The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes.

The relationship between the percentage of rod-shaped rat heart myocytes and ATP, creatine phosphate, creatine and inorganic phosphate content was determined. With these values the free energy of ATP hydrolysis was calculated and found to be 59.2 kJ/mol, a much higher value than found for the perfused rat heart. When, during the isolation procedure, creatine was present in the perfusion medium during the low-calcium period, the total creatine content of the myocytes after isolation was comparable to that found in the perfused rat heart. However, when creatine was absent during this low-calcium perfusion period, total creatine content of the myocytes was significantly lower. This difference is caused by leakage of creatine from healthy cells. The free energy of ATP hydrolysis was not affected by the absence of creatine during the low-calcium perfusion period.

Transmural inhomogeneity of energy metabolism during acute global ischemia in the isolated rat heart: dependence on environmental conditions.

Cardiac energy metabolism is one of the earliest metabolic activities affected when either anoxia or ischemia are induced, as evidenced by the rapid decline of the tissue high-energy phosphate content of creatinephosphate (CrP) and ATP. Several reports deal with the spatial inhomogeneity of these changes and it is generally found, that the subendocardium is more sensitive to ischemia than the subepicardium. The metabolic transmural gradients observed during in vivo ischemia were attributed to both variations in wall tension and collateral flow. Lowe et al. recently presented evidence that in addition to these variations the higher vulnerability of the subendocardium to ischemia could be secondary to an increased metabolic rate.

The change of the free energy of ATP hydrolysis during global ischemia and anoxia in the rat heart. Its possible role in the regulation of transsarcolemmal sodium and potassium gradients.

The timecourse of change of the cytoplasmic free energy of ATP hydrolysis during acute global ischemia and during anoxic perfusion was determined in the isolated rat heart. The timecourse of change of transsarcolemmal Na+ and K+ gradients during anoxia, and of extracellular K+ during ischemia were measured. The free energy of ATP hydrolysis was calculated from the equilibrium of the creatinekinase reaction, taking into account the pH-dependence of the equilibrium constant, and intracellular inorganic phosphate. In control aerobic hearts the mean free energy of ATP hydrolysis was 55.2 kJ/mol. Both during ischemia and anoxia it declines biphasically. The first rapid phase terminates within 4 min into a plateau of about 46 kJ/mol. The duration of this plateau is shorter during anoxia than during ischemia. The second phase of decrease starts after 6 to 8 min during anoxia and after 15 to 20 min during ischemia. After 30 min of anoxia the free energy of ATP hydrolysis has decreased to 31 kJ/mol and after 30 min of ischemia a value of 35.5 kJ/mol is reached. The timecourses of change of measured intracellular Na+ and K during anoxia and of extracellular K+ during ischemia were also biphasic. During anoxia the loss of intracellular K+ was almost equal to the gain of intracellular Na+ at any point. Based on the assumption that the sodium pump is in thermodynamic equilibrium or near-equilibrium during anoxia and ischemia, the time-course of change of Na+ and K+ gradients during anoxia and of extracellular K+ during ischemia were calculated from the respective timecourses of change of the free energy of ATP hydrolysis. Good agreement was observed between calculated and measured changes of Na+ and K+ gradients. It is concluded that the magnitude and direction of change of transsarcolemmal ion-gradients during anoxia and ischemia may be under direct thermodynamic control of myocardial energy metabolism.

Tissue osmolality, metabolic response, and reperfusion in myocardial ischemia.

We studied the effect of tonicity of the perfusate during reperfusion after global ischemia, in both the rat and the porcine heart. After 50 min, tissue osmolality increased by about 40 mOsm/kg. Normotonic as well as hypertonic reperfusion resulted in limited areas of "no-reflow". Metabolic restoration after reperfusion was not dependent on the tonicity of the perfusate, nor was recovery of contractility. Hypertonic reperfusion induced a higher coronary flow rate. In porcine hearts, scatter of metabolic data indicated inhomogeneity of reperfused tissue. The results differ substantially from data obtained after reperfusion of regionally ischemic hearts. Reasons for these differences are discussed.

Boehringer immunoinhibition procedure for creatine kinase-MB evaluated and compared with column ion-exchange chromatography.

