Mar 14, 1995 - Objective: To study creatine kinase isoform variants in muscle. (skeletal and myocardial) damage using a Western blotting procedure.
Clinical Biochemistry, Vol. 28, No. 5, pp. 519-525, 1995 Copyright © 1995 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/95 $9.50 + .00
Pergamon 0009-9120(95)00030-5
Study of the Heterogeneity of Creatine Kinase-MM Isoforms Using Western Blotting INDRA RAMASAMY Department of Biochemistry, Repatriation General Hospital, Daws Road, Daw Park, SA 5041, Australia Objective: To study creatine kinase isoform variants in muscle (skeletal and myocardial) damage using a Western blotting procedure. Design and Methods: The study comprised of 16 patients admitted with chest pain, 14 patients with skeletal muscle damage, and 4 healthy individuals. The creatine kinase-MM (CK-MM) isoforms were separated by isoelectric focusing and detected by electrophoretic transfer to a nitrocellulose membrane and immunoblotting. Binding of the first monoclonal mouse antihuman CK-MM antibody was detected by either a horseradish peroxidase or alkaline phosphatase anti-mouse immunoglobulin conjugate. Results: Three major variants of CK-MM (MM3 pl = 6.8; MM2 pl = 6.4; MM1 pl = 6.2) were detected. Minor sub-bands were detected in 4/16 and 3/14 patients with acute myocardial infarction (AMI) and skeletal muscle damage, respectively. The tissue form (MM0 pl = 7.14) was visible in 6/16 patients with AMI and 4/14 patients with skeletal muscle damage. The appearance of minor variants was therefore of limited diagnostic value in discriminating between patients with and without AMI. Immunoblotting confirms the parallel increase in mass and activity of CK-MM isoforms, thus confirming measurement of isoform activity as an early marker of AMI or reperfusion following thrombolytic therapy. Conclusions: Study of isoform patterns gives more information concerning AMI and skeletal muscle trauma than is currently available from CK isoenzyme analysis. However, because CKMM is also released in skeletal muscle damage, in such instances more discriminant markers of AMI are needed.
forms in ischemic heart disease is useful for early diagnosis of acute myocardial infarction and for noninvasive determination of coronary artery perfusion for infarction patients receiving thrombolytic therapy (4,5). Current analytical methods detect CK isoforms by overlaying the gel with substrate; this can result in decreased resolution as at high enzyme activity individual isoform bands become diffuse or in artefactual bands due to (a) prolonged staining and residual fluorescence in the gel, (b) possible effects of ampholytes on CK activity. It is also possible that both catalytically active and inactive fragments are formed during metabolism and that such fragments appear in the sera of both the well and the sick. This study aims for the further understanding of the breakdown of CK isoforms and to determine the nature of both the active and nonactive breakdown products of CK-MM in patients with myocardial or skeletal muscle damage and otherwise healthy individuals using the technique of Western blotting. Materials and methods P A T I E N T POPULATION
KEY WORDS: creatine kinase; isoforms; Western blotting; heterogeneity. Introduction
nce released in the peripheral blood creatine kinase (CK) loses terminal amino acids by the acO tion of carboxypeptidase present in the blood, yielding enzymatically active fragments (1,2). As breakdown proceeds CK isoforms appear with decreasing molecular masses and faster anodic mobility. Three major isoforms of C K - M M and a total of 14 isoforms using high resolution isoelectric focusing have been identified (3). Measurement of the C K - M M iso-
Manuscript received: Jan 5, 1995; revised and accepted March 14, 1995. C L I N I C A L B I O C H E M I S T R Y , V O L U M E 28, O C T O B E R 1995
