Download Faculty PBL IV

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Faculty PBL IV
Clinical Observations
You are a dental student during your one-week senior hospital orientation. While you were on a
general round with physicians, an obese, middle-aged man was brought to the emergency room
by police officers, who stated that the patient has been involved in an automobile accident. It
appeared that the patient had “blacked out” and caused collision. The patient stated that he had
been short of breath and very dizzy before the crash. Examination raised the suspicion of either a
cerebrovascular accident or myocardial infarction. The patient was admitted for observation, and
blood sample was taken for the assay of creatine kinase (CK) and other plasma enzymes and
forwarded to the laboratory for analysis.
Biochemical Questions
1. In which tissues is CK found and from which can it be readily released into the bloodstream?
2. Would you expect an increased serum or plasma CK activity following a cerebrovascular
accident (stroke)?
3. In determining CK activity, it is necessary to add mercaptoethanol or dithiothreitol to all buffers
and other reagents involved. What does this tell you about the structure of the enzyme?
4. Three major isozymes of CK can be detected by electrophoresis on polyacrylamide gels. The
most anodic of these is known as BB isozymes, the most cathodic is known as MM isozymes and
the intermediate band is known as MB isozymes. Relate these observations on the net charge on
M and B monomers (M stands for muscle and B for brain).
5. Is it possible to quantify the individual dimers by means other than electrophoresis?
6. Since myocardium is muscle, is it reasonable to assume that the major isozyme of myocardium is
of the form MM? Would you expect to find any MB isozymes in the myocardium?
7. Is it possible to relate the increase in serum CK activity to the extent of tissue damage?
8. When tissue damage has occurred and enzymes have been released into circulation, how does the
time course of change in CK activity compare to the time courses measurable for other enzymes.
9. What other assays might have been requested in the present instance?
Study Material:
http://library.med.utah.edu/WebPath/TUTORIAL/MYOCARD/MYOCARD.html
http://www.clinchem.org/cgi/content/abstract/41/9/1266
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=503059
Case Discussion
Myocardial Infarction (MI)
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The pathogenesis can include:



Occlusive intracoronary thrombus - a thrombus overlying a plaque causes 75% of myocardial
infarctions, with superficial plaque erosion present in the remaining 25%.
Vasospasm - with or without coronary atherosclerosis and possible association with platelet
aggregation.
Emboli - from left sided mural thrombosis, vegetative endocarditis, or paradoxic emboli from the
right side of heart through a patent foramen ovale.
In 2000, the European Society of Cardiology and the American College of Cardiology
Consensus group redefined myocardial infarction, with the definition being based on myocyte
necrosis as determined by troponins in the clinical setting of ischemia. The molecular events
during MI relate to the initial ischemic event, reperfusion, and subsequent inflammatory
response. Up to 6 hours following the initial ischemic event, most cell loss occurs via apoptosis.
After that, necrosis predominates. Ischemic endothelial cells express adhesion molecules that
attract neutrophils that subsequently migrate into damaged myocardium.
The gross morphologic appearance of a myocardial infarction can vary. Patterns include:
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Transmural infarct - involving the entire thickness of the left ventricular wall from endocardium
to epicardium, usually the anterior free wall and posterior free wall and septum with extension
into the RV wall in 15-30%. Isolated infarcts of RV and right atrium are extremely rare.
Subendocardial infarct - multifocal areas of necrosis confined to the inner 1/3-1/2 of the left
ventricular wall. These do not show the same evolution of changes seen in a transmural MI.
Laboratory Diagnosis of Myocardial Infarction
A number of laboratory biomarkers for myocardial injury are available. None is completely
sensitive and specific for myocardial infarction, particularly in the hours following onset of
symptoms. Timing is important, as are correlation with patient symptoms, electrocardiograms,
and angiographic studies.
The following biomarkers have been described in association with acute myocardial infarction:
Creatine Kinase - Total:
Creatine kinase (CK) is an enzyme that catalyzes the formation of ATP from ADP and creatine
phosphate:
Creatine-P + ADP
Creatine + ATP
The total CK is a simple and inexpensive test that is readily available using many laboratory
instruments. However, an elevation in total CK is not specific for myocardial injury, because
most CK is located in skeletal muscle, and elevations are possible from a variety of non-cardiac
conditions.
Creatine Kinase - MB Fraction:
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Creatine kinase can be further subdivided into three isoenzymes: MM, MB, and BB. The MM
fraction is present in both cardiac and skeletal muscle, but the MB fraction is much more specific
for cardiac muscle: about 15 to 40% of CK in cardiac muscle is MB, while less than 2% in
skeletal muscle is MB. The BB fraction (found in brain, bowel, and bladder) is not routinely
measured.
The creatine kinase-MB fraction (CK-MB) is part of total CK and more specific for cardiac
muscle that other striated muscle. It tends to increase within 3 to 4 hours of myocardial necrosis,
then peak in a day and return to normal within 36 hours. It is less sensitive than troponins.
The CK-MB is also useful for diagnosis of reinfarction or extension of an MI because it begins
to fall after day, so subsequent elevations are indicative of another event.
It is likely present in all tissues but is released into plasma following injury to some of
1. It is likely present in all tissues but is released at differential rates. The reason for differential
release is not clear. For example, CK is readily released following injury to the brain and the
muscle but not much is released from liver damage. Therefore, assays for CK are of particular
use in diagnosing myocardial infarction even in the presence of liver damage where estimation of
AST, ALT and LDH might be equivocal. As with other laboratory tests, there are concerns with
CK assays, as appreciable damage to major muscles would cause an increase in serum CK.
Repeated intramuscular injections and hypothyroidism may cause increase in serum CK as
would myopathy and neuropathy associated with chronic alcoholism. There are three isozymes
of CK; MM (from skeletal muscle), MB (from cardiac muscle) and BB (from brain).
