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Immunologic detection of myocardial infarction with a radioimmunoassay for MB creatine kinase
141
Clinica Chimica Acta, 83 (1978) 141-149 @ Elsevier/North-Holland Biomedical Press
CCA 9136
IMMUNOLOGIC DETECTION OF MYGCARDIAL INFARCTION A RADIOIMMUNOASSAY FOR MB CREATINE KINASE
ROBERT
ROBERTS
*, BURTON
E. SOBEL and CHARLES
WITH
W. PARKER
Cardiovascular Division, Washington University School of Medicine, St. Louis, MO. (U.S.A.) (Received
August
16th,
1977)
summary Although antibodies to plasma isoenzymes have been induced, a radioimmunoassay (RIA) has not previously been developed in part because of lack of specificity and loss of enzymatic activity incurred during radioactive labelling. A RIA for human MB CK (“myocardial”), a creatine kinase isoenzyme, would provide a specific and quantitative test for myocardial infarction, and perhaps more important, it would aid in elucidating the nature of MB CK protein turnover in the circulation independent of enzymatic activity. Assuming that MB, but not MM (the only other isoenzyme present appreciably in blood from patients with myocardial infarction) would bind to B subunit antibody, we harvested BB antiserum from rabbits immunized with human brain BB purified by chromatography. BB, MB, and MM for use as antigens in the RIA were labelled with radioactive N-succinimidyl 3-( 4-hydroxyphenyl-propionate), a procedure that avoided oxidation of the antigen and yielded 200 000 cpm/pg and 97% of initial CK enzymatic activity. After incubation in 1.6 M Tris, pH 7.4,2% albumin, and 20 mM CH&H#H, labelled BB and MB bound to anti-BB (93 and 56%) and precipitated in 44% (NH4)#04, but 1251-MM in 5000-fold excess did not. Unlabelled MB (20 pg/ml) but not MM (up to 5000 pg/ml) competitively displaced precipitable counts. Development of a sensitive and specific RIA based on this phenomenon accurately detected MB (20 pg/ml) despite excess MM (5000-fold excess) in plasma samples constituted with standard. In patients with myocardial infarction, MB CK determined by the RIA was elevated in all cases.
* Correspondence should be addressed to: Dr. Robert Roberts, Director. Cardiac Care Unit. Washing ton Unhwsity School of Medicine. 860 South Euclid Avenue, St. Louis, MO. 63110, U.S.A.
142
Introduction Despite widespread diagnostic analysis of plasma enzymes, very little is known of factors influencing their rates of disappearance from the circulation [ 11. It is not clear whether disappearance of enzyme activity reflects inactivation, denaturation or removal of intact enzyme molecules. Enzymatic detection of myocardial infarction has been based conventionally on plasma elevations of GOT, LDH, and CK, but recent results indicate that detection with elevated MB CK is the most specific and sensitive approach [ 2,3]. Creatine kinase in human plasma exists in at least three isoenzyme forms (each with molecular weight of approximately 82 000) designated MM, MB and BB on the basis of subunit composition [4]. Plasma from normal subjects contains primarily MM with less than 0.005 I.U./ml of MB and no detectable BB CK [5-71. Analysis of human tissue CK isoenzyme profiles indicate that brain contains only BB [8-lo], skeletal muscle only MM [8,9] and heart, a combination of MM and MB [8-lo]. Nevertheless until recently BB has not been detected in the plasma after brain injury in patients with cerebral infarcts, injury or infection [ll-131. With a chromatographic assay small amounts of BB CK have been detected after cerebral injury [ll]. Release of BB may in part be impeded by the brain barrier or lability of BB CK in cerebrospinal fluid or blood itself. After cerebral damage, plasma MM is often elevated, presumably because of release from skeletal muscle induced by sympathetic stimulation [ 141. Analysis of plasma CK isoenzyme activity after intramuscular injections [ 51, and surgical procedures [lo] demonstrates that plasma MM CK may be markedly elevated. However, elevations of BB or MB do not occur. MB appears consistently in plasma after myocardial infarction but BB does not [9,10]. Although several assays for MB CK activity have been developed [5,8,15], they are not ideally suited for quantitative analysis with large numbers of samples; their sensitivity is somewhat limited; and they rely exclusively on detection of enzyme activity rather than other physical properties of CK isoenzyme molecules. In the present study, human CK isoenzymes were isolated in near pure form from heart and brain and antibodies developed specific for the M and B subunits. Human CK isoenzymes were labelled with lz51 utilizing a procedure which avoids exposure of the isoenzyme to oxidizing agents and provides an antigen with high specific radioactivity without loss of enzyme activity. Dissociation of subunits was prevented by performing the reaction in a high concentration of Tris buffer and 2-mercaptoethanol. A competitive displacement radioimmunoassay was developed specific for the B subunit, reported recently in preliminary form, to permit measurement of plasma MB CK protein concentration after myocardial infarction [ 161. The present communication describes the method and its validation in detail. Materials and methods Isolation
of human
CK isoenzymes
MM and MB isoenzymes from human brain obtained
were prepared from human myocardium and BB at necropsy within six hours of death. Homogenates
143
from human myocardium and brain were prepared as follows: The fresh tissue was trimmed of fat, cut into small pieces with scissors and passed through a pre-cooled meat grinder. Ground tissue was homogenized in a Wring blender containing 2 ml/g of 0.05 M TrisfHCl, pH 7.5, and 0.001 M 2-mercaptoethanol. All preparative procedures were performed at O-4°C. The myocardial homogenate was centrifuged at 31000 Xg for 15 min in a supernatant fraction filtered through 8 layers of cheesecloth. Ninety-five percent ethanol was added in dropwise fashion until the final concentration was 50%, and the mixture was allowed to stand while slowly stirring at 40°C for 30 min. The precipitated material was removed by centrifugation and the supernatant fraction decanted. Again ethanol was added in stepwise fashion until a final concentration of 70% was obtained. The mixture was allowed to stand for 30 min and the resulting precipitate was recovered and saved. With the use of a Dounce homogenizer, the precipi~t~ pellet was suspended in homogenizing medium equal in volume to 50% of the original