Activities of key enzymes in the energy metabolism of human myocardial and skeletal muscle

Summary. Activities of total creatine kinase (CK), its isoenzyme MB (CK-MB), total lactate dehydrogenase (LD) and its isoenzyme LD₁, phosphofructokinase (PFK), asparate aminotransferase (ASAT) and citrate synthase (CS) were determined in skeletal muscle biopsies obtained from physically trained and untrained men and in myocardial biopsies from patients subjected to open heart surgery because of valve disease. The LD₁, ASAT and CS activities were higher in trained than in untrained skeletal muscle and still higher in heart muscle than in either trained or untrained skeletal muscle. The CK-MB activity was higher in trained than untrained skeletal muscle and the myocardial CK-MB activity was similar to that in trained skeletal muscle. Total CK activity was slightly lower in trained than in untrained skeletal muscle and the myocardial CK activity was approximately one third of the skeletal muscle CK. Both the PFK and the total LD activity was of similar magnitude in the different muscle types. In conclusion, as estimated by enzyme activities, the oxidative capacity is 2–3 times larger in myocardial than in skeletal muscle, while the glycolytic capacity as estimated by PFK appears to be the same.     

Differentiating muscle damage from myocardial injury by means of the serum creatine kinase (CK) isoenzyme MB mass measurement/total CK activity ratio

We immunoenzymometrically measured creatine kinase (CK) isoenzyme MB in extracts of myocardium and in homogenates of five different skeletal muscles. CK-MB concentrations in the former averaged 80.9 micrograms/g wet tissue; in the skeletal muscles it varied widely, being (e.g.) 25-fold greater in diaphragm than in psoas. CK-MB in skeletal muscles ranged from 0.9 to 44 ng/U of total CK; the mean for myocardium was 202 ng/U. In sera from 10 trauma and 36 burn patients without myocardial involvement, maximum ratios for CK-MB mass/total CK activity averaged 7 (SEM 1) ng/U and 18 (SEM 6) ng/U, respectively. Except for an infant (220 ng/U), the highest ratio we found for serum after muscular damage was 38 ng/U. In contrast, the mean maximum ratio determined in 23 cases of acute myocardial infarction exceeded 200 ng/U. Among seven determinations performed 8 to 32 h after onset of symptoms, each infarct patient demonstrated at least one ratio greater than or equal to 110 ng/U. Ratios observed after infarct were unrelated to treatment received during the acute phase. We propose a CK-MB/total CK ratio of 80 ng/U as the cutoff value for differentiating myocardial necrosis from muscular injury.     

Biochemical responses of the myocardium and red skeletal muscle to Salmonella typhimurium infection in the rat

Summary. Previous studies with bacterial infections have demonstrated a reduced exercise capacity and equally pronounced catabolic responses in red and white skeletal muscle. In the present study, red skeletal muscle and heart ventricular muscle were compared in a S. typhimurium model in rats. Two days before median lethality was achieved, the activities of one oxidative (cytochrome c oxidase), one glycolytic (glyceraldehyde-3-phosphate dehydrogenase) and one lysosomal (beta-glucuronidase) enzyme were determined in the two tissues. The contents of protein, RNA and DNA were also determined. The oxidative and glycolytic capacity decreased 24–29% in red skeletal muscle but only 7–20% in the myocardium. However, the decrease in oxidative capacity in skeletal muscle and myocardium was statistically correlated. The protein synthetic capacity (RNA) also decreased and was correlated to the protein concentration in both tissues. This metabolic impairment of both skeletal and heart muscle probably contributes to the deterioration of the physical performance capacity previously observed to follow acute infectious diseases. This study emphasizes the importance of the choice of reference, such as ‘wet’ weight, DNA or the entire organ, when evaluating metabolic results in biologic tissues and that biochemical alterations in skeletal muscle biopsies in bacterial infections do not reflect alterations in myocardium reliably.     

