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Creatine kinase B-subunit activity in serum
211
Contributions to Cardiac Disease
Creatine Kinase B.subunit Activity in Serum Three years experience w i t h M - s u b u n i t i m m u n o i n h i b i t o r s and routine application to the diagnosis of acute myocardial infarction ( A M I )
W. GERHARDT, J. WALDENSTROM, R. B I L L S T R O M
a n d S. H O F V E N D A H L
Department of Clinical Chemistry, Lasarettet, 251 87 Helsingborg, Sweden THIS REPORT REVIEWS SOME BASIC FEATURES of the routine photometric method for kinetic determination of creatine kinase (E.C. 2.7.3.2, CK) B-subunit activity in serum (S-CK B) ~ after complete immunoinhibition of C K M-subunit activity by a specific Msubunit inhibitor (anti-M) 2''~.This review is intended as an introduction to papers from our Swedish working group ''5, all based on the same principle of immunoinhibition. This methodology functions equally well in photometry 6'' and in bioluminescence +,9 modes. Photometry. The routine photometry method developed and used by us during three years' is based on rate measurements of S-CK and S-CK B activities in the Scandinavian recommended C K reagent with E D T A '° in the absence or presence of anti-M. Sample residual adenylate kinase (S-AK, E.C. 2.7.4.3) is determined by the same reagent omitting creatine phosphate, and subtracted from the apparent S-CK B activity"L A p a i r of S-CK a n d S-CK B r e s u l t s , c o r r e c t e d f o r S-AK, m a y be o b t a i n e d w i t h i n 25 m i n u t e s . T h e m e t h od is e a s i l y a d a p t a b l e to a u t o m a t i o n . T h r o u g h p u t on t h e L K B R e a c t i o n R a t e A n a l y z e r 2086 is up to 60 samples per hour. Anti-M quality. We have suggested the following q u a l i t y r e q u i r e m e n t s f o r a n t i - M f o r use in t h e d i a g nosis of acute myocardial infarction: complete i m m u n o i n h i b i t i o n w i t h i n 10 m i n u t e s inc u b a t i o n o f s a m p l e in t h e CK r e a g e n t w i t h a n t i - M a t 37°C, w i t h no s i g n i f i c a n t i n h i b i t i o n of S - C K BB a n d a r e c o v e r y o f 48-50 p e r c e n t of S-CK B a c t i v i t y in S-CK MB. A m o n g s e v e r a l a n t i - M p r e p a r a t i o n s f r o m goat, dev e l o p e d b y W i i r z b u r g ~''~ a n d t e s t e d by us to M a r c h
1979, one ( a t p r e s e n t c o m m e r c i a l l y a v a i l a b l e in E u r o p e as M e r c k o t e s t CK-MB 14326, N A C - a c t i v a t e d , initiated with creatine phosphate) fulfilled these specifications. Imprecision: D a y - t o - d a y i m p r e c i s i o n o f CK BB a n d MB c o n t r o l s , a n d w i t h i n - d a y i m p r e c i s i o n o f p a t i e n t s e r a a r e g i v e n in T a b l e 1. D a y - t o - d a y i m p r e c i sion o f S-CK B a t o u r 1978 d i s c r i m i n a t i o n l i m i t o f 12 U/1 f o r A M I , w a s a b o u t 10 p e r cent. F o r m e a s u r e ments below this value bioluminescence measurem e n t s w e r e u s e d 8,9. Upper reference limit for S-CK B: 6 U/1 f o r b o t h sexes as determined by the bioluminescence method, calibrated against the routine photometric proced u r e TM. Clinical application: On t h e b a s i s o f p r e v i o u s d a t a 4'' we f o r m u l a t e d t h e f o l l o w i n g s t r a t e g y f o r o u r 1978 c o r o n a r y c a r e u n i t p a t i e n t s : s a m p l e s w e r e taken every 8 hours after admission and analyzed t h e f o l l o w i n g m o r n i n g . T o t a l S-CK w a s f i r s t m e a s ured. If the value was below a discrimination limit of 360 U / l , t h i s w a s r e p o r t e d a n d S-CK B w a s n o t d e t e r m i n e d . I f S - C K w a s above 360 U/I, S - C K B w a s m e a s u r e d . I f S-CK B w a s b e l o w 12 U / l , t h i s w a s r e p o r t e d . I f i t w a s a b o v e 12 U / l , r e s i d u a l s a m p l e S-AK was measured and subtracted. If corrected S-CK B w a s a b o v e 12 U / l , it i n d i c a t e d a 99 p e r c e n t p r o b a b i l i t y f o r an A M I . T h e a c t u a l r e s u l t s w i t h t h i s s t r a t e g y a r e g i v e n in Ref. 4. For our laboratory, this approach reduced the n u m b e r o f CK B d e t e r m i n a t i o n s to a b o u t 33 p e r c e n t of t h e t o t a l n u m b e r of all CK a n d CK B d e t e r m i n a tions which would otherwise have been performed.
