Myocardial Markers

Release of Intracellular Contents Following Cell Death

Myocardial infarction results in death of myocardial cells served by the effected artery after about 30 minutes of anoxia. The acute inflammatory response, as a consequence of cell death, results in fever, leukocytosis, and increased concentrations of acute phase reactant proteins in the circulation (determined in the clinical laboratory most commonly by the ESR but better by measurement of C-reactive protein). An earlier and more specific consequence of myocardial death is the liberation of intracellular contents and their appearance in the circulation several hours later following diffusion through the interstitium into patent vessels and lymph ducts. Intracellular markers, routinely determined by laboratory testing, are certain enzymes present at high activity in the tissue. The enzymes routinely measured in the clinical laboratory for the purpose of diagnosing and monitoring myocardial infarction include creatine kinase (CK), aspartate amino transferase (sGOT or AST), and lactate dehydrogenase (LDH). These enzymes are present in sufficiently high content in myocardial tissue so that the death of a relatively small amount of tissue results in a substantial increase in measured enzyme activity in serum.

Myocardial cells must die in order for intracellular contents to be released. Brief ischemic episodes (angina pectoris) do not result in the liberation of intracellular material. In contrast, hepatocytes immediately release intracellular proteins upon anoxic insult.

Time Course of Myocardial Enzyme Elevations in Serum Following Infarction

The change in activity of each enzyme in serum following myocardial infarction exhibits a characteristic time course as illustrated in the figure to the right:

Serum CK activity is noticeably elevated earliest, about 6 hours after MI, with maximum activity peaking at about 24 hours and returning to normal within about 2-3 days. Serum activity of AST is noticeably increased after about 12 hours, peaks between 1-2 days, and returns to normal by the 3rd to 5th day. LDH activity increases last, after about 18 hours post MI, peaks between the second and third day and remains elevated for about a week.

Factors Accounting for the Time Course

The properties responsible for the time course of the elevations are
1) the rate of diffusion through damaged tissue into the circulation and
2) the circulating lifetime of the enzymes.

Diffusion Rate and Molecular size
Diffusion rate is determined by molecular size and is related to molecular weight. The molecular weight of CK is 80 KD, of AST is 90 KD, and of LDH is 140 KD. It is evident that CK diffuses out of the necrotic region earliest and most rapidly and LDH latest and most slowly.

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Circulating Lifetime
The approximate circulating half lifes of the different enzymes and isoenzymes are shown below:

	    mitochondrial AST -------  1 hr.
	    LDH-5 (M4)        ------- 12 hrs.
	    CK                ------- 18 hrs.
	    cytoplasmic AST   -------  1 day
	    ALT               -------  2 days
	    LDH-1 (H4)        -------  5 days

The disappearance rates of the different enzymes following their increase from MI are consistent with what is known about their degradation rates and the isoenzyme distribution in cardiac tissue.

The characteristic time course of the enzyme elevations is clinically useful since it demonstrates that the extent of the elevation of a particular enzyme in serum depends upon the time period between when a blood specimen is drawn and when the MI occurred. In a blood specimen drawn only several hours after an MI, CPK provides the most sensitive diagnostic information and may be the only enzyme which is elevated. If MI is not associated with marked chest pain, a not uncommon occurrence, and several days elapse before the symptoms of feeling tired and run down cause a visit to the physician, then LDH may be the only enzyme which is elevated.

Serum enzyme activities are routinely monitored during the post MI period in order to evaluate patients' progress and to obtain diagnostic information about possible complications. Reinfarction will cause the enzymes to increase again, with CK again increasing earliest and LDH last. Heart failure is a complication which is indicated by rapid increases in AST, ALT and LDH (but with no CK increase) from passive liver congestion. When adequate cardiac output is reestablished, LDH activity decreases rapidly in such cases because of the short circulating lifetime of the major liver LDH isoenzyme (LDH5 or M4).

Determination of IsoEnzymes Improves Specificity

The relatively high content of CK, AST and LDH in cardiac tissue accounts for the clinical usefulness of measureing the activity of these enzymes in serum to diagnosis and monitor MI. However, other tissues are also richly endowed with these enzymes. LDH, which catalyzes the terminal step in glycolysis, is present in all cells since glycolsis is a universal metabolic process. Skeletal muscle has a particularly high content of LDH, AST and CPK. Liver has a high content of LDH and AST. Erythrocytes have appreciable LDH and AST activity. It is evident that elevations of these three enzymes are not particularly specific for myocardium.

Improved specificity is provided by determining isoenzymes because isoenzyme distributions are sometimes signficantly different for different tissues and a particular isoenzyme distribution is highly characteristic of a particular tissue.

AST Isoenzymes

AST isoenzymes are of no clinical value. AST activity is exhibited by two isoenzymes; one from the cytosol and the other of mitochondrial origin. Almost all serum AST activity is due to the cytosol isoenzyme, even after damage to tissues with substantial mitochondrial isoenzyme content. Since the mitochondrial isoenzyme has such a short circulating lifetime, its activity is rapidly eliminated.

