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OUR LADY OF FATIMA UNIVERSITYCOLLEGE OF MEDICINE

DEPARTMENT OF BIOCHEMISTRY AUGUST 5, 2010

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Barsaga, Mark Lester A.

Basilio, Rhealyn G.

Baui, Bernard Jr. G.

Bañas, Philip Gideon T.

Belada, Ralph Patrick G.

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Specific Objective– To understand myocardial infarction, and the

biochemical events that lead to it.– To identify enzymes that are significant in the

diagnosis of MI.– To identify isoenzymes and how can they be

use in diagnosis of MI.– To determine the initial rise, peak and descent

of serum levels of enzymes.– To know the significance of proper timing of

enzymes assays in the diagnosis of MI.

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Specific Objective– To know the other proteins not enzymatic in

nature and its significance to MI diagnosis.– To determine enzymes which are of

“therapeutic” value to MI and their mechanism of actions.

– To cite examples of anti-thrombotic drugs that can be use in MI and their mechanism of action.

– To define repurfusion injury and its relation to thrombolytic theraphy.

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INTRODUCTION: Acute myocardial infarction (MI) is defined as the death

or necrosis of myocardial cells due to occlusion in the coronary artery by a thrombus that result to the insufficient or cease of blood flow. (Guyton and Hall 11th edition, pp. 231)

Commonly known as “heart attack” Derived from the words “myocardium” or heart muscle,

and “infarction,” which means tissue death due to oxygen insufficiency.

Approx. 90% occlusion in the blood vessel , blood flow through the affected vessel may cease (total ischemia) and the 02 supply of to myocardium is compromised.

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• Diagram : • 1. occlussion of a branch of the left coronary artery

• 2. myocardial infarction resulting from #1 at the anterior wall of the heart

Ascending Aorta

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• Diagram : • 1. occlussion of a branch of the left coronary artery

• 2. myocardial infarction resulting from #1 at the anterior wall of the heart

Ascending Aorta

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2 Types of Myocardial Infarction

• Transmural - characterized by ischemic necrosis of the full thickness of the affected muscle segment(s).

• Non Transmural – as an area of ischemic necrosis that does not extend through the full thickness of the myocardial wall segment(s)

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Classic Symptoms of MI• Chest pain (angina pectoris) - the most common symptom of acute

myocardial infarction due to ischemia.• Shortness of breath (dyspnea) occurs when the damage to the heart

limits the output of the left ventricle resulting to low cardiac output and less venous return.

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Biochemical Events

In Myocardial Infarction

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Deprivation of blood supply (ischemia) to an area of

myocardium

Shift to anaerobic glycolysis> synthesis of ATP, depletion of

adenine nucleotide pool

pH in heart muscle cells

Inefficient of heart muscles

Death of affected are of heart muscles

Plaque Rupture

Formation of large thrombus in a coronary artery

NADH due to terminal electron transport chain; due to lack of O2

Accumulation of lactic acid and other metabolites in myocardium causing cellular osmolarity & altered membrane permeability

Cessation of contraction

Activation of membrane phospholipases, degradation of proteins by proteases, influx of

Ca ++

Development of atherosclerosis

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Development of atherosclerosis

Deprivation of blood supply (ischemia) to an area of

myocardium

Shift to anaerobic glycolysis> synthesis of ATP, depletion of

adenine nucleotide pool

pH in heart muscle cells

Inefficient contractiion of heart muscles

Death of affected area of the heart muscles

Plaque Rupture

Formation of large thrombus in a coronary artery

NADH due to terminal electron transport chain; due to lack of O2

Accumulation of lactic acid and other metabolites in myocardium causing cellular osmolarity & altered membrane permeability

Cessation of contraction

Activation of membrane phospholipases, degradation of proteins by proteases, influx of

Ca ++

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ENZYME

ISOENZYME(ISOZYME)

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ENZYMES• Enzymes are very important molecules in

biology. Enymes are proteins that help to speed up chemical reactions in the body

• Accelerates reactions in cells• Break old covalent bonds and form new

covalent bonds• Regulate metabolic reaction rates• Biologic catalyst

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ENZYMES

• Catalyst • Substance that increase the rate of a

chemical reaction. It is not change by the reaction. It does not alter the equilibrium constant of a reaction.

