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15. Ahmed M, Dubiel J, Verdon TA, Martin RH: Technetium-99m stannous pyrophosphate myocardial imaging in patients with left ventricular aneurysms. (abstr) Clin Res 123: 168A, 1975 16. O'Rourke R, Righetti A, Schelbert H, Daily P, Ashburn W, Ross J Jr: Usefulness of pre and postoperative Tc-99m-pyrophosphate scans in cardiac surgical patients. (abstr) Am J Cardiol 37: 161, 1976 17. Khentigan A, Gannett M, Lum D, Winchell HS: Effect of prior administration of Sn(II) complexes used in nuclear medicine on in vivo distribution of subsequently administered Tc-99m pertechnetate and Tc99m compounds. J Nucl Med 16: 541, 1975
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55, No 1, JANUARY 1977
18. Klein MS, Coleman RE, Roberts R, Weiss AN: 99m Tc(Sn) pyrophosphate scintigrams in exercise induced angina and calcified valves. (abstr) Am J Cardiol 37: 149, 1976 19. Soin JS, Burdine JA, Beal W: Myocardial localization of 99m-Tcpyrophosphate without evidence of acute myocardial infarction. J Nucl Med 16: 944, 1975 20. Platt MR, Parkey RW, Willerson JT, Bonte FJ, Shapiro W, Sugg WL: Technetium stannous pyrophosphate myocardial scintigrams in the recognition of myocardial infarction in patients undergoing coronary artery revascularization. Circulation 51 & 52 (suppl II):II-141, 1975
Quantification of Infarction in Cross Sections of Canine Myocardium In Vivo with Positron Emission Transaxial Tomography and 11C-Palmitate EDWARD S. WEISS, M.D., SYED A. AHMED, M.D., MICHAEL J. WELCH, PH.D., JOSEPH R. WILLIAMSON, M.D., MICHEL M. TER-POGOSSIAN, PH.D., AND BURTON E. SOBEL, M.D. SUMMARY To assess myocardial infarction quantitatively in 15 mm thick transverse sections of the canine heart in vivo we utilized a new technique, positron emission transaxial tomography (PETT) and cyclotron-produced I'C-palmitate (1'C-P) injected intravenously. Results were compared to regional myocardial creatine phosphokinase (CPK) depletion, diminished "4C-palmitate accumulation in tissue extracts, and infarction estimated morphometrically 48 hours after coronary occlusion. CPK activity and "C-P content declined in parallel In transmural biopsies (N = 44) from normal and ischemic zones (r = .92) in six dogs; and infarct in 10 mm thick cross sections of the
entire left ventricle estimated morphometrically (N = 26) in six other animals correlated with CPK depletion in contiguous 2.5 mm thick slices (r = .92). When the percentage of infarction in 15 mm thick cross sections was assessed tomographically in six other dogs 48 hours after coronary occlusion with 11C-P injected intravenously, results correlated with infarction in corresponding cross sections from the same hearts estimated morphometrically (r = .97, N = 9) and by analysis of CPK depletion (r = .93, N = 9). ThIls, PETT permits estimation of infarction in cross sections of the left ventricle In vivo after intravenous injection of 11C-palmitate.
EVALUATION OF THE EXTENT OF MYOCARDIAL INFARCTION in vivo has become increasingly important because prognosis in patients appears to be influenced by infarct size'`4 and because the extent of irreversible ischemic injury may be amenable to favorable modification5' with interventions implemented during the early evolution of the insult. Although myocardial infarct scintigraphy7 is useful for detection and localization of infarction, quantification of ischemic injury with this technique may be limited because of the dependence of accumulation of tracer on the age of the infarct, and because of inherent limitations leading to disparities between the mass of injured tissue and its twodimensional display.8 9 Precordial ST-segment elevations may reflect serial changes in the electrophysiological response of the heart to ischemia10 '1 but they do not provide a direct reflection of the absolute magnitude of infarction13' 14 and may be influenced by factors not directly related to ischemia such as drug effects, pericarditis, or changes in the concentration of electrolytes in extracellular fluid. Prediction or estimation of infarct size on the basis of
time-activity curves of plasma enzymes such as creatine phosphokinase suffers from the prolonged interval required for acquisition of data prior to construction of projected portions of the curves.'5-'8 The present study was designed to develop and evaluate a method for quantitative estimation of the extent of myocardium undergoing infarction in vivo suitable for prompt, early, and sequential evaluation of the evolution of ischemic injury. Accordingly, we synthesized "IC-palmitate,'9 a shortlived, positron-emitting, cyclotron-produced tracer of the predominant physiological substrate of myocardium;20 injected the material intravenously in closed chest dogs with myocardial infarction; and determined its distribution in ischemic myocardium with computer reconstructed images obtained by positron emission transaxial tomography2' in order to detect, localize, and quantify infarction in vivo. Results were compared to independent estimates of the extent of infarction based on biochemical and morphological analyses of hearts from the same animals. This approach was predicated on several considerations. The positron-emitting tracer employed has a short half-life (20.4 min), permitting frequent sequential studies with a low total body radiation burden. Use of palmitate, a physiological substrate of the heart, permits interpretation of results in terms of the extensive information available characterizing myocardial intermediary metabolism and documenting diminished uptake and oxidation of free fatty
From the Washington University School of Medicine, St. Louis, Missouri. Supported in part by Washington University School of Medicine, USPHS SCOR in Ischemic Heart Disease 1 P17 HL 17646 and NIH Grant 5 P01 HL
13851-13. Address for reprints: Edward S. Weiss, M.D., Cardiovascular Division, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110. Received May 20, 1976; revision accepted July 22, 1976.
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TOMOGRAPHY OF THE HEART/Weiss et al.
acids in zones of ischemia and infarction.21 25 The distribution of positron-emitting isotopes in tissues can be quantified readily with coincidence counting, facilitating spatial resolution and delineation of the distribution of radioactivity in three-dimensional space.21 Furthermore, detection of radiation emitted from the heart is not distorted by the distance of the emitting source from each member of any pair of detectors. Quantification of infarction in a series of cross-sectional slices of myocardium by positron emission transaxial tomography should permit estimation of the entire mass of the infarct based on summation of the amount of infarct detected in each individual cross section. In a preliminary qualitative evaluation of this approach, we verified the externally detectable diminution of myocardial accumulation of "IC-palmitate associated with reduction of coronary flow in isolated perfused rabbit hearts and demonstrated the feasibility of obtaining positron emission transaxial tomographic images of the heart in intact dogs.2" The present study was undertaken to extend these observations to permit quantitative estimation of infarction in vivo and to define the quantitative relationships between computer reconstructed tomograms of infarcts and biochemical and morphological changes in the same tissue. Materials and Methods Animal Preparations
Myocardial infarction was produced in 22 dogs anesthetized with sodium pentobarbital, 30 mg/kg injected intravenously, by ligation of the left anterior descending coronary artery distal to the first septal branch. Immediately after ligation, the thoracotomy was closed. Eighteen animals survived this procedure and were included in the studies performed 48 hours after surgery. Six animals were used to clarify the relationship between myocardial CPK depletion in a cross section of the entire left ventricle and morphometric estimation (see below) of infarction in an adjacent 10 mm thick cross section in the experimental preparation used. Total myocardial CPK activity was assayed in extracts of 2.5 mm thick cross sections. Results were compared to expected activity in the cross sections, calculated as the product of weight and CPK/g of histologically normal, nonischemic myocardium (averaged from four 0.5 g transmural biopsies in each heart). In each dog from this group, three or four pairs of cross sections were analyzed. Six different animals were used to determine the relationship between myocardial CPK depletion 48 hours after coronary occlusion and diminution of regional "4C-palmitate accumulation in myocardium after intravenous injection of 14C-palmitate, five minutes before the animal was killed. Among the six animals studied with 14C-palmitate, myocardial CPK activity was assayed in five to eight 0.5 g transmural myocardial samples obtained with a precooled modified dental drill from ischemic and nonischemic regions of each heart. Results were compared to 14C-palmitate radioactivity in extracts of the same tissue used in a Packard liquid scintillation counter. This isotope was studied to facilitate interpretation of results of subsequent studies in other dogs in which tomography was performed after intravenous injection of "IC-palmitate 48 hours after coronary occlusion.