In determination of creatine kinase isoenzyme MB (CK-MB), the Boehringer immunoinhibition method gives a high and variable blank activity as compared with column-chromatography. Thus a correction must be applied. Furthermore, a second correction of 1% of total creatine kinase activity is necessary to compensate for nonspecific creatine phosphate-dependent activity. As a consequence, two immunoinhibition determinations--one for CK-MB and one for blank activity--and a determination of total creatine kinase are required. Use of the manufacturer's diagnostic criteria, on the basis of which suspected myocardial infarction is confirmed or eliminated, leads to a high frequency of false-negative conclusions.

Infarct size estimation from serial CK MB determinations: peak activity and predictability.

In 198 patients with acute myocardial infarction serial measurements of plasma creatine kinase isoenzyme MB (CK MB) were performed at four hour intervals. In every patient, maximal CK MB activity (peak activity) was compared with calculated total release per litre plasma. In 28 patients (group 1) sufficient plasma samples were available for calculation of the apparent first order inactivation constant kd. Mean apparent kd in group 1 patients was 0 . 085 +/- 0 . 018 h-1 (mean +/- SD). Total release in group 1 was calculated with individual apparent kd values (Q) and with the mean kd value (Q*). In the remaining 170 patients (group 2), Q* only was calculated. A linear relation between peak activity P and total release (both Q and Q*) was found, extending over the whole range of CK MB peak activities that are routinely observed (4-216 U/l). It was immaterial whether a one or a two compartment model was used: both yielded a close linear relation. Though the mean ratio between Q* and peak activity depends on the value of kd chosen for calculation of total release (the ratio increasing with increasing kd), linearity between peak activity and Q* was found for any value of kd up to 0 . 4 h-1. In group 1, shapes of calculated CK MB release curves Q*(t), expressed relative to maximal release Q(40), were sufficiently similar so as to be superimposable; the section of the release curves extending from 12 hours before until two hours after peak time could be tentatively described by a linear time course with a slope of 4 . 2 +/- 0 . 5% per hour (mean +/- SD). We conclude that peak activity of CK MB is a reliable estimate of cumulative CK MB release and may be clinically more practicable than calculation of Q(40). Both the similarity and the large apparently linear section of the calculated enzyme release curves possibly permit early prediction of Q(40), with acceptable precision.

Substrate dependence of reoxygenation arrhythmias in the isolated rat heart.

Isolated rat hearts perfused with various substrates were subjected to oxygen restriction followed by sudden reoxygenation. The incidence of ventricular arrhythmias occurring after reoxygenation appeared to be dependent on the substrate present during oxygen restriction; it was low with glucose (11 mM) and significantly higher with oleic acid (FFA to albumin molar ratio 4), with beta-hydroxy butyrate (11 mM), with acetate (11 mM) or without added substrate. When verapamil (1 muM) was also present in the medium, these arrhythmias were largely prevented. When glucose or verapamil were present during the reoxygenation period only, the incidence of reoxygenation arrhythmias was high. Tissue levels of long-chain acyl-CoA increased during oxygen restriction under all substrate conditions tested. At the moment when reoxygenation was started they were most elevated in hearts perfused with oleic acid. Verapamil did not influence levels of long-chain acyl-CoA. The amount of creatine-kinase (CK) released from the heart after reoxygenation did not correlate with the observed arrhythmias and was greatest in hearts perfused with oleic acid. Verapamil protected against CK release in the absence of added substrate, but not when oleic acid was present. It is concluded that ventricular arrhythmias after reoxygenation are not necessarily caused by FFA or long-chain acyl-CoA.

Determination of creatine kinase isoenzyme MB (CK-MB): comparison of methods and clinical evaluation.

An ion-exchange chromatography method based on the method of Mercer is advocated for the routine determination of serum CK-MB. This method has some prominent advantages over other methods with which it is compared, and which include electrophoresis and an immunological technique. This method proves to be reliable and highly reproducible, while it allows a rather large number of samples to be analyzed within a relatively short period of time. Some parameters of the release pattern of CK-MB after acute myocardial infarction are characterized: normal values, time of first rise, time of peak value and rate-constant of inactivation. The clinical significance of serum CK-MB determination is evaluated.