Chemical Analysis in Acute Myocardial Infarction - OVERVIEW

8/3/2019 Chemical Analysis in Acute Myocardial Infarction - OVERVIEW

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ACUTE MYOCARDIAL INFARCTION

Acute myocardial infarction is defined as death or necrosis of myocardial cells. The diagnosis of AMI was

established by WHO in 1979, requiring the presence of two or more of the following criteria:

1. History of severe and prolong chest pain2. Unequivocal electrocardiographic (ECG) changes such as persistent Q or QS waves and evolving

injury lasting longer than 1 day and

3. Unequivocal initial increase and subsequent decrease in activity of enzymes collected on serialbasis.

Because of the emergence of new biochemical marker such as cardiac troponin (cTn), the European

Society of Cardiology (ESC) and American college of Cardiology (ACC) redefined the criteria for diagnosis

of AMI:

1. Typical increase and gradual decrease of troponin or more rapid increase and decrease ofcreatin kinase CK-MB with at least on of the following:

Ischemic symptoms Development of pathologic Q waves on the ECG ECG changes indicative if ischemic Coronary artery intervention

2. Pathologic finding of an AMIThe more and new guidelines place more emphasis on biochemical markers, particularly cTn, asthe entry criteria for AMI detection relative to CK-MB because this assay has higher clinical and

analytic sensitivity for detection of myocardial damage.


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CARDIAC BIOMARKER

BIOMARKER

Is a substance used as an indicator of a biologic state. It is a characteristic that is objectively measured

and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic

responses to a therapeutic intervention.

CARDIAC MARKERS

Are substances (enzyme) released from heart muscle when it is damaged as a result of myocardial

infarction.

The importance of biomarkers:

1. Diagnosing acute myocardial infarction (AMI)2. Detecting myocardial damage whether due to AMI or other cardiac process3. Risk-stratifying patients4. Commenting on Prognosis5. Stressing interns, confusing residents and worrying cardiology fellows

The important biomarkers in diagnosis of acute myocardial infarction, AMI:

1. Creatine kinase (CK), total activity2. CK-MB isoenzyme, mass3. CK-MB isoforms and isoforms ratio4. Myoglobin (Mb)5. Cardiac troponin I (cTn I)6. Cardiac troponin T (cTn T)7. Lactate dehydrogenase (LDH)8. AST/ALT


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Enzymes in the myocardial cells:

Enzyme Description Physiological functions

Creatine kinase

- consists of two subunits, B (brain type) or M(muscle type), Making three different

isoenzymes: CK-MM, CK-BB and CK-MB

- Responsible for regeneration ofATP

Lactate

dehydrogenase

- Found in cytosol of all human cells- Have 2 subunit which are H (heart) and M

(muscle); combine to form 5 isoenzymes of

LD.

- Catalyze the reduction ofpyruvate to lactate using NAD

Myoglobin

- Ubiquitous small-size heme protein releasedfrom all damaged tissues

- Increases often occur more rapidly than TIand CK

- Not utilized often for AMI- assessment because of its very rapidmetabolism (short plasma half-life) causing

short burst increases that are difficult to

assess clinically

- its lack of specificity for cardiac tissue.

- Oxygen binding protein

Troponin

- is a complex of three regulatory proteins thatis integral to non-smooth muscle contraction

in skeletal as well as cardiac muscle

- Troponin is attached to the tropomyosinsitting in the groove between actin filaments

in muscle tissue

- Troponin has three subunits, TnC, TnT, andTnI

- Troponin-C binds to calciumions to produce a

conformational change in TnI

- Troponin-T binds totropomyosin, interlocking them

to form a troponin-tropomyosin

complex

- Troponin-I binds to actin in thinmyofilaments to hold the

troponin-tropomyosin complex

in place

Aspartate

aminotransferase

(AST)

- Used as surrogate markers of cellulardamage in the past. Very non-specific so not

used for assessment of myocardial damage

any longer

- Catalyze the transfer of anamino group between aspartic

acid and pyruvate to form

oxaloacetate and alanine

Aldolase

- An enzyme of the lyase group- Aldolase is present most significantly in

skeletal and heart muscle.

- Damage to skeletal muscle produces highserum levels of aldolase, particularly in the

case of progressive muscular dystrophy.

- Its effect is reversible cleavageof its substrate into two

compound without hydrolysis.

- It convert the fructose-1,6-diphosphate to

dihydroxyacetone phosphate

and G3P


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CURRENT CARDIAC MARKERS

CREATINE KINASE

Creatine kinase (CK/CPK) is an enzyme expressed in a number of tissues.