Creatine kinase MB in cases of skeletal muscle trauma

Fifty-eight patients admitted through our emergency room with severe skeletal muscle injury but no obvious cardiac contusions were evaluated for creatine kinase isoenzyme MB (CK-MB). When such patients show an above-normal value for total CK, it is a question of whether or not myocardial injury has been sustained along with skeletal muscle injury when (a) there are no obvious chest contusions or (b) the patient is unconscious and unable to complain of chest pain. Whenever there is doubt concerning the cardiac status of a patient, lactate dehydrogenase (LD) isoenzymes, serial electrocardiograms, and CK isoenzymes are ordered. Our study revealed that serum of 8.6% of the trauma victims had CK-MB values exceeding 5.0 EU/L (reflecting abnormal CK-MB concentrations) as part of their increased total CK. All patients had normal electrocardiographic patterns along with negative results for LD isoenzymes; none had sustained any demonstrable myocardial injury. The CK-MB value must be interpreted together with the total CK value for appropriate diagnosis in patients with skeletal muscle trauma.     

Skeletal muscle mitochondrial ATP production rate and walking performance in peripheral arterial disease

This study tested the hypotheses that skeletal muscle mitochondrial ATP production rate (MAPR) is impaired in patients with peripheral arterial disease (PAD) and that it relates positively to their walking performances. Seven untrained patients, eight exercise-trained patients and 11 healthy controls completed a maximal walking test and had muscle sampled from the gastrocnemius medialis muscle. Muscle was analysed for its MAPR in the presence of pyruvate, palmitoyl-L-carnitine or both, as well as citrate synthase (CS) activity. MAPRs were not different between untrained PAD and controls. In contrast, MAPRs (pyruvate) were significantly higher in trained PAD vs. controls. MAPR (pyruvate combinations) was also significantly higher in trained than untrained PAD muscle. MAPR and CS activity were highly correlated with walking performance in patients, but not in controls. These data do not support the hypothesis that isolated mitochondria are functionally impaired in PAD and demonstrate that the muscle mitochondrial capacity to oxidize carbohydrate is positively related to walking performance in these patients.     

Cardiac troponins and creatine kinase content of striated muscle in common laboratory animals

Animal models are important for the investigation of human heart pathology, novel treatments, and medical or surgical interventions for disease. Serum markers of myocardial damage may also be important tools within this field of research. In order to assess the cardiac specificity of widely utilised serum markers, we measured the cardiac troponins and creatine kinase (CK) isoenzymes in cardiac and skeletal muscle samples taken from dog, monkey, pig and rat. These samples were also analysed by immunoblotting for cardiac troponin I (cTnI) and cardiac troponin T (cTnT). The content of cTnI and cTnT in skeletal muscle was below 0.6% of that found in heart for all animal species studied. This low immunoreactivity in skeletal muscle was confirmed by immunoblot analysis. The content of CK was higher in skeletal muscle than in heart muscle for all species. The CK-MB/total CK ratio was lower in skeletal muscle than in cardiac muscle for all species. The differences in CK-MB content of skeletal muscle and heart muscle were much less pronounced than the tissue differences in the amounts of the cardiac troponins. The cardiac troponins are potentially useful serum markers of myocardial damage, with high specificity for myocardial muscle in these common laboratory animals. Creatine kinase-MB is much less cardiac-specific.     

Myoglobin and enzyme adaptations in erector spinae muscles in thoracal scoliosis

Summary. Bilateral biopsies from the erector spinae muscles were taken during surgery from 10 females and two males (mean age 14, range 13–17 years) with thoracal scoliosis for 6 years (range 2–11 years). The biopsies were analysed for myoglobin (MYO), citrate synthase (CS) and creatine kinase MB (CK-MB). The severity of scioliosis was estimavoted by Cobb's angle, the greater the angle the more severe the disease. The convex/concave side ratio (CVX/CCV) was for CS l·3±0·4 (P<0·01), CK 0·9 ± 01 (P<0·05), CK-MB 1·6±0·4 (P<0·01) and for MYO 1·1±0·2 (P>0·05). No significant correlations were found between the CVX/CCV for CS, CK or CK-MB on the one hand and the Cobb's angle on the other. The CVX/CCV for MYO was, however, directly related to the angle (r= 0·80, P<0·01). For the lower range of angles (≤59°) the CVX/CCV for MYO was below unity (0·88, P>0·05) and for the larger angles (>59°) above unity (1·23, P<0·05). In conclusion, a dissociation in the adaptive response of m. erector spinae in scoliosis between mitochondrial enzyme and myoglobin content was demonstrated.     

