RESULTS were presented in previous

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1 Influence of Experimental Myocardial Infarction on Free Amino Acids of Dog Heart By MARTIN CHANIN, PH.D., EUGENE ROBERTS, Ph.D. AND ALFRED GOLDMAN, M.D. Free or eiisily extractable amino acids were determined by two-dimensional pnper chromatography in infarotcd and noninfarcted areas of the left ventricles of dog hearts after ligation of the anterior descending coronary artery. Changes in amino acid content in in fare ted areas were characterized by decreases in some of the major ninhydrin-reactive constituents and appeared coincidentally with histologic signs of myocardial damage. RESULTS were presented in previous communications of determinations by two-dimensional paper chromatography of free or easily extractable amino acids in various normal and neoplastic tissues of several species. 1 " 6 The present communication deals with the study of the influence of experimental myocardial infarction on the amino acid pattern of the left ventricle of dog heart. A preliminary report of some of this work has been made.' MATEIUALS AND METHODS In all instances, fresh specimens were fixed in 75 per cent alcohol immediately after dissection and rapid weighing on a torsion balance. The tissue was minced with seissoi's after immersion in the alcohol. Extracts were prepared by homogenizing the tissue in the alcohol with a ground-glass, motor driven homogenizer, clarifying by ccntrifugation, and evaporating the supernatant fluid in 20 ml. beakers under an infrared lamp in the air stream of a fan. The residue was then taken up in a saturated aqueous solution of picric acid (LOO pl./100 mg. original fresh weight of tissue) and the suspension centrifuged. A total of 75 ft\. of the clear supernutiints were then placed in three applications of 25 n\. each into 18" x 22* sheets of Whatman no. 1 filter paper and in some instances were followed by successive additions of 25 jttl. of 0.05 per cent ammonium molylxlate and From the Departments of Thoracic and Cardiac Surgery and Biochemistry, Medical Research Institute, City of Hope Medical Center, Duurte, Calif. Supported by grant no. 119 from Los Angeles County Heart Association and grant C-256S from the National Cancer Institute, U. S. Public Health Service. Ttecoived for publication July 13, per cent H5O2 to accomplish complete oxidation of cystine and glutathione. Descending two-dimensional ehromatography was then performed in water-saturated phenol and water-saturated lutidine. The picric acid migrated with the front in the lutidine solvent and did not interfere with subsequent detection of the ninhydrin-reactive constituents, which were visualized by spitiying with 0.1 per cent ninhydrin in butanol. Q 111 fa rets were produced in the left ventricle by ligation of the anterior descending coronary artery immediately distal to the circumflex branch. 7 Adjacent sagittal sections of the infarcts and the apparently noninfarcted ventricle were selected for analysis and for histologic study at various times after the ligation, ranging from one-half hour to 6 days. Duplicate determinations of amino acids were made frequently. Twenty-four clogs were employed. RESULTS Typical chromatographic results are shown in figures 1 and 2. For each time interval after ligation it was possible to compare the infarct with a grossly normal area of the same ventricle. Comparisons also were made of the normal and infarcted areas in the hearts of different dogs. Inspection of the chromatograms of the noninfarcted areas of the ventricles of various dogs revealed a remarkable constancy in the pattern of free amino acids from 1 dog to another throughout the period of study (see figs. 1-13, odd numbers). Apparently the experimental procedure did not produce profound effects on the distribution of the free amino acids in various areas of the myocardium which were not directly effected by the ligation and 713 Circulation Retearcl, Volume I V, Xovember 1M8