homogenate. After ceut~fugation at 31000 Xg, the pellet from this resuspended mixture was discarded and the supernatant fraction saved. To separate the MM CK, NaCl, 5 M, was added with rapid stirring to a final concentration of 0.05 M. DEAE Sephadex A-50 was then added (10 ml slurry for 48 mg of protein) and the mixture was stirred for 30 min. This suspension was filtered with a Buchner funnel lined with Whatman’s No. 1 filter paper, and the filtrate was dialyzed against several changes of 0.01 M glycine, NaOH, pH 9.0 prior to freeze drying of the MM fraction. The MB CK present in the homogenate was adsorbed to the DEAE-Sephadex previously added. Accordin&, after the MM filtrate had been obtained, the ~EAE-Sephadex was retained in the Buchner funnel and washed five times with 0.05 M Tris/HCl, pH 7.4, 0.05 M NaCl, and 0.001 M 2-mercaptoethanol. After the DEAE-Sephadex had been washed, MB CK was eluted with 0.05 M Tris/HCl, pH 7.4, containing 0.3 M NaCl. The eluate was dialyzed with glycine buffer, freeze-dried to obtain the MB fraction. To further enrich the MM and MB fractions, further ethanol fractionation and column ~hromato~aphy was performed followed by dialysis, freezed~ing and storage at 0-4°C. CK was extracted from brain in the same fashion as from myocardium with the following exceptions: The final concentration of ethanol was 60% rather than 50%. After filtration of the MM fraction, Sephadex retained in the Buchner funnel was washed five times with Tris/HCl buffer containing 0.1 M NaCl, and subsequently washed once with buffer containing 0.1 M NaCf, and once with buffer containing 0.3 M NaCl. BB CK remained adsorbed to Sephadex under these conditions was then eluted with Tris buffer containing 0.4 M NaCl dialyzed against 0.01 M glycine, NaOH, pH 9.0, and freeze-dried. Polyacrylamide gel electrophoresis [ 17 3 of the human preparations indicated that each isoenzyme was abtained in a sample devoid of activity attributable to other isoenzymes in the initial extract. MM CR averaged 392 I.U./mg of protein, MB 46 I.U./mg, and BB 110 I.U./mg. Specific activity of each isoenzyme was increased more than one hundred-fold over that present in the initial extract and analysis by SDS gel electrophoresis [ 181 with staining for protein showed only one faint contaminating band of the MM and no contaminating bands of BB with two very faint contaminating bands of the MB,
144
Induction of antibodies to CK isoenzymes Utilizing the purified human MM and BB CK mixed with equal volumes of Freund’s complete adjuvant, antibodies to CK isoenzymes were induced in rabbits. Initially, the rabbits were injected subcutaneously with 1 mg of immunogen (0.25 mg/foot pad). Subsequently, they were injected with 0.25 mg weekly for three weeks. All animals were given booster injections of 0.1 mg in complete adjuvant at monthly intervals thereafter. Ten days after each booster injection, the animals were bled and their serum analyzed for antibody activity. Ouchterlony agarose plates, prepared with BB antiserum exhibited single precipitin lines to BB and MB antigen but no precipitin line to MM. Plates prepared with MM antiserum exhibited a single precipitin line to both MB and MM but none to BB. Thus, antibodies to BB CK reacted with BB CK and also crossreacted with MB but did not cross-react with MM indicating specificity for the B subunit. Similarly, MM antibodies were specific for the M subunit. Radioactive labelling of CK isoenzymes Radioiodine ( 12’1) was utilized to radioactively label CK isoenzymes for subsequent use in a competitive displacement radioimmunoassay. When “‘1 was introduced into the isoenzymes by the chloramine-T [19] or lactoperoxidase [20] method, there was marked loss of CK enzyme activity, possibly due to oxidation of essential sulfhydryl groups. To avoid exposing the enzymes to oxidizing agents and contaminants in the radioiodine, the “‘1 was first incorporated into the N-succinimidyl ester 3-( 4-hydroxphenyl-propionate) which in turn was reacted with amino groups on the CK isoenzyme protein. N-Succinimidyl 3-(4-hydroxyphenyl-propionate) was radioiodinated by the method of Bolton and Hunter [21]. The reaction was carried out at room temperature (23°C). N-Succinimidyl 3-( 4-hydroxphenyl-propionate) (0.3 pg) was treated with 5 mCi (lo-20 ~1) of Na1251, 50 pg of chloramine-T and 10 ~1 of 0.25 M phosphate buffer, pH 7.5. The reaction was immediately terminated by the addition of 120 pg of sodium metabisulfite in 10 ~1 of 0.50 M phosphate buffer, pH 7.5 containing 200 pg of KI. The iodinated product was extracted into benzene (0.300 ml X 2 portions) and recovered by evaporation of the solvent under vacuum. The addition of dimethylformamide (5 ~1) before adding the benzene was necessary for full extraction of the ester into the solvent. The residue was then used to label creatine kinase isoenzymes. The labelled residue was combined with 2-8 mg of MM, MB or BB CK in l-2 ml of 0.01 M sodium borate buffer, pH 8.5. After gently shaking the medium for 4 h at 4°C the labelled isoenzymes were then dialyzed against the same buffer containing 0.005 M 2-mercaptoethanol. Radioactivity per mg of labelled CK isoenzymes averaged 200 000 cpm for MM CK and MB CK and 100 000 cpm for BB CK. The maximum loss of enzyme activity resulting from labelling and dialysis was less than 5% for each isoenzyme preparation. Binding affinity The binding antiserum) was by diluting the determinations
and specificity of antiserum affinity and specificity of the BB and MM antibodies (rabbit determined over a wide range of concentrations of the antibody appropriate antiserum over a range of 1 : 15 to 1 : 1000. All were performed in duplicate and carried out in 12 X 75 ml glass
145
