CLIN. CHEM.
33/11, 2039-2042 (1987)
Isoforms of Creatine Kinase Isoenzymes in Serum in Acute MyocardialInfarctionafter
IntracoronaryThrombolysis
Mauro Panteghlnland Franca Paganl
Kineticsof the catalytic activitiesof creatine kinase (CK; EC
2.7.3.2) for three CK-3 and two CK-2 isoformsin serumwere
studied in 20 patients with myocardial infarctionrandomly
assignedto receive either intracoronaryurokinase(groupA)
or conventionaltherapy (groupB). The temporalcharacteris-
ticsof isoformchanges describedwere (a) time at whichthe
isoformactivitiesare significantlygreater than initialvalues,
(b) maximal rate (Ks) at which Isoforms are released into
blood, (c) time lag from onset of pain untilmaximumactivity
value, ( peak value of each serum isoform,and (e) rate (Ku)
at which each isoformis cleared from serum. Thrombolytic
treatment inducedearlier peak times in group A: for CK-33,
7.4 vs 20.0 h;for CK-32, 11.6 vs 24.8; for CK-31, 18.6 vs 34.3;
for CK-22, 9.1 vs 17.8; and for CK-21, 11.8 vs 26.8 (numbers
given are medians; for all isoforms, P.cz0.05). K5 values were
at least twofold greater and the first increase was significantly
earlier in the urokinase group. Consequently, the ratio for
CK-33to CK-31 activitiespeaked significantly earlier in group
A. Isoform peak activities and i#{231} were not significantly
different between the two groups.
AddItIonalKeyphras.s: enzyme kinetics urokinase pt
synthetic modifications . coronary reperfusion . time-activity
curves
Abnormally increased activity of serum creatine kinase
(CK; EC 2.7.3.2) and its CK-2 isoenzyme is considered a
likely indicator of myocardial necrosis (1).’ Recently, it has
been possible to demonstrate multiple forms of CK-3 and
CK-2 isoenzymes in human serum (2,3). In particular, the
M-chain of CK undergoes a post-synthetic modification once
the enzyme reaches the circulation; as a result, five iso-
forms, three of CK-3 (CK-31, CK-32, CK-33) and two of CK-2
(CK-21 and CK-22), can be observed in human sera, whereas
tissue CK-3 and CK-2 exhibit a single isoform alone (CK-33
and CK-22) (4). The patterns of appearance of these isoforms
after their release from the myocardium into the circulation
have been studied in acute myocardial infarction (AM!)
patients and found to be diagnostically helpful (5-7).
Previously, we reported (8, 9) that the kinetics for total
CK and CK-2 in sera from patients assigned to receive early
thrombolytic treatment during AMI is characterized by
higher maximal rate of enzyme release in comparison with
acute infarcts in standard therapy. Furthermore, the reper-
fusion model of early increases in total CK, CK-2 isoenzyme,
and CK-3 isoforms in humans (10-12) and in dogs (13) has
been recently described by other authors.
1st Laboratory of Clinical Pathology, Spedali Civili, Brescia,
Italy.
Presented in part at the 38th national meeting of the AACC,
Chicago, IL, July 1986.
1NonsdJ abbreviations: CK, creatine kinase (ATP:creatine
N-phosphotransferase; EC 2.7.3.2);AMI, acute myocai-dial infarc-
tion; K,,, maximum rate of enzymatic activity increase; Kd, rate of
fractional disappearance of enzyme.
Received February 6, 1987; acceptedAugust 26, 1987.
The present study was designed to further investigate the
serum kinetics of the catalytic activities of three CK-3 and
two CK-2 isoforms in AM! patients undergoing intracoron-
ary thrombolysis and to determine whether changes in
isoform proffles can serve to identif’ successful coronary
recanalization. In particular, we characterized the CK-2
isoform kinetics following urokinase (EC 3.4.21.31; plasmin.
ogen activator)-induced reperfusion, which have not hither-
to been described.