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LDH Isoenzymes

LDH is a tetramer composed of two different subunits, M and H. Five different isoenzymes are possible: M4, M3H, M2H2, MH3, and H4, and all five are present in different tissue in different relative amounts. The five different isoenzymes are resolved by electrophoresis and determination of the LDH isoenzyme distribution by electrophoresis is a commonly performed clinical laboratory test. The predominant LDH isoenzyme in liver and skeletal muscle is M4 (LDH-5) and the predominant cardiac isoenzyme is H4 (LDH-1). The LDH isoenzyme distribution in normal serum is characterized by the major band being MH3 (LDH-2). When total LDH activity is increased from muscle or liver disease, the LDH-5 band becomes dominant. Increased LDH activity from MI is characterized by a predominance of the LDH-1 band. The change from the dominance of the LDH-2 band in normal serum to the dominance of the LDH-1 band is called the "LDH flip" and its occurrence is considered diagnostic for MI. However, the major LDH isoenzyme in erythrocytes is LDH-1 so that hemolysis, either intravascular or in the collected blood specimen, may also cause the LDH flip.
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CK isoenzymes:

CK is a dimer composed of two different subunits, B and M. The three isoenzymes are designated MM, MB and BB. The CK isoenzymes are resolved by electrophoresis but many hospital laboratories conduct CK isoenzyme analysis by a simple procedure involving the use of an antibody against the M subunit which inhibits M activity while the activity of the B subunit is retained.
The BB isoenzyme is present in brain and intestinal smooth muscle, but the BB isoenzyme is rarely present in serum, even in cases of nervous system or intestinal disease. The major CK isoenzyme of both skeletal muscle and myocardium is MM, and both tissues contain the MB isoenzyme. The ratio of MB/MM is, however, different for each tissue. The skeletal muscle content of MB is less than 1% of total CPK activity, whereas the myocardial content of MB is about 25-50% of total CK activity. Normal serum activity is almost entirely from the MM isoenzyme and the MB content of normal serum is usually undetectable. Degenerative muscle disease or crushing muscle injury results in elevated total serum CK activity, but the MB isoenzyme content is less than 5%. Serum MB activity greater than 5% of total is considered diagnostic for MI.

CK and/or LDH isoenzyme determinations are useful when there is question about the tissue source of elevated enzyme activity.

Correspondence Between CK and LDH Isoenzyme Findings

The correspondence between CK and LDH isoenzyme findings in different conditions is illustrated in the figure to the right.

Sample #3 represent results for a control specimen to assure that the technique is working properly and that all bands present are detected.

Sample #8 results are from a normal specimen.

Sample# 1 results are from an MI patient. The specimen was collected at a time when the activity of both LDH and CK were elevated Note the LDH flip and the high relative activity of the MB isoenzyme.

Sample# 2 results are from an MI patient who experienced chest pain only several hours previously. Total CK is significantly elevated with a high relative MB isoenzyme activity.

Sample# 4 results are from a patient with liver disease. Although the LDH isoenzyme pattern is indistinguishable from muscle disease or injury, the absence of at least a trace of CK-MB isoenzyme is inconsistent with the muscle CPK isoenzyme distribution as is the apparently normal total activity.

Sample# 5 results are from an MI patient; the specimen was collected about 2 days post MI so that CK has almost returned to normal activity and the LDH flip is definite.

Sample# 6 results are from an MI patient with the specimen collected about the 1st day post MI; CK activity is definitely elevated with a high relative MB isoenzyme activity and the LDH flip is evident.

Sample# 7 results are from an MI patient with complications of heart failure and passive liver congestion or the patient was involved in an accident as a consequence of the MI, and suffered a crushing muscle injury.

Future Markers for MI

Other intracellular proteins are potentially useful markers for MI. The time course of three, recently proposed, markers are shown, in comparison to that of CK-MB, in the figure to the right.

Myoglobin is a monomeric, low molecular weight, intracellular oxygen transport protein which becomes elevated as early as 4 hours post MI. Myoglobin, however, is not specific for myocardium.
CK-MB isoforms; there are two forms of CK-MB:
- CK-MB2 is the form recently released from tissue and is distinguished by the presence of a C-terminal lysine.
- CK-MB1 is the form present in the circulation for a sufficient period of time for cleavage of the C-terminal lysine by carboxypeptidase.
The two forms are resolved by a specialized piece of electrophoretic equipment. The finding of a considerable fraction of recently released CK-MB2 is diagnostic for MI. CK-MB2 isoform is elevated as early as myoglobin and is more specific. However, the need for specialized electrophoretic equipment is a disadvantage.
TroponinI is a myocardial specific myofilament component. A small amount is free in the cytosol and accounts for relatively early release from dead cells. The slower release of the myofilament component provides for detection for as long as a week following MI. TropininI is myocardial specific, is released as early as CK-MB and remains elevated for as long as LDH and has the potential to serve as a sole marker for MI.