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ENZYMES as CATALYSTIts qualities include:

• Enzymes are very specific - chemical catalysts can react with a variety of substrates. Enzymes catalyze only a single type of reaction, and work only in one or few substrate compounds

• Enzymes are often regulated by (1)concentration of substratres,by binding small molecules or other proteins. (2) By covalent modification of the enzmes amino acids side chain.

• Enzymes are stereo specific - chemical catalyst of a reaction usually leads to a mixture of stereoisomers either the D or L isomer, but not both

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ENZYMES as CATALYST

• Enzymes are Macromolecules - Composed of protein, or in a few cases, RNA.

• Enzymes work under mild conditions – chemical catalyst often require high temperature and/ or pressure to. These conditions of temperature , pressure , and pH characterize enzymatic catalysis, especially with in cells.

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Terminologies• Coenzymes - small non protein ligands that

catalyze reactions• Cofactors – small inorganic ions which act as

activators and/or inhibitors of activity. Mostly metal ions: Cu, Mg, Mn, Fe.

• Active site – portion of enzyme which folds to precisely fit the contours of a substrate via weak electrostatic interactions and facilitates bond reactivity.

• Prosthetic group – large complex organic molecules, which may have catalytic activity.

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ENZYMES

What are the Mechanism used by enzyme to get it easier to the transition state?

• Proximity- enzymes can bring 2 molecules together in solution.

• Orientation - even when two molecules collide with enough energy to cause reaction, they don’t necessarily form the products, they have to be oriented properly. Enzymes bind substrates so that the reactive groups are steered to the reaction that can lead to a reaction.

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ENZYMES

• Reactive amino acid groups – the side chains of amino acids contain a variety of reactive residues.

• Induced fit – enzymes as a flexible. In this regard they are different form solid catalyst, like the metal catalyst s used in chemical hydrogenation.

• Coenzymes and metal ions - coenzymes are bio molecules that provide chemical groups that help catalysis. They are not change during catalysis. Metal ions which act as a activators and or inhibitors of activity. Eg. Cu, Mg, Mn, Fe.

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TYPES of Enzyme Catalayst• Oxidoreductases – catalyzes oxidation reduction

reactions, often using coenzyme as NAD/ FAD• Transferases- catalyze the transfer of functional group• Hydrolyases – catalyzes hydrolytic reactions adds water

across C-C bonds• Lyases – cleave C-C, C-O, C-N and other bonds

generating a C=C bond or ring• Isomerases- the position of a functional group is change

w/ in molecule, but the molecule itself contains the same number and kind of atoms that it did in the beginning. The catalyze the isomerizations.

• Ligases- condensation of two substrate with splitting of ATP. Removes the elements of water from two fuctional groups to form a single bond

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How are Enzymes RegulatedAnchoring enzymes in membranes – in plasma membrane,membrane of mitochondria and chloroplast s, endoplasmic reticulum and nuclear envelop.Inactive precursor Allosteric regulation – change in kinetic property of an enzyme cause by binding to another molecule. The binding of a small molecule to the enzyme alters it conformation so that it carries catalysis more or less efficiently. The first conformation is termed T (tense ) state, the second is called R (relax) state. The higher the concentration of substrate favor the conversion of T to R state.Covalent modificationRegulation of enzyme synthesis

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ISOENZYMESalso known as isozymesare enzymes that differ in amino acid sequence but

catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (for example lactate dehydrogenase (LDH)).

In biochemistry, isozymes are isoforms (closely related variants) of enzymes. In many cases, they are coded for by homologous genes that have diverged over time.