67
After relationships between 14C-palmitate, morphology, and CPK depletion had been examined, six additional animals were studied by positron emission tomography. In each of these, after tomography was completed, the animal was killed and infarction was verified and quantified by two procedures: myocardial CPK analysis and morphometric analysis. CPK analysis was performed in extracts of each of two 2.5 mm thick transverse sections of the entire left ventricle at the level corresponding to the tomographic image and adjacent to a central contiguous 10 mm thick cross section. Morphometric analysis was performed as described below on each central 10 mm thick cross section of the entire ventricle corresponding to the tomographic plane. In several animals more than one tomographic plane was studied with corresponding CPK and morphometric comparisons. For CPK and morphometric analyses, slices of the entire left ventricle were obtained from the rapidly frozen hearts of other animals. The transverse sections were obtained to correspond to tissue planes reflecting the field of view encompassed in each tomographic image based on the known 15 mm thickness of each tomographic section and the distance from the apex of the heart corresponding to each. The distributions of '4C-palmitate and myocardial CPK activity were determined by analysis of both in transmural punch biopsies of normal and ischemic tissue and transverse sections of the entire left ventricle. Preparation of Left Ventricular Biopsies for Analysis of '4C-Palmitate and Myocardial CPK Content
All procedures were performed at 0 to 4° C. Myocardium was minced with scissors, suspended in 0.05 M HepesNaOH buffer, pH 7.4, 10 ml/g, and homogenized in a Virtis homogenizer for two 15 second bursts at half maximum speed. Aliquots of 0.4 ml of homogenate were removed for determination of '4C-palmitate radioactivity in a toluene based fluor after solubilization of protein with Protosol. Myocardial CPK activity was assessed in extracts of whole homogenate of myocardium initially centrifuged for 20 minutes at 30,000 g at O° C. The supernatant fraction was decanted, diluted with Hepes-NaOH buffer, pH 7.4, .05 M such that CPK activity was less than .2 IU/ml. CPK activity was assayed spectrophotometrically as previously described and protein was determined by the Biuret method.'5 CPK depletion per gram of wet weight was determined as follows: CPK activity was measured in four histologically normal regions from each heart, averaged, and designated as CPKN, expressed as IU/g. Total CPK activity in each 2.5 mm transverse section was measured directly and designated as CPKT, also expressed as IU/g. The percent CPK depletion was calculated from [(CPKN- CPKT)/CPKN] X 100. Morphometric Analysis of Infarction
Frozen hearts were sliced into 10 mm thick sections for gross and histological examination. Alternating 2.5 mm thick sections were used for analysis of '4C-palmitate and CPK activity (fig. 1). Each 10 mm section was immersed rapidly in normal saline and photographed with a Graflex 4 X 5" photocopy camera. Subsequently, the slice was fixed with 1.25% glutaraldehyde-l% formaldehyde buffered to pH 7.4 with Ringer's lactate solution and cut into blocks for
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VOL 55, No 1, JANUARY 1977
myocardial biopsies since the short half-life of "IC-palmitate (20.4 min) made destructive analysis of multiple samples for radioactivity impractical. Positron Emission Transaxial Tomography
FIGURE 1. A diagrammatic representation of the sampling technique used to obtain transverse sections of the entire left ventricle for histological analysis in a 10 mm slice, and assessment of myocardial CPK activity and "4C-palmitate radioactivity in adjacent 2.5
slices. The shaded area thetical zone of infarction.
mm
on
the diagram represents
a
hypo-
histology. Sections adjacent to regions analyzed biochemically were stained with hematoxylin and eosin. Each histological section was analyzed to detect infarction, projected, and combined to reconstruct an entire cross section of the heart with the area of necrotic tissue outlined by planimetry. The total area of each 10 mm thick transverse section and the area representing infarction were calculated with a Hewlett-Packard 9100 B calculator coupled to 9107 A digitizer. Calculations of the percentage of infarction in each region were made on the basis of the ratio of outlined infarct to normal myocardium in the entire cross section. Preparation of '"C-Palmitate
"C-palmitate was produced by the reaction of pentadecyl magnesium bromide in diethyl-ether with "CO, produced in the Washington University Medical School cyclotron by the "OB(d,n) 'IC nuclear reaction using boric oxide as the target material. After acidification with 3 ml of N hydrochloric acid, the ether layer (4 ml) was separated and 2 ml of ethanol added. The ether/ethanol solution was boiled to remove the ether. At this stage of the preparation the ethanol contains both the labeled palmitate and pentadecane formed by the hydrolysis of the unreacted Grignard reagent. Eight ml of 4% albumin solution is then added and the solution maintained at 42° C for 5 minutes during which time the pentadecane precipitates. The precipitate is removed from the solution by millipore filtration prior to addition of the remaining "IC-palmitate. Administration of "4C-Palmitate
For studies of the distribution of '4C-palmitate in myocardium after intravenous injection of the tracer, results were obtained by liquid scintillation counting of extracts prepared from biopsies. The tracer was prepared as follows: 250 ,uCi of palmitic-l-14C acid (labeled analogously to the "C-palmitate preparation) in hexane was immersed in a water bath at 900 C until the hexane had been entirely evaporated. Subsequently, ml of absolute ethanol was added to the radioactively labeled material, followed by 4% bovine serum albumin dissolved in 10 ml of 0.15 M NaCl warmed to 52° C. The solubilized '4C-palmitate was used for intravenous injection in the canine preparations. The use of "4C-palmitate permitted analysis of palmitate distribution in
Five to ten millicuries of "C-palmitate in a solution of 4% albumin was injected intravenously in an 8 ml bolus at a rate of 1-2 ml/sec 48 hours after coronary occlusion in lightly anesthesized dogs. Three minutes later, an interval selected to permit clearance of the tracer from the blood pool, the distribution of 1"C-palmitate in myocardium was determined by detection of positron emission during an 8 to 16 minute interval providing detection of 0.5 to 1.0 X 106 counts. At least one and as many as seven tomograms were obtained from each animal corresponding to one or more 15 mm thick cross sections of myocardium. Immediately after completion of the tomograms, 250 ,Ci of '4C-palmitate in 4% albumin were injected intravenously. The animals were killed five minutes later with an overdose of anesthesia. "Cpalmitate was administered in order to permit subsequent analysis of the distribution of this -tracer based on liquid scintillation counting of extracts obtained from transmural punch biopsies and transverse sections of the excised heart. Quantitative assessment of the organ distribution of 11Cpalmitate in myocardium in the studies in vivo was based on a computer reconstructed image of positron emission (511 keV gamma rays) from 15 mm thick cross sections of the heart. Correction factors for attenuation were first obtained with the use of a ring containing 64Cu in a solution of concentrated nitric acid placed within the empty tomograph and then within the same field of view but around the experimental canine preparation. Positron emission from "Cpalmitate accumulated in myocardium after intravenous injection was detected and quantified with a positron emission transaxial tomograph designed at Washington University. This instrumentation employs six banks of 8 Nal crystals to detect positron emission. The detectors are rotated with a programmed pattern of motion around the experimental animal at the level of the heart. Data are analyzed by computer to produce reconstructed cross-sectional images from data profiles from each pair of detectors.26 27 The percentage of infarct in each 15 mm thick cross section of the left ventricle in vivo was estimated by analysis of the digital printout of the 2,500 data points utilized for computer reconstruction of the entire left ventricular cross-sectional tomographic image. In order to define the epicardial and endocardial margins of the ventricular image on the digital printout, a horizontal profile of the image gray scale was obtained on a Tektronix Type 524 wave form monitor. In this profile the region of the ventricular myocardium was defined by inclusion of all data points with values exceeding 50% of the maximum value detected. A Versatec plotter was used to obtain digital printouts of the distribution of 11Cpalmitate radioactivity in each image of normal canine ventricles at the midventricular and A-V valvular levels. The region corresponding to myocardium was delineated with a hand drawn line surrounding those digital values conforming to the count rate criterion. This area correlated well with the oscilloscopic display of the image of myocardium, typically approximately circular, obtained at the midventricular level and with the typically horseshoe-shaped image, obtained at the level of the A-V valves (fig. 2).
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TOMOGRAPHY OF THE HEART/Weiss et al.
69
Arch of oorta
out coronary occlusion (fig. 3). The determination of percentage of infarction represented by these defects was calculated by planimetric measurement of the image area defined in the digital printout. This area was then quantitatively compared to the calculated area between the two circles derived from the minimal diameters across the inner and outer perimeters of the tomogram (fig. 4). A proportionality constant was used for calculation of percentage of infarction at the level of the A-V valves since these tomograms are horseshoe-shaped because of the nonvisualized posterior left atrial border. The area of ventricular myocardium in this region was estimated in ten images from normal dogs and found to average 60% (range = 54 to 67%) of the total area between the two concentric circles at midventricular level. Thus, at this level, the percentage of infarction was estimated by subtracting the planimetered area of the infarct from 0.6 times the area between these concentric circles. Results The Relation between Myocardial CPK Depletion, Diminution of '4C-Palmitate Accumulation, and Morphologically Evident
FIGURE 2.
Representative images obtained by positron emission transaxial tomography after intravenous injection of "C-palmitate in a lightly anesthetized dog. As can be seen, the characteristic distribution of "C-palmitate is circular at the midventricular level, but conforms to a horseshoe-shaped pattern at the level of the A-V valves because the posterior portion of the heart in this region comprises left atrial rather than ventricular myocardium. A, P, R, L refer to anterior, posterior, right, and left, respectively. The central areas within the circle and horseshoe patterns correspond to the left ventricular cavity. The endocardial margin is somewhat blurred because these images were obtained without electrocardiographic gating.