tubes containing 1.6 M Tris buffer, pH 7.6 (200 pl), 2% bovine serum albumin (100 pl), 0.020 M mercaptoethanol (10 pl), 5 pg of rabbit gamma globulin (50 ~1). The gamma globulin and serum albumin minimize nonspecific binding [22]. The high concentration of Tris and 2-mercaptoethanol protects the sulfhydryl groups of the isoenzymes and prevents dissociation into monomers. To this mixture was added the appropriate dilution of antiserum in volumes ranging from 100 ~1 to 5 ~1 (dilutions performed with normal rabbit serum). ‘251-labelled MM (0.1 pg), MB (0.1 pg), and BB CK (0.2 pg) were added such that approximately 25 000 cpm were present in each tube. The total volume was kept constant at 500 ~1 with necessary adjustments being made with Tris buffer. The solutions were then incubated and gently shaken at 4°C for 6 h. Appropriate controls were incubated containing normal rabbit serum rather than rabbit antiserum. Following the incubation period, separation of free from antibodybound labelled CK was accomplished by the addition of cold saturated ammonium sulphate [22] with a final concentration of 44%, and allowed to sit at room temperature for 15 min. The solution was then centrifuged at 2000 Xg for 20 min, the supernatant decanted, and the pellet washed with 50% ammonium sulphate (400 ~1) and again centrifuged. The pellets were counted in a gamma counter (Micromedic Systems, Inc.) until a minimum of 10 000 counts were obtained. The 12’1 counts present in the pellet expressed as a percent of the total number of counts initially present represent percent binding. To optimize conditions for any possible cross-reactivity between the BB antibody and MM CK and BB CK with the MM antibody, determinations were done in which BB antiserum diluted only 1 : 15 was incubated with 4 pg of 12SI-MM and MM antiserum in a dilution of 1 : 15 with 4 pg of 12’I-BB. Procedure for radioimmunoassay To develop a competitive displacement radioimmunoassay for plasma MB CK, BB antiserum was used and the specificity of the BB antiserum for B subunits further established by comparing the ability of unlabelled BB, MB and MM CK to inhibit 13’I-BB binding. The reaction was performed in the same buffer solution used for the binding experiments, but with the exceptions that the dilution of antiserum was kept constant at 1 : 150 and the amount of 12”1-BB CK was kept constant at 0.2 pg containing approximately 25 000 cpm. The antiserum dilution of 1 : 150 was chosen since this concentration of antibody binds about 50% of the 1251-BB [23]. A known amount of unlabelled BB, MB, or MM CK was diluted from 1 : 15 to 1 : 1000 and incubated for 6 h with 12’1-labelled BB. Following incubation, the ammonium precipitated pellet To further deterwas washed, centrifuged and counted for 1251radioactivity. mine the specificity of unlabelled BB or MB to displace 12’I-BB binding in the face of MM CK, inhibition curves were determined for MM incubated with BB or MB in which MM was present in a 25 000 fold-excess over that of unlabelled BB or MB. Determination of MB CK in plasma samples To determine the amount of MB CK present in an unknown sample, a standard MB inhibition curve is run with known amounts of unlabelled MB CK ranging from 200 to 0.1 ng/ml of MB CK. The unlabelled MB CK is incubated
146
with a constant amount of antiserum and “‘1-BB CK as outlined previously under radioimmunoassay procedure. Serial dilutions (3-4) of the unknown sample are made and the amount of inhibition determined at each dilution and compared to the standard curve from which it is possible to calculate the amount of MB CK present expressed as ng/ml. To determine the accuracy of the assay, known amounts of human unlabelled MB CK were added to heat inactivated serum and serial dilutions done and results expected compared to that obtained. In addition, plasma samples were obtained from twenty patients with acute myocardial infarction. At least 15 samples were obtained serially from each patient over a period of 60 h. All samples were assayed in duplicate and assays of enzymatic activity obtained by a kinetic fluorometric assay previously described [5]. All determinations for total CK enzymatic activity were done according to the Rosalki method [ 241. Results Antibody specificity Results of binding experiments using serial dilutions of BB antiserum from 1 : 15 to 1 : 1000 incubated with iodinated MM, MB and CK are shown in Fig. 1. Ninety-three percent of the “‘1-BB was recovered in the pellet in dilutions of 1 : 30, but binding diminished rapidly with only 7% at 1 : 1000, demonstrating that binding was dependent on antibody concentration. Maximum binding of “‘I-MB CK (60%) occurred at 1 : 15 dilutions but again binding was dependent on the concentration of antibody with only 5% binding at ‘251-MM exhibited no such antibody 1 : 1000 dilution. In contradistinction, concentration dependent binding. At all dilutions binding was between 3-5%, similar to that with control (normal rabbit serum). These results demonstrated the antibody is specific for the B subunit rather than the molecule as a whole. Results of binding with MM antiserum showed 94% binding of 12’I-MM and 50% binding of 12’I-MB at dilutions of 1 : 30. Again binding was dependent on the concentration of antibody and returned to control levels at a dilution of 1 : 1000. No specific binding was seen between the MM antiserum and i2’I-BB.
'=I 88 CPK
-
'=I MB CPK
-
lz51 MM
0
I 250
DILUTION
I 500
CPK
---
I
I
750
1000
OF BB ANTISERUM
Fig. 1. Shows the specificity of BB antiserum for BB and MB. Binding of ’ 2SI-BB and ’ 2SI-MB is dependent on the concentration of BB antibody. There is no binding of 1 2 5 I-MM CK at any concentration of BB antibody.
147
The MM antiserum exhibited reactivity with the B subunit.
specificity
for the M subunit
but no cross-
Results of radioimmunoussay Unlabelled MB CK competitively displaced labelled BB CK from binding to the BB antibody which was dependent on the concentration of MB CK as shown in Fig. 2. As can be seen, the inhibition curve is steep between 0.5 and 100 ng/ml with 50% inhibition at 10 ng and complete inhibition of binding at a concentration of 150 ng/ml and above. A similar inhibition curve was seen for unlabelled BB which showed 50% inhibition at 5 ng/ml and complete inhibition at 70 ng/ml and higher. Unlabelled MM CK showed no inhibition of ‘2SI-BB binding, even at 5 pg/ml (2000-fold excess over “‘1-BB CK). Furthermore, the competitive inhibition of unlabelled MB CK was unaltered in the presence of high concentrations of MM CK (5000 molar excess over that of MB CK). Thus, the BB systems as a competitive displacement assay for MB is extremely sensitive, detecting reliably a concentration of 0.5 ng/ml representing enzymatic activity of 1 X lo-’ I.U./ml. Furthermore, the specificity is such that results at this level of sensitivity are unaffected by 5000 molar excess MM. Results of radioimmunoassays performed on heat inactivated serum constituted with known amounts of MB CK ranging from 0.20 ng/ml to 2 pg/ml deviated by less than 3% from those expected. Radioimmunoassay of 300 samples obtained from 20 patients with acute myocardial infarction exhibited elevated MB CK in all cases. A typical MB CK curve from one of the patients is shown in Fig. 3.