TABLE 1
The authors want to thank the Thorsten and Elsa Segerfalks Fonden for generous financial support.
IMPRECISION
OF
CK
DETERMINATIONS
DAY-TO-DAY IMPRECISION TOTAL S-CK
N ............... X (U/l) . . . . . . . . . . SD (U/l) . . . . . . . . CV (%) . . . . . . . . .
N ............... X (U/l) . . . . . . . . . . SD (U/l) . . . . . . . . CV (%) . . . . . . . . .
REFERENCES
Precipath E526
MB Control
BB Control
58 496.7 12.5 2.5
33 607.4 25.8 4.3
30 777.4 30.8 3.9
WITHIN-DAY IMPRECISION TOTAL S-CK Patient A
Patient B
10 348.3 5.2 1.4
10 704.2 5.8 0.8
ACKNOWLEDGEMENT
i. Ljungdahl, L., and Gerhardt, W. Clin Chem. 24, 832, 1978. 2. Wiirzburg, U., Hennrich, N., and Lang, H. K/in Wschr. 54, 357, 1976. 3. Wiiurzburg, U., Hennrich, N., Orth, H.-D., and Lang, H. J. Clin. Chem. Clin. Biochem. 15, 131, 1977. 4. Hofvendahl, S., BillstrSm, R. and Gerhardt, W. Clin. Biochem. This issue. 5. WaldenstrSm, J., Herlitz, J., Hjalmarson, A., Holmbere, S., Swedberg, K., Vedin, A., Waagstern, F., WaldenstrSm A., Wilhelmsen, L. and Wilhelmsson, C. In Preparation.
(co~ti~ed on 2~. £13)
213 We later observed two such cases, which represent an unavoidable source of error. Another source of error could be extracardiac MB, e.g. from skeletal muscle. However, in about 1,000 cases of suspected AMI from two coronary care units (ours and Sahlgrenska's, Gothenburg) during three years we have not observed any such case. E x t r a c a r d i a c CK MB may occur in certain conditions as in one case of carbon monoxide poisoning from our intensive care unit with a total S-CK of 86,000 U/I and S-CK B of 820 U/l, representing 2 per cent CK MB as verified by electrophoresis. This patient exhibited no clinical signs of AMI. A subsequent cardiac scintigram was negative. The very high S-CK B activity indicated t h a t most, if not all, of this patient's S-CK MB came from extracardiac sources, probably skeletal muscle, since the highest CK B value observed in any AMI patient in our two patient series 1'3 was 360 U/1. The data in Table 1 confirm our previous conclusions t h a t : 1. Total CK is an excellent tool for the exclusion of an AMI and can be used as a decision threshold for a meaningful m e a s u r e m e n t of CK B. Such a procedure also rationalizes the work of the labo-. ratory. However, we intend to lower the decision limit to 300 U/1 in the future, since we have recently observed a false negative total CK in an AMI patient. 2. Any positive S-CK B value in the appropriate
Creatine Kinase B-subunit Activity in Serum (continued f~'om p. $ I I ) 6. Gerhardt, W., Ljungdahl, L., BSrjeson, J., Hofvendahl, S., and Hedeniis, B. Clin. Chim. Acts. 78, 29, 1977. 7. Gerhardt, W. and WaldenstrSm. J. Clin. Chem. 25, 1274, 1979. 8. Lundin, A., and Styrelius. Clin. Chim. A c t s . 87, 199, 1978. 9. Lundin, A., Lindberg, K., Nordlander, R., Nyquist, O., and Styrelius I. Proc. Int. Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Brussels 1978. (in press). 10. Scandinavian Committee on Enzymes. Scand. J. Clin. Lab. Invest. 39, 1, 1979.