Maximum rate of oxygen consumption related to succinate dehydrogenase activity in skeletal muscle fibres of chronic heart failure patients and controls

Previous studies indicate that the low maximum rate of oxygen consumption (VO2max) of chronic heart failure (CHF) patients is not because of impaired pump function of the heart. We hypothesize that VO2 during maximum exercise is determined by the total oxidative capacity of skeletal muscle. VO2max of six controls and 14 CHF patients, New York Heart Association class I-III, was determined using an incremental bicycle ergometer test. Cryostat sections of a biopsy from the quadriceps femoris muscle were incubated for succinate dehydrogenase (SDH) using quantitative histochemistry. VO2max (range: 29 ml O2 kg muscle(-1) min(-1) in a class III patient to 118 ml O2 kg muscle(-1) min(-1) in a control subject) correlates with the mean SDH activity of skeletal muscle fibres (r=0.79 or r=0.81, including or excluding oxygen uptake at rest, respectively; P<0.001). The relationship between VO2max and SDH activity is similar to that determined previously using isolated single muscle fibres and myocardial trabeculae under hyperoxic conditions. From the product of SDH activity and the cross-sectional area of the fibre (i.e. spatially integrated SDH activity), it is possible to calculate the maximum oxygen uptake rate per unit muscle fibre length. This uptake rate is linearly related to the number of capillaries per fibre (r=0.76, P<0.001) in all subjects, suggesting that oxidative capacity of skeletal muscle fibres in CHF patients decreases in proportion to the oxygen supply capacity of the microcirculation.     

Effect of hyperthyroidism on fibre-type composition,fibre area,glycogen content and enzyme activity in human skeletal muscle

Summary. Seven hyperthyroid patients were studied by repeated muscle biopsies (vastus lateralis) before and after a period of medical treatment which averaged 10 months. The biopsies were analysed with regard to fibre-type composition, fibre area, capillary density, glycogen content and enzyme activities representing the glycolytic capacity (hexokinase, 6-phosphofructokinase), oxidative capacity (oxoglutarate dehydrogenase, citrate synthase) and Ca²⁺- and Mg²⁺-stimulated ATPase in muscle. In the pretreatment biopsy (hyperthyroid state), there was a significantly lower proportion of type I fibres (30% vs. 41%), a higher capillary density (23%), lower glycogen content (33%), and higher hexokinase activity (32%) compared with the post-treatment biopsy. No significant changes in the activity of the remaining enzymes were observed. The present study indicates that hyperthyroidism induces a transformation from type I to type II fibres in human skeletal muscle. The increase in hexokinase activity probably reflects a higher glucose utilization by skeletal muscle in order to compensate partially for the reduced glycogen content.     

Creatine kinase isoenzymes

Creatine kinase (CK), a widely distributed enzyme in the body, has its highest activities in skeletal muscle and myocardium; when serum CK activities are abnormally increased, injury to these organs must be part of the differential diagnosis. The isoenzyme CK-MB is the most important biochemical test in the diagnosis of acute myocardial infarction.     

Forearm composition and muscle function in trained and untrained limbs

Summary. The influence of a period of training, which lasts for several years, on the proportions of muscle, fat and bone present in the human forearm has been investigated by comparing trained and untrained limbs of nine experienced male tennis players. Ten healthy but untrained males of similar age served as a control group. Computed tomography (CT) scans of the forearm were made at intervals along its length to identify fat, muscle and bone and to calculate the volumes occupied by each of these components. Total forearm volume was greater in the dominant limb compared with the contralateral side in both trained (by 135±59 cm³, mean±SD, P<0·001) and untrained subjects (by 41±45 cm³, P<0·02). Forearm muscle volume was also greater in dominant limbs of trained (by 117±52 cm³, P<0·001) and untrained by 35±41 cm³, P<0·025) subjects. Muscle accounted for 75·4±2·7% of the total volume in the dominant arm of trained subjects compared with 71·4±4·2% in the control group (P<0·05). There was a greater proportion of muscle (P<0·05) and a smaller proportion of fat (P<0·001) in the trained limb compared with the contralateral limb of the same subjects. No differences in proportions of fat, muscle and bone were observed in dominant and non-dominant limbs of the control subjects. Trained subjects were able to exert a greater isometric force with the dominant limb (549±76N) than with the non-dominant limb (496±48N; P<0·005). There was no difference in grip strength between the arms of the untrained group (dominant: 516±107N; non-dominant: 491±91N). The ratio of strength to muscle volume was, however, the same in dominant and non-dominant arms of both groups of subjects.     