1000 t
I
I
0.5
1
2
5 IO 25 MB -CK (ng/ml) I 1 I .008 .016 .032 .08 .I6 0.4 MB-CK
l
RIA
I:
Enrymollc
,I
50
100 150
I 0.8
II 1.6 2.4
0
(mIU/ml)
Fig. 2. Shows competitive displacement of 12 51-BB CK by unlabelled dependent. The Wition curve is steep in the range of O.l5-100 ng.
20
40 TIME
60
80
100
(hr)
MB CK which is concentration
Fig. 3. A plasma CK-time activity curve in a patient with myocardial infarction. Since 66 ng of purified MB enzyme protein was equivalent to 1 m1.U. of enzyme activity, vahws obtained with the ndioimmunoassay method (RIA). expressad initially as ng/ml, wsre converted to enzymatic activity and compared to results obtained by a kinetic fluorometric aawy which m eamues onl~enz~mat&activity.Aacm beseen. v&es obtained with the two methods are in close agraement. Similar agreement was obtained in all 300 samples from the 20 patients studied.
148
Discussion In this study, we have described a radio~munoassay for CK isoenzymes. Antibodies were developed for MM and 38 CK which are specific for the M and B subunits, respectively. Since MB CK, found in the human myocardium, contains both subunits, either antibody can be used to detect MB CK. The BB antibody reacts with BB and cross-reacts with MB CK but not MM even when present in 5000 molar excess over that of MB CK, a ratio far greater than that seen in plasma after myocardial infarction since MB CK usually represents lO-15% of total CK activity [ 51. Detection of plasma MB CK using the BB system is quite specific. Since BB CK activity is not present in normal plasma [5], in patients with cerebral disorders [ 12,131, or those with acute myocardial infarction [ 5,6,8,9]. Thus, in these clinical situations, displacement binding reflects MB exclusively. These impressions were corroborated by the close agreement between enzymatic activity determined by the kinetic ~uorometric method [5] and apparent concentration of protein detected by RIA in samples obtained from patients with acute myocardial infarction. The sensitivity provided by the RIA exceeds that of previously available CK isoenzyme assays be several orders of magnitude. Thus, assays [ 241 based on enzymatic activity [ 241 have a sensitivity in the range of 1 X low2 I.U.fml as opposed to the present assay which detects reliably 1 X lo-’ I.U./ml. Since mean plasma MB CK activity in normal subjects is 1 X 10e3 I.U./ml, a 5-fold increase is necessary for detection of elevations by enzymatic assays in contrast to the RIA which can easily detect normal values as well as modest increases. The increased sensitivity, coupled with the potential for detection of enzymatically inactive MB CK in the circulation should lead to improved estimates of infarct size as well as earlier detection of acute myocardial infarction 1251. The present findings suggest that a similar approach may be useful in differentiation of other clinically important enzymes which exist in multiple forms. Previous assays for plasma enzymes and isoenzymes have been asked on enzymatic activity. Studies evaluating the disappearance of the enzymes from the circulation have been generally restricted to determining the loss of activity. However, since RIA detects the concentration of molecules, it permits evaluation of the rate of isoenzyme protein turnover independent of enzymic activity. Thus, RIA should help to elucidate mechanisms responsible for disappearance of individual CK isoenzymes from the circulation [ 261 as well as aid in elucidating the relative importance of inactivation, denaturation, or removal of CK molecules. Acknowledgements We gratefully acknowledge the technical assistance of Ms. Audrey Painter and the help of Ms. Carole Goode11 in the typing and preparation of this manuscript. Work from the authors’ laboratory was supported in part by the National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease. References 1 Posen, S. (1970) Clin. Chem. 16.71-84 2 Goldberg, D.M. and Winfield. D.A. (1972) Br. Heart J. 34, 597-604
149 3 Konttinen, A. and Sommer. I-I. (1973) Br. Med. J. 1.386-389 4 Dawson, D.M., Eppenberger, H.M. and Kaplan, N.O. (1966) Biochem. Biophys. Res. Common. 21. 346-363 5 Roberts. R.. Henry, P.D., Witteveen. S.A.G.J. and Sobel. B.E. (1974) Am. J. Cardiol. 33,660--654 6 Konttinen. A. and Sommer. H. (1972) Am. J. Cardiol. 29.817-820 7 Roberts, R. and Sob& B.E. (1973) AM. Intern. Med. 79.741-743 8 Roe, C.R.. Limb&d, L.E., Wagner, G.S. and Nerenberg. S.T. (1972) J. Lab. Chin. Med. 80, 577-590 9 Memer. D.W. (1974) CIin. Chem. 20.36-40 10 Roberts, R.. Gowda. K.S.. Ludbrook. P.A. and Sobel. B.E. (1975) Am. J. Cardiol. 36, 433437 11 Nealon. D.A. and Henderson, A.R. (1975) CIin. Chem. 21.1663-1666 12 Cao. A., De Virgilis, S.. LIppi, C. and TrabaIza. N. (1969) CIin. Chim. Acta 23.475478 13 Kotoku. T.. Kawakami, H.. Iwabuchi, T., Sate. T.. Kutsuzama. T. and Nakamura. T. (1971) Tohoku J. Exp. Med. 105.167-175 14 Acheson. J., James, D.C.. Hutchinson. E.C. and Westhead. R. (1965) Lancet i. 1306-1307 15 Henry, P.D.. Roberts, R. and Sobel. B.E. (1975) Clin. Chem. 21.844-849 16 Roberts, R.. Sobel. B.E. and Parker, C.W. (1976) Science 194.865-857 17 Anido, V.. Corn. R.B.. MengoIi, H.F. and Anido, G. (1974) Am. J. CIin. Pathol. 61. 599+06 18 Weber. K. and Osbom. M. (1969) J. Biol. Chem. 244.44064412 19 Hunter. W.M. and Greenwood, F.C. (1962) Nature 194. 495-496 20 MarchaIonis. J.J. (1969) Biochem. J. 113.299-305 21 Bolton. A.E. and Hunter, W.M. (1973) Biochem. J. 133. 629-639 22 Farr, R.S. (1958) J. Infect. Dis. 103, 239-262 23 Parker, C.W. (1972) in Progress in Clinical Pathology (Stefanini. ed.), Vol. IV. Grune and Stratton, New York 24 RosaIki. S.B. (1967) J. Lab. CIin. Med. 69.696-706 25 Painter, A., Sobel. B.E. and Roberts, R. (1977) Am. J. Cardiol. 39.317 (Abstr.) 26 Roberts, R.. Painter, A.A. and Sobel. B.E. (1977) CIin. Res. 26,249A (Abstr.)
