Clin Biochem Vol 25 pp 293301 1992 0009912092 500 + 00
Printed in the USA All rights reserved CppDight c 1992 The Canadian Society of Clinical Chemists
Measurement of Creatine Kinase Isoforms by
Agarose Gel Electrophoresis in the Diagnosis of
Myocardial Infarction
SHUCHU SHIESH 1 WENKAO TING 2 and TJINSHING JAP 1
1Department of Laboratory Medicine Veterans General Hospital and 2Department of Medical
Technology National YangMing Medical College Taipei Taiwan 11217 Republic of China
To evaluate a method to quantitate the isoforms of serum
creatine kinase isoenzyme MM (CKMM) by agarose gel electro
phoresis sera of normal subjects (n 74) and patients with
acute myocardial infarction (n 21) and other diseases (n 67)
were studied The withinassay imprecision (CV) for CKMM1
MM 2 and MM 3 was 19 08 and 22 at the activity of 79
105 and 64 UL (30 °C CKNAC) respectively while the assay
toassay imprecision was 48 32 and 39 respectively
The method could detect 5 UL or more of any CKMM isoform
and was linear with respect to CK activity at values < 1100 UL
Sera from healthy subjects (n 74) contained mainly CKMM1
(~ 485) with lesser amounts of CKMM 2 (~ 306) and
CKMM 3 (~ 208) The central 95percentile reference range
for the ratio of MM3MM 1 was 012134 with 2 049 The sen
sitivity of CKMMJMM~ ratio >13 in the diagnosis of acute myo
cardial infarction employing the first available sample was 90
at a specificity of 91 compared with a sensitivity of 81 and
specificity of 87 for the conventional CKMB assay At CK
MM3MM1 ratio of 16 or more the specificity increased to 96
while sensitivity remained unchanged at 90 This procedure
for the quantitation of serum CKMM isoforms is convenient
practical and suitable for inclusion in the routine panel of car
diac tests
KEY WORDS creatine kinase isoforms agarose gel
electrophoresis myocardial infarction
Introduction
C reatine kinase (CK ATPcreatineNphos
photransferase EC 2732) reversibly catalyzes
the transfer of a phosphate group from ATP to cre
atine It is present in many organs and tissues Two
distinct gene loci were recognized to produce the M
(muscle) and B (brain) subunits resulting in the for
mation of three dimers (12) namely CKBB (CK1)
CKMB (CK2) and CKMM (CK3) In serum the
tissue form of CKMM is successively hydrolyzed
with removal of a positively charged Cterminal
lysine by carboxypeptidase N in each of the two M
subunits and converted from MM3 to MM2 and then
MM~ with acceleration of their anodal migration at
Correspondence ShuChu Shiesh MS Department of
Laboratory Medicine Veterans General Hospital Taipei
Taiwan 11217 Republic of China
Manuscript received December 9 1991 revised March
12 1992 accepted April 13 1992
each step CKMM1 usually predominates in normal
serum however following tissue damage eg the
onset of myocardial infarction MM 3 instead of MM 1
is rapidly released in large quantities into the cir
culation even preceding the release of CKMB
(310)
The analysis of CKMM isoforms in serum offers
early noninvasive detection of myocardial infarction
and of reperfusion after thrombolytic therapy
(891114) Different methods for the separation of
CKMM isoforms are based on the differences of
chargesize detected by chromatofocusing immuno
blot procedures polyacrylamide gel electrophoresis
high voltage cellulose acetate electrophoresis flat
bed isoelectrofocusing and high pressure liquid
chromatography (114) In the present study an
agarose gel electrophoresis method to quantitate the
isoforms CKMM1 MM2 and MM3 and its clinical
application are evaluated
Materials and methods
APPARATUS
The Centrifichem 600 analyzer with pipettor 1000
was from Baker (Baker Instrument Corporation
New York USA) The densitometer was from Corn
ing model 780 (Ciba Coming Diagnostic Corpora
tion MA USA)
REAGENTS
A CK reagent kit was purchased from Boehringer
Mannheim (Mannheim Germany) The other re
agents unless specified were from Coming MOPSO
buffer (3Nmorpholino2hydroxypropane sulfonic
acid cat no 470046) special agarose film (cat no
470104) and Cardiotrac CK isoenzyme substrate
(cat no 470069)
DETERMINATION OF CREATINE KINASE ACTIVITY
The total CK activities were measured by the
method of the Scandinavian Committee on Enzymes
on the Centrifichem 600 analyzer at 30 °C the upper
limit was 120 UL for women and 160 UL for men
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 293
SHIESH TING AND JAP
DETERMINATION OF CREATINE KINASE ISOENZYMES
CK isoenzymes were separated by electrophores
ing 1 ~L of serum on 1 agarose gel plate (Special
film Coming) at 90 V for 20 min in 005 M MOPSO
buffer pH 78 and the formation of NADH was de
tected by overlaying 1 mL of CardiotracCK isoen
zyme reagent (coupled enzymatic Rosalki reaction)
in MES buffer (2Nmorpholino ethane sulfonic acid)
with sucrose for 20 min at 37 °C and drying it at
60 °C in an oven for 25 min Dried gels were scanned
in a microcomputercontrolled densitometer (Com
ing 780) using the fluorescent mode with excitation
wavelength of 365 nm and emission wavelength of
460 nm
DETERMINATION OF CKMM ISOFORMS
Isoforms of CKMM were separated on Titan III
cellulose acetate (Helena Laboratories Beaumont
TX USA) and agarose gel (Corning) electrophore
sis Cellulose acetate electrophoresis was carried out
by applying the sample on a Titan III plate and elec
trophoresing at 800 V for 12 min After incubation
with CK substrate (CKMB isoenzyme reagent kit
from Helena) at 45 °C for 6 min the plate was dried
at 55 °C for 5 min and the fluorescence was scanned
in the densitometer
For agarose gel electrophoresis 1 ~tL of sample
diluted to contain 1501100 UL of CKMM activ
ity was applied to the agarose gel and electropho
resed at 170 V for 45 min using icecooled buffer