2 714 AMINO ACIDS IX IN'FARCTKD VKXTRICLKS V FIGS I''roe aniino acids of left ventricle of dog heart at various times after ligation. The odd numbered figures show the uninfareted areas and the even numbered figures the infarcted areas. / and 2, 2 hours; S anil 4, 4 hours; 5 and 6, S hours..'1 alanine; B taurine; C glutathionc, after oxidation with H»O 2 ; D glutamine; E glyeine; /'' serine; G glutamic acid; H aspartic acid; T ethanolamine phosphoric ester;./ Klutathione, unoxidized. no significant histologic changes were noted in these areas. On the other hand, a comparison of the infarcted areas with a normal area from the ventricle of each dog revealed interesting changes. At one-half hour and 2 hours (figs. 1 and 2 show 2 hour results) after the ligation marked capillary congestion was noted but the histologic changes were minimal. In the 4 hour sample (figs. 3 and 4) in which slight pericarditis was noted but minimal gross abnormality of the muscle fibers in the sections studied, there was a decrease in aspartic acid content by comparison with the control, but otherwise little change in amino acid pattern. At the 8 hour interval (figs. 5 and 6) there was a decrease in the glutathione content as well as in aspartic acid. The contents of the other detectable constituents remained unchanged. At this time segmentation of the muscle fibers, necrosis, loss of cross striations, and extravasation of red corpuscles and polymorphonuclear leukocytes into the infarcted area were noted. Further changes had occurred in the amino acid pattern by the sixteenth hour after ligation (figs. 7 and 8). A marked decrease in the

3 CHANIN, ROBERTS AND GOLDMAN 715 s 10 I FIOH Siiine presentation us figures utid S, 16 liours; 9 and 10, 24 hours; // and 12, 48 hours; IS and 14, 5 days. content of taurine was noted as well as decreases in the levels of alanine, glutamine, glutamic and aspartic acids, glutathione and ethanolamine phosphoric ester. No further significant changes appeared to have taken place at 24 or 48 hours (figs. 9-12). A sample obtained from a dog surviving the ligation for 5 days (fig. 14) showed a great loss of all the constituents in the infarct by comparison with the control area (fig. 13). 12

4 716 AMINO ACIDS IN INPARCTED VENTRICLES A loss of carnosine from the affected areas also was shown, the quantities of ]3-alanlne and histidine detected on chromatograms of acid-hydrolyzed extracts being decreased markedly at 16 hours after ligation and at later time intervals. In another instance, chromatograms were obtained from normal areas (ventricle and atrium) of a single dog heart and from the infarcted portion of the ventricle 5 days after ligation of the coronary artery. Essentially normal patterns were found in the right and left ventricles and right and left atria in the noninfarcted areas, while the concentrations per unit fresh weight of tissue of taurine, alanine, glutamine, glutamic and aspartic acids and glutathionc were markedly decreased in the infarcted area of the left ventricle. In this ease it was concluded that the pattern observed was probably derived, at least in part, from infiltrating leukocytes since the chromatogram showed many points of similarity with the pattern found for dog polymorphonuclear leukocytes. DISCUSSION A number of chemical changes have been reported to take place in cardiac muscle after infarction. 8 ' 9 Especially noteworthy is the recent finding that the enzymes, lactic acid dehydrogenase and oxaloacetic transaminase, are elevated in the seram following myocardial infarction, presumably as a result of the liberation of these enzymes from the damaged muscle into the blood stream. 10 Perhaps the relatively early loss of easily extractable aspartic acid from the infarcts in the present study could be related to a decreased level of transaminase activity in the affected area. However, the degree of damage to the myocardium probably was such that many of the cellular constituents of both high and low molecular weight were lost. The present findings of a loss of free or loosely bound amino acids from the myocardium of the left ventricle beginning between 8 and 16 hours after ligation of the anterior descending coronary artery is consonant with this idea. The mechanism by which the loss occurs is not known, since the nature of the forces by which cells maintain high intracellular concentrations of many constituents is poorly understood. Not all injury to muscle results in the loss of amino acids. Recent determinations of free amino acids and related compoimds in muscle extracts from normal and vitamin E deficient (dystrophic) rabbits have shown that levels of most of the extractable amino acids were elevated in the vitamin deficient animals. 11 It is important to consider the nature of the cell population of the tissue which is being examined in evaluating chemical results obtained in the study of a pathologic process. Each cell type has its characteristic pattern of ammo acids, 1 " 4 and physiologic and structural changes in the cells themselves and in the extracellular elements may affect greatly the results of chemical analyses. SUMMARY Free or easily extractable amino acids were determined by two-dimensional paper chromatography in hearts of dogs at various time intervals up to 6 days after the production of experimental myocardial infarction by ligation of the anterior descending coronary artery, and comparisons were made of the infarcted and noninfarcted areas of the left ventricle. Significant alterations from the normal pattern of amino acids distribution were not found in the nouinfarcted portions of the hearts of the experimental animals. Details are described of changes in amino acid content, characterized by decreases in some of the major ninhydrin-reactive constituents, which began to appear by 8 hours after the infarction coincidentally with liistologic signs of myocardial damage. ACKNOWLEDGMENT? AVc are indebted to Seymour M. Greenstone, M.D., for valuable collaboration in this study, and to Fred Preuss, M.D., for evaluation of the histologic sections. SUMMARIO IN INTEULIXGUA Libere o facilemente extrahibile aminoacidos esseva determinate per bidimensional chromatographia a papiro in cordes canin a varie intervallos (de usque a 6 dies) post le production de infarcimento myocardial experimental