Clinica Chimica Acta, 83 (1978) 141-149 @ Elsevier/North-Holland Biomedical Press
CCA 9136
IMMUNOLOGIC DETECTION OF MYGCARDIAL INFARCTION A RADIOIMMUNOASSAY FOR MB CREATINE KINASE
ROBERT
ROBERTS
*, BURTON
E. SOBEL and CHARLES
WITH
W. PARKER
Cardiovascular Division, Washington University School of Medicine, St. Louis, MO. (U.S.A.) (Received
August
16th,
1977)
summary Although antibodies to plasma isoenzymes have been induced, a radioimmunoassay (RIA) has not previously been developed in part because of lack of specificity and loss of enzymatic activity incurred during radioactive labelling. A RIA for human MB CK (“myocardial”), a creatine kinase isoenzyme, would provide a specific and quantitative test for myocardial infarction, and perhaps more important, it would aid in elucidating the nature of MB CK protein turnover in the circulation independent of enzymatic activity. Assuming that MB, but not MM (the only other isoenzyme present appreciably in blood from patients with myocardial infarction) would bind to B subunit antibody, we harvested BB antiserum from rabbits immunized with human brain BB purified by chromatography. BB, MB, and MM for use as antigens in the RIA were labelled with radioactive N-succinimidyl 3-( 4-hydroxyphenyl-propionate), a procedure that avoided oxidation of the antigen and yielded 200 000 cpm/pg and 97% of initial CK enzymatic activity. After incubation in 1.6 M Tris, pH 7.4,2% albumin, and 20 mM CH&H#H, labelled BB and MB bound to anti-BB (93 and 56%) and precipitated in 44% (NH4)#04, but 1251-MM in 5000-fold excess did not. Unlabelled MB (20 pg/ml) but not MM (up to 5000 pg/ml) competitively displaced precipitable counts. Development of a sensitive and specific RIA based on this phenomenon accurately detected MB (20 pg/ml) despite excess MM (5000-fold excess) in plasma samples constituted with standard. In patients with myocardial infarction, MB CK determined by the RIA was elevated in all cases.
* Correspondence should be addressed to: Dr. Robert Roberts, Director. Cardiac Care Unit. Washing ton Unhwsity School of Medicine. 860 South Euclid Avenue, St. Louis, MO. 63110, U.S.A.
142
Introduction Despite widespread diagnostic analysis of plasma enzymes, very little is known of factors influencing their rates of disappearance from the circulation [ 11. It is not clear whether disappearance of enzyme activity reflects inactivation, denaturation or removal of intact enzyme molecules. Enzymatic detection of myocardial infarction has been based conventionally on plasma elevations of GOT, LDH, and CK, but recent results indicate that detection with elevated MB CK is the most specific and sensitive approach [ 2,3]. Creatine kinase in human plasma exists in at least three isoenzyme forms (each with molecular weight of approximately 82 000) designated MM, MB and BB on the basis of subunit composition [4]. Plasma from normal subjects contains primarily MM with less than 0.005 I.U./ml of MB and no detectable BB CK [5-71. Analysis of human tissue CK isoenzyme profiles indicate that brain contains only BB [8-lo], skeletal muscle only MM [8,9] and heart, a combination of MM and MB [8-lo]. Nevertheless until recently BB has not been detected in the plasma after brain injury in patients with cerebral infarcts, injury or infection [ll-131. With a chromatographic assay small amounts of BB CK have been detected after cerebral injury [ll]. Release of BB may in part be impeded by the brain barrier or lability of BB CK in cerebrospinal fluid or blood itself. After cerebral damage, plasma MM is often elevated, presumably because of release from skeletal muscle induced by sympathetic stimulation [ 141. Analysis of plasma CK isoenzyme activity after intramuscular injections [ 51, and surgical procedures [lo] demonstrates that plasma MM CK may be markedly elevated. However, elevations of BB or MB do not occur. MB appears consistently in plasma after myocardial infarction but BB does not [9,10]. Although several assays for MB CK activity have been developed [5,8,15], they are not ideally suited for quantitative analysis with large numbers of samples; their sensitivity is somewhat limited; and they rely exclusively on detection of enzyme activity rather than other physical properties of CK isoenzyme molecules. In the present study, human CK isoenzymes were isolated in near pure form from heart and brain and antibodies developed specific for the M and B subunits. Human CK isoenzymes were labelled with lz51 utilizing a procedure which avoids exposure of the isoenzyme to oxidizing agents and provides an antigen with high specific radioactivity without loss of enzyme activity. Dissociation of subunits was prevented by performing the reaction in a high concentration of Tris buffer and 2-mercaptoethanol. A competitive displacement radioimmunoassay was developed specific for the B subunit, reported recently in preliminary form, to permit measurement of plasma MB CK protein concentration after myocardial infarction [ 161. The present communication describes the method and its validation in detail. Materials and methods Isolation
of human
CK isoenzymes
MM and MB isoenzymes from human brain obtained
were prepared from human myocardium and BB at necropsy within six hours of death. Homogenates
143
from human myocardium and brain were prepared as follows: The fresh tissue was trimmed of fat, cut into small pieces with scissors and passed through a pre-cooled meat grinder. Ground tissue was homogenized in a Wring blender containing 2 ml/g of 0.05 M TrisfHCl, pH 7.5, and 0.001 M 2-mercaptoethanol. All preparative procedures were performed at O-4°C. The myocardial homogenate was centrifuged at 31000 Xg for 15 min in a supernatant fraction filtered through 8 layers of cheesecloth. Ninety-five percent ethanol was added in dropwise fashion until the final concentration was 50%, and the mixture was allowed to stand while slowly stirring at 40°C for 30 min. The precipitated material was removed by centrifugation and the supernatant fraction decanted. Again ethanol was added in stepwise fashion until a final concentration of 70% was obtained. The mixture was allowed to stand for 30 min and the resulting precipitate was recovered and saved. With the use of a Dounce homogenizer, the precipi~t~ pellet was suspended in homogenizing medium equal in volume to 50% of the original homogenate. After ceut~fugation at 31000 Xg, the pellet from this resuspended mixture was discarded and the supernatant fraction saved. To separate the MM CK, NaCl, 5 M, was added with rapid