Infarction Results of analyses of CPK activity and "4C-palmitate radioactivity in 44 transmural samples of myocardium from normal and ischemic zones from six dogs are shown in figure 5. As can be seen, regions with low '4C-palmitate accumulation were characterized by marked depletion of CPK activity. The correlation between the percentage of CPK depletion and the percentage of diminution of "C-palmitate accumulation was .92. As can be seen in table 1, depletion of CPK in transverse sections of the entire left ventricle correlated closely with necrosis estimated morphologically in adjacent sections. Since 14C-palmitate accumulation was diminished in proportion to myocardial CPK depletion
Tomographic images obtained 48 hours after coronary occlusion revealed appearance of a large anterior defect, which interrupted the circular image noted in animals with-
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Fro;URE 3. Positron emission transaxial tomograms (top panels) obtained at the midventricular level in a normal dog (left) and a dog with an anterior myocardial infarction induced by coronary occlusion 48 hours earlier (right). Printouts of the digital data with lines encompassing normal myocardium drawn as indicated in the text are shown in the bottom two panels. The midventricular imagefrom the dog with the myocardial infarct shows diminution of "Cpalmitate uptake with a defect in the anterior portion of the cross-sectional image (top) and conforming to results apparent on the printout of the digital data shown below.
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CIRCULATION
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VOL 55, No 1, JANUARY 1977
A
B AREA measured AREA hypothetical
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FIGURE 4. The digital printout obtainedfrom a midventricular tomogram in a normal dog is displayed in the left hand panel (A) with a line drawn by hand to include allpoints with values greater than 50% of maximum to define the borders of the myocardial image. Two concentric circles are drawn, based on the minimal endo- to endocardial and epi- to epicardial diameters, to obtain a hypothetical area for the myocardial image. In the right hand panel (B) the method of calculation ofpercentage infarction is indicated. The calculated area, based upon the minimal diameters of the measured image is smaller than the measured area for a noninterrupted myocardial image. The appearance of a defect seen in animals subjected to coronary occlusion is indicated here with crossed-hatched markings. The percentage of infarction in such a tomogram is calculated by subtracting the measured area of the image from the area of the hypothetical image and dividing this difference by the hypothetical area.
throughout ischemic zones (fig. 5), these results suggested that necrosis could be estimated by analysis of myocardial accumulation of palmitate estimated externally. Furthermore, since palmitate-l-'IC and palmitate-I-'4C are likely to
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be equivalent metabolically, it appeared that necrosis could be estimated by external assessment of '1C-palmitate uptake. In six dogs subjected to coronary occlusion but not studied tomographically, myocardial CPK depletion in 2.5 mm thick transverse sections of the left ventricle at multiple levels from apex to base correlated closely with morphometric estimates of infarction in the adjacent 10 mm thick cross section (fig. 6) (r = .92, N = 26 pairs of sections). Thus, the depletion of myocardial CPK activity and 14Cpalmitate accumulation correlated closely with morphometric estimates of infarction in the adjacent transverse section.
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Estimation of Infarction in Cross Sections of the Left Ventricle In Vivo Based on Analysis of Positron Emission Transaxial Tomograms Areas of myocardial infarction were estimated in 15 mm
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serial sections of myocardium represented by tomograms obtained sequentially from apex to base in closed chest dogs.
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TABLE 1. Analyses in Transverse Sedions of the Entire Left Ventricle Obtained from Three Dogs
x 0
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20 40 60 80 % MYOCARDIAL CPK DEPLETION (WIUg) FIGURE 5. 14C-palmitate accumulation and myocardial CPK activity in 44 transmural samples of myocardium from normal and ischemic zones in six dogs. Results are expressed as percentage decreases compared to results in transmural samples from normal myocardium in the same animal. As can be seen, there was a close correlation between CPK diepletion and diminished 14C-palmitate accumulation 48 hours after coronary occlusion. Each symbol refers to data from the same dog. Dashed lines indicate confidence limits drawn as two standard errors of the estimate. The equation for the best-fit regression line (least squares method) is y = .95X 3.43. -
Dog number
A CPK activity % decrease from normal sample
B 14C-palmitate uptake % decrease from normal sample
C Morphological determination of infarct size (%)
1 1 3 2 3 1 2 2 3
0.0 2.5 9.1 10.9 19.0 23.3 26.0 29.6 31.5
5.9 8.8 12.2 14.4 12.3 26.1 20.0 14.8 32.7
0.0 2.0 9.2 16.8 14.3 29.0 18.7 15.5 31.6
The correlation coefficient between results in columns A and C (linear regression least squares method) was .95, and that between results in columns B and C was .94.
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TOMOGRAPHY OF THE HEART/Weiss et al.
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30 40 50 60 20 INFARCT SIZE FROM CPK DEPLETION (%) FIGURE 6. The relationship between infarct size estimated morphologically and infarct size estimated from myocardial CPK depletion in an adjacent transverse section of the entire left ventricle is indicated here. Results obtained 48 hours after coronary occlusion with these two methods correlated closely.
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The typical tomograms obtained at the midventricular (circular) and A-V valvular level (horseshoe-shaped pattern) are shown in figure 2. At least four images were obtained from each dog, but many did not encompass regions of infarction. As can be seen in figure 3, the presence of infarction 48 hours after left anterior descending coronary occlusion in a tomogram corresponding to a 15 mm thick section of the left ventricle at the midventricular level is readily apparent in the image and the digital printout of the data. The anterior defect contrasts with an image from the same level obtained from a normal dog. The percentage of infarction in cross sections from each of six dogs studied tomographically estimated from the area in the tomogram correlated closely with the extent of infarction in the corresponding slice analyzed morphometrically (fig. 7) by an observer unaware of the results obtained tomographically (r = .97, N = 9 pairs of slices) and with infarction estimated from CPK depletion in the adjacent 2.5 mm thick slice analyzed by another observer, also unaware of the tomographic results (r = .93, N = 9 pairs of slices from six dogs). As can be seen, CPK depletion and morphological estimates of infarction correlated closely in sections corresponding to those imaged (r = .92, N = 9 pairs of slices). These results indicate that estimates of infarction in cross sections of the left ventricle obtained by analysis of positron emission transaxial tomograms in vivo correspond closely to estimates of infarction based on analysis of myocardial CPK depletion and to morphometric estimates of infarct size in the corresponding transverse section. Application of the tomographic approach to estimating the entire mass of an infarct is illustrated in figure 8. As can be seen, sequential tomograms corresponding to 15 mm thick cross sections of the heart delineate a volume of infarction that can be calculated from the cross-sectional area representing diminished '1C-palmitate uptake in each cross section multiplied by the thickness of each. Improved results should be available with instrumentation now being developed that will permit 1) simultaneous accumulation and differentiation of data from four sections from each heart, and 2) electrocardiographic gating with reduction of impairment of resolution due to movement of the heart throughout the cardiac cycle.
r s 0.97
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MORPHOLOGICAL INFARCT SIZE (%) FIGURE 7. Myocardial CPK depletion and morphological estimates of infarction in a cross section through the entire left ventricle, compared with the percentage of infarction in a corresponding section delineated by positron emission transaxial tomography in vivo. Results from nine pairs of sections from six different dogs are shown. The correlation coefficients (least squares linear regression) indicate that estimates of infarction based on CPK depletion (N = 9) and morphology correlated closely with estimates obtained in vivo by positron emission transaxial tomography of the corresponding cross section (N = 9) of the left ventricle. The similarity ofslopes in (B) and (C) is reflected by the slope approaching one in (A). Since 100% CPK depletion does not occur within 48 hours (with approximately 10% residual activity, unpublished observations), the slope in (B) would be anticipated to be less than one. The slope in (C) is less than unity probably because the true image of the normal ventricle is not congruent with the concentric circle model utilized for calculations (see fig. 4).
Discussion The use of radioisotopes to detect myocardial injury or ischemia externally has employed intravenous administration of inorganic ions such as 43K, '29Cs, B'Rb, 8tRb, 201TI, 67Ga; 13N labeled NH3;28-" and intracoronary injection of radioisotopes of inert gases such as "Kr and 133Xe.37-" These moieties were selected because of advantages in physical or biological properties but none is a physiological substrate of myocardium. Efforts to utilize organic substrates of the heart as agents for imaging myocardium have involved fatty acid analogs labeled with 131I and *mTc.4o 41 Unfortunately, these derivatives are metabolically altered substrates exhibiting less uptake than the corresponding 'IC-labeled congener.42 Furthermore, quantification of their distribution has been difficult. In the present study we utilized a physiological substrate of the heart by synthesizing radionuclides incorporating positron-emitting 1"C. Previously, we demonstrated that
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CIRCULATION
72
inforct . lW.tI
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thick sections
mm
injection of "C-palmitate
intravenous
farction produced by
in
a
the
the conclusion
at
of the study
are
diagrams comprising the top panel.
palmitate
dog with myocardial
as
be
can
prior
to
in-
study.
examined morp ho-
was
shown
As
a
(left) after
shaded
seen,
zones
in
diminished
corresponding to these shaded areas is evicomprising the bottom panel. The right ven-
diagrammatically,
tricular wall, shown
The transmission images
positron source) c en ter
apex
accumulation
dent in the tomograms
grams.