5 CHANIN, ROBERTS AND GOLDMAN 717 per ligation del antero-descendente arteria coronari. Comparationes esseva facite inter le infarcite e le non-infarcite areas del ventriculo sinistre. Alterationes significative del distribution normal de aminoacidos non esseva trovate in le non-infarcite portiones del cordes del animales experimental. Es describite in detalio le alterationes del contento de aminoacidos, characterisate per reductiones de certes del major constituentes ninhydrino-reactive, le quales comenciava manifestar se 8 horas post le infarcimento, in coincidentia con signos histologic de lesiones myocardial. REFERENCES 1 ROBERTS, E., AND TISHKOFF, G. H.: Distribution of free iimino acids in mouse epidermis in various phases of growth as determined by paper partition chronmtography. Science 109: 14, , AND FRAJJKEL, S.: Fit-e amino acids in normal and neoplastiu tissues. Cancer Res. 9: 645, ,, AND HARMAN, P. J.: Amino acids of nervous tissue. Proc. Soc. Exper. Biol. & Med. 74: 383, A, KARNOFSKY, D. A., AND FRANKEL, S.: Influence of cortisone on free hydroxyproline in the developing chick embryo. Proc. Soc. Exper. Biol. <fe Med. 76: 2S9, RONZONI, E., ROBERTS, E., FRANKEL, S., AND RAMASARMA, G. B.: Influence of administration of ACTH on urinary amino acids. Proc. Soc. Exper. Biol. & Med. 82: 496, ROBERTS, E., CHANIN, M., AND GREENSTONE, S. M.: Free amino acid patterns in cardiac muscle of several species. Fed. Proc. 14: 269, GOLDMAN, A., GREENSTONE, S. M., PREUSS, F. S., STRAUSS, S. H., AND CHANG, E.: Experimental methods for producing a collateral circulation to the heart directly from the left ventricle. J. Thoracic Surg. 31: 364, YOKOYAMA, H. 0., JENNINGS, R. B., CLABOUQH, G. F., AND WORTMAN, W. B.: Histochemical studies of early experimental myocardial infarction. Arch. Pathol. 69: 347, WACHSTEIN, M., AND MEISEL, E.: Succinic dehydrogenase activity in myocardial infarction and in induced myocardial necrosis. Am. J. Path. 31: 353, WROBLEWSKI, F., AND LADUE, J. S.: Lactic dehydrogenase activity in blood. Proc. Soc. Exper. Biol. & Med. 90: 210, TALLAN, H. H.: Free amino acids of normal and vitamin E-deficient rabbits. Proc. Soc. Exper. Biol. <fc Med. 89: 553, 1955.