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tropic effect with only a small increase in heart rate. In addition, they suggest that the effective concentrations of isoprenaline and dobutamine should be 0.1 and 0 . 3 , re~~ spectively. Rat hearts were perfused hypoxically [N2+02+C02 (90:5:5 ) ] for 6h retrograde via the aorta (Langendorff, 1895). The perfusate was assayed for the release of several cardiac enzymes as described by Hearse et al. (1974). Fig. 1 shows the effect of both isoprenaline and dobutamine on the release of creatine kinase (which was representative of all the enzymes studied). In both instances enzyme release was increased. Analysis of the curves revealed that, during the 6 h of hypoxia alone, hearts released 930+67 units (pmol of creatine phosphate formed/min) of creatine kinase/g dry wt. of heart, but that the inclusion of dobutamine or isoprenaline increased this to I428 -I 142 (P,0.0025) and 1221L-142 (P,O.O4) respectively. Our results therefore confirm that, in contrast with isoprenaline, dobutamine can exert a strong inotropic effect without greatly increasing heart rate, but that, despite this, both compounds cause an increase in tissue damage as assessed by enzyme release. This work was supported in part by grants from the British Heart Foundation and the Vandervell Foundation. Braunwald, E. & Maroko, P. R. (1974) Circulation 50, 206-208 Ewen, L. M. & Griffiths, J. (1971) Amer. J. Clin. Pathol. 56, 614-622 Hearse, D. J. & Chain, E. B. (1972) Biochern. J. 128, 1125-1133 Hearse, D. J. & Humphrey, S. M. (1975) J. Mol. Cell. Cardiol. in the press Hearse, D. J., Humphrey, S. M. & Chain, E. B. (1974) J. Mol. Cell. Cardiol. 5, 395-407 Hearse, D. J., Humphrey, S. M., Nayler, W. G., Slade, A. & Border, D. (1975) J. Mol. Cell. Cardiol. in the press Jewitt, D., Birkhead, J., Mitchell, A. & Dollery, C. T. (1974) Lancet ii, 363-367 Krebs, H. A. & Henseleit, K. (1932) Hoppe-Seyler’s Z. Physiol. Chem. 210, 33-66 Langendorff, 0. (1895) Pjugers Arch. Gesamte Physiol. Menschen Tieren 61, 291-332 Neely, J. R., Liebermeister, H., Battersby, E. J. & Morgan, H. E. (1967) Amer. J. Physiol. 212, 804-814 Shell, W. E., Kjekshus, J. K. & Sobel, B. E. (1971) J. Clin. Invest. 50, 2624-2625 Sobel, B. E. 8~Shell, W. E. (1972) Circulation 45,471-482 Tuttle, R. R. & Mills, J. (1973) Belg. Patent 798051 Tuttle, R. R., Pollock, G. D., Todd, G. & Tust, R. (1973) Circulation 48, 132

Creatine Kinase Isoenzymes : their Separation, Quantitative Determination and Use in the Assessment of Acute Myocardial Infarction EDWARD A. OGUNRO, DAVID J. HEARSE and JOHN P. SHILLINGFORD Cardiovascular Research Unit,Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, U.K.

The appearance in the serum of various cytoplasmic enzymes has formed the basis of a varietyof clinical diagnosticprocedures for the detectionoftissuedamage. By virtueof the characteristic enzyme profiles found in different tissue types it is often possible to identify the site of tissue damage by the appearance of specific enzymes in the serum (Goldberg, 1971). In this way, acute myocardial infarction is associated with a marked rise in serum creatine kinase activity (Ewen & Griffiths, 1971). However, creatine kinase is not specific to the heart, and thus, although it is possible to distinguish myocardial damage from hepatic damage, it is not possible to distinguish it from skeletal-muscle damage. In an attempt to increase the specificity of these diagnostic procedures, isoenzyme profiles have been examined. Creatine kinase has a dimeric structure: the monomeric subunits are of two types designated the M and B forms and the various combinations of these forms result in the existence of three isoenzymes MM, BB and MB.

