleft. (A); The blank (lower portion) is demonstrated
in a
similar way as the test (upper portion), except without the substrate creatine
phosphate; (1) normal serum with total CK activity of 33 U/liter; (2) patients
serum with total CK activity of 146 U/liter (B) (a) and (b): patient’s serum with
total CK activity of 146 U/lIter, October 1, and 210 U/liter. October 5. respectively. In spite of the Increased total CK activity, absolute CK activity in the cathodic band was not above normal
(Pharmacia Fine Chemicals) was developed for 2 h with
phosphate buffer (67 mmol/liter, pH 7.4). A strip of TOYO
filter paper No. 50 was placed on top of the gel surface to directly absorb the separated
protein fractions. To detect CK
activity, another strip of filter paper soaked in the reaction
solution was then placed over the first sheet. After incubation
for 60 mm at 37 #{176}C,
sites of CK activity could be seen under
ultraviolet light (360 nm). Gel filtration was also performed
on a Sephadex G-200 column (1.8 X 45cm) at 4#{176}C,
which was
eluted with phosphate buffer (67 mmol/liter, pH 7.4) at a flow
rate of 9-10 ml/h. The relative molecular mass (Mr) of the
patient’s serum CK was estimated by comparisons with
standard proteins (Boehringer Mannheim GmbH Biochemica).
CK-B activity was determined by use of an antibody against
the CK-M monomer. This antibody, which only recently became available (E. Merck, Merck-i-Test CK-MB 14301), was
used according to the vendor’s instruction sheet.
Immunoelectrophoresis was carried out on agar gels against
rabbit anti-whole human serum, anti-IgG, anti-IgA, anti-IgM,
and anti-13-lipoprotein (Behringwerke, Hoechst-Japan Co.,
Tokyo). After the agar plate was washed with isotonic saline,
CK activity was detected both by the fluorescence method and
by tetrazolium staining as in the methods used in electrophoretic CK isoenzyme analysis. The plates were then incubated for 60 mm at 37 #{176}C.
The heat stability and response to urea, to 6 g/liter sodium
10
20
Fraction
30
40
Number
Fig. 3. Pattern of elution of patient’s serum creatine kinase
isoenzymes from Sephadex G-200 column (1 .8 X 45 cm)
Elutionwas with phosphatebuffer (67 mmol/liter, pH 7.4) at a flow rate of 9-10
mi/h, and fractions were collected at 4 #{176}c.
Samples of these wereassayed for
total CK activity (#{149}-#{149}).
The protein concentration is also shown (0
0).
Left to right, peaks of serum protein show gM, I9G, and albumin, respectively.
The result obtainedhere agrees with that represented in Figure 2
deoxycholate,
to 50 mmol/liter
ethylenediaminetetraacetic
acid (EDTA), and to 10 mmol/liter
fl-mercaptoethanol
were
examined. All reagents were analytical grade.
Results
The atypical serum CK isoenzyme, “macro-CK,”
migrated
toward the cathode (Figure 1). This band does not appear
when creatine phosphate
is omitted from the incubation
medium. “Macro-CK”
was also present in the patient’s pencardial fluid.
Molecular
size
investigation.
Thin-layer
gel filtration
patterns of patient’s serum and normal serum are shown in
Figure 2. The position
of the fractions
was observed by ultraviolet
fluorescence.
Normal
CK was found between the
albumin
and IgG fractions,
while “macro-CK”
was found
between the IgG and 1gM fractions, suggesting
a greater relative molecular mass. Figure 3 shows the profile of Sephadex
G-200 elution. The patient’s serum shows a peak of CK activity in fraction 23, as well as in the normal CK fraction, no.
31. This fraction 23, developed with thin-layer gel filtration,
emitted a fluorescence, appearing between the IgG and 1gM
fractions. Figure 4 shows the regression line of the relationship
between elution volume (Ve) and relative molecular mass of
four proteins used for calibration purposes on the Sephadex
CLINICAL CHEMISTRY.
Vol. 24, No. 11, 1978
2055
50
Albumin
Normal
CK
E
Aidolase
a
>
40
Kate lase
Macro-CK”
30
Ferritin
a
i
itil
I
10
I
I
I
lilt]
5
10
6
Mo I. W t.