The following procedure was identical to that of CK
isoenzymes The withinassay imprecision was as
sessed by analyzing 7 aliquots of a sample on one
agarose plate while the assaytoassay imprecision
was assessed by 11 separate assays The relation
ship between intensity of fluorescence and enzyme
activity for every CKMM isoform on the agarose gel
was evaluated by serial dilution of a serum with
high CK activity
the onset of chest pain were evaluated for clinical
sensitivity and specificity
SAMPLE COLLECTION
Blood samples were collected in evacuated con
tainers without anticoagulant Blood was allowed to
clot and serum was separated by centrifugation
Tests were performed immediately after serum sep
aration If the assay had to be delayed the serum
was promptly stored at 20 °C and assayed within 3
days
A
R
SUBJECTS
Blood samples were collected from 74 normal sub
jects (17 women and 57 men aged from 37 to 79
years with an average of 56 years) with normal
results of biochemical and hematological tests and
no signs of systemic diseases such as those of liver
pancreas heart and skeletal muscle to establish the
reference intervals of CK isoforms Blood samples
were also collected from 88 patients including those
with acute myocardial infarction (n 21) chronic
ischemic heart disease (n 12) other heart disease
(n 16) renal failure (n 6) liver and gastroin
testinal disease (n 9) pulmonary disease (n 4)
muscle disease (n 7) brain disease (n 3) and
miscellaneous diseases (n 10) Total CK activity
CK isoenzymes and CKMM isoforms were ana
lyzed Serial samples from patients with myocardial
infarction were analyzed but only the results of the
first available samples collected less than 10 h after
Figure 1 The densitometric scanning pattern of serum
CKMM isoforms separated by high voltage cellulose ac
etate (A) and agarose gel electrophoresis (B) with total CK
activity of 214 UL and 217 UL respectively
294 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
ISOFORMS OF CREATINE KINASE ISOENZYME MM
Figure 2 CK agarose gel electrophoresis (50 mM MOPSO buffer pH 78 170 V 45 min) The sera were applied on the
bottom and migrated toward the top
STATISTICAL ANALYSIS
Linear regression nonparametric statistical tests
and one way analysis of variance (ANOVA) were
performed on a personal computer (IBMPC) utiliz
ing the software package BMDP (BMDP Statistical
Software Los Angeles CA USA)
Results
SEPARATION OF CKMM ISOFORMS
BY ELECTROPHORESIS
The densitometric scans of CKMM isoforms after
electrophoresis on cellulose acetate and agarose gel
are shown in Figure 1 The separation of isoforms by
high voltage electrophoresis on cellulose acetate was
abandoned due to the high background noise mak
ing the quantitation irreproducible especially at
low CK activity The fluorescence of CKMM iso
forms obtained after agarose gel electrophoresis is
demonstrated in Figure 2 The withinassay impre
cision (coefficient of variation) was 22 08 and
19 for CKMM 3 MM2 and MM 1 at the isoform
activity of 64 105 and 79 UL respectively while
the assaytoassay imprecision was 39 32 and
48 for MM3 MM2 and MM at the isoform activ
ity of 273 359 and 186 UL respectively The de
termination of every isoform was linear to at least
800 UL as shown in Figure 3 Samples with CK
MM activity <150 UL required duplicate applica
tion on the agarose plate while those with CKMM
activity >1100 UL required dilution to avoid sub
strate depletion during incubation
STABILITY OF SERUM CKMM ISOFORMS
Serum from a patient with acute myocardial in
farction was aliquoted into polyethylene tubes im
mediately after separation and stored frozen at
20 °C The distribution of CKMM isoforms on the
fresh sample was 127 425 and 448c~ of total
CKMM content for MM~ MM2 and MM3 respec
tively and was 133 413c~ and 454 after 24h
freezing for MM MM2 and MM 3 respectively The
patterns of CKMM isoforms analyzed immediately
7 days 1 month and 4 months after serum separa
tion are presented in Figure 4
CKMM ISOFORMS IN NORMAL CONTROLS
The reference interval of serum CKMM isoforms
in 74 healthy individuals is shown in Table 1 The
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 295
SHIESH TING AND JAP
1RO0
1000
J
D
U
'
01 0 2 04 08 08 1 0
OILUTION
Figure 3 Linearity of the isoforms of CKMM determined by agarose gel electrophoresis A serum with total CKMM
of 1700 UL was diluted with normal saline to various activities and electrophoresis was performed simultaneously in all
samples
CK isoenzyme analysis of these normal controls re
vealed that only MM isoenzyme was present
CKMM ISOFORMS IN DISEASES
The CKMM 3 level was the first to reach a peak
after the onset of chest pain in acute myocardial
infarction followed by MM2 and MM~ (Figure 5)
The changes of serum CK total activity CKMB
and CKMM3MM 1 ratio in a patient with acute
myocardial infarction who was treated with strep
tokinase 6 hours after the onset of chest pain are
shown in Figure 6 The elevation of serum CKMM s
and MM3MM ~ ratio in acute myocardial infarction
occurred earlier than that of total CK and CKMB
Among 21 patients with acute myocardial infarc
tion 19 had a MM3MM 1 ratio greater than the up
per reference limit of 13 while 58 of 67 patients
with other diseases had a MM3MM 1 ratio < 13 (Fig
ure 7) A ratio of CKMMsMM1 greater than 13
provided a sensitivity of 90 and specificity of 91
in detecting acute myocardial infarction whereas
CKMB greater than 39 of total CK activity pro
vided a sensitivity of 81 and a specificity of 87
The cutoff value of MM3MM1 ratio >16 yielded a
specificity of 96 without loss of sensitivity The
CKMM2MM ~ ratio was also elevated significantly
in acute myocardial infarction (Figure 8) Specificity
could be increased to 94 through combining MM2