clinical situation indicates an AMI with a probability of about 99%. Our results with the given sampling routine and methodology should be applicable to a clinical situation with a prevalence of AMI in the range 40-60%. This prevalence will be found in most coronary care units, and also commonly in the clinical situation in which the clinician suspects an AMI and requests an ECG, and determination of S-CK, and S-CK B. ACKNOWLEDGEMENT The authors want to thank the Thorsten and Elsa Segerfalk's Foundation for generous financial support. REFERENCES 1. Ljungdahl, L. Serum Creatine Kinase B-subunit Ac-
2. 3. 4. 5. 6.
tivity in Acute Myocardial Infarction (M.D. Thesis) Helsingborg-Lund, 1978. Ljungdahl, L., Hofvendahl, S., Gerhardt, W., and BSrjesson, J. Clin. Chim A c t s 78, 43, 1977. Gerhardt, W., Ljungdahl, L., and Hofvendahl, S. Clinical Enzymology Symposia 2 (Burlina, A. and Galzigha, L. eds). Piccin Medical Books (in press). Gerhardt, W., WaldenstrSm, J., BillstrSm, R., and Hofvendahl, S. Clin. Bioehem. This issue. Ljungdahl, L. and Gerhardt, W. Clin. Chem. 24, R32, 1978. Urdal, P. and Landaas, S. Clin. Chem. 25, 461, 1979. ~
4r
Contributions to Cardiac Disease
Creatine Kinase B.subunit Activity in Serum Three years experience w i t h M - s u b u n i t i m m u n o i n h i b i t o r s and routine application to the diagnosis of acute myocardial infarction ( A M I )
W. GERHARDT, J. WALDENSTROM, R. B I L L S T R O M
a n d S. H O F V E N D A H L
Department of Clinical Chemistry, Lasarettet, 251 87 Helsingborg, Sweden THIS REPORT REVIEWS SOME BASIC FEATURES of the routine photometric method for kinetic determination of creatine kinase (E.C. 2.7.3.2, CK) B-subunit activity in serum (S-CK B) ~ after complete immunoinhibition of C K M-subunit activity by a specific Msubunit inhibitor (anti-M) 2''~.This review is intended as an introduction to papers from our Swedish working group ''5, all based on the same principle of immunoinhibition. This methodology functions equally well in photometry 6'' and in bioluminescence +,9 modes. Photometry. The routine photometry method developed and used by us during three years' is based on rate measurements of S-CK and S-CK B activities in the Scandinavian recommended C K reagent with E D T A '° in the absence or presence of anti-M. Sample residual adenylate kinase (S-AK, E.C. 2.7.4.3) is determined by the same reagent omitting creatine phosphate, and subtracted from the apparent S-CK B activity"L A p a i r of S-CK a n d S-CK B r e s u l t s , c o r r e c t e d f o r S-AK, m a y be o b t a i n e d w i t h i n 25 m i n u t e s . T h e m e t h od is e a s i l y a d a p t a b l e to a u t o m a t i o n . T h r o u g h p u t on t h e L K B R e a c t i o n R a t e A n a l y z e r 2086 is up to 60 samples per hour. Anti-M quality. We have suggested the following q u a l i t y r e q u i r e m e n t s f o r a n t i - M f o r use in t h e d i a g nosis of acute myocardial infarction: complete i m m u n o i n h i b i t i o n w i t h i n 10 m i n u t e s inc u b a t i o n o f s a m p l e in t h e CK r e a g e n t w i t h a n t i - M a t 37°C, w i t h no s i g n i f i c a n t i n h i b i t i o n of S - C K BB a n d a r e c o v e r y o f 48-50 p e r c e n t of S-CK B a c t i v i t y in S-CK MB. A m o n g s e v e r a l a n t i - M p r e p a r a t i o n s f r o m goat, dev e l o p e d b y W i i r z b u r g ~''~ a n d t e s t e d by us to M a r c h
