proton nmr spectroscopy in canine myocardial infarction

MAGNETIC RESONANCE IN MEDICINE 4, 555-566 (1987)

Proton NMR Spectroscopy in Canine Myocardial Infarction*

TODD RICHARDS,? DIMITER TSCHOLAKOFF, AND CHARLES B. HIGGINS

Department of Radiology, University of California, San Francisco Medical Center, San Francisco, California 94143

Received August 15, 1986; revised November 25, 1986

Proton NMR spectroscopy was used to study the relationship between proton relaxation times and other resonances in the proton spectra, such as lipids, creatine, and choline/ carnitine in subacute (8-day-old) myocardial infarctions. Eight mongrel dogs received operative ligation of the left anterior descending coronary artery (four were permanently occluded, four were occluded for 1 h and reperfused) and were sacrificed 8 days later so that tissue samples could be prepared for NMR spectroscopy. The results of this study indicate that (for the core of infarcted tissue) the lipids do not contribute directly to the increased bulk relaxation times associated with myocardial infarction and that the lipid peaks (2.3, 1.2, 0.8 ppm) and creatine peak (3.0 ppm) are more specific to the kind of infarct than to the relaxation times. Therefore, analysis of the proton spectrum of myocardial tissue may Serve as a method for tissue characterization. o 1987 Academic press, Inc.

INTRODUCTION

Myocardial infarction is associated with prolongation of proton NMR relaxation times (I). The initial assumption that an increase in total tissue water content is responsible for the changes in the relaxation times in acute myocardial infarctions may be too simplistic. A dissociation between TI and water changes in 4-h-old myocardial infarcts was observed by Canby et al. (2). In order to better define the mechanisms involved with changes in proton relaxation times, Evanochko et al. (3) studied triglyceride levels in 24-h infarcts with proton NMR spectroscopy. They observed an increase in triglyceride levels in moderately ischemic myocardium but not in severely ischemic myocardium. Other investigators have found lipid accumulation only in myocardium adjacent to infarcted tissue (4 ) .

The purposes of the current study are (1) to determine the contribution of mobile lipid levels to increased relaxation times in subacute (8day-old) myocardial infarctions; (2) to compare the lipid levels in occlusive and reperfused myocardial infarctions; and (3) to determine if there are differences in the proton NMR spectral resonances among normal, occlusive infarct, and reperfused infarct.

METHODS

Animal Preparation Eight mongrel dogs had operative ligation of the left anterior descending coronary

artery. In four dogs the coronary artery remained permanently occluded. The myo-

* Supported by NIH Grant lROlHL32283. t Present address: Department of Radiology, University of Washington, Seattle, WA 98195.

555 0740-3194J87 $3.00 Copyiigbt 0 1987 by Academic Prss, Inc. All rights of reproduction in any form reservd

556 RICHARDS, TSCHOLAKOF’F, AND HIGGINS

cardial infarction created in this way is referred as occlusive infarct. In the other four dogs the arterial ligation was removed slowly after a period of 1 h of coronary artery occlusion. This type of myocardial infarction is referred to as reperfused infarct. To reduce cardiac arrhythmias, procainamide (0.2 g) was injected im and lidocaine was given as a continuous intravenous infusion (2 mg/min) during the occlusion and reperfusion procedure. Eight days later the animals were sacrificed and their hearts were excised. At autopsy myocardial infarctions were grossly visible and were located in the anterior wall of the left ventricle. At this point in time the myocardial infarcts were easily differentiated from normal myocardium. Therefore, microscopic tissue evaluation was not done. Three out of four dogs with reperfused myocardial infarcts had multiple areas of hemorrhage located within the pale, infarcted myocardial tissue. Four-millimeter-thick tissue samples were harvested from the core of the myocardial infarcts and from normal myocardium (posterior left ventricular wall) for NMR measurements. All pericardial fat was removed. Myocardial tissue samples were placed in 5-mm NMR tubes with deuterium oxide (to aid in shimming) covering the entire sample.

