CLIN. CHEM.

22/2, 173-175 (1976)

Creatine Kinase Isoenzyme Patterns in

Human Tissue Obtained at Surgery

Swei H. Tsung

Chromatography on DEAE-Sephadex A-50 was applied Material and Methods

to study the quantitative distribution of creatine kinase
isoenzymes in extracts of human tissue obtained during Tissue obtained during surgery was homogenized
surgery. The results are compared with those deter- in a Virtis 45 homogenizer (1 g wet weight in 10 ml)
mined by an immunological method [Clin. Chim. Acta within 20 mm of its removal. The buffer used was as
58, 223 (1 975)] Conflicting . results for some organs as described by Klein et al. (7); per liter it contained
reported by the two methods are probably attributable 0.25 mol of sucrose, 10 mmol of tnis(hydroxymeth-
to postmortem autolysis. yl)aminomethane (pH 7.4), 1 mmol of [ethylenebis-
(oxyethylenenitrilo)]tetraacetate (pH 7.4), and 1
AddItIonal Keyphrases: intermethod comparison #{149}column
mmol of mercaptoethanol. Cellular debris was re-
chromatography #{149}reference values #{149}stability in tissues
moved by centrifuging twice at 4 #{176}C for 20 mm
Creatine kinase (CK, EC 2.7.3.2) isoenzymes have (16 000 X g) and the clear supernatant fluid was
been intensively investigated, in part because of the stored at -20 #{176}C
until used.
recent development of sufficiently sensitive and The supernatant fluid of tissue extracts was chro-
rapid techniques (1-4). However, there is little infor- matographed by discontinuous-gradient elution from
mation regarding the isoenzyme patterns in human DEAE-Sephadex A-50 (Pharmacia Labs. Inc.,
tissue. An earlier study by Dawson and Fine (5), by a Piscataway, N. J. 08854) as described by Yasmineh et
less sensitive technique, reported the presence of CK al. (2), with slight modification. One-milliliter ali-
in a few human organs other than brain, heart, or quots of tissue extracts containing not more than 3 U
skeletal muscle. More recently, study by the sensitive of CK activity were loaded on 0.6 X 6 cm micro col-
immunological method (6) showed CK to be ubiqui- umns of the anion exchanger equilibrated with 0.1
tous in human organs. Unfortunately, the specimens mol/liter NaCl-50 mmol/liter tris(hydroxymethyl)-
were obtained at autopsy, 12-24 h after death, and aminomethane (pH 8.0), CK isoenzymes were consec-
the distribution of CX isoenzymes found might not utively eluted with solutions containing 0.1 mol/liter

be representative of the situation during life. In this NaCl-50 mmol/liter tris(hydroxymethyl)amino-

communication, I report the use of a sensitive chro- methane (pH 8.0), 0.2 mol/liter NaC1 (pH 8.0), and
matognaphic method to study the distribution of CK 0.4 mol/liter NaCl (pH 7.0). Instead of depending on
isoenzymes in tissue obtained during surgery. a fixed elution volume of 15 for isoenzyme MM, 11
for MB, and 8 ml for BB, I determined the elution
Clinical Chemistry Section, Laboratory Service/113, VA Center, volume by electrophoresing CK isoenzymes to avoid
Wood (Milwaukee), Wis. 53193; and the Department of Pathology, overdilution of each fraction and to increase the sen-
The Medical College of Wisconsin, Milwaukee, Wis. 53233.
sitivity. For example, electrophoresis of extracts from
Ed note: cf. the paper by Yasmineh et al., in the January 1976
issue of this journal. stomach showed mainly BB isoenzymes with small
Received Aug. 1, 1975; accepted Nov. 10, 1975. peaks of MB and MM; respective elution volumes of

