INTRODUCTION
Cardiac troponins challenge enzymes as cardiac markers. The combination of myoglobin and cardiac troponin I (cTnI) or T (cTnT) is a powerful combination for diagnosing myocardial damage in the routine laboratory and this panel considerably improves the efficiency of the laboratory in the detection of myocardial damage (1). However there are still some unresolved issues, and an enzyme marker, i.e. glycogen phosphorylase isoenzyme BB (GP-BB), based on its function as a key enzyme of glycogenolysis may help to solve some of the remaining shortcomings of current routine diagnosis (2). One of these limitations is that in the final analysis creatine kinase (CK)-MB mass, CK isoforms, myoglobin, cTnI and cTnT all are not sufficiently sensitive within the first 3-4 h after the onset of infarct-related symptoms (1). They all have a roughly comparable early sensitivity for acute myocardial infarction (AMI), and all are markedly more sensitive than CK and CK-MB activity measurements (1). Nonetheless they are not suitable markers for the very early diagnosis of AMI (1,3,4). The diagnostic performance of the electrocardiogram (ECG) is still clearly superior to that of biochemical markers during the first 3 h after chest pain onset. However, it is noteworthy that thereafter biochemical markers were more accurate for AMI diagnosis than the ECG (3,4). But still up to 40-50% of patients who are subsequently diagnosed as having sustained an AMI may have non-diagnostic ECG with no or non-specific changes at hospital admission, and there is certainly need for other alternative more accurate diagnostic methods (3,4). A laboratory parameter with a sufficiently high sensitivity within the first 3 h from chest pain onset could be a diagnostic breakthrough. Another disadvantage of all currently available markers is that they do not specifically indicate ischaemic myocardial damage, because they are released whenever cardiomyocytes are damaged, e. g. by inflammation, toxins or trauma, and not only after ischaemic myocardial damage.

BIOCHEMISTRY OF GLYCOGENPHOSPHORYLASE
Glycogen phosphorylase (GP) is one of the best studied enzymes in biochemistry (2). It is a glycolytic enzyme which plays an essential role in the regulation of carbohydrate metabolism by mobilization of glycogen. It catalyses the first step in glycogenolysis in which glycogen is converted to glucose-1-phosphate. The physiological role of muscle phosphorylase is to provide the fuel for the energy supply required for muscle contraction. Its activity is allosterically regulated by the binding of adenosine monophosphate (AMP) and phosphorylation (2). Phosphorylase kinase converts GP b into its more active form GP a.
GP exists in the human cardiomyocyte in association with glycogen and the sarcoplasmatic reticulum (SR) and forms a macromolecular complex (5,6), the so-called SR-glycogenolysis complex (Fig. I). The degree of association of GP with this complex depends essentially on the metabolic state of the myocardium. With the onset of tissue hypoxia, when glycogen is broken down and disappears, GP-BB becomes soluble and can move from the peri-SR compartiment directly into the extracellular fluid, if the cell membrane permeability is simultaneously increased (5-8). A high GP-BB concentration gradient is immediately formed in the compartment of the SR-glycogenolytic complex. This may be the reason for the high efflux rate of this enzyme. In contrast to other cytosolic proteins, this gradient may be at least partly also be realized via the T-tubuli system. Interestingly, in experimental studies and in patients with AMI the released GP-BB was exclusively found in its b form (8).
GP exists as a dimer under normal physiological conditions. The dimer is composed of two identical subunits. Three GP isoenzymes are found in human tissues, they are named after the tissue in which they are preferentially expressed, GP-LL (liver), GP-MM (muscle), and GP-BB (brain) (2). Adult human skeletal muscle contains only one isoenzyme, GP-MM. GP-LL is the predominant isoenzyme in human liver and all other human tissues except for heart, skeletal muscle, and brain. The isoenzyme BB is the predominant isoenzyme of human brain. Its molecular weight as a monomer is approximately 94 kD. In the human heart the isoenzymes BB and MM are found, but GP-BB is the predominant isoenzyme in myocardium as well.
The three human isoenzymes are encoded by three distinct genes and have a different structure (9). The proteins are 846 (LL), 842 (MM) and 862 (BB) amino acids long. Amino acids 1-830 match and differences are mainly found at the C-terminus. The C-terminal end of the protein is the catalytic domain. Consequently the three different isoenzymes also differ in functional properties. These functional differences of GP isoenzymes, for example, reflect the differences of metabolism in heart and skeletal muscle. In pairwise sequence comparison the brain-type protein is 80% identical to the liver-type and 83% identical to the muscle-type. GP-BB has 21 and 16 additional amino acid residues on its C-terminal portion that are not present on the MM and LL isoenzymes, respectively. It was therefore possible to produce specific antibodies for the detection of the human GP-BB isoenzyme (10,11).
The highest amounts of GP-BB are found in human brain and heart, where its concentrations are comparable (11). Although immunoblot, electrophoresis, and northern blot data are partly conflicting, there is evidence that GP-BB isoenzyme might not be restricted to brain and heart in humans (2,9,11,12). Lower GP-BB concentrations have been reported in leukocytes, spleen, kidney, bladder, testis, digestive tract, and aorta. However, in all these tissues GP-LL is by far the predominant isoenzyme.

EFFLUX OF GP FROM THE MYOCARDIUM AFTER HYPOXIA OR SUBSTRATE DEPLETION
The release of GP from the myocardium after hypoxia or substrate depletion has been studied in isolated perfused rat and rabbit hearts. The GP release in these experiments correlated with the remaining myocardial glycogen content. In the same experimental model the addition of imipramine under aerobic conditions to a cardioplegic perfusion solution only caused a release of CK, but not of GP. The myocardial glycogen content remained uneffected as well. It is well established that imipramine in a certain concentration range causes a selective increase in the plasma membrane permeability without leading to myocardial hypoxia. On the other hand, the stimulation of glycogenolysis by high doses of epinephrine did not cause a decrease in myocardial GP activity, although the glycogen content of the tissue was greatly diminished by the added epinephrine (13-15). These experimental results allow to conclude that the release of GP-BB requires both conditions, a burst in glycogenolysis and a concomitantly increased plasma membrane permeability. Selective stimulation of glycogenolysis or selective induction of plasma membran damage is not sufficient for rapidly releasing GP from myocardium.