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Human tissue-distributionstudies(Smith, 1972) have revealed that the MM isoenzyme occurs predominantly in skeletal muscle and myocardium, the BB isoenzyme occurs mainly in the brain and to a smaller extent in other tissues and the MB isoenzyme is found mainly in the myocardium. The prospect of increasing diagnostic specificity by measuring the relative proportions in which the three creatine kinase isoenzymes appear in the serum has focused considerable attention on the need for good isolation, separation and assay techniques. Each isoenzyme has been shown (Wood, 1963) to possess a different charge at pH8.6, and a variety of procedures for electrophoretic and ion-exchange separation and quantitative determination have been reported (Somer & Konttinen, 1972; Roe et al., 1972; Klein et al., 1973; Mercer, 1974). Some of these procedures have been used for experimental and clinical studies (Sapsford et al., 1974; Wagner et al., 1974). Clinical requirements demand a rapid and accurate separation and quantitative determination of isoenzymes from small samples of serum. Although good electrophoretic separations have been reported, accurate quantitative determination, particularly for low isoenzyme activities, has proved difficult. The use of tetrazolium dyes for detection on either gels or cellulose acetate paper has proved to be time-consuming, insensitive and subject to artifacts (Somer & Konttinen, 1972). Detection on cellulose acetate by apposition and fluorimetric scanning techniques is inaccurate and irreproducible (Roberts et al., 1974). The objective of the present studies was to establish a procedure for the rapid separation and quantitative determination, to a high degree of accuracy, reproducibility and sensitivity, of isoenzymes from small (10-20pI) samples of serum.

Incubation time (min)

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40

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' 70 80

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' 90

'

'

'

'

100 110 I20 130

Total creatine kinase activity (rnunits/rnl) Fig. 1 . Characterizationof creatine kinase isoenzyme assay (a)Linearity offluorescence intensity with incubation time: 0 , BB isoenzyme; 8, MB ;soenzyme; 0,MM isoenzyme. (b) Linearity of fluorescence intensity against NADH concentration: k,! NADH on cellulose acetate paper and detected by fluorimetric scanning; 0, NADH in aqueous solution. (c) Relationship between fluorescence intensity and total enzyme activity for each isoenzyme separated on an agarose gel: o, BB isoenzyme; o,MB isoenzyme; m, M M isoenzyme.

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;250

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3 v)

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Time after infarction (h)

Fig. 2. Time-course of creatiiie kinase activity in the serum after acute myocardia infarction O , Total enzyme release; 0 , MM isoenzyme release; m, MB isoenzyme release. The inset illustrates an isoenzyme separation on agarose gel ; the broken line indicates the origin.

Our procedure involved the modification of a variety of techniques (Roe et al., 1972; Klein el af.,1973; Somer & Konttinen, 1972). Isoenzymes were separated electrophoretically (IlOV; 25-35mA/gel; 10°C; 35min) on an agarose gel [1.2% (w/v) agarose in 0.025~-veronalbuffer, pH8.6, containing 5 m - E D T A and 3.8mM-NaN3] measuring 8.0cm x 8.0cm x0.2cm. Serum samples (10-20p1, depending upon enzyme activity, dissolved in agarose at 43°C to give the same concentration agarose as in the gel) were introduced into the gel slots before electrophoresis. After electrophoresis the isoenzyme gel was overlayed (43°C) with a substrate gel [1.2% (w/v) agarose in 0.05~-triethanolamine buffer, pH 7.0, containing optimal concentrations of the substrates required for the NADP-linked analysis of creatine kinase as described by Oliver (1955)l. The preparation was incubated at 37°C for 60-120min. The isoenzyme bands were located (inset in Fig. 2u) by NADPH fluorescence (350nm excitation) under U.V. light and were cut away from the gel. The NADPH was eluted into 2.50ml of 0.1 M-triethanolamine buffer, pH7.0, by vigorous shaking for lOmin and was determined fluorimetrically (excitation 350nm, emission 450nm). This procedure was then characterized and compared with an established technique of cellulose acetate electrophoresis and fluorimetric scanning (Klein et al., 1973). To compare the linearity of detection of the two procedures, NADH concentrat ion was compared with fluorescence intensity. Fig. 1 (b)reveals that fluorimetric scanningis only linear when less than 6.0nmol of NADH is present per electrophoretic band; however, the elution technique remains linear up to 60nmol. Since routine isoenzyme separations yield bands with sufficient activity to produce in excess of 6.0nmol per incubation period, it is clear that fluorimetric scanning is suitable only for qualitatitve assessments. Further characterization studies of our procedure revealed that the lOmin agitation period allowed quantitative extraction of the NADH (98.5 % after 5min agitation). The preparative procedures of sample-slot filling and overlay formation (which involved