CLINICAL CHEMISTRY, Vol. 22, No. 2, 1976 173

 Table 1 Between-run
. Precision of CK lsoenzymes Table 2. CK Concentration and lsoenzyme
Assay by Column Chromatography in a Special Patterns (in Percent of Total Activity CK)
MB-Fortified Serum Control in Human Tissue
Percentage of MM MB BB
Recovery, CK activity,
MM MB BB % Tissue U/g wet tissue %
Mean 91.8 7.3 0.9 105.6 Skeletal muscle
SD 1 .08 0.78 0.27 2.34 (Gastrocnemi us) 3281 100 0 0
CV,% 1.2 10.6 30. 2.2 Skeletal muscle
n = 25 fortified serum samples with total CK activity ranging from (external intercostal) 1894 99 <1 <1
2295 to 2610 U/liter. Heart (right atrium) 402 78 22 0
Heart (right atrium) 356 76 22 2
Brain 157 0 0 100
Urinary bladder 162 2 6 92
Lung 13.4 30 1 69
4, 4, and 15 ml were obtained. The electrophoresis
Lung 9.2 24 1 75
was performed at pH 8.8 on angarose film by the
Lung 8.7 16 0 84
CK-isoenzyme procedure of Corning ACI, Palo Alto,
Lung 14 35 1 64
Calif. 94303. A 1- to 2-id sample containing CK was
Prostate 10 3 4 93
applied and electrophoresed (25 #{176}C, 25 mm, 250 V). Prostate 8 4 2 94
After electrophoresis, the films were layered with the
Uterus 47 2 1 97
CK reagent, blotted gently, incubated at 37 #{176}C for 20
Uterus 30 2 3 95
mm, and dried in an oven at 56 #{176}C.
Electrophoretic
Thyroid 28 26 1 73
patterns were scanned with a fluorometer (G.K.
Thyroid 36 4 0 96
Turner Associates, Palo Alto, Calif. 94303). Peak
Pancreas 3 14 1 85
areas were recorded with a recorder from Beckman
Stomach 86 2 4 94
Instruments, Inc., Fullerton, Calif. 92634.
Stomach 120 3 2 95
CK activity was estimated at 30 #{176}C with a centnifu-
Stomach 160 4 1 95
gal analyzer (GEMSAEC; Electronucleonics Inc.,
Colon 148 3 ‘1 96
Fairfield, N. J. 00706) by the kinetic method of Ros-
Colon 125 4 0 96
alki (8), with reagents from Calbiochem, La Jolla,
Ileum 161 3 1 96
Calif. 92037.
Kidney 15 12 0 88
Results Kidney 21 8 0 92
Liver 3.8 90 6 4
Column reproducibility was evaluated by repeated
Spleen 7 74 0 26
analysis of a serum pool fortified with MB isoen-
Salivary gland 6 44 0 56
zymes from an extract of heart tissue. A sample vol-
Bile 14.6 38 2 60
ume of 1 ml, containing about 2500 U of CX per liter,
(U/liter)
was applied to each of 25 ion-exchange columns. The
Placenta 1.7 15 22 63
results (Table 1) proved satisfactory.
Placenta 2.8 48 6 46
Table 2 summarizes results of the isoenzyme stud-
ies on tissue extracts. Skeletal muscle contained the
greatest concentration of CK activity, almost exclu-
sively MM isoenzyme, which agreed with previous
observations (2, 9). Myocardial tissue contained the
second greatest activity, of which 76-78% was MM ney and stomach was very stable: only 10% loss 72 h
isoenzyme, 22% MB isoenzyme, and 0-2% BB isoen- after the specimens were obtained from surgery. At
zyme. CK activity in brain was exclusively attribut- zero time, the isoenzyme distribution was 4% MM,
able to BB isoenzyme. Urinary bladder, lung, pros- 4% MB, and 92% BB for kidney and 4% MM, 1% MB,
tate, uterus, thyroid, pancreas, stomach, intestine, and 95% BB for stomach, as compared to 1% MM,
and kidney contained primarily BB isoenzyme, with 99% BB for both kidney and stomach when analyzed
activities ranging from 3 to 162 U/g wet weight. In 72 h later. In contrast, skeletal muscle lost half of its
contrast, liver and spleen contained a higher percent- CK activity in 10 h.
age of MM isoenzyme than of BB. Salivary gland and
bile contained both MM and BB isoenzymes, with
Discussion
more BB than MM. Placenta was most variable in The CK isoenzyme distribution I found for some
CK isoenzyme content. human tissues-such as skeletal muscle, heart, un-
I assessed the stability of CK activity in kidney, nary bladder, brain, stomach, and spleen-agreed
stomach, and skeletal muscle. The tissue was stored with that obtained by the immunological method (6).
at room temperature in a Petri dish with sufficient However, the values differed somewhat in the case of
moisture to obviate dehydration. CK activity in kid- prostate, uterus, pancreas and intestine, and differed