MM 1 ratio >13 with MMsMM ~ ratio >13
Two sera from patients with pneumonia were
found to contain extra MM isoforms Their relative
positions as compared with CKMM~ MM2 and
MM 3 are shown in Figure 9 The sera were ana
lyzed freshly and again after 4 days at 20 °C with
consistent results
EFFECTS OF MACROCK ISOENZYMES
During the period of this study (6 months) 2 cases
with macroCK type I and 6 cases with macroCK
type II by CK isoenzymes analysis were found The
macroCK types I or II did not interfere with the
CKMM isoforms since type I migrated more anod
ically than CKMM 1 and type II migrated toward
the cathode (Figure 9)
296 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
A
ISOFORMS OF CREATINE KINASE ISOENZYME MM
C
B
i
D
Figure 4 The stability of CKMM isoforms of a serum with CK activity of 818 UL A fresh sample B 7 days after
storing at 20 °C C one month later D four months later
Discussion
Wevers et al (12) first demonstrated the multiple
isoforms of CK isoenzymes in human serum With
agarose gel or cellulose acetate electrophoresis
three isoforms of CKMM (MM12 3) and two isoforms
of CKMB (MB12) were commonly reported
(161213) A fou~h MM band running anodal to
MM1 noted by Wevers et al (2) was considered as
an artifact resulting from interaction between CK
and carrier ampholytes during isoelectrofocusing
Another MM isoform denoted as MMX (more anodic
than MM 1) was observed with the Helena agarose
gel system especially in longstored samples (14)
Others have detected 4 to 21 CKMM isoforms in
human serum and tissues using polyacrylamide gel
isoelectrophoresis or chromatofocusing techniques
(111520) With agarose gel electrophoresis we
found three isoforms of MM in most cases Six MM
isoforms were observed in a patient with pneumonia
and necrotizing myocarditis one day prior to his
death The other case was a pneumonia patient in
whom an extra CKMM isoform migrating anodal
to MM1 was observed The analysis of CK isoen
TABLE 1
Reference Intervals of CKMM Isoforms Expressed as a Percentage of Total
CKMM in Serum of Normal Subjects
25th 975th
Mean SD Percentile Percentile
CK at 30 °C 834 UL 202 UL 54 UL 133 UL
CKMM 1 485 100 270 628
CKMM2 306 39 210 365
CKMM3 208 69 88 368
CKMMJMM 049 030 012 134
CKMMaMM2 067 017 040 106
CKMM2MM 1 068 025 030 131
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 297
SHIESH TING AND JAP
A
IB 16
IM3MMI 5o
B 10
16
C 5
O1
Figure 5 The change of CKMB and MM3MM ratio
in a patient with acute myocardial infarction (A) 8 h (B)
16 h (C) 30 h after the onset of chest pain
zymes revealed a single CKMM isoenzyme in these
two sera The appearance of extra MM isoforms was
not the effect of macroCK or ageing of the samples
The clinical significance of these extra isoforms
needs further investigation
The reference interval of serum CKMM isoforms
has been reported by several authors (162123)
Our results are in accordance with the finding of
Morelli et al (21) obtained by agarose gel isoelec
trofocusing Although the distribution of CKMM
isoforms varies in these reports the main pattern of
predominant MM1 plus only a small amount of the
tissue form MM 3 is found consistently
During the course of acute myocardial infarction
t Total CK _ _ CKMM3MM 1
6834 ' • ~CKMB
24oo ~ \ 90
\ t
' \
0 88
i ~ 0918 rr
6oo ~
0
\
0 \ \~
I \ \ ~ 05
800 ' ' \
A O3 6
\ r i ' \l
Ireptokinase
_ di2
HOURS AFTER ONSET OF CHEST PAIN
Figure 6 Total CK activity CKMB and CKMM3
MM ratio as a function of time in a patient with acute
myocardial infarction following streptokinase treatment
14 ~
k
10 (9
MM3 reaches a peak first followed by MM2 and then
MM1 The MMJMM1 ratio rises even earlier than
the isoform peaks An MMJMM1 ratio of 10 with
cellulose acetate electrophoresis has been reported
as the cutoff point for the detection of the patholog
ical release of MM3 from tissue (24) In our study
the MM3MM 1 ratio was 012149 in normal sub
jects 010136 in the first available serum of pa
tients with acute myocardial infarction while the
MM2MM 1 ratio was 0314 in normal subjects and
0348 in patients with acute myocardial infarc
tion The changes of the ratios MM3MM ~ and MM2
MM~ in acute myocardial infarction as compared
with the other diseases were statistically signifi
cant (p < 0001)
The CK isoforms in serum were reported to be
unstable (92225) and blood samples should be col
lected in EDTA tubes to inactivate carboxypeptidase
N and to prevent the conversion of isoforms Others
observed that CKMM isoforms were stable at least
3 months at 20 °C (216) Our results demon
strated that CKMM isoforms remain unchanged
over 7 days at 20 °C then 10 of total CKMM
was converted from MM3 to MM1 and no further
change was noticed for at least 4 months
The determination of serum CK isoforms is be
lieved to be an alternate noninvasive method to de
tect and follow acute myocardial infarction and suc
cessful reperfusion (89) According to Abendschein
(9) CKMM 3 increased dramatically within 1 h af
ter coronary occlusion and returned to baseline level
after 4 h Panteghini et al (23) reported that the
MM3MM1 ratio was raised 3 h after the onset of
acute myocardial infarction and peaked at 93 h
earlier than the peaking of isoforms It was also re
ported that CKMB2 the tissue form of CKMB was
298 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
O0 (t)
3
(3
0
0
0
h3
(~
o
>
¢r
¢r
Normal
AMI
Chronic
ischernic
disease
Other
heart
Muscle
Renal
GI
Lung
Brain
Misc•
CKMM2MM 1 Ratio ~
• ~' '~r~mm3wv 1 Ratio
b u b b b b o I
I I I I I I I ' ~ bu o b 0 b b 0 b b b b o ~ (~
I • ~ & Normal
• • I • lmOOnOmlOO I AMI • • I • '~ • •
I
' ~'~ Chronic I In • elm• •
~ ~ i5chemic
~~ msease
~ Other ~po • • • ov IPI • '~ hea rt O
~~
• '~ IVlU~Cl~ oo• 00 ooo • • ~