1979, one ( a t p r e s e n t c o m m e r c i a l l y a v a i l a b l e in E u r o p e as M e r c k o t e s t CK-MB 14326, N A C - a c t i v a t e d , initiated with creatine phosphate) fulfilled these specifications. Imprecision: D a y - t o - d a y i m p r e c i s i o n o f CK BB a n d MB c o n t r o l s , a n d w i t h i n - d a y i m p r e c i s i o n o f p a t i e n t s e r a a r e g i v e n in T a b l e 1. D a y - t o - d a y i m p r e c i sion o f S-CK B a t o u r 1978 d i s c r i m i n a t i o n l i m i t o f 12 U/1 f o r A M I , w a s a b o u t 10 p e r cent. F o r m e a s u r e ments below this value bioluminescence measurem e n t s w e r e u s e d 8,9. Upper reference limit for S-CK B: 6 U/1 f o r b o t h sexes as determined by the bioluminescence method, calibrated against the routine photometric proced u r e TM. Clinical application: On t h e b a s i s o f p r e v i o u s d a t a 4'' we f o r m u l a t e d t h e f o l l o w i n g s t r a t e g y f o r o u r 1978 c o r o n a r y c a r e u n i t p a t i e n t s : s a m p l e s w e r e taken every 8 hours after admission and analyzed t h e f o l l o w i n g m o r n i n g . T o t a l S-CK w a s f i r s t m e a s ured. If the value was below a discrimination limit of 360 U / l , t h i s w a s r e p o r t e d a n d S-CK B w a s n o t d e t e r m i n e d . I f S - C K w a s above 360 U/I, S - C K B w a s m e a s u r e d . I f S-CK B w a s b e l o w 12 U / l , t h i s w a s r e p o r t e d . I f i t w a s a b o v e 12 U / l , r e s i d u a l s a m p l e S-AK was measured and subtracted. If corrected S-CK B w a s a b o v e 12 U / l , it i n d i c a t e d a 99 p e r c e n t p r o b a b i l i t y f o r an A M I . T h e a c t u a l r e s u l t s w i t h t h i s s t r a t e g y a r e g i v e n in Ref. 4. For our laboratory, this approach reduced the n u m b e r o f CK B d e t e r m i n a t i o n s to a b o u t 33 p e r c e n t of t h e t o t a l n u m b e r of all CK a n d CK B d e t e r m i n a tions which would otherwise have been performed.
TABLE 1
The authors want to thank the Thorsten and Elsa Segerfalks Fonden for generous financial support.
IMPRECISION
OF
CK
DETERMINATIONS
DAY-TO-DAY IMPRECISION TOTAL S-CK
N ............... X (U/l) . . . . . . . . . . SD (U/l) . . . . . . . . CV (%) . . . . . . . . .
N ............... X (U/l) . . . . . . . . . . SD (U/l) . . . . . . . . CV (%) . . . . . . . . .
REFERENCES
Precipath E526
MB Control
BB Control
58 496.7 12.5 2.5
33 607.4 25.8 4.3
30 777.4 30.8 3.9
WITHIN-DAY IMPRECISION TOTAL S-CK Patient A
Patient B
10 348.3 5.2 1.4
10 704.2 5.8 0.8
ACKNOWLEDGEMENT
i. Ljungdahl, L., and Gerhardt, W. Clin Chem. 24, 832, 1978. 2. Wiirzburg, U., Hennrich, N., and Lang, H. K/in Wschr. 54, 357, 1976. 3. Wiiurzburg, U., Hennrich, N., Orth, H.-D., and Lang, H. J. Clin. Chem. Clin. Biochem. 15, 131, 1977. 4. Hofvendahl, S., BillstrSm, R. and Gerhardt, W. Clin. Biochem. This issue. 5. WaldenstrSm, J., Herlitz, J., Hjalmarson, A., Holmbere, S., Swedberg, K., Vedin, A., Waagstern, F., WaldenstrSm A., Wilhelmsen, L. and Wilhelmsson, C. In Preparation.