Sixteen patients with AMI (12 men, 4 women; ages 48-88 y). Diagnosis of AMI was based on clinical history, changes in ECG, and characteristic changes in serum enzymes (creatine kinase, lactate dehydrogenase, CK-MB, lactate dehydrogenase isoenzymes). Two patients received thrombolytic therapy. Fourteen non-AMI patients (12 men, 2 women; ages 69-82 y). This group included post-operative skeletal muscle trauma (aneurysm repair, total hip replacement, gastrectomy, laminectomy, anterior resection for colorectal cancer), post motor vehicle trauma and chest infection. Samples were collected 24-48 h after trauma. Four (4 men; ages 24-26 y) healthy individuals sampled 12-24 h after exercise. Serum for CK isoform analysis was allowed to clot 519
RAMASAMY
Thin layer flat bed isoelectric focusing (IEF) with pH 6 - 8 Biolyte (Bio Rad, CA, USA) was performed on the Biophoresis electrophoresis cell (Bio Rad). Polyacrylamide gels (100 x 100 x 0.6 mm (T = 4.85%, C = 0.15%) containing 10% w/v ampholyte were polymerised with N, N, N', N'-tetramethylethylenediamine, a m m o n i u m persulphate, and riboflavin -5'-phosphate. Gels were prefocused at 400 V for 15 min. Following the addition of 15 ~1 sample to each lane, gels were electrophoresed at 1400 V for 1 h, with 20 mmol/L Lysine, 20 mmol/L Arginine, 2 mol/L ethanolamine as the catholyte and 1 mol/L orthophosphoric acid as the anolyte. Surface pH measurements were carried out with a flat tip pH probe (Activon Products, Aus).
in Tris-buffered saline, pH 7.4 (TBS). After blocking the nonspecific binding sites, the filters were incubated overnight at room temperature with either mouse monoclonal antibody against the h u m a n creatine kinase M subunit or mouse monoclonal antibody against the h u m a n creatine kinase B subunit (Biospacific, CA, USA) at a dilution of 1:1000 (3.7 ~tg/ml) or 1:3000 (0.46 ~g/ml), respectively. The membranes were washed and incubated for 2 h at room temperature with horseradish peroxidase conjugated sheep anti-mouse immunoglobulin (at a dilution of 1:1000) in 1% blocker in TBS. Conjugate antibody was obtained from two different sources (Amersham, or Silenus, Vic, Aus). In between incubations the membranes were washed in 0.5% Tween 20 in TBS (two 15 min washes). The optimum antibody concentration for the primary and conjugate antibody were determined by dot blots of purified h u m a n CK-MM (Calbiochem, USA) titrated against several dilutions of antibody (1:100-1:100,000).
WESTERN BLOTTING
CHEMILUMINESCENT DETECTION
Following IEF the gels were washed in transfer buffer (25 mmol/L Tris, 192 mmol/L Glycine, pH 8.3) and transferred to nitrocellulose m e m b r a n e optimised for chemiluminescent detection (Hybond - ECL, Amersham, UK), at 50 V, 150 mA for 2 h using a mini Trans-Blot cell (Bio Rad).
Detection of the membrane bound second antibody was enhanced by chemiluminescence (BM Chemiluminescence Western blotting kit, Boehringer Mannheim, Germany). The method used luminol as the s u b s t r a t e for horseradish peroxidase, w i t h 4-iodophenol as the enhancer. Incubation was at room temperature for 60 s. Exposure time of the blot to the X-ray film (Kodak, USA) varied from 5 to 60 rain. An alternative method was to transfer proteins to nitrocellulose membranes. Binding of the first antibody was detected by alkaline phosphatase conju-
and centrifuged within 1 h of collection, stored at - 4 0 °C and analysed within 1 week. ISOELECTRIC FOCUSING
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F i g u r e 1 - - S e n s i t i v i t y of detection by dot blot analysis. Different a m o u n t s of purified h u m a n CK-MM were directly dotted onto t h e m e m b r a n e (Dilutions from 300 pg down to 10 pg). A f t e r i n c u b a t i o n w i t h a n t i - C K - M M a n t i b o d y (a) peroxidase l i n k e d or (b) a l k a l i n e p h o s p h a t a s e l i n k e d a n t i - m o u s e i m m u n o g l o b u l i n was used as secondary antibody. 520
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Figure 2a -- Chemiluminescent immunoblotting of CKMM variants from serum of patient FL following AMI. Insert shows changes in total CK activity. Arrows indicate time at which sequential samples in lane a, b, c were taken. gated goat anti-mouse immunoglobulin antibody and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as substrate (Bio Rad).