NMR Measurements

NMR measurements were performed (1-2 h after autopsy) on a 5.6-T spectrometer (proton frequency 240 Mhz) interfaced to a Nicolet 1 180 computer. All experiments were performed with the sample at room temperature. TI relaxation time was measured using the inversion-recovery pulse sequence with six to seven different delays ranging from 0.1 to 4 s. The intensities or areas were fit to a three-parameter equation described by Levy and Peat (5 ) to calculate T I . T2 relaxation time was measured using the Hahn spin-echo pulse sequence with six different delay times ranging from 10 to 150 ms. Because the T2 relaxation curve was not a single exponential, the fast T2 component was calculated by fitting the first three points separately to a single exponential equation. Bulk TI and T2 were calculated from the total integrated area of the free induction decay (FID). Water TI and T2 values were calculated from the intensity of the water peak. Lipid TI and T2 values were calculated from the integrated area under the lipid methylene peak at 1.2 ppm (water referenced at 4.7 ppm).

The lipid/water ratios were measured with a one-pulse sequence (no water suppression) with a 5-s delay and 20 to 40 averages. The ratios were then calculated by integrating the areas under the lipid peak at 1.2 ppm and the water peak at 4.7 ppm. The water-suppressed proton spectrum was measured using the pulse sequence devised by Plateau and Gueron (6) (sometimes referred to as “jump and return”) with a 400 ps delay between the two 90” pulses. Peak identification was made by assigning water to 4.7 ppm and using parts-per-million assignments based on previous reports (3, 7, 8,20). The areas under the peaks were evaluated using curve-fitting s o h a r e developed at our laboratory. Groups of overlapping peaks were extracted and baseline corrected by allowing the user to define the edges of the group. The software then draws a straight line between the two endpoints and uses the line as the baseline reference. For example, the methylene and terminal methyl peaks were baseline corrected as a group by defining one edge at the 1.5 ppm valley and the other edge at about 0.5 ppm. This assumes that the baseline is straight between the two endpoints and also that the peaks do not

PROTON SPECTROSCOPY OF MYOCARDIAL INFARCTION 557

extend beyond these points. This type of baseline correction could possibly give an underestimation of the peak areas. The extracted part of the spectrum is then passed on to the curve-fitting subroutine which fits the peaks to a superposition of one or more Lorentzian-Gaussian curves by minimizing the x2 difference. The fitted parameters were peak area, linewidth, and frequency for each resonance. Peak area ratios were calculated relative to the methylene peak ( 1.2 ppm) and the choline/carnitine peak (3.2 ppm). Spin-echo proton spectra were also measured in order to acquire a spectrum without the broad proton lines. Lactate appears as an inverted peak when a 90-180” delay of 68 ms was used.

Water TI and T2 relaxation time measurements were made on normal myocardium with and without deuterium oxide in the NMR tube in order to measure the effect of deuterium oxide on relaxation times. Experiments were done at 200 MHz on a Bruker system with four separate samples of fresh cow heart. Data were acquired with a block size of 2K, a sweep width of 5000 Hz, and an intercycle delay of 5 s, in the high- resolution 5-mm proton probe. The measurements were first made on the tissue without deuterium and then deuterium was added to the sample with the use of a long-needle syringe. The relaxation time measurements were then repeated.

All of the data were analyzed to test for significant differences between the means of control and infarcted samples by performing the paired t test. The paired test was used because tissue samples were taken from the infarcted area and the normal area of the same heart. This test could only statistically evaluate the difference between infarcted and control values. Therefore, analysis of variance (ANOVA) and Tukey’s HSD test were used to make multiple comparisons (9) between controls, occlusive infarct, and reperfused infarct data. Tukey’s test was used only if the ANOVA test was significant at the P < 0.05 level.

RESULTS

TI and Tr Relaxation

The six different delay intensity values fit very well on the monoexponential curve for all measurements (control and infarcted). Using Tukey’s HSD test, a significant difference (P < 0.05) was found for the following comparisons: (i) the water T1’s of reperfused infarcts were greater than those of controls, (ii) the bulk Tl’s of occlusive infarcts were greater than those of controls, and (iii) the water T2 values of the occlusive infarcts were greater than control values (Table 1). None of the relaxation time comparisons showed a significant difference between the two types of infarcts.