CLINICAL RESULTS
Acute myocardial infarction
In a pilot study on the early sensitivity of GP-BB for AMI diagnosis distinct differences in early sensitivities of GP-BB in comparison with myoglobin, CK-MB mass, CK and cTnT were noted within the first 2-3 h after AMI onset (10). In the majority of AMI patients, GP-BB increased between 1 and 4 h after the onset of chest pain. Therefore, GP-BB may be a very important marker for the early diagnosis of AMI. In a German multicenter study (unpublished results) the early sensitivities of myoglobin and GP-BB for AMI diagnosis were found to be equivalent. However, in this study a different assay was used for GP-BB determination than originally developed by Rabitzsch et al. (10). According to these preliminary results, GP-BB is at least as sensitive than myoglobin or heart fatty acid-binding protein. GP-BB usually peaks before CK, CK-MB or cTnT and returns within the reference interval within 1-2 days after AMI onset. A comparison of GP-BB and myoglobin time courses during the acute phase of AMI is shown in Figure II. Similar to soluble markers, such as myoglobin and CK-MB, it was demonstrated that GP-BB time courses in AMI patients are markedly influenced by the fact whether early reperfusion of the infarct-related coronary artery occurs. The well established so-called "washout" phenomenon after successful thrombolysis leads to a more rapid increase in GP-BB, earlier and usually higher peak values (10). Therefore, GP-BB may be useful, alongside with other soluble myocardial proteins, to non-invasively assess the effectiveness of thrombolytic therapy.

Acute coronary syndrome
The application of GP-BB is not restricted to conventional AMI. As demonstrated by receiver operator characteristics (ROC) curve analysis the diagnostic performance of GP-BB to detect acute coronary syndrome at hospital admission was significantly greater than that of CK and CK-MB. An early release of GP-BB was demonstrated in patients with Braunwald class III unstable angina who showed ST-T alterations at rest in the admission ECG. In these patients who all presented with a short delay from chest pain onset all markers were significantly higher than in the rest (16). But of all tested markers only GP-BB was increased above the upper reference limit in the majority at hospital admission.

Coronary artery bypass grafting (CABG)
GP-BB is also a sensitive marker for the detection of perioperative myocardial damage and infarction in patients undergoing CABG. GP-BB more accurately reflects myocardial ischemia than CK-MB does. Due to inevitable myocardial ischemia and damage, GP-BB also increases moderately in uncomplicated patients. However, GP-BB rapidly returns to baseline values (17). In contrast to CK-MB, a correlation was found between aortic crossclamping time which reflects myocardial ischemia in these patients and GP-BB release. GP-BB also more accurately reflected ischemic myocardial damage than CK-MB in patients with emergency CABG (17).

Diagnostic specificity
GP-BB is not a heart-specific protein and thus its specificity is limited. However, increases in GP-BB are specific for ischemic myocardial damage when damage to the brain can be excluded. The diagnostic specificity of GP-BB for myocardial damage in non-traumatized chest pain patients was in the range of CK-MB (10). This suggests sufficient diagnostic specificity in clinical settings in which GP-BB determination would be ordered by the physician.

CONCLUSION
Although, these first clinical results have to be confirmed in a larger number of patients, there is no doubt that GP-BB is an interesting and promising marker for the detection of ischemic myocardial damage. This is explained by its function as a key enzyme of glycogenolysis. GP-BB has a high early diagnostic sensitivity for the detection of acute coronary syndrome and could be useful for early risk stratification in these patients. GP-BB was also a sensitive marker for the detection of perioperative myocardial ischemia and infarction in patients undergoing CABG. The early GP-BB release is a specific marker of ischemic myocardial damage. The diagnostic specificity of GP-BB seems to be sufficient for clinical practice. A future scenario for the laboratory testing for myocardial damage could be the combination of a highly specific marker, such as cTnI or cTnT, and GP-BB measurement, which combines cardiac specificity with high early sensitivity and specificity for ischemic myocardial damage.

REFERENCES

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RELAZIONI

Approccio clinico al paziente con sospetta sindrome coronarica acuta
M. Galvani*, D. Ferrini, F. Ottani
Unità di Ricerca Cardiovascolare, Fondazione Myriam Zito Sacco, Forlì
Divisione di Cardiologia, Ospedale G.B. Morgagni, Forlì

Gli "enzimi cardiaci" nell'era delle troponine: cosa salvare
M. Panteghini*, C. cuccia, F. Pagani, G. Bonetti
Laboratorio Analisi Chimico Cliniche 1, Spedali Civili
Divisione di Cardiologia, Spedali Civili e Cattedra di Cardiologia, Università, Brescia

CK-MB: dalla misura dell'attività catalitica a quella della concentrazione proteica
M. Zaninotto
Servizio di Medicina di Laboratorio, Azienda Ospedaliera di Padova, Padova

La determinazione delle isoforme della CK-MB nel nuovo panorama biochimico
Sara Altinier
Centro di Ricerca Biomedica, Castelfranco Veneto (TV)

Glycogen phosphorylase isoenzyme BB: a novel enzyme marker for the diagnosis of ischemic myocardial damage
Johannes Mair
Institut für Medizinische Chemie & Biochemie, University of Innsbruck, Austria

Abstracts

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