2. One would expect an increase in serum brain isozymes (BB) following stroke.
3. The necessity of adding mercaptoethanol or dithiothreitol to the assay mixture suggests
that the enzyme contains cysteine residues, which need to be kept in reduced state for
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expression of full enzyme activity. CK contains Cys at the active site of the enzyme,
which is required for catalysis in the formation of ATP.
4. Electrophoresis on paper or on non-denaturing polyacrylamide gels would separate the
proteins on the basis of size and charge. The three isoenzyme are essentially of the same
size, so their separation would be based primarily on charge. BB isozymes being the most
anodic would carry a net negative charge on the molecule whereas MM enzyme being
least anodic or cathodic might carry a net positive charge or a negative charge, which is
less than BB. The isoenzyme, MB, is expected to carry a net charge on its molecule,
which is intermediate between MM and BB isoenzymes.
5. Since CK isoenzymes differ in terms of overall net charge, another method of separating
them is by glass beads coated with anion exchange resin. The isoenzymes would be
bound to the resin from which they could be eluted with high sodium chloride
concentrations. In fact, MB isozymes can be eluted with 3.8 M NaCl. The lack of
sensitivity and time constraints of analysis make the electrophoretic and ion exchange
method unsuitable as good diagnostic tools, so a radioimmunoassay has been developed
based upon [125I] labeled antibody to the B subunit. The antibody reacts with MB
isozymes but not with MM isozymes even when the latter is present at 20, 000 molar
excess. This immunoassay is very sensitive and requires small samples. In addition, this
method determines actual amount of the enzyme from antigen-antibody complex and is
not influenced by associated contaminants or enzyme inhibitors in plasma.
6. Myocardium is muscle but it is cardiac muscle and CK isoenzyme is MB and not MM. In
fact only source of MB in plasma is cardiac muscle so MB has been generally designated as
myocardial enzyme.
7. See Fig below as to other enzyme assays. LDH (lactate dehydrogenase) and HBDH (betahydroxybutyrate dehydrogenase) are other enzymes tested following myocardial
infarction. LDH is a tetramer of two non-identical chains (H and M) so it exists as 5
possible isozymes (H4 (LDH1), H3M (LDH2), H2M2 (LDH3), HM3 (LDH4), M4
(LDH5)). LDH1 and LDH2 are cardiac muscle specific isozymes. Increase in the ratio of
plasma specific activity (LDH1/LDH2) is an indicator of myocardial infarction. LDH is
also known as an indicator of silent myocardial infarction because elevated level of these
isozymes persists in plasma for over 2 weeks following the infarct.
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8.
It is important to remember that all three isozymes exist in the heart tissue. The
concentration of CK in normal myocardium is relatively constant as is the fraction of MB
isozyme. As seen in Fig. 3 the increase in plasma of LDH1/LDH2 increases and it is estimated
that the extent of this increase is indicative of size of the infarct.
9. See fig. above
Troponin:
Another protein (not an enzyme) which is an excellent indicator of myocardial infarction is
cardiac troponin I (cTnI). cTnI is the inhibitory protein which interacts with other troponin
components in a thin filament.
F-actin-----------Tropomycin
Tn-I……………..Tn-T
Tn-C
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Troponin is a spherical protein consisting of three subunits, Tn-T, a tropomycin binding subunit;
Tn-C a calcium binding subunit and TnI an inhibitory subunit. Each of these subunits interacts
with at least one other subunit. They are released into the bloodstream with myocardial injury.
They are highly specific for myocardial injury--more so than CK-MB--and help to exclude
elevations of CK with skeletal muscle trauma. Troponins will begin to increase following MI
within 3 to 12 hours, about the same time frame as CK-MB. However, the rate of rise for early
infarction may not be as dramatic as for CK-MB.
Troponins will remain elevated longer than CK--up to 5 to 10 days for troponin I and up to 2
weeks for troponin T. This makes troponins a superior marker for diagnosing myocardial
infarction in the recent past--better than lactate dehydrogenase (LDH). However, this continued
elevation has the disadvantage of making it more difficult to diagnose reinfarction or extension
of infarction in a patient who has already suffered an initial MI. Troponin T lacks some
specificity because elevations can appear with skeletal myopathies and with renal failure.
(Kumar and Cannon, Part I, 2009)
Myoglobin:
Myoglobin is a protein found in skeletal and cardiac muscle which binds oxygen. It is a very
sensitive indicator of muscle injury. However, it is not specific for cardiac muscle, and can be
elevated with any form of injury to skeletal muscle. The rise in myoglobin can help to determine
the size of an infarction. A negative myoglobin can help to rule out myocardial infarction. It is
elevated even before CK-MB. (Kumar and Cannon, Part I, 2009)
BNP:
B-type natriuretic peptide (BNP) is released from ventricular myocardium. BNP release can be
stimulated by systolic and diastolic left ventricular dysfunction, acute coronary syndromes,
stable coronary heart disease, valvular heart disease, acute and chronic right ventricular failure,
and left and right ventricular hypertrophy secondary to arterial or pulmonary hypertension. BNP
is a marker for heart failure.
CRP:
C-reactive protein (CRP) is an acute phase protein elevated when inflammation is present. Since
inflammation is part of atheroma formation, then CRP may reflect the extent of atheromatous
plaque formation and predict risk for acute coronary events. However, CRP lacks specificity for
vascular events.
References:
Kumar A, Cannon CP. Acute coronary syndromes: diagnosis and management, part I. Mayo Clin
Proc. 2009;84:917-938.
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Kumar A, Cannon CP. Acute coronary syndromes: Diagnosis and management, part II. Mayo
Clin Proc. 2009;84:1021-1036.