Using the paired t test, a significant difference was found for the following comparisons: (1) water and bulk TI values of occlusive infarcts were greater than normal myocardium values, (2) water and bulk T2 values of occlusive infarcts were greater than those of normal, and (3) lipid T2 values of reperfused infarcts were less than those for normal (Table 2).

Lipid/Water Ratio

The water peak areas in the one-pulse sequence spectra were at least 200 times larger than the lipid peak areas. The reperfused infarct lipidlwater ratio was significantly

558 RICHARDS, TSCHOLAKOFF, AND HIGGINS

TABLE 1

Multicomparison Test with ANOVA and Tukey’s HSD

Relaxation time data

n Control n Reper. inf. n Occ. inf.

7 1.85 k 0.19 4 2.74 k 0.79** 4 8 I .76 k 0.28 4 2.18k0.35 3 6 0.77 k 0.42 3 0.66 k 0.27 3 7 5 6 f 9 3 113k76 4 6 5 6 f 10 3 122 f 78 2 5 41 k 11 3 2 5 t 5 3

n = 8 n = 4 (38.2 f 9)/10000 (28.8 f 11)/10000

Peak area ratios relative to methylene peak area

Control Reper. inf. PPm n = 8 n = 4

2.47 f 0.22 2.37 t 0.28 * 0.51 k0.15 139 f 42** 105 + 8 2 7 k 2 1

n = 4 (26 k 16)/1oooO

Occ. inf n = 4

3.2 0.19 f 0.07 0.22 f 0.04 0.17 f 0.10 3.0 0.29 f 0.15 0.31 t0.13 0.078 k 0.036 * 2.3 0. I 1 f 0.07 0.06 k 0.03 0.15 k 0.02 2.0 0.35 k 0.21 0.30 f 0.15 0.39 f 0.20 0.8 0.11tO.04 0.16 f 0.06 0.29 f 0.1 1 ****t

Peak area ratios relative to choline peak area

Control Reper. inf. PPm n = 8 n = 4

Occ. inf. n = 4

3.0 1.5 f 0.42 1.4 f 0.44 0.50 f 0.14***stt 2.3 0.58 f 0.20 0.26 f 0.09 1.06 + 0.40***ttt 2.0 2.0 t 1.0 1.4 k 0.66 2.45 k 0.37 1.2 6.2 f 2.7 4.7 k 1 .o 7.45 f 3.2 0.8 0.67 f 0.36 0.74 f 0.20 2.33 f 1.7**

Note. T, values are in seconds, T2 values are in milliseconds, W = water, B = bulk, L = lipid peak at 1.2 ppm, lw = lipidwater ratio, reper. inf. = reperfused infarct, and occ. inf. = occlusive infarct. “n” is the number of measurements.

* = P < 0.05 Tukey’s test with respect to controls. ** = P < 0.025.

*** = P < 0.005.

= P < 0.025. ttt = P < 0.005.

t = P < 0.05 Tukey’s test with respect to reper. inf.

less (P < 0.02) than that of the normal myocardium (Table 2). The lipid/water ratio average for the occlusive infarcts was also less than that for normal myocardium but the difference did not reach statistical significance.


TABLE 2

Paired Statistical Test

Occlusive infarct average Reperfused infarct average difference difference

Relaxation time differences (infarct minus controls)

T2W ms T2B ms T2L ms TiW s TIB s TIL s Lipid/water ratio

3.2 ppm 3.0 ppm 2.2 ppm 2.0 ppm 0.8 ppm

87.5 f 36 ( P i 0.02) 51.5 f 75 55.3 f 80

-20.3 k 7.6 (P < 0.05) -0.13 f 1.2

57.0 k 1.4 (P < 0.02) -7.0 f 14 0.75 kO.14 (P<0.005) 0.68 * 0.22 (P < 0.02) 0.27 k 0.38

0.043 f 0.15

(P < 0.02)

-0.41 f 0.59 (-7.Of 13)/10000 (-14.6 f 5.3)/10000

Peak area ratio differences relative to 1.2 ppm (methylene)

-0.032 f 0.16 -0.25 f 0.02

0.0026 f 0.10 0.0086 f 0.30 0.164 f 0.13

0.040 f 0.017 (P < 0.02) 0.050 f 0.079

-0.021 k 0.035 -0.019 k0.153

0.067 f 0.026 (P< 0.02)

Note. The following equation was used to calculate the paired difference: (infarct area ilinfarct area 1.2 ppm) - (contr. area ilcontr. area 1.2 ppm).