(1)
Fig. 4. Estimate of relativemolecular mass of patient’s serum
creatine kinase isoenzymes
Elution volume (V,) of proteinsof knownrelative molecularmassandpatient’s
serum CI( isoenzymesfrom Sephadex G-200column(1.8 X 45cm) are plotted
vs. the logarithm of relative molecular mass.The four calibration proteinswere:
albumin,Mr 67 000; aldolase, M, 158 000; catalase, Mr 240 000; ferritin, M,
540 000.
140
120
N6CI
100
150
80
concen,,a,,on
300
4
1.
0
80
40
20
(6)
(7)
buffer
(50 mmol/liter,
pH 3.4). The protein
con-
centration was determined
by absorbance
at 280 nm. However,
the results showed no increase of protein precipitate or decrease in CK activity in the supernatant
fluid.
Ion-exchange
column chromatography.
Figure 5 shows the
elution pattern of the patient’s serum with CK activity 228
U/liter. CK activity was observed only in the CK-MM fraction
(fractions
1-4) in spite of the fact that CK-B activity was
observed in the patient’s serum. The enzyme activity in the
eluates showed 231 U/liter and these fractions 1-3 were rethey migrated
in the same position
as shown
in Figure 1 (A-2). Evidently, no bound “B” activity remained
2
4
8
Fraction
8
10
12
14
16
Number
Fig. 5. Elution pattern of patient’s serum creatine kinase isoenzymes from DEAE-Sepharose CL-6B mini-column (5 X 60
mm)
The serum was diluted with tris(hydroxymethyl)aminomethane
buffer (50
mmol/liter, pH 7.0) before it was applied to the column; we keptinmindthat
serum containsabout 150mmol of sodiumchlorideper liter. Sample was applied
and four1-mifractions
of10 mmol/llter
sodium chloride
bufferwerecollected
(fractions
2-5).Another three 1-mi fractions of 150mmol/liter sodium chloride
buffer werecollected (fractions 6-8). Finally, el,t 1-mI fractions (usually three
1-mI)of 300 mmoi/llter sodiumchloride buffer werecollected (fractIons 9-16,
usually 9-11)
G-200 column. The results indicate that the relative molecular
mass of “macro-CK”
is approximately
325 000. By this
method, that of normal CK was estimated to be approximately
80 000.
investigation.
CK-B
activity
was deter-
mined to be 20 U/liter by use of an antibody against CK-M
monomer. This result suggests that “macro-CK” would have
CK-B activity, because the lowest limit of visual detectability
of enzyme activity is at 5 U/liter by our electrophoretic technique.
Immunoelectrophoresis
against anti-whole human serum,
anti-IgG, anti-IgA, anti-IgM, and anti-3-lipoprotein
failed to
show any precipitin lines containing CK activity. Furthermore, to investigate the possibility that “macro-CK” might
be linked to serum immunoglobulin, the serum was also incubated overnight at room temperature with rabbit antiserum against IgG, IgA, and 1gM. Increasing volumes of the
patient’s serum were added to 0.1 ml of the rabbit anti-serum
according to the method of Harada et al. (10). The samples
were centrifuged at 3000 rpm and the CK activity in the su2056
(5)
lent’s serum after heating at 56 #{176}C
for 5 mm and 10 mm, respectively; (7): the
patient’s serum
electrophoresed;
0
Immunological
(4)
Fig. 6. Inhibitory effects of temperature (56 #{176}C)
and urea on the
patient’s serum creatine kinase isoenzymes
The cathode is at the bottom of the figure. (1), (2), (3), and (4): treatmentof patient’sserum with 1, 2, 3, and 4 mol/liter urea,respectively. (5) and (6): the pat-
cine/HC1
a
0
(3)
pernatant
fluid was determined. After washing with saline
three times, the precipitate was completely dissolved in gly-
160
4
(2)
CLINICAL CHEMISTRY,
Vol. 24, No. 11, 1978
on the column.
Physico-chemical
treatment.