Materials and Methods
Patients and Clinical Protocol
Ten men (ages 43 to 61 years) admitted to the coronary-
care unit of our hospital because of AM! underwent intra-
coronary thrombolysis. In addition, 10 conservatively treat-
ed patients (eight men and two women, ages 41 to 70 years)
with documented acute transmural myocardial infarction
(14) served as a control group to define the temporal course
of isoform activities from admission through hospital dis-
charge. These groups were the same patients evaluated for
other serum enzymatic characteristics in a previous study
(8); for more details about these patients and the modalities
of the urokinase infusion, cf reference 8.
Peripheral venous blood samples were obtained for deter-
mination of serum enzymes in both groups immediately
after admission to the hospital, every 2 h for the first day,
every 6 h for the following 72 h, and every 24 h for the
following three days. Within 2 h of collection, the nonhemo-
lyzed sera were frozen at -20 #{176}C in hermetically sealed
plastic containers and analyzed within 24 h.
Enzymatic Methods
Total CK activity was measured by the method recom-
mended by the Scandinavian Society Committee on En-
zymes (15), with reagents from Boehringer, Mannheim,
F.R.G., and a Cobas Bio analyzer (F. Hoffmann-La Roche
and Co., Ltd., Basle, Switzerland). Isoenzymes of CK and
their isoforms were separated on Titan ifi cellulose acetate
membrane in a “Zip Zone” electrophoresis chamber (Helena
Labs., Beaumont, TX 77704) with CK reagents from Boeh-
ringer (15). Separation of CK isoenzymes was performed
according to the manufacturer’s instructions. Serum CK
isoforms were separated by the technique described previ-
ously (6). Briefly, 0.5 L of sample was applied and electro-
phoresed in Ths-barbital, pH 8.8, at 4#{176}C for 50 mm at 300
mV; after electrophoresis, the strips were layered with the
CK reagent, blotted gently, incubated at 37 #{176}C for 20 mm,
and dried. The method yields results that vary linearly with
activity concentration, up to 500 U/L per band; samples with
values greater than this were diluted with saline. The
separated isoenzymes and isoforms were quantified by fluo-
rimetric scanning of the tracings with a Helena “Cliniscan”
densitometer. Fractions of each isoenzyme or isoform were
expressed as percentage of total CK activity in the original
serum sample; the absolute enzyme activity was calculated
by multiplying measured total CK by the percentage of CK
CLINICALCHEMISTRY, Vol. 33, No. 11, 1987 2039
attributable to each isoenzyme or isoform. Precision data for
CK isoform fractionation were obtained by using human
sera with normal (83 UIL) and high (1259 UIL) CK activi-
ties. The CV in serial analyses (n = 16) was between 2.2%
and 9.0%; between-day analysis of 10 observations of the
same specimens gave a CV between 2.4% and 11.7%. All the
bandswere verified to have true CK activity by incubating
celluloseacetate membranes containing identical samples,
one with and one without creatine phosphate substrate.
There was no fluorescencewhen the substrate-deletedsolu-
tion was used. Isoform nomenclature followed accepted
practice (12).
Data Analysis
Because values for the senun enzymes were not normally
distributed, we used nonparametric statistical tests for the
statistical analyses, and we used nonparametric group tests
to compare two data groups (Wilcoxon rank sum test) (16).
The level of significance was set at 0.05. The relationship of
two continuous variables was tested by linear least squares
regression analysis. In particular, the rate of increase (K0)
and the clearance rate (fractional disappearance rate, Kd)
for each enzyme activity were calculated by linear regres-
sion analysis from the linear portions of the ascending and
declining slopes of the time-activity curves, plotted semilo-
garithmically. The points used in calculations were the
corrected isoform activities, that is, the measured activities
minus individual baseline values. The minimum number of
values used to determine Ka and Kd was three and five,
respectively.