3.0 ppm 2.4 ppm 2.0 ppm 1.2 ppm 0.8 ppm

Peak area ratio differences relative to 3.2 ppm (choline + carnitine)

- 1. I f 0.532 (P < 0.05) -0.082 * 0.3 1 0.353 f 0.36 -0.185 f0 .17 0.329 f 0.72 -0.41 k 0.90 0.906 f 4.29 -1.12+0.50 (P<0.05)

1.54+ 1.29 0.198 f 0. I 1 (P < 0.05)

Note. The following equation was used to calculate the paired difference: (infarct area i/infarct area 3.2 ppm) - (contr. area i/contr. area 3.2 ppm). There were four pairs of data for each comparison.

Water Suppressed Proton Spectroscopy

Using the jump and return pulse sequence to suppress the water a number of resonances were visible in the proton spectrum (Figs. 1 and 2). The peaks were identified as choline + carnitine at 3.2 ppm, creatine (CR) at 3.0 ppm, lipid -CH2-CO protons at 2.3 ppm, lipid allylic protons -CH2CH= at 2.0 ppm, lipid methylene (-CH2-) at 1.2 ppm, and lipid terminal methyls at 0.8 ppm. The resonances at 2.3 and 2.0 ppm may contain contributions from glutamate and the resonance at 1.2 ppm has a contribution from lactate.

The ANOVA and Tukey’s test of the peak area data yielded the following results: (1) the ratio of CR/methylene peak area of the occlusive infarct was significantly less than the ratio for normal myocardium, (2) the ratio of 0.8 peak/methylene peak area of the occlusive infarct was greater than both the ratios for normal myocardium and


Cholinel Carnitine

-CHp-C=

4 3 2 1 0 -1 PPM

FIG. 1. Water-suppressed proton spectra of excised myocardium. The top spectrum was recorded from normal myocardium and the bottom spectrum was recorded from occlusive infarcted myocardium taken from the same animal. The spectra were acquired using the jump and return sequence of Plateau and Gueron (6) with block size 4096, sweep width +3000 Hz, spectral frequency 240 Mhz, 90-90" delay 400 ps, intercycle delay 7 s, and 20 transients.

reperfused infarct, (3) the ratio of CR/3.2 peak area for the occlusive infarct was less than ratios for normal myocardium and reperfused infarct, (4) the ratio of 2.3/3.2 peak area for the occlusive infarct was greater than the ratios for normal myocardium and the reperfused infarct, and ( 5 ) the ratio of 0.8/3.2 peak area for the occlusive infarct was greater than the ratio for normal myocardium (Table 1).

The paired analysis of the peak area data yielded the following results: (1) the ratio of 3.2/methylene peak area for reperfused infarcts was greater than the ratio for normal myocardium, (2) the ratio of 0.8/methylene peak area for reperfused infarcts was greater than the ratio for normal myocardium, (3) the ratio of 0.8/3.2 peak area for

PROTON SPECTROSCOPY OF MYOCARDIAL INFARCTION 56 1

l ' ' ' ' I ' ' i ~ I ' ' ' ' I ' ' ' ' I ' 4 3 2 1 0 PPM 4 3 2 i 0 PPM

FIG. 2. Water-suppressed proton spectra of normal (top) and infarcted (bottom) tissue taken from an animal with reperfused infarct. Same acquisition parameters as Fig. 1.

reperfused infarcts was greater than the ratio for normal myocardium, (4) the ratio of CR/3.2 peak area for occlusive infarcts was less than the ratio for normal myocardium, and (5) the ratio of methylene/3.2 peak area for occlusive infarcts was less than the ratio for normal myocardium (Table 2).