Inhibitory effects of temperature and urea are shown in Figure 6. The “macro-CK”
retained
75% of its original activity after heating at 56 #{176}C
for
5 mm, and 15% after heating for 10 mm at the same temperature. Normal CK activity disappeared entirely after heating
for only 5 mm. Inhibition studies were also carried out with
urea. Normal CK showed only slight inhibition by 2 mol/liter
urea, whereas “macro-CK”
condition.
was inhibited
entirely
by the same
Treatment
of patient’s serum with 6 g/liter sodium deoxycholate, 10 mmol/liter
fl-mercaptoethanol,
and (or) 50
mmol/liter EDTA had no effect on the apparent molecular
mass or electrophoretic
mobility of “macro-CK.”
Discussion
In analyzing for CK isoenzymes by electrophoresis, nonspecific reactions often create problems;
e.g., the reaction
catalyzed
by myokinase
(2,8),nonspecific staining by tetrazolium (2, 8), or nonspecific
fluorescent substances in the albumin zone (especially in the serum of patients
on hemodialysis treatment)
(8). However, it is apparent
that “macroCK” is not present as a result of a nonspecific
reaction,
because the cathodic band referred
to in this paper does not
appear without creatine phosphate.
CK isoenzyme
bands having cathodic mobility in tissue
homogenate
have been described
in association
with the mitochondria
and myofibrillar
extracts of rabbit (11),beef (12),
and rat (13) myocardium,
and rat skeletal muscle (13). CK
activity associated with the mitochondria
(m-CK) is more heat
labile and more inhibited
by urea than is the enzyme in the
supernatant fraction (11). The special features of “macro-CK”
are that it is more heat stable and more inhibited
by urea than
is normal CK-MM (Figure 6). Such heat stability contrasts
with that of m-CK.
There is some disagreement concerning the amount of
m-CK and its relative molecular mass. Sobel et a!. (11 ) reported that the molecular weight of m-CK is approximately
80 500, which is near to that of CK in the cytoplasm, whereas
Farrell et al. (12) reported approximately
250 000. In a short
communication previously published concerning an atypical
cathodic band (14), we had estimated the molecular weight
of m-CK according to Sobel et al. and our data confirmed their
findings. As shown in Figure 4, the relative molecular mass of
normal CK was estimated to be about 80 000, which is in good
agreement with reports by other investigators (1, 11 ). But we
estimated “macro-CK” to have a relative molecular mass of
about 325 000. This finding suggests that “macro-CK” may
be a tetramer of normal CK.
Increased serum enzyme activity after irradiation
has been
widely reported. MacWilliam et a!. (15) suggested that this
increase could originate from the damaged cytoplasm of heart
We acknowledge the help of the members of the Department of
Radiology, Showa University, School of Medicine, in supplying
specimens from the patient,
technical
and also Mr. S. Ishizawa for his skilled
assistance.
References
1. Dawson,
D. M., Eppenberger,
ofcreatine
comparative enzymology
H. M., and Kaplan, N. 0., The
kinases. II. Physical and chemical
these findings make it seem most unlikely that “macro-CK”
properties. J. Biol. Chem. 242, 210 (1967).
2. Somer, H., and Konttinen, A., Demonstration of serum creatine
kinase isoenzymes by a fluorescence technique. Clin. Chim. Acta 40,
133 (1972).
3. Takahashi, K., Shutta, K., Matsuo, B., et al., Serum creatine kinase
isoenzymes in Duchenne muscular dystrophy.
Clin. Chim. Acta 75,
435 (1977).
4. Nealon, D. A., and Henderson, A. R., Measurement of brain-specific creatine kinase isoenzyme activity in serum. Clin. Chem. 21, 1663
(1975).
5. Yuu, H., Gomi, K., and Ishii, T., Separation of serum creatine kinase isoenzymes by ion-exchange column chromatography. Jpn. J.
Clin. Pathol., Suppl., 24, 195 (1976). Abstract.
6. Yuu, H., Diagnostic significance of serum creatine kinase isoen-
originated
zyme, MB fraction
muscle. In our case, the increase in CK activity was slight, so
. it could not reflect profound tissue damage. Taken together,
from the mitochondria.
There have been a few reports (16,17)concerning abnormal
electrophoretic
patterns of serum CK isoenzyme bands. In
them all, an abnormal
fraction has been found between the
CK-MM
and CK-MB
position.
It has been
mentioned
that
a CK species with abnormal electrophoretic
mobility gives rise
to a false positive CK-MB in column chromatography.