to
coronary occlusion 48 hours
Regions of infarction evident when the heart logically
depicting
transaxial tomograms
from base (right)
panel
and
th e thorax and
each level
at are
to
used
obtlain
to
is not visualized in the tomo-
(obtained with the
from base
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of
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are
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jection of "C-palmitate and acquisition of data for
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myocardial ischemia under
could be detected
11C-palmitate
in
to
the
physiological
albumin in these studies
physiological
myocardium raphy. vivo
qualitatively
perfusate of isolated conditions of controlled flow."1 The high
11C-palmitate added
substrate in viva
suggested would
by positron
The distribution of
was
demonstrated in
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present study
we
solutions
with
rabbit hearts
extraction of
with
equimolar
that this radionuclide of
permit imaging of
a
normal
emission transaxial tomog-
"IC-palmitate pilot
studies
in myocardium in (data not shown) by
with conventional instrumentation. In the demonstrated that the distribution of this
analyzed quantitatively by positron emission transaxial tomography. The diminished uptake of "C-palmitate in tomographic images was found to correspond to decreased "IC-palmitate uptake and to independent indices of ischemic injury in analogous experimental preparations. In our previous study with isolated perfused hearts we tracer
could be
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1, JANUARY 1977
showed that the diminished extraction of "IC-palmitate was a function of the duration of low flow and an index of metabolic manifestation of ischemia rather than a reflection simply of decreased delivery of the tracer to myocardium.Y The present results indicate a similar relationship between decreased palmitate accumulation demonstrated by PETT in areas exhibiting diminished CPK content in myocardium. The two correlations 1) between CPK depletion and histologic necrosis, and 2) between decreased accumulation of both "1C- and '1C-palmitate and CPK depletion, support the interpretation that under the conditions selected (48 hours after coronary occlusion), diminished 'IC-palmitate accumulation detectable tomographically reflects infarction. Thus, although CPK content and '1C-palmitate accumulation are independently regulated, both are affected under these particular conditions as a result of infarction. Reconstructive tomography offers several advantages in spatial resolution. For example, the technique delineates and separates the display of anterior and posterior regions and provides a cross-sectional representation avoiding the superimposition encountered with conventional imaging techniques. This facilitates quantification of the distribution of tracer and hence assessment of metabolism in a cylindrical volume of tissue. It also permits quantitative assessment of reduced accumulation of tracer and representation of a cold zone in three-dimensional space. In the present study quantification of the percentage of myocardial infarction in 15 mm thick cross sections of the heart was determined by analysis of the digital printout of the data points obtained by computer reconstruction. A close correlation was obtained between the percentage of infarction estimated by analysis of each tomogram and histological estimates of infarction in the corresponding transverse section of the left ventricle. Determination of the total mass of ischemic ventricular myocardium by positron emission transaxial tomography with '1C-palmitate depends on imaging geometrically contiguous regions of the heart without overlap. This presently requires movement of the experimental animal in 15 mm increments in a cephalad direction to obtain sequential images of the entire left ventricle. The rapid decay of activity encountered with the short-lived '1C-nuclide requires progressively longer sampling intervals to obtain sufficient counts for computer reconstruction with sequential images. Accordingly, in this study, nongated images were used to permit the acquisition of the maximal amount of counts after a single injection of 11C-palmitate even though the use of nongated images resulted in diminished resolution. The myocardial infarctions studied in this investigation resulted from coronary occlusion of 48 hours' duration. This interval was chosen because histological indices of infarction and CPK depletion are well established within 48 hours after occlusion.5 Well circumscribed areas of ischemia can be detected as soon as 20 minutes after coronary occlusion by positron emission transaxial tomography with "IC-palmitate2' but unlike the case in which the occlusion is maintained for longer intervals, diminished uptake of "IC-palmitate is completely reversible with reperfusion. Thus, the capability for early recognition of ischemia after experimental coronary occlusion may presage significant potential for early clinical detection of jeopardized myocardium. In addition, the short half-life of '1C (20.4 min) permits se-
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TOMOGRAPHY OF THE HEART/Weiss et al. quential studies in an individual organism facilitating repeated examinations during the early evolution of acute myocardial ischemic injury. The differentiation of ischemia from infarction should be possible by analysis of results of sequential studies in the same experimental animal or patient, once the duration of ischemia sufficient to abolish "IC-palmitate accumulation and required to produce infarction has been defined. Results in this study suggest that positron emission transaxial tomography utilizing cyclotronproduced, short-lived, positron-emitting radionuclides of physiological substrates of myocardium will facilitate objective evaluation of the efficacy of potentially therapeutic interventions on the extent of infarction ultimately sustained after an ischemic insult. Acknowledgment We appreciate the assistance of Mrs. Carol Higgins, Mr. Nizar Mullani, Ms. Alice Robison, and Ms. Maria Straatmann in biochemical, radiochemical, and tomographic procedures and Ms. Carolyn Lohman in preparing this
manuscript.
References 1. Page DL, Caulfield JB, Kastor JA, DeSanctis RW, Sanders CA: Myocardial changes associated with cardiogenic shock. N Engl J Med 285: 133, 1971 2. Harnarayan C, Bennett MA, Pentecost BL, Brewer DB: Quantitative study of infarcted myocardium in cardiogenic shock. Br Heart J 32: 728, 1970 3. Sobel BE, Bresnahan GF, Shell WE, Yoder RD: Estimation of infarct size in man and its relation to prognosis. Circulation 46: 640, 1972 4. Norris RM, Whitlock RML, Barratt-Boyes C, Small CW: Clinical mea-
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surement of myocardial infarct size. Modification of a method for the estimation of total creatine phosphokinase release after myocardial infarction. Circulation 51: 614, 1975 Maroko PR, Kjekshus JK, Sobel BE, Watanabe T, Covell JW, Ross J Jr, Braunwald E: Factors influencing infarct size following experimental coronary artery occlusions. Circulation 43: 67, 1971 Shell WE, Sobel BE: Protection of jeopardized ischemic myocardium by reduction of ventricular afterload. N Engl J Med 291: 481, 1974 Willerson JT, Parkey RW, Bonte FJ, Meyer SL, Atkins JM, Stokely EM: Technetium stannous pyrophosphate myocardial scintigrams in patients with chest pain of varying etiology. Circulation 51: 1046, 1975 Ter-Pogossian MM: Limitations of present radionuclide methods in the evaluation of myocardial ischemia and infarction. Circulation 53 (suppl I): 1-119, 1976 Ter-Pogossian MM, Hoffman EJ, Weiss ES, Coleman RE, Phelps ME,
Welch MJ, Sobel BE: Positron emission reconstruction tomography for the assessment of regional myocardial metabolism by the administration of substrates labeled with cyclotron produced radionuclides. In Proceedings from the Conference on Cardiovascular Imaging and Image Processing Theory and Practice, vol 72, edited by Harrison DC, Sandler H, Miller HA. Palos Verdes Estates, Society of Photo-Optical Instrumentation Engineers, 1975, p 277 Maroko PR, Libby P, Covell JW, Sobel BE, Ross J Jr, Braunwald E: Precordial S-T segment elevation mapping: An atraumatic method for assessing alterations in the extent of myocardial ischemic injury. Am J Cardiol 29: 223, 1972 Kjekshus JK, Maroko PR, Sobel BE: Distribution of myocardial injury and its relation to epicardial ST-segment changes after coronary artery occlusion in the dog. Cardiovasc Res 6: 490, 1972 Reid PR, Taylor DR, Kelly DT, Weisfeldt ML, Humphries JO, Ross RS, Pitt B: Myocardial-infarct extension detected by precordial ST-segment mapping. N Engl J Med 290: 123, 1974 Ross J Jr: Electrocardiographic ST-segment analysis in the characterization of myocardial ischemia and infarction. Circulation 53 (suppl I): I73, 1976
14. Muller JE, Maroko PR, Braunwald E: Evaluation of precordial electrocardiographic mapping as a means of assessing changes in myocardial ischemic injury. Circulation 52: 16, 1975 15. Shell WE, Lavelle JF, Covell JW, Sobel BE: Early estimation of myo-