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transient heating of the enzyme to 43°C) results in a loss of less than 10% of the total enzyme activity. The relationship between the incubation time after overlay and the resultant fluorescence intensity was linear up to 2h (Fig. la), and fluorescence intensity against enzyme activity was linear up to 80munits/ml total activity (Fig. lc). It was possible to detect and accurately determine creatine kinase in samples (10-20~1) with a total activity as low as 0.75munit and the reproducibility of MB/MM ratio determinations was +1.7%. By using our procedure we have monitored the time-course of serum creatine kinase isoenzyme changes immediately after suspected acute myocardial infarction in a series of patients. A typical result is illustrated in Fig. 2. Isoenzyme activities rise rapidly after the onset of symptoms, peak after approx. 20h and thereafter steadily decline. The appearance of the MB isoenzyme, which would not be expected after skeletal-muscle damage, indicated a myocardial involvement, and this was subsequently confirmed by other diagnostic procedures. This work was supported in part by grants from the British Heart Foundation and the Vandervell Foundation.We gratefully acknowledgeDr. M. Hobart for hisadvice and discussion. Ewen, L. M. & Gtiffiths, J. (1971) Amer. J. Clin. Puthol. 56, 614-622 Goldberg, D. M. (1971) Ann. Clin. Biochem. 8, 195-200 Klein, M. S., Shell, W. E. & Sobel, B. E. (1973) Cardiovasc. Res. 7, 412-418 Mercer, D. W. (1974) Clin. Chem. 20, 3640 Oliver, I. T. (1955) Biochem. J. 61, 116-122 Roberts, R., Henry, P. D., Witteeveen, S. A. G. J. & Sobel, B. E. (1974) Amer. J. Curdiol. 33, 650-654 Roe,C. R., Limbird, L. E., Wagner, G. S. & Nerenberg, S. T. (1972) J. Lab. Clin. Med. 80,577590

Sapsford, R. N., Blackstone, E. H., Kirklin, J. W., Karp, R. B., Kouchoukos, N. T., Pacifico, A. D., Roe, C. R. & Bradley, E. L. (1974) Circulation 49, 1190-1199 Smith, A. F. (1972) Clin. Chim. Actu 39, 351-359 Somer, H. & Konttinen, A. (1972) Clin. Chim. Actu 40,133-138 Wagner, G. S., Roe, C. R., Limbird, L. E., Rosati, R. A. &Wallace, A. G. (1974) Circulation 48, 263-269 Wood, T. (1963) Biochem. J. 89,210-220

An Improved Extraction Procedure for the Endotoxin from Microcystis aeruginosa NRC-1 PETER RABIN and ANDRG DARBRE Department of Biochemistry, King’s College, Strand, London WC2R 2LS, U.K. It has been reported that in many parts of the world cattle and birds die from drinking water containing a bloom of the toxic bluegreen alga Microcystis aeruginosa (Grant & Hughes, 1953; Ingram & Prescott, 1954; Schwimmer & Schwimmer, 1955; Gorham, 1964). Some toxic strains of this alga have been isolated (Hughes et al., 1958). The ‘fastdeath factor’ (Bishop et al., 1959) and ‘microcystin’ (Rama Murthy & Capindale, 1970) have certain properties in common. Both groups of workers showed that the toxin was a polypeptide containing D-serine, L-ornithine and some protein L-amino acids. Because the toxin was insensitive to most proteolytic enzymes, they concluded that the peptide was circular. Isolation of microcystin by the method of Rama Murthy & Capindale (1970) was lengthy, and in our hands the material isolated was non-toxic. Thus we developed a new procedure for the isolation of the toxin. This has shortened the time of extraction from 10 to 3 daysandgave a yield of 1Omg of toxin/lOg batch of freeze-dried cells. This compared well with the yield obtained by Rama Murthy & Capindale (1970). Toxicity was 1975