Function: it catalyses the conversion of creatine to phosphocreatine degrading ATP to ADP

Creatine kinase isoenzymesIn the cells, the cytosolic CK enzymes consists of two subunits which can be either B (brain types)

or M ( muscle type). There are therefore three different isoenzymes:

CK1=CK-BB=brain type

CK2=CK-MB=cardiac type

CK3=CK-MM=muscle type

Skeletal muscle express CK-MM (98%) and low level of CK-MM at 70% and CK-MB at 25-30%.

Diagnostic uses:

Clinically, creatine kinase is assayed in blood tests as a marker of myocardial infarction. Inmyocardial infarction, it begins to rise in 4-8 hours and reaches its maximum level in 12-24 hours

and return to normal level after two or three days.

Normal level:

Male: 38-174 U/L

Female: 26-140U/L

CK-MB (CK2) ISOFORMS

The primary indicator (gold standard) used to diagnose a heart attack because it exists in thehighest amount in the heart.

If CK-Mb makes up more than 5% of total CK level, a heart attack is suspected.

The CK-MB fraction exists in two isoforms called 1 and 2 identified by electrophoretic

methodology.

If one part of CK-MB (CK-MB2) is greater than another part (CK-MB1) by ratio of 1.5 or more,

then this is indicative that a heart attack has occurred.


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The mitochrondrial creatine kinase isoenzymes:

In addition to those three cytosolic CK isoforms, there are two mitochrondrial creatine kinase

isienzymes:

The ubiquitous CKMT1 (present in non-muscle tissues)The sarcomeric CKMT2 (present in sarcomeric skeletal and cardiac)

While mitochrondrial creatine kinase is directly involved in formation of phosphor-creatine from

mitochrondrial ATP, cytosolic CK regenerate ATP from ADP. This happens at intracellular sites where ATP

is used in the cell.

Picture show Mitochrondrial and

cytoplasmic isoenzyme of creatine

kinase. In the motochrondria, mitCK

catalyze the formation of creatine

phosphate; it is transported to the

cytoplasm for storage of high-energy

phosphate bonds. With muscle

contraction, cytoplasmic CK catalyze

the formation of ATP.


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Electrophoresis of Creatine kinase

Electrophoresis is the migration of charged molecules in a media upon application of an electric

field.

The rate of migration depends on the charged of the molecule, its molecular mass and the strength

of the electric field.Usually electrophoresis is used for the separation of protein. The most commonly used matrix is

agarose. Agarose is used mostly for the separation or larger macromolecules, including proteins

and their complexes. Protein can be visualized after electrophoresis by treating the gel with a stain

such as Coomassive blue.

The picture show the pattern of migration of

different creatine kinase isoenzymes. The

molecules being sorted are dispensed into a well

in the gel material. The gel is placed in an

electrophoresis chamber, which is thenconnected to a power source. When the electric

current is applied, the larger molecules of CK

isoenzyme move more slowly through the gel

while the smaller molecules move faster and

further away from the negative charge. The

different sized molecules of CK isoenzymes form

distinct bands on the gel.

The picture also show the normal pattern of CK

isoenzyme migration and also the pathologic

condition that suggest AMI.

A gel electrophoresis


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LACTATE DEHYDROGENASE

An enzyme that catalyzes the conversion of lactate to pyruvate. This is an important step in energy

production in cells. Many different types of cells in the body contain this enzyme. Some of the

organs relatively rich in LDH are the heart, kidney, liver, and muscle.

Increase absorption of UV maximum at 340 nm

Serum LDH-isoenzyme patterns are determined by the agar gel electrophoresis and quantitative

determination by densitometry.

Lactate dehyrogenase isoenzyme Locations

LDH-1 (4H) In the heart

LDH-2 (3H1M) In the reticuloendothelial system

LDH-3 (2H2M) In the lungs

LDH-4 (1H3M) In the kidneys

LDH-5 ( 4M) In the liver and striated muscle

LDH-2 is usually the predominant form in the serum. A LDH-1 higher than LDH-2 suggests AMI

The picture beside show a various

pattern of LDH serum

electrophoresis. From the picture

the normal pattern and pathologic

pattern (AMI) can be compared and

determined.


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TROPONIN

Troponin is a protein that is integral to muscle contraction in skeletal and cardiac muscle but not smooth

muscle. Troponin is attached to the protein tropomyosin and lies within the groove between actin

filament in the muscle tissue.

The function of troponin:

When the muscle cell is stimulated to contract by an action potential calcium channel open in the

sarcoplasmin reticulum and release calcium into the sarcoplasm.

Some of this calcium attaches to troponin, causing a conformational changes that moves tropomyosin

out of the way so that the cross bridges can attach to actin and produce muscle contraction.

Individual subunits serve different functions:

Troponin C: binds to calcium ions to produce a conformational changes in troponin.