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cardial damage in conscious dogs and patients with evolving acute myocardial infarction. J Clin Invest 52: 2579, 1973 16. Sobel BE, Larson KB, Markham J, Cox JR Jr: Empirical and physiological models of enzyme release from ischemic myocardium. In Computers in Cardiology. Long Beach, IEEE Computer Society, 1974, p 189 17. Roberts R, Henry PD, Sobel BE: An improved basis for enzymatic estimation of infarct size. Circulation 52: 743, 1975 18. Sobel BE, Roberts R, Larson KB: Estimation of infarct size from serum MB creatine phosphokinase activity: Applications and limitations. Am J Cardiol 37: 474, 1976 19. Welch MJ: The role of the chemist in nuclear medicine. In Yearbook of Nuclear Medicine, vol 4. Chicago, Year Book Medical Publishers, 1969, p 6 20. Neely JR, Bowman RH, Morgan HE: Effects of ventricular pressure development and palmitate on glucose transport. Am J Physiol 216: 804, 1969 21. Weiss ES, Hoffman EJ, Phelps ME, Welch MJ, Henry PD, TerPogossian MM, Sobel BE: External detection and visualization of myocardial ischemia with "'C-substrates in vitro and in vivo. Circ Res 39: 24, 1976 22. Evans JR: Importance of fatty acid in myocardial metabolism. Circ Res 15 (suppl II): 11-96, 1964 23. Scheuer J, Brachfeld N: Myocardial uptake and fractional distribution of palmitate-1-C" by the ischemic dog heart. Metabolism 15: 945, 1966 24. Scott JC, Finkelstein LJ, Spitzer JJ: Myocardial removal of free fatty acids under normal and pathological conditions. Am J Physiol 203: 482, 1962 25. Oram JF, Bennetch SL, Neely JR: Regulation of fatty acid utilization in isolated perfused rat hearts. J Biol Chem 248: 5299, 1973 26. Ter-Pogossian MM, Phelps ME, Hoffman EJ, Mullani NA: A positronemission transaxial tomograph for nuclear imaging (PETT). Radiology 114: 89, 1975 27. Phelps ME, Hoffman EJ, Mullani NA, Ter-Pogossian MM: Application of annihilation coincidence detection to transaxial reconstruction tomography. J Nucl Med 16: 210, 1975 28. Hurley PJ, Cooper M, Reba RC, Poggenburg KJ, Wagner HN Jr: 4"KCI: A new radiopharmaceutical for imaging the heart. J Nucl Med 12: 516, 1971 29. Yano Y, Van Dyke D, Budinger TF, Anger HO, Chu P: Myocardial uptake studies with "'Cs and the scintillation camera. J Nucl Med 11: 663,
1970 30. Zaret BL, Strauss HW, Martin ND, Wells HP Jr, Flamm MD Jr: Noninvasive regional myocardial perfusion with radioactive potassium. Study of patients at rest, with exercise and during angina pectoris. N Engl J Med 288: 809, 1973 31. Martin ND, Zaret BL, McGowan RL, Wells HP Jr, Flamm MD: Rubidium-81: A new myocardial scanning agent. Radiology 111: 651, 1974 32. Love WD, Burch GE: Estimation of the rates of uptake of Rb" by the heart, liver and skeletal muscle of man with and without cardiac disease. Int J Appl Radiat Isot 3: 207, 1958 33. Strauss HW, Harrison K, Langan JK, Lebowitz E, Pitt B: Thallium-201 for myocardial imaging. Relation of thallium-201 to regional myocardial perfusion. Circulation 51: 641, 1975 34. Love WD, Ishihara Y, Lyon LD, Smith RO: Differences in the relationships between coronary blood flow and myocardial clearance of isotopes of potassium, rubidium and cesium. Am Heart J 76: 353, 1968 35. Harper PV, Lathrop KA, Krizek H, Lembares N, Stark V, Hoffer PB: Clinical feasibility of myocardial imaging with "-NH,. J Nucl Med 13: 278, 1972 36. Kramer RJ, Goldstein RE, Hirshfeld JW Jr, Roberts WC, Johnston GS, Epstein SE: Accumulation of Gallium-67 in regions of acute myocardial infarction. Am J Cardiol 33: 861, 1974 37. Ross RS, Ueda K, Lichtlen PR, Rees JR: Measurement of myocardial blood flow in animals and man by selective injection of radioactive inert gas into the coronary arteries. Circ Res 15: 28, 1964 38. Pitt A, Friesinger GC, Ross RS: Measurement of blood flow in the right and left coronary artery beds in humans and dogs using the "'3xenon technique. Cardiovasc Res 3: 100, 1969 39. Sullivan JM, Taylor WJ, Elliott WC, Gorlin R: Regional myocardial blood flow. J Clin Invest 46: 1402, 1967 40. Bonte FJ, Graham KD, Moore JG, Parkey RW, Curry GC: Preparation of ""'Tc-oleic acid complex for myocardial imaging. (abstr) J Nucl Med 14: 381, 1973 41. Robinson GD Jr, Lee AW: Radioiodinated fatty acids for heart imaging: Iodine monochloride addition compared with iodide replacement labeling. J Nucl Med 16: 17, 1975 42. Poe ND, Robinson GD, MacDonald NS: Myocardial extraction of variously labeled fatty acids and carboxylates. (abstr) J Nucl Med 14: 440, 1973
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Quantification of infarction in cross sections of canine myocardium in vivo with positron emission transaxial tomography and 11C-palmitate. E S Weiss, S A Ahmed, M J Welch, J R Williamson, M M Ter-Pogossian and B E Sobel Circulation. 1977;55:66-73 doi: 10.1161/01.CIR.55.1.66 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1977 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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CIRCULATION
15. Ahmed M, Dubiel J, Verdon TA, Martin RH: Technetium-99m stannous pyrophosphate myocardial imaging in patients with left ventricular aneurysms. (abstr) Clin Res 123: 168A, 1975 16. O'Rourke R, Righetti A, Schelbert H, Daily P, Ashburn W, Ross J Jr: Usefulness of pre and postoperative Tc-99m-pyrophosphate scans in cardiac surgical patients. (abstr) Am J Cardiol 37: 161, 1976 17. Khentigan A, Gannett M, Lum D, Winchell HS: Effect of prior administration of Sn(II) complexes used in nuclear medicine on in vivo distribution of subsequently administered Tc-99m pertechnetate and Tc99m compounds. J Nucl Med 16: 541, 1975
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18. Klein MS, Coleman RE, Roberts R, Weiss AN: 99m Tc(Sn) pyrophosphate scintigrams in exercise induced angina and calcified valves. (abstr) Am J Cardiol 37: 149, 1976 19. Soin JS, Burdine JA, Beal W: Myocardial localization of 99m-Tcpyrophosphate without evidence of acute myocardial infarction. J Nucl Med 16: 944, 1975 20. Platt MR, Parkey RW, Willerson JT, Bonte FJ, Shapiro W, Sugg WL: Technetium stannous pyrophosphate myocardial scintigrams in the recognition of myocardial infarction in patients undergoing coronary artery revascularization. Circulation 51 & 52 (suppl II):II-141, 1975
Quantification of Infarction in Cross Sections of Canine Myocardium In Vivo with Positron Emission Transaxial Tomography and 11C-Palmitate EDWARD S. WEISS, M.D., SYED A. AHMED, M.D., MICHAEL J. WELCH, PH.D., JOSEPH R. WILLIAMSON, M.D., MICHEL M. TER-POGOSSIAN, PH.D., AND BURTON E. SOBEL, M.D. SUMMARY To assess myocardial infarction quantitatively in 15 mm thick transverse sections of the canine heart in vivo we utilized a new technique, positron emission transaxial tomography (PETT) and cyclotron-produced I'C-palmitate (1'C-P) injected intravenously. Results were compared to regional myocardial creatine phosphokinase (CPK) depletion, diminished "4C-palmitate accumulation in tissue extracts, and infarction estimated morphometrically 48 hours after coronary occlusion. CPK activity and "C-P content declined in parallel In transmural biopsies (N = 44) from normal and ischemic zones (r = .92) in six dogs; and infarct in 10 mm thick cross sections of the
entire left ventricle estimated morphometrically (N = 26) in six other animals correlated with CPK depletion in contiguous 2.5 mm thick slices (r = .92). When the percentage of infarction in 15 mm thick cross sections was assessed tomographically in six other dogs 48 hours after coronary occlusion with 11C-P injected intravenously, results correlated with infarction in corresponding cross sections from the same hearts estimated morphometrically (r = .97, N = 9) and by analysis of CPK depletion (r = .93, N = 9). ThIls, PETT permits estimation of infarction in cross sections of the left ventricle In vivo after intravenous injection of 11C-palmitate.
EVALUATION OF THE EXTENT OF MYOCARDIAL INFARCTION in vivo has become increasingly important because prognosis in patients appears to be influenced by infarct size'`4 and because the extent of irreversible ischemic injury may be amenable to favorable modification5' with interventions implemented during the early evolution of the insult. Although myocardial infarct scintigraphy7 is useful for detection and localization of infarction, quantification of ischemic injury with this technique may be limited because of the dependence of accumulation of tracer on the age of the infarct, and because of inherent limitations leading to disparities between the mass of injured tissue and its twodimensional display.8 9 Precordial ST-segment elevations may reflect serial changes in the electrophysiological response of the heart to ischemia10 '1 but they do not provide a direct reflection of the absolute magnitude of infarction13' 14 and may be influenced by factors not directly related to ischemia such as drug effects, pericarditis, or changes in the concentration of electrolytes in extracellular fluid. Prediction or estimation of infarct size on the basis of
time-activity curves of plasma enzymes such as creatine phosphokinase suffers from the prolonged interval required for acquisition of data prior to construction of projected portions of the curves.'5-'8 The present study was designed to develop and evaluate a method for quantitative estimation of the extent of myocardium undergoing infarction in vivo suitable for prompt, early, and sequential evaluation of the evolution of ischemic injury. Accordingly, we synthesized "IC-palmitate,'9 a shortlived, positron-emitting, cyclotron-produced tracer of the predominant physiological substrate of myocardium;20 injected the material intravenously in closed chest dogs with myocardial infarction; and determined its distribution in ischemic myocardium with computer reconstructed images obtained by positron emission transaxial tomography2' in order to detect, localize, and quantify infarction in vivo. Results were compared to independent estimates of the extent of infarction based on biochemical and morphological analyses of hearts from the same animals. This approach was predicated on several considerations. The positron-emitting tracer employed has a short half-life (20.4 min), permitting frequent sequential studies with a low total body radiation burden. Use of palmitate, a physiological substrate of the heart, permits interpretation of results in terms of the extensive information available characterizing myocardial intermediary metabolism and documenting diminished uptake and oxidation of free fatty
From the Washington University School of Medicine, St. Louis, Missouri. Supported in part by Washington University School of Medicine, USPHS SCOR in Ischemic Heart Disease 1 P17 HL 17646 and NIH Grant 5 P01 HL
13851-13. Address for reprints: Edward S. Weiss, M.D., Cardiovascular Division, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110. Received May 20, 1976; revision accepted July 22, 1976.