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Figure 2b - - Western blot of CK-MM variants of samples from Figure 2a, with alkaline phosphatase as the second antibody and photometric detection. Lane d is a representative Western blot of sera from a healthy individual 24 h after exercise; minor variants are indicated by arrows. Insert shows densitometric tracing of the individual isoform enzyme activity in lane a, b, c measured by the Rep ® CK electrophoresis method.
E N Z Y M E ASSAYS
Total CK was d e t e r m i n e d on the Hitachi 911 (Boehringer Mannheim, Reference interval 0 - 1 2 0 IU/L). C K - M B was determined by an immunoinhibition method (Boehringer Mannheim) on the Cobas Bio (Roche Diagnostic Systems Inc, Montclair, NJ, USA) and lactate dehydrogenase isoenzymes (Titan Gel LDH, Helena Labs, USA) and individual C K MM isoform activity (Rep ® CK Isoforms, Helena Labs.) by an electrophoretic method. Results This study characterizes CK isoforms by Western blotting. Two methods of antigen detection, photometric detection of the alkaline phosphatase second antibody conjugate and chemiluminescent detection of the horseradish peroxidase conjugate were examined. The horseradish peroxidase substrate, luminol, showed similar sensitivity to the alkaline phosphatase substrate (Fig. 1) but was a more rapid form of detection. A positive response was observed with C L I N I C A L B I O C H E M I S T R Y , V O L U M E 28, O C T O B E R 1995
10 ng of antigen by dot blots, though 30 ng appeared as darker spots (Fig. 1). A total CK of 142 IU/L was detected by Western blots (Fig. 2). However, artefactual bands, the result of nonspecific binding by the second antibody were observed w i t h certain batches of t h e peroxidase conjugate. The color formed by the reaction of alkaline phosphatase with 5-bromo-4-chloro-3-indolyl p h o s p h a t e t e n d e d to fade, compared w i t h t h e e n h a n c e d l u m i n e s c e n t method which gave p e r m a n e n t densitometrically scanable photographic records. To allow for the presence of artefactual bands "control" Western blots substituting either mouse antibody against the B subunit or 1% blocker in TBS, for mouse anti-CKMM was t a k e n through the same procedure. Similar results were seen with two different commercial sources of the h o r s e r a d i s h peroxidase conjugate (Silenus, Amersham) further confirming specificity of binding. All Western blots were carried out in duplicate. Three major isoforms were detected by both methods in 16 patients with AMI, 14 patients with skel521
RAMASAMY
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Figure 2c -- Chemiluminescent immunoblotting of CKMM variants from serum of patients SL, EA following AMI. Lane a,b = Patient SL; Lane c,d = Patient EA. Insert shows changes in total CK activity. Arrows show times at which sequential samples were taken. etal muscle t r a u m a , and 4 h e a l t h y individuals after exercise. Table 1 lists the average value of the isoelectric points observed. Despite differences in methodology the results are in agreement with previous authors (6,7,8). Isoforms are listed with arabic numbers, beginning with the band having the fastest mobility; the unmodified form is therefore MM3. The changes in serum C K - M M sub-band pattern after AMI is shown in Fig. 2 a,b,c,d. An initial cathodal shift with an increase in MM3 during increase in total CK activity was followed by an anodal shift (MM1 > MM3) during CK activity decrease. The time course of the appearance of MM3 in the circulation, therefore, provides an index of release of MM3 from muscle. In Fig. 2c, patient SL, the relative proportions of C K - M M isoforms (MM2 > MM3), suggests t h a t myocardial enzyme release has ended. The anodal shift was in 4 patients accompanied by the appearance of further sub-bands (Fig. 2d) which were the slowest of the sub-bands to appear. The tissue form of the isoenzyme was detected in 6/16 patients in the early hours after onset of the chest pain. One patient's serum contained 2 additional variants of C K - M M isoforms. 522