Results
The data for serum CK-3 and CK-2 isoforms are presented
in Table 1. The findings regarding total CK and CK-2
isoenzyme have been already published in a previous paper
(8) and are used here only for comparison. Times to peak for
serum CK isoform activities, measured from the onset of
chest pain, were significantly earlier in the urokinase-
treated patients, as compared with those receiving conven-
tional therapy (P <0.05 to 0.001). On the other hand, the
peak activities were similar in the two groups, although the
average peaks were lower in the control group.
The maximal rate of release for the isoformsin the treated
group was always more than twofold that in the control
group (P <0.05 to 0.001). To determine the exact start of
increasing isoform activities, we determined the time at
which the enzyme activities were significantly greater than
the initial values. This first increase was significantly
earlier in the urokinase group (P <0.05 to 0.001).
We also calculated the Kd of CK isoforms from each of the
serum curves. These were not statistically different between
the two groups.
Table 1 also showsthe ratio of CK-33 to CK-31 activities,
which peaked significantly earlier (P <0.05) in the uroki-
nase group. Again, in both groups, the ratio peaked signifi-
cantly earlier (P <0.01) than did CK-33, the isoform with
fastest appearance, alone (a difference of 2.2 h for the
urokinase group, 10.1 h for the control group). As in the
control group, the time to initial increase of CK-33 in the
reperfused group tended to be earlier than that of other
enzymatic variables, but not significantly.

Table 1. Data for CK-3 and CK-2 Isoforms In Serum in the Patients Studied, According to the Presence or Absence
of Thrombolytlc Therapy
Median(and range)
Isorm Control group Uroklnas group
Time of firstincrease,h after onset
CK-33 4.3 (3.5-7.1) 1.6 (1.1_2.3)8
CK-32 5.6 (3.6-7.6) 2.7 (1,8_4,0)b
CK-31 8.3 (3.8-13.8) 4.0 (2.9_5.5)d
CK-22 4.3 (2.8-7.9) 2.5 (1,2_47)d
CK-21 10.1 (4.9-13.3) 5.3 (3.9-10.6)”

CK-33 0.145 (0.095-0.215) 0.445 (0.230-0.51 1)

CK-32 0.077 (0.048-0,140) 0.210 (0.135-0.490) a
CK-31 0.053 (0.033-0.103) 0.112 (O.037-0.310)#{176}
CK-22 0.117 (0.049-0.415) 0.451 (0,250_0.824)b
CK-21 0.067 (0.041-0.100) 0.185 (O.0750.46O)”
Timeof peak, h after onset
CK-33 20.0 (10.0-29.4) 7.4 (5.8.-17.2)
CK-32 24.8 (10.5-30.1) 11.6 ($.6_21.$)b
CK-3, 34.3 (17.5-43.3) 18.6 (12,2_24.1)a
CK-22 17.8 (8.6-27.4) 9.1 (5.7_13.6)c
CK-21 26.8 (10.9-29.1) 11.8 (1#{216}.6_18,5)d
CK-33/CK-31ratio 9.9 (4.0-12.3) 5.2 (3,o7,g)d
Peak activity,U/L
CK-33 388 (127-945) 647 (267-3207)
CK-32 596 (276-1753) 960 (341-1765)
CK-31 610 (260-2860) 875 (283-1434)
CK-22 184 (54-567) 367 (74-837)
CK-21 61(8-149) 80(16-233)
h
CK-33 -0.047 (-0.034 to -0.118) -0.063 (-0.035 to -0.085)
CK-32 -0.033 (-0.026 to -0.041) -0.042 (-0.027 to -0.052)
CK-31 -0.021 (-0.005 to -0.030) -0.021 (-0.017 to -0.028)
CK-22 -0.047 (-0.033 to -0.149) -0.070 (-0.045 to -0.175)
CK-21 -0.026 (-0.010 to -0.039) -0.031 (-0.008 to -0.047)
&b.c.dsignificanuy
different( P <0.001, b P <0.01, ‘P <0.02. “P <0.05) fromcontrolgroup.

