Creatine Kinase Isoenzymes and Troponins in MI Workshop
What are the steps of a typical enzyme immunoassay?
1. Put patient sample into incubation well coated with anti-CK-MB antibody 2. After incubation and washing of the well, enzyme labeled anti-CK-M antibody is added to form an immunocomplex between CK-MB and the two antibodies. 3. Washing the well removes unbound CK-MM and CK-BB, but immunobound CK-MB remains 4. Enzyme substrate is added and a reaction between the enzyme labeled immunocomplex takes place, which results in a product that is highly fluorescent 5. Measurement of the intensity of fluorescent is quantitatively related to the concentration of CK-MB in the patient's serum.
What are nonspecific reactions to myocardial injury associated with?
1. polymorphonuclear leukocytosis appears within a few hours after the onset of pain, persisting for 3 to 7 days, reaching levels of 12k to 15k leukocytes/μl 2. Erythrocyte sedimentation rate rises more slowly than the white blood cell count, peaking during the first week, and remaining elevated for up to 1-2 weeks.
How many isoenzymes does CK have?
4 isoenzymes 3 cytoplasmic 1 mitochondrial Each isoenzyme is a dimer composed of two subunits of the M (muscle type), Mt (mitochondrial) type, or Brain (B) type.
What is the timeline for CK activity appearing in blood?
4 to 6 hours after onset of chest pain, reaching peak at 24 to 36 hours. It returns to baseline after 72 hours. Peak levels typically 10 to 20 times the upper limit of normal.
What is the current european society guidelines for diagnosis of MI?
A focus on cardiac troponins: 1. Changes in specific biomarkers: - typical rise or gradual fall of cardiac troponin or - more rapid rise and fall of CK-MB with at least one of the following: 2. Clinical presentation - ischemic symptoms - development of pathologic Q waves on the ECG - ECG changes indicative of ischemia (ST segment elevation or deprssion) or - coronary artery intervention (angioplasty)
What predominant isoenzyme of CK is in the brain and colon?
BB This CK is also called *CK1* It does not get past the blood brain barrier and therefore cannot show up in serum.
Why was CK used as a diagnostic test?
Based on principle that CK can be released and appears in blood whenever cell membrane damage occurs
Why is Ck-MB not as specific for cardiac injury?
Because a small amount is found in skeletal muscle. CK-MB is not a good test!!!
Why is it possible to use CK2 (CK-MB) as a diagnostic for MI?
Because significant amounts of MB are found only in the heart, so a rise in percentage of MB (CK2) above normal levels is very specific for acute infraction.
Why are serum enzymes/cardiac specific proteins a good way to diagnose MI?
Because they are released in large quantities into the blood from necrotic heart muscle following MI. The temporal pattern of enzyme release is of diagnostic importance.
How quickly can ELISA detect the heart isoforms?
Between 5 to 30 minutes.
What does CK do in the mitochondria?
CK reacts with creatine and ATP to form ADP and creatine phosphate (CP) ADP is recycled into the mitochondria and CP diffuses into the cytoplasm, where it is a substrate for cytoplasmic CK. The cytoplasmic CK catalyzes the reaction between CP and ADP, to form ATP
What form of creatine kinase is cardiac specific?
CK-MB
What is another reason MB isoenzyme can be elevated?
CK-MB (CK2) could be elevated during skeletal muscle regeneration.
What are troponins?
Cardiac troponin I (cTn-I) and T (cTn-T) are cardiac regulatory proteins that control the calcium mediated interaction of actin and myosin. Both have cytosolic or early releasable and structural pools, with most existing in the structural pool
How is mitochondrial CK (mtCK) and cytosolic CK connected?
Creatine-phosphocreatine (PCr/Cr)-shuttle or circuit. The PCr generated by mtCK in mitochondria is shuttled to cytosolic CK that is coupled to ATP-dependent processes (ATPases). The cytosolic CK renenerates ATP, which can be used by the ATPase that it is bound to.
What is the behavior of creatine kinase (CK) after an MI?
creatine kinase (CK) rises within 8-24 hours and generally returns to normal by 48-72 hours, unless it is a big infarction, in which case it takes longer *Cardiac Non-specific*
What does 800 U/l of total CK indicate?
moderate sized infarction.
What is cardiac injury?
Disruption of normal cardiac myocyte membrane integrity resulting in the release into the extracellular space of intracellular constituents including detectable levels of biologically active cytosolic and structural proteins such as troponin, creatine kinase, myoglobin, heart type fatty acid binding protein, and lactate dehydrogenase. Injury is usuaully irreversible.
What does electrophoretic separation delinate when testing for isoforms of MM/MB?
Electrophoretic separations only delinate two forms: 1. MB2 represents the tissue form and lysine minus B chain 2. MB1 includes the small amount of lysine minus M chain-lysine plus B chain and the conversion product.
T/F: CK-MB is the only isoenzyme with multiple isoforms
False. CK-MM (CK3) also has two isoforms.
What does troponin T (Tn-T) do?
IT is the tropomyosin-binding component. Two isoforms of Tn-T exist in adult human cardiac tissue: Tn-T1 and Tn-T2. There are two additional isoforms in fetal heart tissue.
What are the levels of CK-MB that indicates pathologies?
If MB < 1% of total serum CK - Indicative of skeletal muscle nerosis If MB > 3% of total CK - indicative of MI If MB ~1.5% of total CK - Normal levels
What is acute myocardial infarctoin?
Imbalance between myocardial oxygen supply and demand resulting in injury and eventual death of myocytes. Interrupted blood supply to the heart leads to gross necrosis of the myocardium.
What is the difference between immunochemical separation vs column and electrophoretic methods?