Troponin T: binds to tropomyosin, interlocking them to form a troponin-troponyosin complex.

Troponin I: binds to actin in thin myofilaments to hold the troponin-tropomyosin complex in place.

Diagnostic use:

certain subtype of troponin ( cardiac troponin I and T) are very sensitive and specific indicators of

damage to the heart muscle (myocardium).it is important to note that cardiac troponins are marker of

all heart muscle damage, not just myocardial infarction.


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Normal range:

cTNT: < 0.1 ng/mL

nTNI: < 0.4 ng/mL

Detection of cardiac troponin:

Cardiac troponin T and I are measured by immunoassay methods. An immunoassay is a biochemical test

that measures the concentration of a substances in a biological fluid using the reaction of an antibody

antigen interaction.

The assay takes advantage of the specific binding of an antibody ton its antigen. Detecting the quantity

of antibody or antigen can be achieved by a variety of methods. One of them is to label either the

antigen antibody. The label may consist of an enzyme immunoassay, radioisotope or fluorescence.

Troponin will begin to increase following myocardial infarction eithin (2-4) hours. Approximate peak

12 hours, about the same time framne as CK-MB.

Troponin will remain elevated longer than CK up to 5 to 9 days for troponin I and up to 2 weeks for

troponin T.


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MYOGLOBIN

Myoglobin is a protein found in skeletal and cardiac muscle which binds oxygen.

Clinical interpretations:

Serum concentrations of myoglobin rise above the reference values as early as 1 hours after the

occurrence of an AMI with peak activity in the range of 4 to 12 hours.

Myoglobin is cleared rapidly and has a reduced clinical sensitivity after 12 hours.

The role of myoglobin in the detection of AMI is within the first 0 t0 4 hours. The time period in

which the CK-2 and cardiac troponin are still within their normal values.

A negative myoglobin can help to rule out myocardial infarction.it is elevated even before CK-MB.

However, it is not specific for cardiac muscle, and can be elevated with any form of injury to skeletal

muscle.

Serum myoglobin levels were measured in normal subjects and patients by means of a newlydeveloped radioimmunoassay.

Myoglobin ranged between 6 and 85 ng/ml.


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ASPARTATE TRANSAMINASE

Aspartate transaminase (AST) also called serim glutamic oxaloacetic transaminase or aspartate

aminotransferase is enzyme associated witH liver parenchymal cells.

Aspartate transaminase isoenzymes

Two isoenzymes are present in humans:

GOT1 - the cytosolic isoenzyme derives mainly from red blood cells and heart.

GOT2 - the mitichrondrial isoenzyme is predominantly present in liver.

Clinical significance:

It is raised in acute liver damage. It is also present in red blood reds and cardiac muscle and may be

elevated due to those sources as well.

Clinical interpretation:

The serum activity of AST begins to rise about 6 to 12 hours after myocardial infarction and usually

reaches its maximum value in about 24 to 48 hours.

It is usually return to normal 4 to 6 days after the infarction.

AST was defined as biochemical marker for the diagnosis of acute myocardial infarction. This was

the first used. However the used of AST for such a diagnosis is now redundant and has been

superseded by the cardiac troponins.

AST is commonly measured clinically as part of diagnostic liver function tests.

A maximum increase of 20 times normal usually indicates severe viral hepatitis, severe trauma. A

high level of 10 to 20 times normal may indicate a heart attack or alcoholic cirrhosis of the liver.


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SUMMARY ON IMPORTANT CARDIAC MARKERS

Test Sensitivity and

specitivity

Normal range Rise in Approximate

peak

Return

norma

CK-MB It is relatively specificwhen skeletal muscle

damage is not present

Immunoassay CK-MBmass< 6%

Electrophoresis method:

CK-MB (


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OTHER RECENT CARDIAC MARKERS:

C-REACTIVE PROTEIN (CRP)

CRP is a protein found in serum or plasma at levels during a inflammatory processes.

It is a sensitive marker of acute and chronic inflammation and infection.

CRP levels are useful in predicting the risk for the thrombotic event such as blood clot causing MI.

CRP level normally found in serum is (0.1 2.5 mg/L). Patients who have persistent CRP level

between 4 and 10 mg/L, with clinical evidence of low-grade inflammation, should be considered to

be at risk for thrombosis.

HOMOCYSTEINE

Homocysteine is an amino acid.

According to the American Heart Association, too much homocysteine in the blood is related to a

higher risk of coronary heart disease, stroke, and peripheral vascular disease, and that it may also

have an effect on atherosclerosis.

The normal fasting level for plasma is 5-15 micromol/L.

Chemical Analysis in Acute Myocardial Infarction - OVERVIEW

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