A decrease in the creatine peak (relative to methylene) and an increase in the lipid terminal methyl peak (relative to methylene) were consistently evident and observed in the proton spectra of occlusive infarcts (Fig. 1). The increase in the terminal methyl peak was also visible in the reperfused infarct spectra (Fig. 2).


Cholinel Carnitine

4 3 2 1 0 PPM

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 PPM

FIG. 3. Proton spin-echo spectra of the normal (top) and occlusive infaraed (bottom) myocardium samples in Fig. 1. The spectra were acquired using a Hahn spinzcho sequence with block size 4096, weep width

spectral frwluency 240 Mhz, 90- 180' delay of 68 ms, intercycle delay of 5 s, and 40 transients.

Spin-Echo Spectroscopy The spectra obtained using spin-echo spectroscopy (Figs. 3 and 4) were vastly dif-

ferent from the non-T2 weighted spectra (Figs. 1 and 2) because the T2 relaxation times of the proton resonances were not the same. The methyl lactate peak appears inverted ( 1.3 ppm) in a spin-echo spectrum at a 90- 180" delay of 68 ms (Fig. 3). Most of the spectra showed little or no contamination of lactate to the lipid resonance at a 7 of 68 ms; however, this information could not be used to assess the amount of lactate contamination to the lipid resonance in the non-T2 weighted spectrum.

The spin-echo spectra of the reperfused infarcts and paired controls correlated well with the lipid methylene T2 results. The lipid T2 relaxation time was shorter in the r e p e r f d infarcts than in the controls and this would cause the lipid peak to be smaller in the spin-echo spectrum of the reperfused infarcts (Fig. 4) than in the normal myocardium.

The Eflect of Deuterium Oxide on Relaxation Times The results of the individual measurements and averages are shown in Table 3. The

TI measurements of tissue with deuterium were not significantly different (using the paired and unpaired t test) from the tissue without deuterium. However, a significant difference was found (P < 0.05) for the T2 measurements.


-Clip-

4 3 2 1 0 PPM

FIG. 4. Proton spin-echo spectra of the normal (top) and reperfused infarcted (bottom) myocardium samples in Fig. 2. Same acquisition parameters as in Fig. 3.

DISCUSSION

The current study indicates that water as well as bulk TI and TZ values are prolonged for infarcted compared to normal myocardium. The presence of deuterium oxide in these experiments affected the T2 meaurements; however, it is likely that it increased the Tz of both infarct and normal myocardium. Such an artifact of T2 prolongation caused by deuterium oxide would tend to diminish the differences between the T2 of normal and infarcted myocardium. The increase of TI and T2 relaxation times of ischemic tissue found in this study is consistent with previous studies where T, and Tz were measured (1-4). The Carr-Purcell-Meiboom-Gill (ZO) spin-echo train is usually used in the imaging experiment to measure T2 to avoid errors due to molecular diffusion; however, the Hahn spin-echo experiment will not give diffusion errors (diffusion term = 0.00003) when the main magnetic field is shimmed to a linewidth of 20 Hz and no magnetic field gradients are pulsed between the 90 and the 180" delays.

It has been suggested that the increase in lipids in the infarcted tissue influences the prolongation of bulk T2 relaxation time measured in the proton image (Z-4). The lipid/water ratios found in the current study indicate that the mobile lipid levels in the 8-day-old myocardial infarctions do not make any important contribution to the prolongation of relaxation times of infarcted myocardium because the lipid peak area was at least 200 times smaller than the water peak area. Also, the lipid/water ratio actually decreased for infarcted tissue compared to normal myocardium. The lipid


TABLE 3

The Effect of Deuterium Oxide (D. 0.) on Relaxation Times

Sample Ti without D. 0. Ti with D. 0. No. (s) (4

1 1.34 1.77 2 1.73 1.86 3 1.63 1.60 4 1.65 1.77

Average 1.58 f 0 . 1 7 1 . 7 5 f 0 . 1 1

Sample T2 without D. 0. T2 with D. 0. No. (ms) (ms)

1 28 2 25 3 28 4 25

64 34 34 57

Average 26 f 1.6 4 7 + 16

methylene relaxation times were shorter for reperfused infarcts than for normal myocardium.