~ Renal • 0 •
O c~
~ GI m • o~ lid • • LL
~
d Lun~I
• 8 • ~
~ Brain • • • •
'1
Misc 1~ •
cn Z
~o
z > ~ 3o ~ 0
j
C
SHIESH TING AND JAP
4 MacroCK I
MM 1
1 1 1 ~ MM 2
I
I I MM 3
~ ~ ~ r j Applicat ion
point
MacroCK II
1 2 3 4
Figure 9 The migrating positions of CKMM isoforms
and other variants Arrows point to the positions of the
extra CKMM isoforms Lane 1 typical CKMM isoforms
in serum Lanes 2 and 3 extra CKMM isoforms found in
patients with pneumonia lane 4 macroCK types I II and
CKMM123
elevated earlier than the CKMB during the
course of cardiac damage (232627) The elevation of
the CKMB2 isoform in serum is more cardiac
specific than CKMM3 which is increased in both
myocardial and skeletalmuscle injuries (161921)
However the elevation of the MB 2 isoform occurs
more slowly and in lesser degree than the MM3 iso
form Furthermore the assay requires a technique
with sufficient sensitivity to detect the small change
of CKMB isoform In conjunction with the clinical
examination CKMM isoform analysis appears to
have significant advantages over CKMB isoforms
in clinical application
With the use of conventional agarose gel electro
phoresis for the CK isoenzymes eight specimens can
be analyzed simultaneously to quantitate CKMM
isoforms within 15 h without additional apparatus
The method is precise simple free from interference
by macroCK and hightemperatureinduced arti
facts and applicable for routine use in the clinical
laboratory
References
1 Wevers RA Olthuis HP Van Niel JCC Van Wilgen
burg MGM Soons JBJ A study on the dimeric struc
ture of creatine kinase (EC 2732) Clin Chim Acta
1977 75 37785
2 Wevers RA Wolters RJ Soons JBJ Isoelectric focus
ing and hybridization experiments on creatine kinase
(EC 2732) Clin Chim Acta 1977 78 2716
3 Chapelle JP Heusghem C Further heterogeneity
demonstrated for serum creatine kinase isoenzyme
MM Clin Chem 1980 26 45762
4 Falter H Michelutti L Mazzuchin A Whiston W
Studies on the subbanding of creatine kinase MM
and the CK conversion factor Clin Biochem 1981 14
37
5 Yasmineh WG Yamada MK Cohn JN Postsynthetic
variants of creatine kinase MM J Lab Clin Med 1981
98 10918
6 Panteghini M Pagani F Isoforms of creatine kinase
isoenzymes in serum in acute myocardial infarction
after intracoronary thrombolysis Clin Chem 1987 33
2O3942
7 Panteghini M Serum isoforms of creatine kinase
isoenzymes Clin Biochem 1988 21 21118
8 Wu AHB Creatine kinase isoforms in ischemic heart
disease Clin Chem 1989 35 713
9 Abendscheim DR Rapid diagnosis of myocardial in
farction and reperfusion by assay of plasma isoforms
ofcreatine kinase isoenzymes Clin Biochem 1990 23
399407
10 Armbruster DA The genesis and clinical significance
of creatine kinase isoforms Lab Med 1991 22 325
34
11 Chapelle JP Serum creatine kinase MM subband de
termination by isoelectric focusing a potential
method for the monitoring of myocardial infarction
Clin Chim Acta 1984 137 27381
12 Wu AHB Gornet TG Wu VH Brockie RE Nishikawa
A Early diagnosis of acute myocardial infarction by
rapid analysis of creatine kinase isoenzyme3(CK
MM) subtypes Clin Chem 1987 33 35862
13 Apple FS Sharkey SW Werdick M Elsperger KJ
Tilbury RT Analysis of creatine kinase isoenzymes
and isoforms in serum to detect reperfusion after
acute myocardial infarction Clin Chem 1987 33
50711
14 Wu AHB Creatine kinase isoform analysis Clin
Chem News 1991 17 1920
15 Vaidya H Dietzler DN Leykam JF Ladenson JH
Purification of five creatine kinaseMM variants from
human heart and skeletal muscle Biochim Biophys
Acta 1984 790 2307
16 Annesley TM Strongwater SL Schnitzer TJ MM
subisoenzymes of creatine kinase as an index of dis
ease activity in polymyositis Clin Chem 1985 31
4026
17 Leykam JF Dietzler DN Ladenson JH Purification
of human creatine kinase isoenzymes for monoclonal
antibody production Clin Chem 1985 29 1219
18 SiragEldin E Gercken G Harm K The isoelectric fo
cusing of creatine kinase variants II the heterogene
ity of creatine kinase in human serum with normal
and elevated catalytic concentrations J Clin Chem
Clin Biochem 1986 24 84760
19 Apple FS Hellsten Y Clarkson PM Early detection
of skeletal muscle injury by assay of creatine kinase
MM isoforms in serum after acute exercise Clin Chem
1988 34 11024
20 Williams J Williams KM Marshall T Heterogeneity
of serum creatine kinase isoenzyme MM in myocardi
al infarction clinical significance and postsynthetic
conversion ofabnormal subbands Clin Chem 1989
35 20610
21 Morelli RL Carlson CJ Emilson B Abendschein DR
Rapaport E Serum creatine kinase MM isoenzyme
subbands after acute myocardial infarction in man
Circulation 1983 67 12839
22 Wu AHB Gornet TG Measurement ofcreatine kinase
MM subtype by anionexchange liquid chromatogra
phy Clin Chem 1985 31 18415
23 Panteghini M Calarco M Serum isoforms of creatine
300 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
ISOFORMS OF CREATINE KINASE ISOENZYME MM
kinase MM and MB in myocardial infarction An ap
praisal of quantitative clinical and pathophysiologi
cal information Scand J Clin Lab Invest 1987 47
3259
24 Panteghini M Serum isoforms of creatine kinase MM
and MB as an index for the monitoring of acute myo
cardial infarction Clin Chem 1986 32 1135
25 Chapelle JP Bertrand A Heusghem C The protec
tion of creatine kinase MM subbands by EDTA dur
ing storage Clin Chim Acta 1981 115 25562
26 Puleo PR Guadagno PA Roberts R Perryman B Sen
sitive rapid assay of subforms of creatine kinase MB
in plasma Clin Chem 1989 35 14525
27 Puleo PR Guadagno PA Roberts R et al Early diag
nosis of acute myocardial infarction based on assay for
subforms of creatine kinaseMB Circulation 1990 82
75964
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 301