2040 CLINICALCHEMISTRY, Vol. 33, No. 11, 1987

 Finally, in both groups of patients, CK-33 activity in-
creased faster than CK-32 and CK-31 (P <0.05), and CK-22
activity increased faster than CK-21 (P <0.05). Also, within
each group, CK-33 and CK-22 activities decreased faster
than CK-32, CK-31, and CK-21 activities (P <0.01).
DiscussIon
Reperfusion of ischemic myocardium accelerates the en-
try of cardiac enzymes released from necrotic cells into the
circulating blood (17-19). Consequently, the process of fibri-
nolytic dissolution of coronary clot leads to characteristic
time-activity profiles of myocardial enzymes, including im-
mediate appearance, higher Ka, and earlier peak activity (8,
9, 20). Our previous results in different AMI populations
indicate that the consistency of CK isoform changes in
serum is related to the uniform kinetics of isoform conver-
sion and to the uniform kinetics of elimination of each
isoform from serum, despite differences in infarct location
and functional status of patients (6, 7). Thus, analysis of the
possible temporal changes in CK isoform patterns from sera
of patients submitted to successful thrombolytic therapy
may provide a reliable index of myocardial reperfusion.
Five kinds of isoform characteristics have been considered
in this study. The initial upward phase of the serum curves
and the peak catalytic concentrations occurred significantly
earlier in the urokinase-treated patients, and the maximum
rate of increase of serum CK isoforms in the urokinase-
treated patients was from two- to fourfold greater than in
the control group. Conversely, there were no significant
treatment-related differences in the peak activity values,
and the Kd analysis showed that the isoforms were eliminat-
ed at the same rate for both groups of studied patients.
Therefore, three characteristics of the time-dependent
change in serum isoforms seem potentially useful as indexes
of reperfusion: (a) first increase, (b) K0, and (c) time to peak.
In particular, earlier time to peak for the ratio of CK-3W31
activities in both groups of patients confirms this ratio to be
an earlier indicator of acute enzyme release from necrotic
myocardium, also after therapeutic thrombolysis (5, 7, 10).
However, the utility of the time to peak is diminished by the
frequent blood sampling necessary to establish the peak.
Consequently, the first increase and the rate of increase of
the different isoforms appear to be more useful indexes of
coronary reperfusion, being based on a simple calculation
requiring only two or three determinations, respectively.
In studies on dogs, the amount of CK in serum was twice
as high per gram of infarcted myocardium after reperfusion
as for animals in permanent occlusion (21). Nevertheless, in
more recent clinical studies as well as in the present work,
the peak serum enzyme activities did not differ significantly
according to the presence or absence of thrombolytic thera-
py-thus showing the independence of the total amount of
enzyme released from the type of therapy accomplished (8-
10, 17, 18,22).
The temporal characteristics of CK isoforms we have
described enlarge previous findings suggesting that the
thrombolytic therapy affects CK isoform profiles (washout
phenomenon) (10-12). The major differences between the
current work and those previously published are the man-
ner in which thrombolysis was achieved (urokinase in the
present work, streptokinase and (or) tissue plasminogen
activator in previous studies) and the characterization of
CK-2 isoform proffles, which have not hitherto been de-
scribed. In general, the manner in which thrombolysis is
achieved is not significant in terms of the effect of enzyme
release (11); the similarity of our findings to those of other
authors (10, 12) confirms this.
Our electrophoretic method showed sufficient sensitivity
for the simultaneous detection of CK-3 and CK-2 isoforms,
and the reproducibility of the isoform analyses was satisfac-
tory over the entire range of CK activities encountered (6,
7). This is important because the specificity of CK-33 or of
any CK-3 isoform as a criterion for the diagnosis of AMI is
less than that of CK-2 isoforms, owing to the fact that
quantities of CK-33 may be released from tissues other than
the heart (23,24).
Delineation of the relative efficacy of new specific throm-
bolytic agents requires reliable methods for noninvasive
detection of reperfusion (25). The information presented
here supports the use of CK-3 and CK-2 isoform determina-
tion as an early marker of therapeutic reperfusion in AMI
patients.
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