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acids in zones of ischemia and infarction.21 25 The distribution of positron-emitting isotopes in tissues can be quantified readily with coincidence counting, facilitating spatial resolution and delineation of the distribution of radioactivity in three-dimensional space.21 Furthermore, detection of radiation emitted from the heart is not distorted by the distance of the emitting source from each member of any pair of detectors. Quantification of infarction in a series of cross-sectional slices of myocardium by positron emission transaxial tomography should permit estimation of the entire mass of the infarct based on summation of the amount of infarct detected in each individual cross section. In a preliminary qualitative evaluation of this approach, we verified the externally detectable diminution of myocardial accumulation of "IC-palmitate associated with reduction of coronary flow in isolated perfused rabbit hearts and demonstrated the feasibility of obtaining positron emission transaxial tomographic images of the heart in intact dogs.2" The present study was undertaken to extend these observations to permit quantitative estimation of infarction in vivo and to define the quantitative relationships between computer reconstructed tomograms of infarcts and biochemical and morphological changes in the same tissue. Materials and Methods Animal Preparations
Myocardial infarction was produced in 22 dogs anesthetized with sodium pentobarbital, 30 mg/kg injected intravenously, by ligation of the left anterior descending coronary artery distal to the first septal branch. Immediately after ligation, the thoracotomy was closed. Eighteen animals survived this procedure and were included in the studies performed 48 hours after surgery. Six animals were used to clarify the relationship between myocardial CPK depletion in a cross section of the entire left ventricle and morphometric estimation (see below) of infarction in an adjacent 10 mm thick cross section in the experimental preparation used. Total myocardial CPK activity was assayed in extracts of 2.5 mm thick cross sections. Results were compared to expected activity in the cross sections, calculated as the product of weight and CPK/g of histologically normal, nonischemic myocardium (averaged from four 0.5 g transmural biopsies in each heart). In each dog from this group, three or four pairs of cross sections were analyzed. Six different animals were used to determine the relationship between myocardial CPK depletion 48 hours after coronary occlusion and diminution of regional "4C-palmitate accumulation in myocardium after intravenous injection of 14C-palmitate, five minutes before the animal was killed. Among the six animals studied with 14C-palmitate, myocardial CPK activity was assayed in five to eight 0.5 g transmural myocardial samples obtained with a precooled modified dental drill from ischemic and nonischemic regions of each heart. Results were compared to 14C-palmitate radioactivity in extracts of the same tissue used in a Packard liquid scintillation counter. This isotope was studied to facilitate interpretation of results of subsequent studies in other dogs in which tomography was performed after intravenous injection of "IC-palmitate 48 hours after coronary occlusion.
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After relationships between 14C-palmitate, morphology, and CPK depletion had been examined, six additional animals were studied by positron emission tomography. In each of these, after tomography was completed, the animal was killed and infarction was verified and quantified by two procedures: myocardial CPK analysis and morphometric analysis. CPK analysis was performed in extracts of each of two 2.5 mm thick transverse sections of the entire left ventricle at the level corresponding to the tomographic image and adjacent to a central contiguous 10 mm thick cross section. Morphometric analysis was performed as described below on each central 10 mm thick cross section of the entire ventricle corresponding to the tomographic plane. In several animals more than one tomographic plane was studied with corresponding CPK and morphometric comparisons. For CPK and morphometric analyses, slices of the entire left ventricle were obtained from the rapidly frozen hearts of other animals. The transverse sections were obtained to correspond to tissue planes reflecting the field of view encompassed in each tomographic image based on the known 15 mm thickness of each tomographic section and the distance from the apex of the heart corresponding to each. The distributions of '4C-palmitate and myocardial CPK activity were determined by analysis of both in transmural punch biopsies of normal and ischemic tissue and transverse sections of the entire left ventricle. Preparation of Left Ventricular Biopsies for Analysis of '4C-Palmitate and Myocardial CPK Content
All procedures were performed at 0 to 4° C. Myocardium was minced with scissors, suspended in 0.05 M HepesNaOH buffer, pH 7.4, 10 ml/g, and homogenized in a Virtis homogenizer for two 15 second bursts at half maximum speed. Aliquots of 0.4 ml of homogenate were removed for determination of '4C-palmitate radioactivity in a toluene based fluor after solubilization of protein with Protosol. Myocardial CPK activity was assessed in extracts of whole homogenate of myocardium initially centrifuged for 20 minutes at 30,000 g at O° C. The supernatant fraction was decanted, diluted with Hepes-NaOH buffer, pH 7.4, .05 M such that CPK activity was less than .2 IU/ml. CPK activity was assayed spectrophotometrically as previously described and protein was determined by the Biuret method.'5 CPK depletion per gram of wet weight was determined as follows: CPK activity was measured in four histologically normal regions from each heart, averaged, and designated as CPKN, expressed as IU/g. Total CPK activity in each 2.5 mm transverse section was measured directly and designated as CPKT, also expressed as IU/g. The percent CPK depletion was calculated from [(CPKN- CPKT)/CPKN] X 100. Morphometric Analysis of Infarction
Frozen hearts were sliced into 10 mm thick sections for gross and histological examination. Alternating 2.5 mm thick sections were used for analysis of '4C-palmitate and CPK activity (fig. 1). Each 10 mm section was immersed rapidly in normal saline and photographed with a Graflex 4 X 5" photocopy camera. Subsequently, the slice was fixed with 1.25% glutaraldehyde-l% formaldehyde buffered to pH 7.4 with Ringer's lactate solution and cut into blocks for
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myocardial biopsies since the short half-life of "IC-palmitate (20.4 min) made destructive analysis of multiple samples for radioactivity impractical. Positron Emission Transaxial Tomography
FIGURE 1. A diagrammatic representation of the sampling technique used to obtain transverse sections of the entire left ventricle for histological analysis in a 10 mm slice, and assessment of myocardial CPK activity and "4C-palmitate radioactivity in adjacent 2.5
slices. The shaded area thetical zone of infarction.
mm
on
the diagram represents
a
hypo-
histology. Sections adjacent to regions analyzed biochemically were stained with hematoxylin and eosin. Each histological section was analyzed to detect infarction, projected, and combined to reconstruct an entire cross section of the heart with the area of necrotic tissue outlined by planimetry. The total area of each 10 mm thick transverse section and the area representing infarction were calculated with a Hewlett-Packard 9100 B calculator coupled to 9107 A digitizer. Calculations of the percentage of infarction in each region were made on the basis of the ratio of outlined infarct to normal myocardium in the entire cross section. Preparation of '"C-Palmitate
"C-palmitate was produced by the reaction of pentadecyl magnesium bromide in diethyl-ether with "CO, produced in the Washington University Medical School cyclotron by the "OB(d,n) 'IC nuclear reaction using boric oxide as the target material. After acidification with 3 ml of N hydrochloric acid, the ether layer (4 ml) was separated and 2 ml of ethanol added. The ether/ethanol solution was boiled to remove the ether. At this stage of the preparation the ethanol contains both the labeled palmitate and pentadecane formed by the hydrolysis of the unreacted Grignard reagent. Eight ml of 4% albumin solution is then added and the solution maintained at 42° C for 5 minutes during which time the pentadecane precipitates. The precipitate is removed from the solution by millipore filtration prior to addition of the remaining "IC-palmitate. Administration of "4C-Palmitate
For studies of the distribution of '4C-palmitate in myocardium after intravenous injection of the tracer, results were obtained by liquid scintillation counting of extracts prepared from biopsies. The tracer was prepared as follows: 250 ,uCi of palmitic-l-14C acid (labeled analogously to the "C-palmitate preparation) in hexane was immersed in a water bath at 900 C until the hexane had been entirely evaporated. Subsequently, ml of absolute ethanol was added to the radioactively labeled material, followed by 4% bovine serum albumin dissolved in 10 ml of 0.15 M NaCl warmed to 52° C. The solubilized '4C-palmitate was used for intravenous injection in the canine preparations. The use of "4C-palmitate permitted analysis of palmitate distribution in
Five to ten millicuries of "C-palmitate in a solution of 4% albumin was injected intravenously in an 8 ml bolus at a rate of 1-2 ml/sec 48 hours after coronary occlusion in lightly anesthesized dogs. Three minutes later, an interval selected to permit clearance of the tracer from the blood pool, the distribution of 1"C-palmitate in myocardium was determined by detection of positron emission during an 8 to 16 minute interval providing detection of 0.5 to 1.0 X 106 counts. At least one and as many as seven tomograms were obtained from each animal corresponding to one or more 15 mm thick cross sections of myocardium. Immediately after completion of the tomograms, 250 ,Ci of '4C-palmitate in 4% albumin were injected intravenously. The animals were killed five minutes later with an overdose of anesthesia. "Cpalmitate was administered in order to permit subsequent analysis of the distribution of this -tracer based on liquid scintillation counting of extracts obtained from transmural punch biopsies and transverse sections of the excised heart. Quantitative assessment of the organ distribution of 11Cpalmitate in myocardium in the studies in vivo was based on a computer reconstructed image of positron emission (511 keV gamma rays) from 15 mm thick cross sections of the heart. Correction factors for attenuation were first obtained with the use of a ring containing 64Cu in a solution of concentrated nitric acid placed within the empty tomograph and then within the same field of view but around the experimental canine preparation. Positron emission from "Cpalmitate accumulated in myocardium after intravenous injection was detected and quantified with a positron emission transaxial tomograph designed at Washington University. This instrumentation employs six banks of 8 Nal crystals to detect positron emission. The detectors are rotated with a programmed pattern of motion around the experimental animal at the level of the heart. Data are analyzed by computer to produce reconstructed cross-sectional images from data profiles from each pair of detectors.26 27 The percentage of infarct in each 15 mm thick cross section of the left ventricle in vivo was estimated by analysis of the digital printout of the 2,500 data points utilized for computer reconstruction of the entire left ventricular cross-sectional tomographic image. In order to define the epicardial and endocardial margins of the ventricular image on the digital printout, a horizontal profile of the image gray scale was obtained on a Tektronix Type 524 wave form monitor. In this profile the region of the ventricular myocardium was defined by inclusion of all data points with values exceeding 50% of the maximum value detected. A Versatec plotter was used to obtain digital printouts of the distribution of 11Cpalmitate radioactivity in each image of normal canine ventricles at the midventricular and A-V valvular levels. The region corresponding to myocardium was delineated with a hand drawn line surrounding those digital values conforming to the count rate criterion. This area correlated well with the oscilloscopic display of the image of myocardium, typically approximately circular, obtained at the midventricular level and with the typically horseshoe-shaped image, obtained at the level of the A-V valves (fig. 2).