stirring to a final concentration of 0.05 M. DEAE Sephadex A-50 was then added (10 ml slurry for 48 mg of protein) and the mixture was stirred for 30 min. This suspension was filtered with a Buchner funnel lined with Whatman’s No. 1 filter paper, and the filtrate was dialyzed against several changes of 0.01 M glycine, NaOH, pH 9.0 prior to freeze drying of the MM fraction. The MB CK present in the homogenate was adsorbed to the DEAE-Sephadex previously added. Accordin&, after the MM filtrate had been obtained, the ~EAE-Sephadex was retained in the Buchner funnel and washed five times with 0.05 M Tris/HCl, pH 7.4, 0.05 M NaCl, and 0.001 M 2-mercaptoethanol. After the DEAE-Sephadex had been washed, MB CK was eluted with 0.05 M Tris/HCl, pH 7.4, containing 0.3 M NaCl. The eluate was dialyzed with glycine buffer, freeze-dried to obtain the MB fraction. To further enrich the MM and MB fractions, further ethanol fractionation and column ~hromato~aphy was performed followed by dialysis, freezed~ing and storage at 0-4°C. CK was extracted from brain in the same fashion as from myocardium with the following exceptions: The final concentration of ethanol was 60% rather than 50%. After filtration of the MM fraction, Sephadex retained in the Buchner funnel was washed five times with Tris/HCl buffer containing 0.1 M NaCl, and subsequently washed once with buffer containing 0.1 M NaCf, and once with buffer containing 0.3 M NaCl. BB CK remained adsorbed to Sephadex under these conditions was then eluted with Tris buffer containing 0.4 M NaCl dialyzed against 0.01 M glycine, NaOH, pH 9.0, and freeze-dried. Polyacrylamide gel electrophoresis [ 17 3 of the human preparations indicated that each isoenzyme was abtained in a sample devoid of activity attributable to other isoenzymes in the initial extract. MM CR averaged 392 I.U./mg of protein, MB 46 I.U./mg, and BB 110 I.U./mg. Specific activity of each isoenzyme was increased more than one hundred-fold over that present in the initial extract and analysis by SDS gel electrophoresis [ 181 with staining for protein showed only one faint contaminating band of the MM and no contaminating bands of BB with two very faint contaminating bands of the MB,
144
Induction of antibodies to CK isoenzymes Utilizing the purified human MM and BB CK mixed with equal volumes of Freund’s complete adjuvant, antibodies to CK isoenzymes were induced in rabbits. Initially, the rabbits were injected subcutaneously with 1 mg of immunogen (0.25 mg/foot pad). Subsequently, they were injected with 0.25 mg weekly for three weeks. All animals were given booster injections of 0.1 mg in complete adjuvant at monthly intervals thereafter. Ten days after each booster injection, the animals were bled and their serum analyzed for antibody activity. Ouchterlony agarose plates, prepared with BB antiserum exhibited single precipitin lines to BB and MB antigen but no precipitin line to MM. Plates prepared with MM antiserum exhibited a single precipitin line to both MB and MM but none to BB. Thus, antibodies to BB CK reacted with BB CK and also crossreacted with MB but did not cross-react with MM indicating specificity for the B subunit. Similarly, MM antibodies were specific for the M subunit. Radioactive labelling of CK isoenzymes Radioiodine ( 12’1) was utilized to radioactively label CK isoenzymes for subsequent use in a competitive displacement radioimmunoassay. When “‘1 was introduced into the isoenzymes by the chloramine-T [19] or lactoperoxidase [20] method, there was marked loss of CK enzyme activity, possibly due to oxidation of essential sulfhydryl groups. To avoid exposing the enzymes to oxidizing agents and contaminants in the radioiodine, the “‘1 was first incorporated into the N-succinimidyl ester 3-( 4-hydroxphenyl-propionate) which in turn was reacted with amino groups on the CK isoenzyme protein. N-Succinimidyl 3-(4-hydroxyphenyl-propionate) was radioiodinated by the method of Bolton and Hunter [21]. The reaction was carried out at room temperature (23°C). N-Succinimidyl 3-( 4-hydroxphenyl-propionate) (0.3 pg) was treated with 5 mCi (lo-20 ~1) of Na1251, 50 pg of chloramine-T and 10 ~1 of 0.25 M phosphate buffer, pH 7.5. The reaction was immediately terminated by the addition of 120 pg of sodium metabisulfite in 10 ~1 of 0.50 M phosphate buffer, pH 7.5 containing 200 pg of KI. The iodinated product was extracted into benzene (0.300 ml X 2 portions) and recovered by evaporation of the solvent under vacuum. The addition of dimethylformamide (5 ~1) before adding the benzene was necessary for full extraction of the ester into the solvent. The residue was then used to label creatine kinase isoenzymes. The labelled residue was combined with 2-8 mg of MM, MB or BB CK in l-2 ml of 0.01 M sodium borate buffer, pH 8.5. After gently shaking the medium for 4 h at 4°C the labelled isoenzymes were then dialyzed against the same buffer containing 0.005 M 2-mercaptoethanol. Radioactivity per mg of labelled CK isoenzymes averaged 200 000 cpm for MM CK and MB CK and 100 000 cpm for BB CK. The maximum loss of enzyme activity resulting from labelling and dialysis was less than 5% for each isoenzyme preparation. Binding affinity The binding antiserum) was by diluting the determinations
and specificity of antiserum affinity and specificity of the BB and MM antibodies (rabbit determined over a wide range of concentrations of the antibody appropriate antiserum over a range of 1 : 15 to 1 : 1000. All were performed in duplicate and carried out in 12 X 75 ml glass
145
tubes containing 1.6 M Tris buffer, pH 7.6 (200 pl), 2% bovine serum albumin (100 pl), 0.020 M mercaptoethanol (10 pl), 5 pg of rabbit gamma globulin (50 ~1). The gamma globulin and serum albumin minimize nonspecific binding [22]. The high concentration of Tris and 2-mercaptoethanol protects the sulfhydryl groups of the isoenzymes and prevents dissociation into monomers. To this mixture was added the appropriate dilution of antiserum in volumes ranging from 100 ~1 to 5 ~1 (dilutions performed with normal rabbit serum). ‘251-labelled MM (0.1 pg), MB (0.1 pg), and BB CK (0.2 pg) were added such that approximately 25 000 cpm were present in each tube. The total volume was kept constant at 500 ~1 with necessary adjustments being made with Tris buffer. The solutions were then incubated and gently shaken at 4°C for 6 h. Appropriate controls were incubated containing normal rabbit serum rather than rabbit antiserum. Following the incubation period, separation of free from antibodybound labelled CK was