(co~ti~ed on 2~. £13)
213 We later observed two such cases, which represent an unavoidable source of error. Another source of error could be extracardiac MB, e.g. from skeletal muscle. However, in about 1,000 cases of suspected AMI from two coronary care units (ours and Sahlgrenska's, Gothenburg) during three years we have not observed any such case. E x t r a c a r d i a c CK MB may occur in certain conditions as in one case of carbon monoxide poisoning from our intensive care unit with a total S-CK of 86,000 U/I and S-CK B of 820 U/l, representing 2 per cent CK MB as verified by electrophoresis. This patient exhibited no clinical signs of AMI. A subsequent cardiac scintigram was negative. The very high S-CK B activity indicated t h a t most, if not all, of this patient's S-CK MB came from extracardiac sources, probably skeletal muscle, since the highest CK B value observed in any AMI patient in our two patient series 1'3 was 360 U/1. The data in Table 1 confirm our previous conclusions t h a t : 1. Total CK is an excellent tool for the exclusion of an AMI and can be used as a decision threshold for a meaningful m e a s u r e m e n t of CK B. Such a procedure also rationalizes the work of the labo-. ratory. However, we intend to lower the decision limit to 300 U/1 in the future, since we have recently observed a false negative total CK in an AMI patient. 2. Any positive S-CK B value in the appropriate
Creatine Kinase B-subunit Activity in Serum (continued f~'om p. $ I I ) 6. Gerhardt, W., Ljungdahl, L., BSrjeson, J., Hofvendahl, S., and Hedeniis, B. Clin. Chim. Acts. 78, 29, 1977. 7. Gerhardt, W. and WaldenstrSm. J. Clin. Chem. 25, 1274, 1979. 8. Lundin, A., and Styrelius. Clin. Chim. A c t s . 87, 199, 1978. 9. Lundin, A., Lindberg, K., Nordlander, R., Nyquist, O., and Styrelius I. Proc. Int. Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Brussels 1978. (in press). 10. Scandinavian Committee on Enzymes. Scand. J. Clin. Lab. Invest. 39, 1, 1979.
clinical situation indicates an AMI with a probability of about 99%. Our results with the given sampling routine and methodology should be applicable to a clinical situation with a prevalence of AMI in the range 40-60%. This prevalence will be found in most coronary care units, and also commonly in the clinical situation in which the clinician suspects an AMI and requests an ECG, and determination of S-CK, and S-CK B. ACKNOWLEDGEMENT The authors want to thank the Thorsten and Elsa Segerfalk's Foundation for generous financial support. REFERENCES 1. Ljungdahl, L. Serum Creatine Kinase B-subunit Ac-
2. 3. 4. 5. 6.
tivity in Acute Myocardial Infarction (M.D. Thesis) Helsingborg-Lund, 1978. Ljungdahl, L., Hofvendahl, S., Gerhardt, W., and BSrjesson, J. Clin. Chim A c t s 78, 43, 1977. Gerhardt, W., Ljungdahl, L., and Hofvendahl, S. Clinical Enzymology Symposia 2 (Burlina, A. and Galzigha, L. eds). Piccin Medical Books (in press). Gerhardt, W., WaldenstrSm, J., BillstrSm, R., and Hofvendahl, S. Clin. Bioehem. This issue. Ljungdahl, L. and Gerhardt, W. Clin. Chem. 24, R32, 1978. Urdal, P. and Landaas, S. Clin. Chem. 25, 461, 1979. ~
4r