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Arch of oorta
out coronary occlusion (fig. 3). The determination of percentage of infarction represented by these defects was calculated by planimetric measurement of the image area defined in the digital printout. This area was then quantitatively compared to the calculated area between the two circles derived from the minimal diameters across the inner and outer perimeters of the tomogram (fig. 4). A proportionality constant was used for calculation of percentage of infarction at the level of the A-V valves since these tomograms are horseshoe-shaped because of the nonvisualized posterior left atrial border. The area of ventricular myocardium in this region was estimated in ten images from normal dogs and found to average 60% (range = 54 to 67%) of the total area between the two concentric circles at midventricular level. Thus, at this level, the percentage of infarction was estimated by subtracting the planimetered area of the infarct from 0.6 times the area between these concentric circles. Results The Relation between Myocardial CPK Depletion, Diminution of '4C-Palmitate Accumulation, and Morphologically Evident
FIGURE 2.
Representative images obtained by positron emission transaxial tomography after intravenous injection of "C-palmitate in a lightly anesthetized dog. As can be seen, the characteristic distribution of "C-palmitate is circular at the midventricular level, but conforms to a horseshoe-shaped pattern at the level of the A-V valves because the posterior portion of the heart in this region comprises left atrial rather than ventricular myocardium. A, P, R, L refer to anterior, posterior, right, and left, respectively. The central areas within the circle and horseshoe patterns correspond to the left ventricular cavity. The endocardial margin is somewhat blurred because these images were obtained without electrocardiographic gating.
Infarction Results of analyses of CPK activity and "4C-palmitate radioactivity in 44 transmural samples of myocardium from normal and ischemic zones from six dogs are shown in figure 5. As can be seen, regions with low '4C-palmitate accumulation were characterized by marked depletion of CPK activity. The correlation between the percentage of CPK depletion and the percentage of diminution of "C-palmitate accumulation was .92. As can be seen in table 1, depletion of CPK in transverse sections of the entire left ventricle correlated closely with necrosis estimated morphologically in adjacent sections. Since 14C-palmitate accumulation was diminished in proportion to myocardial CPK depletion
Tomographic images obtained 48 hours after coronary occlusion revealed appearance of a large anterior defect, which interrupted the circular image noted in animals with-
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Fro;URE 3. Positron emission transaxial tomograms (top panels) obtained at the midventricular level in a normal dog (left) and a dog with an anterior myocardial infarction induced by coronary occlusion 48 hours earlier (right). Printouts of the digital data with lines encompassing normal myocardium drawn as indicated in the text are shown in the bottom two panels. The midventricular imagefrom the dog with the myocardial infarct shows diminution of "Cpalmitate uptake with a defect in the anterior portion of the cross-sectional image (top) and conforming to results apparent on the printout of the digital data shown below.
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AREA OF DEFECT
(measured by subtraction)
FIGURE 4. The digital printout obtainedfrom a midventricular tomogram in a normal dog is displayed in the left hand panel (A) with a line drawn by hand to include allpoints with values greater than 50% of maximum to define the borders of the myocardial image. Two concentric circles are drawn, based on the minimal endo- to endocardial and epi- to epicardial diameters, to obtain a hypothetical area for the myocardial image. In the right hand panel (B) the method of calculation ofpercentage infarction is indicated. The calculated area, based upon the minimal diameters of the measured image is smaller than the measured area for a noninterrupted myocardial image. The appearance of a defect seen in animals subjected to coronary occlusion is indicated here with crossed-hatched markings. The percentage of infarction in such a tomogram is calculated by subtracting the measured area of the image from the area of the hypothetical image and dividing this difference by the hypothetical area.
throughout ischemic zones (fig. 5), these results suggested that necrosis could be estimated by analysis of myocardial accumulation of palmitate estimated externally. Furthermore, since palmitate-l-'IC and palmitate-I-'4C are likely to
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be equivalent metabolically, it appeared that necrosis could be estimated by external assessment of '1C-palmitate uptake. In six dogs subjected to coronary occlusion but not studied tomographically, myocardial CPK depletion in 2.5 mm thick transverse sections of the left ventricle at multiple levels from apex to base correlated closely with morphometric estimates of infarction in the adjacent 10 mm thick cross section (fig. 6) (r = .92, N = 26 pairs of sections). Thus, the depletion of myocardial CPK activity and 14Cpalmitate accumulation correlated closely with morphometric estimates of infarction in the adjacent transverse section.
0.92/
C-I
Estimation of Infarction in Cross Sections of the Left Ventricle In Vivo Based on Analysis of Positron Emission Transaxial Tomograms Areas of myocardial infarction were estimated in 15 mm
"
'40
serial sections of myocardium represented by tomograms obtained sequentially from apex to base in closed chest dogs.
C3
, 20 Q
~
~
TABLE 1. Analyses in Transverse Sedions of the Entire Left Ventricle Obtained from Three Dogs
x 0
0
20 40 60 80 % MYOCARDIAL CPK DEPLETION (WIUg) FIGURE 5. 14C-palmitate accumulation and myocardial CPK activity in 44 transmural samples of myocardium from normal and ischemic zones in six dogs. Results are expressed as percentage decreases compared to results in transmural samples from normal myocardium in the same animal. As can be seen, there was a close correlation between CPK diepletion and diminished 14C-palmitate accumulation 48 hours after coronary occlusion. Each symbol refers to data from the same dog. Dashed lines indicate confidence limits drawn as two standard errors of the estimate. The equation for the best-fit regression line (least squares method) is y = .95X 3.43. -
Dog number
A CPK activity % decrease from normal sample
B 14C-palmitate uptake % decrease from normal sample
C Morphological determination of infarct size (%)
1 1 3 2 3 1 2 2 3
0.0 2.5 9.1 10.9 19.0 23.3 26.0 29.6 31.5
5.9 8.8 12.2 14.4 12.3 26.1 20.0 14.8 32.7
0.0 2.0 9.2 16.8 14.3 29.0 18.7 15.5 31.6
The correlation coefficient between results in columns A and C (linear regression least squares method) was .95, and that between results in columns B and C was .94.
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TOMOGRAPHY OF THE HEART/Weiss et al.