accomplished by the addition of cold saturated ammonium sulphate [22] with a final concentration of 44%, and allowed to sit at room temperature for 15 min. The solution was then centrifuged at 2000 Xg for 20 min, the supernatant decanted, and the pellet washed with 50% ammonium sulphate (400 ~1) and again centrifuged. The pellets were counted in a gamma counter (Micromedic Systems, Inc.) until a minimum of 10 000 counts were obtained. The 12’1 counts present in the pellet expressed as a percent of the total number of counts initially present represent percent binding. To optimize conditions for any possible cross-reactivity between the BB antibody and MM CK and BB CK with the MM antibody, determinations were done in which BB antiserum diluted only 1 : 15 was incubated with 4 pg of 12SI-MM and MM antiserum in a dilution of 1 : 15 with 4 pg of 12’I-BB. Procedure for radioimmunoassay To develop a competitive displacement radioimmunoassay for plasma MB CK, BB antiserum was used and the specificity of the BB antiserum for B subunits further established by comparing the ability of unlabelled BB, MB and MM CK to inhibit 13’I-BB binding. The reaction was performed in the same buffer solution used for the binding experiments, but with the exceptions that the dilution of antiserum was kept constant at 1 : 150 and the amount of 12”1-BB CK was kept constant at 0.2 pg containing approximately 25 000 cpm. The antiserum dilution of 1 : 150 was chosen since this concentration of antibody binds about 50% of the 1251-BB [23]. A known amount of unlabelled BB, MB, or MM CK was diluted from 1 : 15 to 1 : 1000 and incubated for 6 h with 12’1-labelled BB. Following incubation, the ammonium precipitated pellet To further deterwas washed, centrifuged and counted for 1251radioactivity. mine the specificity of unlabelled BB or MB to displace 12’I-BB binding in the face of MM CK, inhibition curves were determined for MM incubated with BB or MB in which MM was present in a 25 000 fold-excess over that of unlabelled BB or MB. Determination of MB CK in plasma samples To determine the amount of MB CK present in an unknown sample, a standard MB inhibition curve is run with known amounts of unlabelled MB CK ranging from 200 to 0.1 ng/ml of MB CK. The unlabelled MB CK is incubated
146
with a constant amount of antiserum and “‘1-BB CK as outlined previously under radioimmunoassay procedure. Serial dilutions (3-4) of the unknown sample are made and the amount of inhibition determined at each dilution and compared to the standard curve from which it is possible to calculate the amount of MB CK present expressed as ng/ml. To determine the accuracy of the assay, known amounts of human unlabelled MB CK were added to heat inactivated serum and serial dilutions done and results expected compared to that obtained. In addition, plasma samples were obtained from twenty patients with acute myocardial infarction. At least 15 samples were obtained serially from each patient over a period of 60 h. All samples were assayed in duplicate and assays of enzymatic activity obtained by a kinetic fluorometric assay previously described [5]. All determinations for total CK enzymatic activity were done according to the Rosalki method [ 241. Results Antibody specificity Results of binding experiments using serial dilutions of BB antiserum from 1 : 15 to 1 : 1000 incubated with iodinated MM, MB and CK are shown in Fig. 1. Ninety-three percent of the “‘1-BB was recovered in the pellet in dilutions of 1 : 30, but binding diminished rapidly with only 7% at 1 : 1000, demonstrating that binding was dependent on antibody concentration. Maximum binding of “‘I-MB CK (60%) occurred at 1 : 15 dilutions but again binding was dependent on the concentration of antibody with only 5% binding at ‘251-MM exhibited no such antibody 1 : 1000 dilution. In contradistinction, concentration dependent binding. At all dilutions binding was between 3-5%, similar to that with control (normal rabbit serum). These results demonstrated the antibody is specific for the B subunit rather than the molecule as a whole. Results of binding with MM antiserum showed 94% binding of 12’I-MM and 50% binding of 12’I-MB at dilutions of 1 : 30. Again binding was dependent on the concentration of antibody and returned to control levels at a dilution of 1 : 1000. No specific binding was seen between the MM antiserum and i2’I-BB.
'=I 88 CPK
-
'=I MB CPK
-
lz51 MM
0
I 250
DILUTION
I 500
CPK
---
I
I
750
1000
OF BB ANTISERUM
Fig. 1. Shows the specificity of BB antiserum for BB and MB. Binding of ’ 2SI-BB and ’ 2SI-MB is dependent on the concentration of BB antibody. There is no binding of 1 2 5 I-MM CK at any concentration of BB antibody.
147
The MM antiserum exhibited reactivity with the B subunit.
specificity
for the M subunit
but no cross-
Results of radioimmunoussay Unlabelled MB CK competitively displaced labelled BB CK from binding to the BB antibody which was dependent on the concentration of MB CK as shown in Fig. 2. As can be seen, the inhibition curve is steep between 0.5 and 100 ng/ml with 50% inhibition at 10 ng and complete inhibition of binding at a concentration of 150 ng/ml and above. A similar inhibition curve was seen for unlabelled BB which showed 50% inhibition at 5 ng/ml and complete inhibition at 70 ng/ml and higher. Unlabelled MM CK showed no inhibition of ‘2SI-BB binding, even at 5 pg/ml (2000-fold excess over “‘1-BB CK). Furthermore, the competitive inhibition of unlabelled MB CK was unaltered in the presence of high concentrations of MM CK (5000 molar excess over that of MB CK). Thus, the BB systems as a competitive displacement assay for MB is extremely sensitive, detecting reliably a concentration of 0.5 ng/ml representing enzymatic activity of 1 X lo-’ I.U./ml. Furthermore, the specificity is such that results at this level of sensitivity are unaffected by 5000 molar excess MM. Results of radioimmunoassays performed on heat inactivated serum constituted with known amounts of MB CK ranging from 0.20 ng/ml to 2 pg/ml deviated by less than 3% from those expected. Radioimmunoassay of 300 samples obtained from 20 patients with acute myocardial infarction exhibited elevated MB CK in all cases. A typical MB CK curve from one of the patients is shown in Fig. 3.
1000 t
I
I
0.5
1
2
5 IO 25 MB -CK (ng/ml) I 1 I .008 .016 .032 .08 .I6 0.4 MB-CK
l
RIA
I:
Enrymollc
,I
50
100 150
I 0.8
II 1.6 2.4
0
(mIU/ml)
Fig. 2. Shows competitive displacement of 12 51-BB CK by unlabelled dependent. The Wition curve is steep in the range of O.l5-100 ng.