Figure 2d -- Chemiluminescent immunoblotting of CKMM variants from serum of patient PE following AMI. Arrows indicate minor variants. Insert shows changes in total CK activity. Arrows, in insert, show times at which sequential samples in lane a, b were taken. Lane c is a "control" Western blot of sample in lane a. The effect of prolonged elevation of total CK is shown in Fig. 3 a,b. The anodal shift shown in Fig. 2 is not evident, but a persistent increase in MM3, suggesting continuous release of the isoform with a more equal distribution of sub-bands was observed. The reintensification of MM3, for patient RS, is due to reinfarction. One episode of chest pain was identified for patient GB and the increase in MM3 is attributed to newly released CK from skeletal muscle. Fig. 4 shows the representative isoelectric focusing p a t t e r n s from p a t i e n t s with skeletal muscle TABLE 1 Isoelectric Points
of CK-MM Isoforms* pI CK-MM3 CK-MM2 CK-MM1
6.8 6.4 6.2
* A n average of 10 values. CLINICAL BIOCHEMISTRY, VOLUME 28, OCTOBER 1995
CREATINE KINASE-MM AND WESTERN BLOTTING
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Figure 3a - - Chemiluminescent immunoblotting of CKMM variants from serum of patient RS following AMI. Insert shows changes in total CK activity. Arrows indicate time at which sequential samples in lane a, b were taken. Lane c is a "control" Western blot of sample in lane a. An episode of chest pain was identified on Day 0 and reinfarction of Day 3. trauma. Minor CK sub-bands focusing between the three major isoforms were identified in 3/14 patients in this group; a f u r t h e r 4 patients demonstrated the tissue form and the slow sub-band was identified in 1 patient. 1/4 h e a l t h y individuals demonstrated all 8 variants of C K - M M isoforms.
Discussion Total CK activity is a composite of the activity of the various isoforms. The ratio of the untransformed form of CK to CK-MM1 reflects the time during which enzyme is being released from injured myocardium, t r a n s i t time while it is being transported through lymphatics, and rate of elimination of CKMM1 from the circulation. The more prolonged h a l f life is associated with the modified forms (9). Modification of CK alters some physicochemical properties of the enzyme. It causes an increase in the a p p a r e n t e n e r g y of a c t i v a t i o n a n d M i c h a e l i s Menten constants increase in the order CK-MM3 to CK-MM1, suggesting the original form has the lowest affinity for the substrate (10). This study shows t h a t CK activity and mass undergo conversion proportionally. Previous studies CLINICAL BIOCHEMISTRY, VOLUME 28, OCTOBER 1995
Figure 3b -- Chemiluminescent immunoblotting of CKMM variants from serum of patient GB following AMI. Insert shows changes in total CK activity. Arrows indicate time at which sequential samples in lane a, b, c were taken. One episode of chest pain was identified on Day 0. suggest t h a t CK-MM2 and CK-MM1 are the stages in the degradation of the enzyme t h a t still have enzymatic activity; we confirm t h a t further major enzymatically inactive forms are not found in the serum. Conversion of MM3 to MM1 and clearance of MM1 from serum, contributes to the decrease in CK activity. The theory of Wevers et al. does not explain the n a t u r e of several minor sub-bands of CK-MM (11). Williams et al. demonstrated t h a t in the presence of glutathione the minor bands converted to MM1, MM2, or MM3 (12). The mechanism is unknown, but other isoenzyme patterns are known to be modified by sulfhydryl group with glutathione (Enz - SH + GSSG ~ Enz-SS-G + GSH). This modification causes an anodal shift in isoenzyme distribution. Other authors report on 6 (14) or 11 (15) isoforms of C K - M M by isoelectric focusing. Williams et al. (3) identified 11 minor sub-bands and report on the presence of two sub-bands (pI 7.5, 7.35) associated with patients with AMI alone. This study reports on a total of 8 isoforms. Furthermore, sub-bands associated with AMI were not detected. These differences can be due to the different histories of the patient population in each study. The n a t u r e of the cathodically migrating forms is debatable. The isoform designated the tissue form in this investigation, appeared early d u r i n g tissue necrosis a n d bound to anti-CK-MM antibodies, suggesting it will 523
