Cardiology 64: 222 230 (1979)

Normalization o f the Measurement o f Cardiac Creatine Phosphokinase A ctivity1 J.A . Spath, jr., M .H. Gee and P.A. Gwirtz Department o f Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

Key Words. Creatine phosphokinase • Myocardial CPK • Myocardial ischemia • Pericardial tamponade • Cardiac water Abstract. Cardiac creatine phosphokinase (CPK) activity in international units (IU) was normalized to homogenate soluble protein (IU/mg), to tissue wet weight (IU/100 mg) or to tissue dry weight (IU/mg). Samples o f cardiac tissue were excised from sham-operated cats and dogs or from cats after 4 h o f severe pericardial tamponade and dogs after 4 h o f circumflex artery ligation. In sham-operated cats and dogs, cardiac CPK activity was about 30 IU/mg homogenate soluble protein. 200 IU/100 mg wet tissue, and 9.5 IU/mg dry tissue. In cats, after tamponade, the CPK activity o f subepicardial and subendocardial tissue o f the left ventricle was decreased 24 35% if the enzyme activity was referenced to wet weight or dry weight. Similarly, in dogs, after circumflex artery ligation, the CPK activity o f posterior papillary muscles was reduced 24-30% if the enzyme activity was referenced to tissue wet or dry weight. In both cats subjected to 4 h o f tamponade and dogs after 4 h o f circumflex artery ligation, significant loss o f CPK activity could not be demonstrated if the cardiac enzyme activity was normalized only to the soluble protein content o f homogenates. We conclude that normalizing cardiac CPK activity to tissue wet and dry weight may permit early assessment o f myocardial injury in experimental models o f myocardial ischemia.

The activity o f creatine phosphokinase (CPK) decreases within ischemic cardiac tissue (1, 2, 8). Since some investigators (2, 3) have correlated the decrease in cardiac CPK activity with infarct size, this measurement has become an important tool in experimental studies assessing the efficacy o f different 1 Supported in part by a grant (HL 20283-01) from the National Heart, Lung and

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Blood Institute, by Young Investigator Pulmonary Research Grant (HL 19498-01), an American Lung Association Grant, and a grant-in-aid from the Delaware Heart Association.

Cardiac CPK and Water Content


therapeutic modalities in the preservation of the ischemic myocardium. One problem in relating cardiac CPK activity to infarct size in the experimental animal is the variability of the measurement in normal tissue. Reported values for normal cardiac CPK activity in the dogs vary from 18 to 30 international units (lU)/mg homogenate soluble protein (2, 6). The large variability in the measurements limits the usefulness o f cardiac CPK as an index of ischemic injury, since small changes in CPK activity tend not to reach statistical signifi­ cance. The purpose of the present study was to assess the effectiveness o f proce­ dures used to normalize measurements of cardiac CPK activity. We found that the common method o f normalizing activity to homogenate soluble protein may not detect enzyme loss early in the course o f ischemic injury. Moreover, we show that normalizing CPK activity to tissue wet weight and dry weight reveals enzyme loss from ischemic tissue, eliminates the need for chemical determina­ tion of homogenate protein, and provides important information on changes in water content o f ischemic tissue.

Methods Surgical Procedures 11 cats (average weight 2.3 kg) and 9 dogs (average weight 15.5 kg) were anesthetized with intravenous sodium pentobarbital (30 mg/kg). The animals were placed on an inter­ mittent positive pressure respirator. The heart was exposed through an incision in the left

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sixth intercostal space in dogs and through a midline sternotomy in cats. Catheters were placed in the left common carotid artery to monitor mean arterial blood pressure. An additional catheter was placed directly into the left atrium o f dogs and in the right external jugular vein o f cats to record left atrial pressure, and central venous pressure, respectively. Catheters were attached to appropriate transducers and pressures continuously recorded on a direct writing oscillograph. Pericardial tamponade was induced in cats in which a 4- to 5-mm incision was made in the pericardial sac overlying the left coronary artery. A cannula was secured in the wall o f the pericardium. The cannula consisted o f two opposed Teflon discs with a short section o f 23-gauge stainless steel tubing placed through and flush with the lower disc. The lower disc was positioned within the pericardial sac and the upper disc was screwed tightly against the lower disc forming a water-tight seal with the interposed pericardial surface. Tamponade was produced by introducing 1 3 -1 6 ml o f 0.9% NaCl at 37 °C into the pericardial sac. There­ after, saline was added or withdrawn through the pericardial cannula to maintain the mean arterial blood pressure at 40 mm Hg for 4 h. A t the end o f 4 h, all excess fluid was removed from the pericardial sac and the cat monitored for an additional hour prior to removal o f the heart for analysis o f cardiac CP K activity. In sham-operated cats, the pericardial cannula



was positioned as described, but no fluid was added or removed from the pericardial space for 5 h. In dogs the circumflex branch o f the left coronary artery was cleared o f surrounding tissue about 1 cm from its origin and a 2-0-silk ligature placed under the vessel. After obtaining baseline measurements o f pressures, I ml o f 2% lidocainc hydrochloride was topically applied to the dissected region o f the circumflex artery to minimize postocclusion arrhythmias. Sham-operated animals were subjected to all procedures except ligation o f the circumflex artery.