20
40 TIME
60
80
100
(hr)
MB CK which is concentration
Fig. 3. A plasma CK-time activity curve in a patient with myocardial infarction. Since 66 ng of purified MB enzyme protein was equivalent to 1 m1.U. of enzyme activity, vahws obtained with the ndioimmunoassay method (RIA). expressad initially as ng/ml, wsre converted to enzymatic activity and compared to results obtained by a kinetic fluorometric aawy which m eamues onl~enz~mat&activity.Aacm beseen. v&es obtained with the two methods are in close agraement. Similar agreement was obtained in all 300 samples from the 20 patients studied.
148
Discussion In this study, we have described a radio~munoassay for CK isoenzymes. Antibodies were developed for MM and 38 CK which are specific for the M and B subunits, respectively. Since MB CK, found in the human myocardium, contains both subunits, either antibody can be used to detect MB CK. The BB antibody reacts with BB and cross-reacts with MB CK but not MM even when present in 5000 molar excess over that of MB CK, a ratio far greater than that seen in plasma after myocardial infarction since MB CK usually represents lO-15% of total CK activity [ 51. Detection of plasma MB CK using the BB system is quite specific. Since BB CK activity is not present in normal plasma [5], in patients with cerebral disorders [ 12,131, or those with acute myocardial infarction [ 5,6,8,9]. Thus, in these clinical situations, displacement binding reflects MB exclusively. These impressions were corroborated by the close agreement between enzymatic activity determined by the kinetic ~uorometric method [5] and apparent concentration of protein detected by RIA in samples obtained from patients with acute myocardial infarction. The sensitivity provided by the RIA exceeds that of previously available CK isoenzyme assays be several orders of magnitude. Thus, assays [ 241 based on enzymatic activity [ 241 have a sensitivity in the range of 1 X low2 I.U.fml as opposed to the present assay which detects reliably 1 X lo-’ I.U./ml. Since mean plasma MB CK activity in normal subjects is 1 X 10e3 I.U./ml, a 5-fold increase is necessary for detection of elevations by enzymatic assays in contrast to the RIA which can easily detect normal values as well as modest increases. The increased sensitivity, coupled with the potential for detection of enzymatically inactive MB CK in the circulation should lead to improved estimates of infarct size as well as earlier detection of acute myocardial infarction 1251. The present findings suggest that a similar approach may be useful in differentiation of other clinically important enzymes which exist in multiple forms. Previous assays for plasma enzymes and isoenzymes have been asked on enzymatic activity. Studies evaluating the disappearance of the enzymes from the circulation have been generally restricted to determining the loss of activity. However, since RIA detects the concentration of molecules, it permits evaluation of the rate of isoenzyme protein turnover independent of enzymic activity. Thus, RIA should help to elucidate mechanisms responsible for disappearance of individual CK isoenzymes from the circulation [ 261 as well as aid in elucidating the relative importance of inactivation, denaturation, or removal of CK molecules. Acknowledgements We gratefully acknowledge the technical assistance of Ms. Audrey Painter and the help of Ms. Carole Goode11 in the typing and preparation of this manuscript. Work from the authors’ laboratory was supported in part by the National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease. References 1 Posen, S. (1970) Clin. Chem. 16.71-84 2 Goldberg, D.M. and Winfield. D.A. (1972) Br. Heart J. 34, 597-604
149 3 Konttinen, A. and Sommer. I-I. (1973) Br. Med. J. 1.386-389 4 Dawson, D.M., Eppenberger, H.M. and Kaplan, N.O. (1966) Biochem. Biophys. Res. Common. 21. 346-363 5 Roberts. R.. Henry, P.D., Witteveen. S.A.G.J. and Sobel. B.E. (1974) Am. J. Cardiol. 33,660--654 6 Konttinen. A. and Sommer. H. (1972) Am. J. Cardiol. 29.817-820 7 Roberts, R. and Sob& B.E. (1973) AM. Intern. Med. 79.741-743 8 Roe, C.R.. Limb&d, L.E., Wagner, G.S. and Nerenberg. S.T. (1972) J. Lab. Chin. Med. 80, 577-590 9 Memer. D.W. (1974) CIin. Chem. 20.36-40 10 Roberts, R.. Gowda. K.S.. Ludbrook. P.A. and Sobel. B.E. (1975) Am. J. Cardiol. 36, 433437 11 Nealon. D.A. and Henderson, A.R. (1975) CIin. Chem. 21.1663-1666 12 Cao. A., De Virgilis, S.. LIppi, C. and TrabaIza. N. (1969) CIin. Chim. Acta 23.475478 13 Kotoku. T.. Kawakami, H.. Iwabuchi, T., Sate. T.. Kutsuzama. T. and Nakamura. T. (1971) Tohoku J. Exp. Med. 105.167-175 14 Acheson. J., James, D.C.. Hutchinson. E.C. and Westhead. R. (1965) Lancet i. 1306-1307 15 Henry, P.D.. Roberts, R. and Sobel. B.E. (1975) Clin. Chem. 21.844-849 16 Roberts, R.. Sobel. B.E. and Parker, C.W. (1976) Science 194.865-857 17 Anido, V.. Corn. R.B.. MengoIi, H.F. and Anido, G. (1974) Am. J. CIin. Pathol. 61. 599+06 18 Weber. K. and Osbom. M. (1969) J. Biol. Chem. 244.44064412 19 Hunter. W.M. and Greenwood, F.C. (1962) Nature 194. 495-496 20 MarchaIonis. J.J. (1969) Biochem. J. 113.299-305 21 Bolton. A.E. and Hunter, W.M. (1973) Biochem. J. 133. 629-639 22 Farr, R.S. (1958) J. Infect. Dis. 103, 239-262 23 Parker, C.W. (1972) in Progress in Clinical Pathology (Stefanini. ed.), Vol. IV. Grune and Stratton, New York 24 RosaIki. S.B. (1967) J. Lab. CIin. Med. 69.696-706 25 Painter, A., Sobel. B.E. and Roberts, R. (1977) Am. J. Cardiol. 39.317 (Abstr.) 26 Roberts, R.. Painter, A.A. and Sobel. B.E. (1977) CIin. Res. 26,249A (Abstr.)