Repeatability of fibre type and enzyme activity measurements in human skeletal muscle

Summary. In order to assess the variability in repeated determination of human muscle fibre type distribution, fibre area and enzyme activity measurements, two biopsies were taken within 10 days in the same vastus lateralis for 12 females and 13 males, and in the right and in the left muscles for 25 other subjects (13 females and 12 males). Within muscle, intraclass reliability coefficients were 0·88, 0·82 and 0·56 for type I, IIa and IIb per cent fibres, respectively, and ranged from 0·74 to 0·82 for fibre areas and from 0·71 to 0·90 for enzyme markers of different metabolic pathways. Correlations between right and left muscle measurements were also high for fibre areas (from 0·85 to 0·91) and enzyme activities (from 0·71 to 0·87), except for phosphofructokinase (r=0·63). In contrast, the right and left thigh muscle correlation reached 0·67, 0·40 and 0·64 for type I, IIa and IIb fibre distribution, respectively. Thus, the variation in muscle sampling and technical procedures reached about 15% of the total variation (i.e. total differences between subjects) for the proportion of fibre type I and IIa and about 20–25% for fibre areas and enzyme activities. On the other hand, the technical error for the proportion of fibre type I and IIa is about 6–7%. This implies that differences brought about by any experimental treatment on these skeletal muscle characteristics in human studies have to be of a relatively large magnitude before being detectable. On the other hand, fibre areas and enzyme activities measured in single needle biopsy sample, from one of the vastus lateralis muscles, are quite representative of the other vastus lateralis. Similarity in fibre type proportion between right and left vastus lateralis cannot be postulated, however, without investigating both muscles.     

The specificity of biochemical markers of cardiac damage: a problem solved.

This paper reviews the tissue specificity of cardiac troponin I (cTnl), cardiac troponin T (cTnT) and creatine kinase (CK) MB in human and animal heart and skeletal muscles. Studies reveal that CK-MB can be expressed up to 20% of total CK activity in human skeletal muscle; and therefore is not 100% specific for the heart. One cTnl isoform has been described and shown to be 100% specific for the heart. While one to four cTnT isoforms are expressed in diseased and regenerating human skeletal muscle, these isoforms are not the same as the cTnT isoforms expressed in the human heart and are not detected by the cTnT diagnostic assays used in clinical practice. Representative cases are described demonstrating the role of monitoring cardiac troponins in blood for differentiating false positive CK-MB increases due to skeletal muscle injury. Further, sufficient reactivity and tissue specificity of cTnl and cTnT assays are demonstrated for use as markers of myocardial injury in laboratory animals. Monitoring cTnl and cTnT concentrations in the circulation appears poised as the new standards for detection of myocardial injury.     

Creatine kinase isoenzyme MB assay by electrophoresis

A method for determination of the creatine kinase isoenzyme MB (CK-MB) is reported: separation of the isoenzymes was done by electrophoresis and the activity of the isoenzyme bands quantitated by scanning fluorometry. Total CK activity was used for calculation of CK-MB level. The precision of the method was satisfactory: coefficient of variation 5-10%. Its accuracy good: CK-MB was consistently found in high concentrations in tissue extracts of myocardium, but was virtually absent in skeletal muscle and could not be demonstrated in serum from patients with skeletal muscle damage. The sensitivity of the method fitted its clinical use: CK-MB was undetectable (less than 5 U/l) in normal sera, below 30 U/l in seventy-six out of seventy-seven patients in whom the diagnosis of acute myocardial infarction (AMI) was disproved, and above 30 U/l in all seventy-two patients with AMI according to WHO criteria. The CK-MB concentration in serum rises to a maximum about 20 h after onset of clinical symptoms of AMI and reaches baseline levels 20-30 h later. The electrophoretic CK-MB method is easy, fast and reliable and is considered as an important diagnostic test for AMI.     