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Printed in the USA All rights reserved CppDight c 1992 The Canadian Society of Clinical Chemists
Measurement of Creatine Kinase Isoforms by
Agarose Gel Electrophoresis in the Diagnosis of
Myocardial Infarction
SHUCHU SHIESH 1 WENKAO TING 2 and TJINSHING JAP 1
1Department of Laboratory Medicine Veterans General Hospital and 2Department of Medical
Technology National YangMing Medical College Taipei Taiwan 11217 Republic of China
To evaluate a method to quantitate the isoforms of serum
creatine kinase isoenzyme MM (CKMM) by agarose gel electro
phoresis sera of normal subjects (n 74) and patients with
acute myocardial infarction (n 21) and other diseases (n 67)
were studied The withinassay imprecision (CV) for CKMM1
MM 2 and MM 3 was 19 08 and 22 at the activity of 79
105 and 64 UL (30 °C CKNAC) respectively while the assay
toassay imprecision was 48 32 and 39 respectively
The method could detect 5 UL or more of any CKMM isoform
and was linear with respect to CK activity at values < 1100 UL
Sera from healthy subjects (n 74) contained mainly CKMM1
(~ 485) with lesser amounts of CKMM 2 (~ 306) and
CKMM 3 (~ 208) The central 95percentile reference range
for the ratio of MM3MM 1 was 012134 with 2 049 The sen
sitivity of CKMMJMM~ ratio >13 in the diagnosis of acute myo
cardial infarction employing the first available sample was 90
at a specificity of 91 compared with a sensitivity of 81 and
specificity of 87 for the conventional CKMB assay At CK
MM3MM1 ratio of 16 or more the specificity increased to 96
while sensitivity remained unchanged at 90 This procedure
for the quantitation of serum CKMM isoforms is convenient
practical and suitable for inclusion in the routine panel of car
diac tests
KEY WORDS creatine kinase isoforms agarose gel
electrophoresis myocardial infarction
Introduction
C reatine kinase (CK ATPcreatineNphos
photransferase EC 2732) reversibly catalyzes
the transfer of a phosphate group from ATP to cre
atine It is present in many organs and tissues Two
distinct gene loci were recognized to produce the M
(muscle) and B (brain) subunits resulting in the for
mation of three dimers (12) namely CKBB (CK1)
CKMB (CK2) and CKMM (CK3) In serum the
tissue form of CKMM is successively hydrolyzed
with removal of a positively charged Cterminal
lysine by carboxypeptidase N in each of the two M
subunits and converted from MM3 to MM2 and then
MM~ with acceleration of their anodal migration at
Correspondence ShuChu Shiesh MS Department of
Laboratory Medicine Veterans General Hospital Taipei
Taiwan 11217 Republic of China
Manuscript received December 9 1991 revised March
12 1992 accepted April 13 1992
each step CKMM1 usually predominates in normal
serum however following tissue damage eg the
onset of myocardial infarction MM 3 instead of MM 1
is rapidly released in large quantities into the cir
culation even preceding the release of CKMB
(310)
The analysis of CKMM isoforms in serum offers
early noninvasive detection of myocardial infarction
and of reperfusion after thrombolytic therapy
(891114) Different methods for the separation of
CKMM isoforms are based on the differences of
chargesize detected by chromatofocusing immuno
blot procedures polyacrylamide gel electrophoresis
high voltage cellulose acetate electrophoresis flat
bed isoelectrofocusing and high pressure liquid
chromatography (114) In the present study an
agarose gel electrophoresis method to quantitate the
isoforms CKMM1 MM2 and MM3 and its clinical
application are evaluated
Materials and methods
APPARATUS
The Centrifichem 600 analyzer with pipettor 1000
was from Baker (Baker Instrument Corporation
New York USA) The densitometer was from Corn
ing model 780 (Ciba Coming Diagnostic Corpora
tion MA USA)
REAGENTS
A CK reagent kit was purchased from Boehringer
Mannheim (Mannheim Germany) The other re
agents unless specified were from Coming MOPSO
buffer (3Nmorpholino2hydroxypropane sulfonic
acid cat no 470046) special agarose film (cat no
470104) and Cardiotrac CK isoenzyme substrate
(cat no 470069)
DETERMINATION OF CREATINE KINASE ACTIVITY
The total CK activities were measured by the
method of the Scandinavian Committee on Enzymes
on the Centrifichem 600 analyzer at 30 °C the upper
limit was 120 UL for women and 160 UL for men
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 293
SHIESH TING AND JAP
DETERMINATION OF CREATINE KINASE ISOENZYMES
CK isoenzymes were separated by electrophores
ing 1 ~L of serum on 1 agarose gel plate (Special
film Coming) at 90 V for 20 min in 005 M MOPSO
buffer pH 78 and the formation of NADH was de
tected by overlaying 1 mL of CardiotracCK isoen
zyme reagent (coupled enzymatic Rosalki reaction)
in MES buffer (2Nmorpholino ethane sulfonic acid)
with sucrose for 20 min at 37 °C and drying it at
60 °C in an oven for 25 min Dried gels were scanned
in a microcomputercontrolled densitometer (Com
ing 780) using the fluorescent mode with excitation
wavelength of 365 nm and emission wavelength of
460 nm
DETERMINATION OF CKMM ISOFORMS
Isoforms of CKMM were separated on Titan III
cellulose acetate (Helena Laboratories Beaumont
TX USA) and agarose gel (Corning) electrophore
sis Cellulose acetate electrophoresis was carried out
by applying the sample on a Titan III plate and elec
trophoresing at 800 V for 12 min After incubation
with CK substrate (CKMB isoenzyme reagent kit
from Helena) at 45 °C for 6 min the plate was dried
at 55 °C for 5 min and the fluorescence was scanned
in the densitometer
For agarose gel electrophoresis 1 ~tL of sample
diluted to contain 1501100 UL of CKMM activ
ity was applied to the agarose gel and electropho
resed at 170 V for 45 min using icecooled buffer