Immunoassays measure mass isoenzyme concentrations Column and electrophoretic methods measure enzymatic activity.
Where is the most CK activity in the body found?
In the skeletal and heart muscles. Muscle CK functions within the cytoplasm and mitochondria of the cell.
What are the isoforms of CK-MB (CK2)? What are their differences?
Isoform 1 and Isoform 2 They result from postsynthetic modification of the primary protein structure of CK-MB.
What is CPaseN?
It is a serum protease, carboxypeptidase-N, that sequentially cleaves the two lysines off of MB and MM isoenzymes.
How does CK-Mt compare to cytoplasmic CK?
It is also a dimer of two identical subunits of Mt type. The molecular mass of CK-Mt is idential to that of cytoplasmic isoenzyme, but the amino acid composition, electrophoretic mobility, and chromatographic and immunological properties are different. A unique property of CK-Mt is its ability to form aggregates of high molecular mass.
What does Creatine Kinase do?
It is an energy transfer enzyme that catalyzes the reversible biochemical reaction of transformation of creatine into creatine phosphate.
When is troponin I, Tn-I, elevated?
It is eleveated 4 hours after MI It stays up for 7 days in 60% of patients *Cardiac specific*
What is the ratio of MM to MB in normal serum?
It is similar to the ratio found in skeletal muscle, 0-2% CK2 (MB) and the rest MM (CK3)
What does troponin C (Tn-C) do?
It is the calcium binding component. Only two major isoforms of Tn-C are found in the human heart and skeletal muscles. The heart isoform is identical with the slow twitch skeletal muscle isoform.
What does troponin I (Tn-I) do?
It is the inhibitory subunit. This subunit is found only in the heart. Elevated serum levels of the cardiac isoform of Tn-I is also predictive of adverse effects of unstable angina or MI.
What is the traditional means of estimating infarction size?
It is traditionally estimated using total CK rather than MB, partially due to very rapid rise and fall in MB values following an infarction.
Where is CK-MB2 found?
It originates from cardiac tissue and is named according to its relatively slow anodic migration in electrophoresis.
What is the behavior of lactate dehydrogenase (LDH) after an MI?
It rises later, at 24-48 hours, and remains elevated for up to 7-14 days. *cardiac non-specific*
What does electrophoretic testing do to identify isoenzymes of CK?
It separates CK isoenzymes in an electrical field by differences in their net charge on cellulose acetate, agarose, and polyacrylamide gels.
What predominant isoenzyme of CK is in heart muscle?
MB - This is the most specific for MI Also called *CK2*
What is the difference between isoforms MB1 and MB2?
MB1 isoform is more negatively charged than MB2 (because the lysine has been cleaved off)
What predominant isoenzyme of CK is in skeletal muscle?
MM This CK is also called *CK3* About 0-2% of skeletal muscle CK is also MB isoform: CK2
What is the predominant CK in the heart?
MM (CK3), but cardiac muscle form (MB) makes up 10-15% of CK in heart muscle.
What are isoenzymes?
Multiple forms of an enzyme family that catalyzes the same biochemical reaction but differ in their physical and chemical properties.
Where is Mt CK mostly found?
Muscle, brain, and colon
What are normal levels of CK and MB?
Normal levels of total serum CK is ≤ 130 U/l Normal levels of MB ≤ 3 U/l - the rise in absolute MB value above normal is diagnostic of myocardial necrosis
When is LDH levels useful to test?
Only when initital CK or CK-MB elevation might have been missed.
When is Tn-T2 elevated?
Serum levels of Tn-T2 increase within 4 hours of acute myocardial infraction and remains high for up to 14 days.
When is CK-MB (CK2) have most diagnostic use?
The CK-MB isoforms have greater diagnostic value (especially as cardiac markers during early periods after myocardial infarction) in the range of 3-6 hours after the onset of chest pains.
How reliable is Tn-T2 for detection of myocardial infarction?
The appearance of Tn-T2 in serum is 100% sensitive and 95% specific for detection of MI. Levels of this rise within 0-4 hours, generally returning to normal by 14 days unless there is recurrance. *Cardiac Specific*
What does the cleavage of lysine from MB isoforms do?
The cleavage of the lysine changes the charge but not the enzymatic activity of the molecule.
What are the differences between CK MM, MB, BB?
The cytoplasmic isoforms, MM, MB, and BB catalyze the same reaction with minimal differences in kinetic properties, such as pH optimum and substrate affinity. Their differences exist in thermal stability, chromatographic behavior on ion-exchange columns, electrophoretic mobility, and immunological properties.
What is the behavior of serum aminotransferases (AST and ALT) after an MI?
Their time course of elevation is intermediate between CK and LDH, so it doesn't have much advantage and it is *Cardiac non-specific*
What are the subunits of troponin?
There are 3 troponin subunits: Troponin C (Tn-C) Troponin I (Tn-I) Troponin T (Tn-T)
How can you tell the difference between cTn-I and cTn-T from their respective skeletal muscle isoforms?
They are encoded by different genes and therefore have difference sequences, giving them cardiac specificity.
How well do electrocardiographic/pathologic correlations to MI?
They are excellent correlations and not very specific, so it should not be used as the sole basis for the diagnosis of infraction.
When are troponin elevations not specific for cardiac injury?
Troponin elevations are almost always specific for cardiac injury, except for the infrequent analytical false positives caused by fibrin interference and/or cross-reacting antibodies.
What is the preferred test for diagnosis of MI? Why?
Troponins are preferred for the diagnosis of MI because of their increased sensitivity and specificity compared to CK-MB and other markers. Troponin is more reliable for a consistent test than CK-MB because there is more of it in the heart per gram of myocardium and a greater percentage depleted from the heart by cardiac injury arrives in the blood.