174 CLINICAL CHEMISTRY, Vol. 22, No. 2, 1976

completely for thyroid gland, liver, kidney, and lung. Reference
Wretou and Pfleiderer (6) found MM isoenzyme pre-
1. Mercer, D. W., Separation of tissue and serum creatine kinase
dominating in thyroid and kidney; I found BB isoen- isoenzymes by ion-exchange column chromatography. Clin. Chem.
zyme predominating in these two organs. In contrast 20, 36 (1974).
to their results, four lung specimens tested contained 2. Yasmineh, W. G., and Hanson, N. Q., Electrophoresis on cellu-
lose acetate and chromatography on DEAE-Sephadex A-SO com-
60-84% BB isoenzyme with slight variability in CK
pared in the estimation of creatine kinase isoenzymes. Clin. Chem.
isoenzyme composition, and one liver specimen con- 21, 381 (1975).
tamed primarily MM isoenzyme. The conflicting re- 3. Nealson, D. A., and Henderson, A. R., Separation of creatine ki-
sults noted are likely attributable to postmortem au- nase isoenzymes in serum by ion-exchange column chromatogra-
phy (Mercer’s method, modified to increase sensitivity). Clin.
tolysis. Sobel et al. (10) found low CK activity values
Chem. 21, 392 (1975).
in heart autopsy specimens obtained 4-27 h after 4. Henry, P. D., Roberts, R., and Sobel, B. E., Rapid separation of
death and had to extrapolate to the time of death to plasma creatine kinase isoenzymes by batch adsorption on glass
beads. Clin. Chem. 21, 844 (1975).
obtain values comparable to those obtained in fresh
5. Dawson, D. M., and Fine, I. H., Creatine kinase in human tis-
surgical specimens. Dawson and Fine (5) also en-
sue. Arch. Neurol. (Chicago) 16, 175 (1967).
countered the instability of the enzyme activity in 6. Wretou, V. J., and Pfleiderer, G., Quantitation of creatine ki-
autopsy tissue. nase isoenzymes in human tissue. Clin. Chim. Acta 58, 223 (1975).
My stability study indicates that the BB isoen- 7. Klein, M. S., Shell, W. E., and Sobel, B. E., Serum creatine
zyme is more stable than the MM. Our experience phosphokinase (CPK) isoenzymes after intramuscular injections,
surgery and myocardial infarction. Cardiovasc. Res. 7, 412 (1973).
with sera from patients with acute myocandial infarct
8. Rosalki, S. B., An improved procedure for serum creatine phos-
suggests that MB is also less stable than MM. Thus, phokinase determination. J. Lab. Clin. Med. 69, 696 (1967).
the number of hours after death at which the autopsy 9. Roberts, R., Henry, P. D., Witteeveen, S. A. G. J., and Sobel, B.
specimens were obtained will affect the isoenzyme E., Quantification of serum creatine phosphokinase isoenzyme ac-
tivity. Am. J. Cardiol. 33, 650 (1974).
pattern in tissue. Moreover, the cause of death may
10. Sobel, B. E., Bresnahan, G. F., Shell, W. E., and Yoder, R. D.,
be related to decomposition of organs and a change in Estimation of infarct size in man and its relation to prognosis. Cir-
isoenzyme pattern-for instance, sepsis. culation 46, 640 (1972).

CUNICAL CHEMISTRY, Vol. 22, No. 2. 1976 175