Calf muscle adaptation in intermittent claudication. Side-differences in muscle metabolic characteristics in patients with unilateral arterial disease

Summary. The adaptation of enzyme activities, notably in the oxidative metabolism, and of prerequisites for tissue transport of oxygen in the claudication leg was evaluated by comparing muscle biopsies from the gastrocnemius muscle of the claudication and the symptom-free leg of seven patients with unilateral claudication. The claudication leg had higher activities of a marker enzyme for mitochondrial oxidative capacity, citrate synthase (CS), as well as of the MB and the mitochondrial isoenzyme of creatine kinase (CK), which are considered to be involved in the transfer of high energy phosphate from the mitochondria to the resynthesis of ATP in the cytoplasm. The difference between claudication and healthy leg in activities of these CK isoenzymes were well correlated with the corresponding side difference in CS activity. No significant differences between claudication and healthy leg were found in distribution of muscle fibre types or fibre dimension, capillary density or myoglobin content, nor was there any side difference in phosphofructokinase or lactate dehydrogenase. Side differences tended to be greater in those patients with the most advanced obstructive arterial disease as estimated from non-invasive pressure measurements. It is concluded that in reasonably physically-active patients, the mode of ischaemia to which the claudication leg is subjected leads to a metabolic adaptation characterized by increased activities of enzymes involved in the oxidative metabolism, but no significant adaptation of either the conditions for local oxygen transport, as estimated by myoglobin content, and capillary density, or capacity for anaerobic metabolism.     

Dynamics of creatine kinase shuttle enzymes in the human heart

Myocardial cytoplasmic creatine kinase subunits M and B, mitochondrial CK (CKMIT), and citrate synthase (CS) were determined in 10 locations of the normal human heart (n = 8) and in papillary muscles of patients operated on for mitral regurgitation (n = 6). Compared to atrial biopsies, septal and left ventricular biopsies showed higher activities for CS (P less than 0.0001), total CK (P less than 0.05) and CKMIT (P less than 0.0001). CKM was evenly distributed. CKB activity in the right septum and left ventricular locations were 0.5-1% of total CK and 4-5 times lower than those of the atria and the right ventricular free wall. Activities of CS, CKB and CKMIT in right septal biopsies did not differ from those in left ventricular locations. The activities of CS, total CK, and CKM in papillary muscle from patients operated on for mitral regurgitation did not differ from that of healthy papillary muscle. CKMIT was about 40% lower (P less than 0.02), whereas CKB was 15-20 times higher (P less than 0.0001) than in the healthy heart. In conclusion, adaptations within the creatine kinase system occur in the human heart in health and disease. Small amounts of CKB in the normal left ventricle, as opposed to the right ventricular free wall, might be related to differences in myocardial perfusion during the cardiac cycle. In disease, a decreased CKMIT and dramatically increased CKB may indicate a stressed intracellular energy transfer. CK enzyme activities in right septal biopsy specimens may be used as an indication of metabolic stress on the myocardium of the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Change in concentrations of myogenic components of serum during 93 h of strenuous physical exercise

Effects of 93 h of long, strenuous ranger training on activities of creatine kinase (CK) and lactate dehydrogenase (LD), along with their isoenzymes, and on concentration of myosin light chain were examined in sera of young soldiers. Total CK activity in serum was measured before, during, and after the training. Throughout, total CK activity in serum increased steadily. At the end of the training, activity of CK-MB was increased but its activity ratio to total CK remained unchanged; the activity ratio of LD1/LD2 also was not increased, although total LD activity was increased. Myosin light chain was increased by about fourfold at the end of the training and remained high for three days thereafter. However, its concentration was much lower than in myopathies such as polymyositis and Duchenne muscle dystrophy. The increased activities in serum of total CK and CK-MB isoenzyme on strenuous physical exercise evidently were of noncardiac origin. Although CK activity was comparable with that seen in myopathies accompanied by disintegration of skeletal muscle, the relatively low concentration of myosin light chain in serum suggests minimal skeletal muscle damage.     