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30 40 50 60 20 INFARCT SIZE FROM CPK DEPLETION (%) FIGURE 6. The relationship between infarct size estimated morphologically and infarct size estimated from myocardial CPK depletion in an adjacent transverse section of the entire left ventricle is indicated here. Results obtained 48 hours after coronary occlusion with these two methods correlated closely.
w
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INFARCT SIZE
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MORPHOLOGICAL INFARCT SIZE
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The typical tomograms obtained at the midventricular (circular) and A-V valvular level (horseshoe-shaped pattern) are shown in figure 2. At least four images were obtained from each dog, but many did not encompass regions of infarction. As can be seen in figure 3, the presence of infarction 48 hours after left anterior descending coronary occlusion in a tomogram corresponding to a 15 mm thick section of the left ventricle at the midventricular level is readily apparent in the image and the digital printout of the data. The anterior defect contrasts with an image from the same level obtained from a normal dog. The percentage of infarction in cross sections from each of six dogs studied tomographically estimated from the area in the tomogram correlated closely with the extent of infarction in the corresponding slice analyzed morphometrically (fig. 7) by an observer unaware of the results obtained tomographically (r = .97, N = 9 pairs of slices) and with infarction estimated from CPK depletion in the adjacent 2.5 mm thick slice analyzed by another observer, also unaware of the tomographic results (r = .93, N = 9 pairs of slices from six dogs). As can be seen, CPK depletion and morphological estimates of infarction correlated closely in sections corresponding to those imaged (r = .92, N = 9 pairs of slices). These results indicate that estimates of infarction in cross sections of the left ventricle obtained by analysis of positron emission transaxial tomograms in vivo correspond closely to estimates of infarction based on analysis of myocardial CPK depletion and to morphometric estimates of infarct size in the corresponding transverse section. Application of the tomographic approach to estimating the entire mass of an infarct is illustrated in figure 8. As can be seen, sequential tomograms corresponding to 15 mm thick cross sections of the heart delineate a volume of infarction that can be calculated from the cross-sectional area representing diminished '1C-palmitate uptake in each cross section multiplied by the thickness of each. Improved results should be available with instrumentation now being developed that will permit 1) simultaneous accumulation and differentiation of data from four sections from each heart, and 2) electrocardiographic gating with reduction of impairment of resolution due to movement of the heart throughout the cardiac cycle.
r s 0.97
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71
20
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40
60
MORPHOLOGICAL INFARCT SIZE (%) FIGURE 7. Myocardial CPK depletion and morphological estimates of infarction in a cross section through the entire left ventricle, compared with the percentage of infarction in a corresponding section delineated by positron emission transaxial tomography in vivo. Results from nine pairs of sections from six different dogs are shown. The correlation coefficients (least squares linear regression) indicate that estimates of infarction based on CPK depletion (N = 9) and morphology correlated closely with estimates obtained in vivo by positron emission transaxial tomography of the corresponding cross section (N = 9) of the left ventricle. The similarity ofslopes in (B) and (C) is reflected by the slope approaching one in (A). Since 100% CPK depletion does not occur within 48 hours (with approximately 10% residual activity, unpublished observations), the slope in (B) would be anticipated to be less than one. The slope in (C) is less than unity probably because the true image of the normal ventricle is not congruent with the concentric circle model utilized for calculations (see fig. 4).
Discussion The use of radioisotopes to detect myocardial injury or ischemia externally has employed intravenous administration of inorganic ions such as 43K, '29Cs, B'Rb, 8tRb, 201TI, 67Ga; 13N labeled NH3;28-" and intracoronary injection of radioisotopes of inert gases such as "Kr and 133Xe.37-" These moieties were selected because of advantages in physical or biological properties but none is a physiological substrate of myocardium. Efforts to utilize organic substrates of the heart as agents for imaging myocardium have involved fatty acid analogs labeled with 131I and *mTc.4o 41 Unfortunately, these derivatives are metabolically altered substrates exhibiting less uptake than the corresponding 'IC-labeled congener.42 Furthermore, quantification of their distribution has been difficult. In the present study we utilized a physiological substrate of the heart by synthesizing radionuclides incorporating positron-emitting 1"C. Previously, we demonstrated that
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CIRCULATION
72
inforct . lW.tI
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,
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Il
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FIGURE
8.
Positron
of 15
series
emnission
thick sections
mm
injection of "C-palmitate
intravenous
farction produced by
in
a
the
the conclusion
at
of the study
are
diagrams comprising the top panel.
palmitate
dog with myocardial
as
be
can
prior
to
in-
study.
examined morp ho-
was
shown
As
a
(left) after
shaded
seen,
zones
in
diminished
corresponding to these shaded areas is evicomprising the bottom panel. The right ven-
diagrammatically,
tricular wall, shown
The transmission images
positron source) c en ter
apex
accumulation
dent in the tomograms
grams.
to
coronary occlusion 48 hours
Regions of infarction evident when the heart logically
depicting
transaxial tomograms
from base (right)
panel
and
th e thorax and
each level
at are
to
used
obtlain
to
is not visualized in the tomo-
(obtained with the
from base
indicatle
atten ua tio n
to
apex
th e loca tio n
fac to rs
use
of
"Cu
as a
shown in the
are
of th e
h eart with in
p rio r to in tra veno us in
jection of "C-palmitate and acquisition of data for
emission tomog-
raphy.
myocardial ischemia under
could be detected
11C-palmitate
in
to
the
physiological
albumin in these studies
physiological
myocardium raphy. vivo
qualitatively
perfusate of isolated conditions of controlled flow."1 The high
11C-palmitate added
substrate in viva
suggested would
by positron
The distribution of
was
demonstrated in
rectilinear
scans
present study
we
solutions
with
rabbit hearts
extraction of
with
equimolar
that this radionuclide of
permit imaging of
a
normal
emission transaxial tomog-
"IC-palmitate pilot
studies
in myocardium in (data not shown) by
with conventional instrumentation. In the demonstrated that the distribution of this
analyzed quantitatively by positron emission transaxial tomography. The diminished uptake of "C-palmitate in tomographic images was found to correspond to decreased "IC-palmitate uptake and to independent indices of ischemic injury in analogous experimental preparations. In our previous study with isolated perfused hearts we tracer
could be
VOL 55, No
1, JANUARY 1977
showed that the diminished extraction of "IC-palmitate was a function of the duration of low flow and an index of metabolic manifestation of ischemia rather than a reflection simply of decreased delivery of the tracer to myocardium.Y The present results indicate a similar relationship between decreased palmitate accumulation demonstrated by PETT in areas exhibiting diminished CPK content in myocardium. The two correlations 1) between CPK depletion and histologic necrosis, and 2) between decreased accumulation of both "1C- and '1C-palmitate and CPK depletion, support the interpretation that under the conditions selected (48 hours after coronary occlusion), diminished 'IC-palmitate accumulation detectable tomographically reflects infarction. Thus, although CPK content and '1C-palmitate accumulation are independently regulated, both are affected under these particular conditions as a result of infarction. Reconstructive tomography offers several advantages in spatial resolution. For example, the technique delineates and separates the display of anterior and posterior regions and provides a cross-sectional representation avoiding the superimposition encountered with conventional imaging techniques. This facilitates quantification of the distribution of tracer and hence assessment of metabolism in a cylindrical volume of tissue. It also permits quantitative assessment of reduced accumulation of tracer and representation of a cold zone in three-dimensional space. In the present study quantification of the percentage of myocardial infarction in 15 mm thick cross sections of the heart was determined by analysis of the digital printout of the data points obtained by computer reconstruction. A close correlation was obtained between the percentage of infarction estimated by analysis of each tomogram and histological estimates of infarction in the corresponding transverse section of the left ventricle. Determination of the total mass of ischemic ventricular myocardium by positron emission transaxial tomography with '1C-palmitate depends on imaging geometrically contiguous regions of the heart without overlap. This presently requires movement of the experimental animal in 15 mm increments in a cephalad direction to obtain sequential images of the entire left ventricle. The rapid decay of activity encountered with the short-lived '1C-nuclide requires progressively longer sampling intervals to obtain sufficient counts for computer reconstruction with sequential images. Accordingly, in this study, nongated images were used to permit the acquisition of the maximal amount of counts after a single injection of 11C-palmitate even though the use of nongated images resulted in diminished resolution. The myocardial infarctions studied in this investigation resulted from coronary occlusion of 48 hours' duration. This interval was chosen because histological indices of infarction and CPK depletion are well established within 48 hours after occlusion.5 Well circumscribed areas of ischemia can be detected as soon as 20 minutes after coronary occlusion by positron emission transaxial tomography with "IC-palmitate2' but unlike the case in which the occlusion is maintained for longer intervals, diminished uptake of "IC-palmitate is completely reversible with reperfusion. Thus, the capability for early recognition of ischemia after experimental coronary occlusion may presage significant potential for early clinical detection of jeopardized myocardium. In addition, the short half-life of '1C (20.4 min) permits se-
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TOMOGRAPHY OF THE HEART/Weiss et al. quential studies in an individual organism facilitating repeated examinations during the early evolution of acute myocardial ischemic injury. The differentiation of ischemia from infarction should be possible by analysis of results of sequential studies in the same experimental animal or patient, once the duration of ischemia sufficient to abolish "IC-palmitate accumulation and required to produce infarction has been defined. Results in this study suggest that positron emission transaxial tomography utilizing cyclotronproduced, short-lived, positron-emitting radionuclides of physiological substrates of myocardium will facilitate objective evaluation of the efficacy of potentially therapeutic interventions on the extent of infarction ultimately sustained after an ischemic insult. Acknowledgment We appreciate the assistance of Mrs. Carol Higgins, Mr. Nizar Mullani, Ms. Alice Robison, and Ms. Maria Straatmann in biochemical, radiochemical, and tomographic procedures and Ms. Carolyn Lohman in preparing this
manuscript.
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Quantification of infarction in cross sections of canine myocardium in vivo with positron emission transaxial tomography and 11C-palmitate. E S Weiss, S A Ahmed, M J Welch, J R Williamson, M M Ter-Pogossian and B E Sobel Circulation. 1977;55:66-73 doi: 10.1161/01.CIR.55.1.66 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1977 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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