The following procedure was identical to that of CK
isoenzymes The withinassay imprecision was as
sessed by analyzing 7 aliquots of a sample on one
agarose plate while the assaytoassay imprecision
was assessed by 11 separate assays The relation
ship between intensity of fluorescence and enzyme
activity for every CKMM isoform on the agarose gel
was evaluated by serial dilution of a serum with
high CK activity
the onset of chest pain were evaluated for clinical
sensitivity and specificity
SAMPLE COLLECTION
Blood samples were collected in evacuated con
tainers without anticoagulant Blood was allowed to
clot and serum was separated by centrifugation
Tests were performed immediately after serum sep
aration If the assay had to be delayed the serum
was promptly stored at 20 °C and assayed within 3
days
A
R
SUBJECTS
Blood samples were collected from 74 normal sub
jects (17 women and 57 men aged from 37 to 79
years with an average of 56 years) with normal
results of biochemical and hematological tests and
no signs of systemic diseases such as those of liver
pancreas heart and skeletal muscle to establish the
reference intervals of CK isoforms Blood samples
were also collected from 88 patients including those
with acute myocardial infarction (n 21) chronic
ischemic heart disease (n 12) other heart disease
(n 16) renal failure (n 6) liver and gastroin
testinal disease (n 9) pulmonary disease (n 4)
muscle disease (n 7) brain disease (n 3) and
miscellaneous diseases (n 10) Total CK activity
CK isoenzymes and CKMM isoforms were ana
lyzed Serial samples from patients with myocardial
infarction were analyzed but only the results of the
first available samples collected less than 10 h after
Figure 1 The densitometric scanning pattern of serum
CKMM isoforms separated by high voltage cellulose ac
etate (A) and agarose gel electrophoresis (B) with total CK
activity of 214 UL and 217 UL respectively
294 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
ISOFORMS OF CREATINE KINASE ISOENZYME MM
Figure 2 CK agarose gel electrophoresis (50 mM MOPSO buffer pH 78 170 V 45 min) The sera were applied on the
bottom and migrated toward the top
STATISTICAL ANALYSIS
Linear regression nonparametric statistical tests
and one way analysis of variance (ANOVA) were
performed on a personal computer (IBMPC) utiliz
ing the software package BMDP (BMDP Statistical
Software Los Angeles CA USA)
Results
SEPARATION OF CKMM ISOFORMS
BY ELECTROPHORESIS
The densitometric scans of CKMM isoforms after
electrophoresis on cellulose acetate and agarose gel
are shown in Figure 1 The separation of isoforms by
high voltage electrophoresis on cellulose acetate was
abandoned due to the high background noise mak
ing the quantitation irreproducible especially at
low CK activity The fluorescence of CKMM iso
forms obtained after agarose gel electrophoresis is
demonstrated in Figure 2 The withinassay impre
cision (coefficient of variation) was 22 08 and
19 for CKMM 3 MM2 and MM 1 at the isoform
activity of 64 105 and 79 UL respectively while
the assaytoassay imprecision was 39 32 and
48 for MM3 MM2 and MM at the isoform activ
ity of 273 359 and 186 UL respectively The de
termination of every isoform was linear to at least
800 UL as shown in Figure 3 Samples with CK
MM activity <150 UL required duplicate applica
tion on the agarose plate while those with CKMM
activity >1100 UL required dilution to avoid sub
strate depletion during incubation
STABILITY OF SERUM CKMM ISOFORMS
Serum from a patient with acute myocardial in
farction was aliquoted into polyethylene tubes im
mediately after separation and stored frozen at
20 °C The distribution of CKMM isoforms on the
fresh sample was 127 425 and 448c~ of total
CKMM content for MM~ MM2 and MM3 respec
tively and was 133 413c~ and 454 after 24h
freezing for MM MM2 and MM 3 respectively The
patterns of CKMM isoforms analyzed immediately
7 days 1 month and 4 months after serum separa
tion are presented in Figure 4
CKMM ISOFORMS IN NORMAL CONTROLS
The reference interval of serum CKMM isoforms
in 74 healthy individuals is shown in Table 1 The
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 295
SHIESH TING AND JAP
1RO0
1000
J
D
U
'
01 0 2 04 08 08 1 0
OILUTION
Figure 3 Linearity of the isoforms of CKMM determined by agarose gel electrophoresis A serum with total CKMM
of 1700 UL was diluted with normal saline to various activities and electrophoresis was performed simultaneously in all
samples
CK isoenzyme analysis of these normal controls re
vealed that only MM isoenzyme was present
CKMM ISOFORMS IN DISEASES
The CKMM 3 level was the first to reach a peak
after the onset of chest pain in acute myocardial
infarction followed by MM2 and MM~ (Figure 5)
The changes of serum CK total activity CKMB
and CKMM3MM 1 ratio in a patient with acute
myocardial infarction who was treated with strep
tokinase 6 hours after the onset of chest pain are
shown in Figure 6 The elevation of serum CKMM s
and MM3MM ~ ratio in acute myocardial infarction
occurred earlier than that of total CK and CKMB
Among 21 patients with acute myocardial infarc
tion 19 had a MM3MM 1 ratio greater than the up
per reference limit of 13 while 58 of 67 patients
with other diseases had a MM3MM 1 ratio < 13 (Fig
ure 7) A ratio of CKMMsMM1 greater than 13
provided a sensitivity of 90 and specificity of 91
in detecting acute myocardial infarction whereas
CKMB greater than 39 of total CK activity pro
vided a sensitivity of 81 and a specificity of 87
The cutoff value of MM3MM1 ratio >16 yielded a
specificity of 96 without loss of sensitivity The
CKMM2MM ~ ratio was also elevated significantly
in acute myocardial infarction (Figure 8) Specificity
could be increased to 94 through combining MM2