In the
present investigation, it is very interesting that CK activity
was observed
only in the CK-MM fraction by ion-exchange
column chromatography,
in spite of the fact that CK-B activity was observed in the patient’s
serum by use of an antibody against the CK-M monomer. With our method, the
possibility
of CK-MB or CK-BB carryover into CK-MM can
be neglected.
This evidence suggests that the superficial
charge of “macro-CK”
may be similar to that of CK-MM or
that it may carry a positive charge.
However,
Witteveen
et al. (18) reported
an as-yet-unidentified CK, chromatographically
behaving as CK-MM and
immunologically
indistinguishable from CK-MB in human heart
tissue. They suggested that any CK, for example m-CK, or
another CK-MM that has a different subunit structure (19),
would
be measured
as CK-MB
with
the
immunological
method, because the antibodies only block the normal CK-M
subunit. In our case, CK-B activity observed is believed to be
due to CK-MB originating from the cytoplasm
of the myocardium;
however, it is not certain whether this patient’s
serum really showed CK-B activity.
In conclusion,
the evidence
thus obtained
suggests
that
“macro-CK”
may be a tetramer of normal CK. However, the
possibility
of CK combined with some substance
(relative
molecular
mass about 240 000) carrying a positive charge
cannot be entirely
excluded. Furthermore, it is not certain
whether
“macro-CK”
really involves CK-B activity. We are
continuing
our investigation
of sera from identical cases in
which the patient is exposed to radiation
in the thoracic
re-
gion.
(CK-MB)
in acute myocardial
infarction.
J. Jpn.
Soc. Intern.
Med. 67, 476 (1978).
7. Rosalki, S. B., An improved procedure for serum creatine phosphokinase determination. J. Lab. Gun. Med. 69, 696 (1967).
8. Yuu, H., Studies on creatine kinase isoenzyme analysis. Jpn. J.
Gun. Pathol. 24, 997 (1976).
9. Yuu, H., Hosoya, J., and Ishii, T., Chromatographic
and electro-
phoretic separation of
creatine
kinase isoenzymes
compared.
Phys-
ico-Chem. Biol. 21, 111 (1977). Abstract.
10. Harada, K., Nakayama, T., Kitamura, M., and Sugimoto, T.,
Immunological
and electrophoretical approaches to macroamylase
analysis. Gun. Chim. Acta 59, 291 (1975).
11. Sobel, B. E., Shell, W. E., and Klein, M. S., An isoenzyme of creatine phosphokinase
associated with rabbit heart mitochondria.
J.
Mol. Cell. Cardiol. 4, 367 (1972).
12. Farrell, Jr., E. C., Baba, N., Brierley, G. P., and Grumer, H. D.,
On the creatine phosphokinase of heart muscle mitochondria. Lab.
Invest. 27, 159 (1972).
13. Sholte, H. R., On the triple localization of creatine kinase in heart
and skeletal muscle cells of the rat: Evidence for the existence of
myofibrillar and mitoehondrial isoenzymes. Biochim. Biophys. Acta
305, 413 (1973).
14. Yuu, H., Takagi, Y., Senju, 0., et al., A case of atypical creatine
kinase isoenzyme pattern. Igaku No Ayumi 105, 78 (1978).
15. MacWilliam, L. D., and Bhakthan, N. M. G., Radiation-induced
enzyme efflux from rat heart: Sedentary animals. Recent Adv. Stud.
Card. Struct. Metab. 9, 447 (1976).
16. Velletri, K., Griffith, W. C., and Diamond, I., Abnormal electrophoretic mobility of a creatine kinase MM isoenzyme. Clin. Chem.
21, 1837 (1975).
17. Sax, S. M., Moore, J. J., Giegel, J. L., and Welsh, M., Atypical
increase in serum creatine kinase activity in hospital patients. Clin.
Chem. 22, 87 (1976).
18. Witteveen, S. A. G. J., Hollaar, L., and van der Laarse, A., A
comparison of two creatine kinase MB determinations:
A chromatographic versus an immunological method. Clin. Chim. Acta 83, 297
(1978).
19. Wevers, R. A., Olthuis, H. P.,van Niel,J. C. C., et al., A study on
the dimeric structure of creatine kinase (EC 2.7.3.2). Clin. Chim. Acta
75, 377 (1977).
CLINICALCHEMISTRY, Vol. 24,No. 11,1978
2057