Sampling o f Cardiac Tissues A t the conclusion o f each experiment, the heart was excised and the CPK activity o f the tissue assayed. In cats, subepicardial and subendocardial tissue (100 mg) from the anterior surface o f the left ventricle was prepared for analysis o f CPK activity. In dogs, tissue (100 mg) from the anterior and posterior papillary muscles o f the left ventricle were taken for the determination o f CPK activity. In both species, additional samples o f tissue were taken adjacent to the tissue sampled for analysis o f CPK activity. These tissues were weighed, placed in 80 °C' oven for 48 h and reweighed to determine tissue dry weight.

Chemical Analyses Samples o f ventricular tissue were homogenized twice in 0.25 4/ sucrose (1:10, w:v) using a Tekmar® homogenizer at 24 X 103 rpm for 15 sec each time. The homogenates were centrifuged at 16,000 g for 30 min at 4 °C and the supernatants analyzed for CPK activity using the method o f Rosalki (7). Protein concentration o f cardiac homogenates was determined using the method o f I.owry er al. (4).

Reference o f CPK Activity and Statistical Analyses The CPK activity o f homogenates o f myocardial tissue is expressed in IU o f activity. 1 IU o f CPK is that activity which converts 1.0 ¿¿mole creatine phosphate per milliliter o f sample per minute at 30 °C. The CPK activity was normalized in three ways: lU/mg homogenate soluble protein, IU/100 mg wet weight, and lU/mg dry tissue. Student’s t test was used to test the level o f significance o f the difference between means. A probability o f less than 0.05 was taken as indicative o f a significant difference between means. To evalute variations in the measurement o f homogenate protein content and tissue water content, the coefficients o f variation o f each o f these measurements were calculated for tissue taken from sham-operated and experimental animals. The coefficient o f variation o f a parameter is the standard deviation as a percentage o f the mean value o f the measurements.


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Table 1 presents the hemodynamic effects o f pericardial tamponade in cats and circumflex artery ligation in dogs. Cats subjected to sham pericardial tamponade showed stable mean arterial blood pressure and central venous

Cardiac CPK and Water Content


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pressure for 5 h. However, infusion o f 13 16 ml of saline into the pericardial sac o f cats subjected to tamponade reduced the mean arterial blood pressure to 40 mm Hg and increased the central venous pressure to 13 cm 1120 , Thereafter, the central venous pressure progressively declined for 4 h. Mean arterial blood pressure and mean left atrial pressure did not change significantly for 4 h after ligation o f the circumflex artery in dogs (table I). Brief occlusion o f the vessel prior to permanent ligation did produce slight transient changes. For example, in i dog, arterial pressure declined 5 mm Hg and left atrial pressure increased 4 cm H 20 . These changes disappeared within 5 min of ligation. 5 sham-operated dogs exhibited stable hemodynamic parameters for the entire period of observation. At the end of 4 h, dogs in which the coronary artery was ligated could not be distinguished from sham-operated animals on the basis o f the recorded hemodynamic parameters. In sham-operated cats the CPK activities o f subepicardial and subendo­ cardial tissue were similar and were about 32 IU/mg homogenate protein, about 200 IU/100 mg wet tissue and about 9 IU/mg dry tissue. The water content and the homogenate soluble protein content o f tissue taken from sham-operated cats were similar in the subepicardium and subendocardium (table II). Variations in measured water or protein content were slight, being 5-6% for the former and 11 12% for the latter parameter. Moreover, duplicate measurements o f enzyme activity or homogenate soluble protein agreed within 5%. The cardiac water content and the homogenate protein content o f subepi­ cardial and subendocardial tissue excised after tamponade were not different from each other or from those o f sham-operated cats. Again, the variation in the measurement of water content was slight, i.e. 4-6% , and the variation for homogenate protein content was only 10%. However, compared to sham-operated cats, there was a significant (p

Normalization of the measurement of cardiac creatine phosphokinase activity.

Cardiology 64: 222 230 (1979) Normalization o f the Measurement o f Cardiac Creatine Phosphokinase A ctivity1 J.A . Spath, jr., M .H. Gee and P.A. Gw...
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