MM 1 ratio >13 with MMsMM ~ ratio >13
Two sera from patients with pneumonia were
found to contain extra MM isoforms Their relative
positions as compared with CKMM~ MM2 and
MM 3 are shown in Figure 9 The sera were ana
lyzed freshly and again after 4 days at 20 °C with
consistent results
EFFECTS OF MACROCK ISOENZYMES
During the period of this study (6 months) 2 cases
with macroCK type I and 6 cases with macroCK
type II by CK isoenzymes analysis were found The
macroCK types I or II did not interfere with the
CKMM isoforms since type I migrated more anod
ically than CKMM 1 and type II migrated toward
the cathode (Figure 9)
296 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
A
ISOFORMS OF CREATINE KINASE ISOENZYME MM
C
B
i
D
Figure 4 The stability of CKMM isoforms of a serum with CK activity of 818 UL A fresh sample B 7 days after
storing at 20 °C C one month later D four months later
Discussion
Wevers et al (12) first demonstrated the multiple
isoforms of CK isoenzymes in human serum With
agarose gel or cellulose acetate electrophoresis
three isoforms of CKMM (MM12 3) and two isoforms
of CKMB (MB12) were commonly reported
(161213) A fou~h MM band running anodal to
MM1 noted by Wevers et al (2) was considered as
an artifact resulting from interaction between CK
and carrier ampholytes during isoelectrofocusing
Another MM isoform denoted as MMX (more anodic
than MM 1) was observed with the Helena agarose
gel system especially in longstored samples (14)
Others have detected 4 to 21 CKMM isoforms in
human serum and tissues using polyacrylamide gel
isoelectrophoresis or chromatofocusing techniques
(111520) With agarose gel electrophoresis we
found three isoforms of MM in most cases Six MM
isoforms were observed in a patient with pneumonia
and necrotizing myocarditis one day prior to his
death The other case was a pneumonia patient in
whom an extra CKMM isoform migrating anodal
to MM1 was observed The analysis of CK isoen
TABLE 1
Reference Intervals of CKMM Isoforms Expressed as a Percentage of Total
CKMM in Serum of Normal Subjects
25th 975th
Mean SD Percentile Percentile
CK at 30 °C 834 UL 202 UL 54 UL 133 UL
CKMM 1 485 100 270 628
CKMM2 306 39 210 365
CKMM3 208 69 88 368
CKMMJMM 049 030 012 134
CKMMaMM2 067 017 040 106
CKMM2MM 1 068 025 030 131
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 297
SHIESH TING AND JAP
A
IB 16
IM3MMI 5o
B 10
16
C 5
O1
Figure 5 The change of CKMB and MM3MM ratio
in a patient with acute myocardial infarction (A) 8 h (B)
16 h (C) 30 h after the onset of chest pain
zymes revealed a single CKMM isoenzyme in these
two sera The appearance of extra MM isoforms was
not the effect of macroCK or ageing of the samples
The clinical significance of these extra isoforms
needs further investigation
The reference interval of serum CKMM isoforms
has been reported by several authors (162123)
Our results are in accordance with the finding of
Morelli et al (21) obtained by agarose gel isoelec
trofocusing Although the distribution of CKMM
isoforms varies in these reports the main pattern of
predominant MM1 plus only a small amount of the
tissue form MM 3 is found consistently
During the course of acute myocardial infarction
t Total CK _ _ CKMM3MM 1
6834 ' • ~CKMB
24oo ~ \ 90
\ t
' \
0 88
i ~ 0918 rr
6oo ~
0
\
0 \ \~
I \ \ ~ 05
800 ' ' \
A O3 6
\ r i ' \l
Ireptokinase
_ di2
HOURS AFTER ONSET OF CHEST PAIN
Figure 6 Total CK activity CKMB and CKMM3
MM ratio as a function of time in a patient with acute
myocardial infarction following streptokinase treatment
14 ~
k
10 (9
MM3 reaches a peak first followed by MM2 and then
MM1 The MMJMM1 ratio rises even earlier than
the isoform peaks An MMJMM1 ratio of 10 with
cellulose acetate electrophoresis has been reported
as the cutoff point for the detection of the patholog
ical release of MM3 from tissue (24) In our study
the MM3MM 1 ratio was 012149 in normal sub
jects 010136 in the first available serum of pa
tients with acute myocardial infarction while the
MM2MM 1 ratio was 0314 in normal subjects and
0348 in patients with acute myocardial infarc
tion The changes of the ratios MM3MM ~ and MM2
MM~ in acute myocardial infarction as compared
with the other diseases were statistically signifi
cant (p < 0001)
The CK isoforms in serum were reported to be
unstable (92225) and blood samples should be col
lected in EDTA tubes to inactivate carboxypeptidase
N and to prevent the conversion of isoforms Others
observed that CKMM isoforms were stable at least
3 months at 20 °C (216) Our results demon
strated that CKMM isoforms remain unchanged
over 7 days at 20 °C then 10 of total CKMM
was converted from MM3 to MM1 and no further
change was noticed for at least 4 months
The determination of serum CK isoforms is be
lieved to be an alternate noninvasive method to de
tect and follow acute myocardial infarction and suc
cessful reperfusion (89) According to Abendschein
(9) CKMM 3 increased dramatically within 1 h af
ter coronary occlusion and returned to baseline level
after 4 h Panteghini et al (23) reported that the
MM3MM1 ratio was raised 3 h after the onset of
acute myocardial infarction and peaked at 93 h
earlier than the peaking of isoforms It was also re
ported that CKMB2 the tissue form of CKMB was
298 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
O0 (t)
3
(3
0
0
0
h3
(~
o
>
¢r
¢r
Normal
AMI
Chronic
ischernic
disease
Other
heart
Muscle
Renal
GI
Lung
Brain
Misc•
CKMM2MM 1 Ratio ~
• ~' '~r~mm3wv 1 Ratio
b u b b b b o I
I I I I I I I ' ~ bu o b 0 b b 0 b b b b o ~ (~
I • ~ & Normal
• • I • lmOOnOmlOO I AMI • • I • '~ • •
I
' ~'~ Chronic I In • elm• •
~ ~ i5chemic
~~ msease
~ Other ~po • • • ov IPI • '~ hea rt O
~~
• '~ IVlU~Cl~ oo• 00 ooo • • ~
~ Renal • 0 •