The lipid/water ratio of the reperfused infarcts was less than the ratio for normal myocardium. This information alone does not allow one to differentiate between a decrease in the lipid or an increase in the water peak area. However, the decreased ratio of methylene/3.2 (Table 2) indicates that there probably was a decrease in the lipids instead of an increase in the water protons. No significant change in the lipid/ water ratio of the occlusive infarct was detected even though there was a large change in water and bulk TI and T2 relaxation times. Thus, the lipid/water ratio did not correlate with the relaxation times. Lactate contribution to the lipid resonance at 1.2 ppm in the non-T2 weighted proton spectra is a possible source of error. The observed lipid/water ratio could decrease in infarcted tissue compared to normal tissue because contaminating lactate produced at infarction might disperse in 8 days whereas lactate produced at sacrifice would persist in healthy animals examined by NMR a few hours later. However, there is no known method for editing lactate in the one-pulse experiment. When using the spin echo to edit lactate the results could still be misleading because the peak areas are heavily T2 weighted.

The lipid peak in all of the spin-echo spectra was very small because the tissue was sampled from the core of the myocardial infarction where ischemia was severe. Blood flow measurements are needed to correlate the results with the extent of ischemia (3).

The most interesting observations in the water-suppressed proton spectra were changes in the ratio of -CH2-C0/3.2 and 3.0/3.2, which were different for the occlusive and the reperfused infarcts (Table 1). This data along with the change in the ratio of terminal methyl (Table 1 ) suggest that there was a change in lipid structure because the -CH2-CO relative peak area varies with the type of lipid in the proton spectrum


(8). Hydrolysis of lipids by enzymes has been shown to occur in ischemic myocardium (11). The mechanism responsible for the decrease in the creatine peak area ratios (Tables 1 and 2) remains unclear. Evanochko et al. (3) also observed a decrease in creatine in severely ischemic myocardium.

Myocardial tissue characterization has been done using bulk TI and T2 relaxation times (12-16); however, these measurements lack the specificity needed to differentiate between types of infarcts. Additional parameters are needed for more specific myocardial tissue characterization. The current study suggests that the lipid resonances (2.3, 1.2, 0.8 ppm) and the creatine resonance (3.0 ppm) may be potentially useful for differential tissue characterization. The proton spectrum of impaired myocardium is considerably different than that of normal myocardium. These differences have been expressed as a ratio of peak areas within the spectrum. However, to date we have only made proton spectroscopic measurements of myocardial tissue in vitro. In order to make the measurements noninvasively in vivo there are some technical difficulties to overcome, such as motion of the heart, main-field inhomogeneity, and a pulse sequence which allows a non-T2 weighted acquisition with water suppression and spatial selection. Some techniques that may potentially overcome these difficulties are DRESS (1 7), SPARS (18) (may be partially T2 weighted), and phase-encoded spectroscopy along a tissue column (19) with short TE.

In summary, the results of this study indicate that for the core of the infarction (1) the lipids do not contribute directly to the increased bulk relaxation times associated with myocardial infarction, (2) lipid relaxation time was shorter for the reperfused infarcts than for normal myocardium, and (3) the lipid peaks (2.3, 1.2,0.8 ppm) and creatine peak (3.0 ppm) are more specific to the kind of infarct than are the water or bulk relaxation times. Therefore, semiquantitative analysis of the proton spectra of abnormal compared to normal tissue might serve as a method for tissue characterization of the myocardium.

ACKNOWLEDGMENTS

The authors thank Chris P. Anson and Robert Golden for help with computer programming to analyze the data, Nikita Derugin for help in tissue preparation, and Eric Shankland for help on the 200 CXP spectrometer.

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proton nmr spectroscopy in canine myocardial infarction

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