RAMASAMY
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Figure 4 - - Chemiluminescent immunoblotting of CK-MM variants from sera of 3 patients following post-operative skeletal muscle trauma. Lane a = Repair of aortic aneurysm (total CK = 1407 IU/L); Lane b = Gastrectomy (total CK = 1387 IU/L); Lane c = Repair of aortic aneurysm (total CK = 556 (IU/L). Arrows indicate minor variants. not interfere in th e i m m unoi nhi bi t i on test. Mitochondrial CK, an isoenzyme associated with a bad outcome has a similar alkaline isoelectric point (15). The two forms, mitochondrial CK and the tissue form of C K - M M can be distinguished by t hei r different reactivities towards the anti-CK-MM antibody. The inconsistent appearance of the observed minor sub-bands and t h e i r presence in both skeletal and myocardial muscle damage m a k e t he i r appearance a laboratory curiosity r a t h e r t h a n of diagnostic significance. F u r t h e r m o r e , C K - M M isoform distribution changes similarly in patients with AMI and skeletal muscle t r a u m a . In p atien ts with AMI and secondary skeletal muscle damage, and p ati e nt s with AMI and subsequent reinfarction the sub-band distribution is similar and more even, reflecting continuous MM3 release from tissue. The MM subform distribution in such patients therefore lacks specificity. In these instances C K - M B m e a s u r e m e n t by mass (16) or the newly developed assays for troponin T and troponin I (17) m a y be more discriminating. Use of an immunochemical r e a g e n t for fractionation of C K - M M isoforms can be useful for early diagnosis of AMI and for monitoring perfusion status (18,19,20). P r e p a r i n g an immunochemical t h a t discriminates b etwee n the MM isoforms is challenging, as t h e r e is one lysine residue difference in the 80,000 dalton protein. The technique described here can be valuable in testing antibody reactivity to the various isoforms. The analysis of C K - M M isoforms is an earlier i n d i c a to r of m y o c a r d i a l i n f a r c t i o n and c o r o n a r y reperfusion (21,22). T he pr e s ent study shows t h a t 524
isoform activity and mass change concurrently, th u s f u r t h e r validating m e a s u r e m e n t of isoform activity and MM3/MM1 ratio (21) as early indicators of AMI. However, because MM3 is also released in skeletal muscle disease and converted similarly to MM2 and MM1, m e a s u r e m e n t of C K - M B and other m a r k e r p r o t e i n s , t r o p o n i n T and t r o p o n i n I, m a y h a v e higher clinical specificity. References 1. Abendschein DR, Serota H, Plummer Jr TH, et al. Conversion of MM creatine kinase isoforms in human plasma by carboxypeptidase N. J Lab Clin Med 1987; 110: 798-806. 2. Michelutti L, Falter H, Certossi S, et al. Isolation and purification of creatine kinase conversion factor from human serum and its identification as carboxypeptidase. N Clin Biochem 1987; 20: 21-29. 3. Williams J, Williams KM, Marshall T. Heterogeneity of serum creatine kinase isoenzyme MM in myocardial infarction. Clin C h e m 1989; 35: 206-210. 4. Wu AHB, Gornet TG, Wu VH, et al. Early diagnosis of acute myocardial infarction by rapid analysis of creatine kinase isoenzyme-3 subtypes. Clin C h e m 1987; 33: 358-362. 5. Morelli RM, Emilson B, Rapaport E. MM-CK subtypes diagnose reperfusion early after myocardial infarction. A m J Med Sc 1987; 293: 139-149. 6. Wevers RA, Olthuis HP, van Niel JCC, et al. A study on the dimeric structure of creatine kinase. Clin C h i m Acta 1977; 75: 377-385. 7. Chapelle JP, Hensghem C. Further heterogeneity demonstrated for serum creatine kinase MM. Clin C h e m 1980; 26: 457-462. 8. Morell RL, Carlson CJ, Emilson B, Abendschein DR, CLINICAL BIOCHEMISTRY, VOLUME 28, OCTOBER 1995
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