O c~
~ GI m • o~ lid • • LL
~
d Lun~I
• 8 • ~
~ Brain • • • •
'1
Misc 1~ •
cn Z
~o
z > ~ 3o ~ 0
j
C
SHIESH TING AND JAP
4 MacroCK I
MM 1
1 1 1 ~ MM 2
I
I I MM 3
~ ~ ~ r j Applicat ion
point
MacroCK II
1 2 3 4
Figure 9 The migrating positions of CKMM isoforms
and other variants Arrows point to the positions of the
extra CKMM isoforms Lane 1 typical CKMM isoforms
in serum Lanes 2 and 3 extra CKMM isoforms found in
patients with pneumonia lane 4 macroCK types I II and
CKMM123
elevated earlier than the CKMB during the
course of cardiac damage (232627) The elevation of
the CKMB2 isoform in serum is more cardiac
specific than CKMM3 which is increased in both
myocardial and skeletalmuscle injuries (161921)
However the elevation of the MB 2 isoform occurs
more slowly and in lesser degree than the MM3 iso
form Furthermore the assay requires a technique
with sufficient sensitivity to detect the small change
of CKMB isoform In conjunction with the clinical
examination CKMM isoform analysis appears to
have significant advantages over CKMB isoforms
in clinical application
With the use of conventional agarose gel electro
phoresis for the CK isoenzymes eight specimens can
be analyzed simultaneously to quantitate CKMM
isoforms within 15 h without additional apparatus
The method is precise simple free from interference
by macroCK and hightemperatureinduced arti
facts and applicable for routine use in the clinical
laboratory
References
1 Wevers RA Olthuis HP Van Niel JCC Van Wilgen
burg MGM Soons JBJ A study on the dimeric struc
ture of creatine kinase (EC 2732) Clin Chim Acta
1977 75 37785
2 Wevers RA Wolters RJ Soons JBJ Isoelectric focus
ing and hybridization experiments on creatine kinase
(EC 2732) Clin Chim Acta 1977 78 2716
3 Chapelle JP Heusghem C Further heterogeneity
demonstrated for serum creatine kinase isoenzyme
MM Clin Chem 1980 26 45762
4 Falter H Michelutti L Mazzuchin A Whiston W
Studies on the subbanding of creatine kinase MM
and the CK conversion factor Clin Biochem 1981 14
37
5 Yasmineh WG Yamada MK Cohn JN Postsynthetic
variants of creatine kinase MM J Lab Clin Med 1981
98 10918
6 Panteghini M Pagani F Isoforms of creatine kinase
isoenzymes in serum in acute myocardial infarction
after intracoronary thrombolysis Clin Chem 1987 33
2O3942
7 Panteghini M Serum isoforms of creatine kinase
isoenzymes Clin Biochem 1988 21 21118
8 Wu AHB Creatine kinase isoforms in ischemic heart
disease Clin Chem 1989 35 713
9 Abendscheim DR Rapid diagnosis of myocardial in
farction and reperfusion by assay of plasma isoforms
ofcreatine kinase isoenzymes Clin Biochem 1990 23
399407
10 Armbruster DA The genesis and clinical significance
of creatine kinase isoforms Lab Med 1991 22 325
34
11 Chapelle JP Serum creatine kinase MM subband de
termination by isoelectric focusing a potential
method for the monitoring of myocardial infarction
Clin Chim Acta 1984 137 27381
12 Wu AHB Gornet TG Wu VH Brockie RE Nishikawa
A Early diagnosis of acute myocardial infarction by
rapid analysis of creatine kinase isoenzyme3(CK
MM) subtypes Clin Chem 1987 33 35862
13 Apple FS Sharkey SW Werdick M Elsperger KJ
Tilbury RT Analysis of creatine kinase isoenzymes
and isoforms in serum to detect reperfusion after
acute myocardial infarction Clin Chem 1987 33
50711
14 Wu AHB Creatine kinase isoform analysis Clin
Chem News 1991 17 1920
15 Vaidya H Dietzler DN Leykam JF Ladenson JH
Purification of five creatine kinaseMM variants from
human heart and skeletal muscle Biochim Biophys
Acta 1984 790 2307
16 Annesley TM Strongwater SL Schnitzer TJ MM
subisoenzymes of creatine kinase as an index of dis
ease activity in polymyositis Clin Chem 1985 31
4026
17 Leykam JF Dietzler DN Ladenson JH Purification
of human creatine kinase isoenzymes for monoclonal
antibody production Clin Chem 1985 29 1219
18 SiragEldin E Gercken G Harm K The isoelectric fo
cusing of creatine kinase variants II the heterogene
ity of creatine kinase in human serum with normal
and elevated catalytic concentrations J Clin Chem
Clin Biochem 1986 24 84760
19 Apple FS Hellsten Y Clarkson PM Early detection
of skeletal muscle injury by assay of creatine kinase
MM isoforms in serum after acute exercise Clin Chem
1988 34 11024
20 Williams J Williams KM Marshall T Heterogeneity
of serum creatine kinase isoenzyme MM in myocardi
al infarction clinical significance and postsynthetic
conversion ofabnormal subbands Clin Chem 1989
35 20610
21 Morelli RL Carlson CJ Emilson B Abendschein DR
Rapaport E Serum creatine kinase MM isoenzyme
subbands after acute myocardial infarction in man
Circulation 1983 67 12839
22 Wu AHB Gornet TG Measurement ofcreatine kinase
MM subtype by anionexchange liquid chromatogra
phy Clin Chem 1985 31 18415
23 Panteghini M Calarco M Serum isoforms of creatine
300 CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992
ISOFORMS OF CREATINE KINASE ISOENZYME MM
kinase MM and MB in myocardial infarction An ap
praisal of quantitative clinical and pathophysiologi
cal information Scand J Clin Lab Invest 1987 47
3259
24 Panteghini M Serum isoforms of creatine kinase MM
and MB as an index for the monitoring of acute myo
cardial infarction Clin Chem 1986 32 1135
25 Chapelle JP Bertrand A Heusghem C The protec
tion of creatine kinase MM subbands by EDTA dur
ing storage Clin Chim Acta 1981 115 25562
26 Puleo PR Guadagno PA Roberts R Perryman B Sen
sitive rapid assay of subforms of creatine kinase MB
in plasma Clin Chem 1989 35 14525
27 Puleo PR Guadagno PA Roberts R et al Early diag
nosis of acute myocardial infarction based on assay for
subforms of creatine kinaseMB Circulation 1990 82
75964
CLINICAL BIOCHEMISTRY VOLUME 25 AUGUST 1992 301
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