Minimal myocardial injury after uncomplicated coronary bypass surgery. Various sources of overestimation

Myocardial injury after aorto-coronary bypass surgery was estimated in 72 patients from total release into plasma of cardiac creatine kinase (CK-MB) and alpha-hydroxybutyrate dehydrogenase (HBD). Activities of CK-MB were determined both by immuno-inhibition of CK-M units and by ion-exchange chromatography. After correction for per-operative hemolysis, the estimates based on HBD were in agreement with the estimates based on CK-MB as determined by the ion-exchange method. Both enzymes indicated a mean loss of only about 2 gram-equivalents of myocardium. Such minimal injury was also found in metabolic and ultrastructural studies of myocardial biopsies in the same patients, as reported earlier. However, approximately two-fold larger estimates of injury were obtained from plasma CK-MB activities determined by immuno-inhibition. This apparent extra release of CK-MB runs parallel with massive release of CK-activity from skeletal muscle damaged by surgery. Taking also into account the various calculation methods used by different authors, overestimates as large as 10-20 gram-equivalents of lost myocardium after uncomplicated bypass surgery, as published in the literature, can be explained.     

Creatine kinase MB isoforms in patients with skeletal muscle injury: ramifications for early detection of acute myocardial infarction.

We measured total creatine kinase (CK), CK-MB isoenzyme, and the MB isoforms in 202 serum and plasma samples from nine groups of patients and normal individuals: 39 with acute myocardial infarction (MI), divided according to time between the onset of chest pain and blood collection (1-6 h, 7-12 h, and 13-48 h); 26 with chest pain for whom an MI was ruled out, sampled at admission; 17 undergoing bypass surgery or cardiac catheterization, sampled within 6 h after either procedure; 17 with acute skeletal muscle injury, sampled within 8 h after injury; 30 marathon runners immediately after a race; 17 runners and other athletes > 12 h after training or a race; 12 with cerebral injury or seizures, sampled at admission; 8 with closed head injury, sampled at admission; and 38 normal subjects. CK-MB (relative index) and MB isoforms (MB2/MB1) were respectively increased in 15% and 75% of MI patients 1-6 h after onset, 94% and 94% after 7-12 h, and 88% and 8% after 12 h, and in 87% and 82% of cardiac surgery patients. MB isoforms were increased in most patients with acute skeletal muscle trauma and in subjects examined after exercise, but were within normal limits in patients for whom MI was ruled out, patients with cerebral trauma, and normal individuals. The relative index of MB/total CK was normal in essentially all individuals in the last groups, including those with acute skeletal muscle trauma. We concluded that the CK-MB isoform ratio is increased in both acute skeletal muscle injury and MI. The isoform ratio is most useful for distinguishing recent from old (> 12 h) injury.     

Diagnostic use of CK-MM and CK-MB isoforms for detecting myocardial infarction

Following acute myocardial infarction, total CK and CK-MB levels begin to rise 5 to 6 hours after the onset of chest pain. The serial profile of the rise and fall of both activities is nearly always indicative of AMI. The recent increase in the use of thrombolytic agents in an attempt to attain reperfusion of occluded coronary arteries alters the enzyme profiles observed in blood after AMI. After successful reperfusion a washout phenomenon occurs in which early restoration of blood flow to damaged myocardium causes an early rise in total CK and MB levels above the normal range 2 to 4 hours after AMI, with earlier and higher peak enzyme values. Recently reports have appeared describing numerous serum and plasma CK-MM and CK-MB isoform patterns after AMI. Following release from injured myocardium CK-MM3 and CK-MB2 (designated the tissue isoforms) are converted in the circulation to post-translation products (MM2, MM1, MB1, respectively). Studies have now shown that CK-MM isoform patterns provide a unique means of assessing the time of onset of necrosis and a monitor of the duration of enzyme release from the site of injury. Following AMI, MM3, the MM3/MM1 ratio, or both rises and peaks earlier than either total CK or CK-MB levels. During successful reperfusion, the rate of rise of CK-MM3 is more rapid and the MM3/MM1 ratio peaks earlier than without reperfusion. However, any concomitant release of CK-MM3 from skeletal muscle would decrease the clinical utility of MM isoforms in detecting myocardial damage. Recent advances in technology have shown that CK-MB2 rise parallels the CK-MM increase and also rises earlier than total CK and total MB levels and provides increased specificity for the myocardium. The full potential of the diagnostic utility of MM and MB isoforms will not be realized until a reliable, sensitive, simple, and rapid quantitative assay becomes available.