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Aspartate Aminotransferase• sensitivity progressively decay and higher degree of

specificity• Highest concentration found in cardiac tissue, liver, and

skeletal muscle• Involved in the transfer of amino group between aspartate

and alpha keto acids• Low levels of AST are normally found in the blood

– Appears in blood 6-10 hours– peaks at 12-48 hours– returns to normal in 3-4 days

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Lactate Dehydrogenase• Lactate dehydrogenase catalyses the conversion of

pyruvate to lactate. • Lactate Dehydrogenase levels are also high in tissue

breakdown or hemolysis. It can mean cancer, meningitis, encephalitis, or HIV.

– Appears in blood 8-10 hours– Peaks at 72 hours– Back to normal in 8-12 days

• It is not as specific as troponin.• Has 2 monomers: H (for heart) M (for muscle)

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Lactate Dehydrogenase Isozyme

• 5 isozymes: HHHH (I1) predominates heart tissue HHHM (I2) blood serum HHMM (I3)

HMMM (I4) MMMM (I5) liver• Lactate Dehydrogenase 1 (LDH-1) isozyme is

normally found in the heart muscle and Lactate Dehydrogenase-2 (LDH-2) is found predominately in blood serum.

• A high LDH-1 level to LDH-2 suggest MI.

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Creatine Kinase Also known as creatine phosphokinase Creatine kinase is released when any muscle cell undergoes

necrosis, which makes it a specific and sensitive marker for any muscle injury.

An enzyme that catalyzes the muscle reaction between creatine phosphate and adenosine diphosphate (ADP), yielding adenosine triphosphate (ATP) and creatine. Adenosine triphosphate, along with magnesium, provides the energy for muscle contraction. Creatine phosphate, the substrate of CK, is a rather unstable compound that is autohydrolyzed to creatinine. Although creatinine has no real function, its predictable concentration in muscle, as well as its known rate of excretion by the kidney, makes it a valuable marker for renal function measurements.

The sensitivity of the CK determination is limited because the increase in serum CK is relatively small, lasts only a short time after the onset of pain and may be obscured by differences in normal serum levels.

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Creatine Kinase Isozyme• This enzyme is found in heart muscle (CK-MB),

skeletal muscle (CK-MM), and brain (CK-BB).  • Creatine kinase is increased in over 90% of

myocardial infarctions. However, it can be increased in muscle trauma, physical exertion, postoperatively, convulsions, delirium tremens and other conditions.

• Time sequence after myocardial infarction – begins to rise 4-6 hours – Peaks at 24 hours– returns to normal in 2-3 days

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Creatine Kinase IsozymeCK-MB resides in the cytosol and facilitates high energy

phosphates into and out of mitochondria. It is distributed in a large number of tissues even in the skeletal muscle. Since it has a short duration, it cannot be used for late diagnosis of acute MI but can be used to suggest infarct extension if levels rise again. This is usually back to normal within 2–3 days.

It is relatively specific when skeletal muscle damage is not present.

CK-MB is the most sensitive and specific marker of MI. The blood levels of CK-MB rise by 6-8 hrs and peak at 12-48 hrs later.

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Creatine Kinase• Creatine kinase isozymes.Combine at

random to give three isozymes:– • CK-MM (primarily muscle)– • CK-MB (hybrid)– • CK-BB (primarily brain)

• The CK-MB has its highest concentration in heart muscle

• CK-MB >5% of total CPK strongly suggests myocardial infarction

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Creatine Kinase

• This test is becoming more popular. MB2 is released from heart muscle and converted in blood to MB1.

– A level of MB2 equal or greater than 1.0 U/L and an MB2/MB1 ratio equal or greater than 1.5 indicates myocardial infarction.

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ENZYMES Initial Rise Peak Level Descent

Aspartate Aminotransferase

6-10 Hrs 12-48 Hrs 3-4 Days

Lactate dehydrogenase

8-10 Hrs 72 Hrs 8-12 Days

Creatinine Kinase 4-6 Hrs 24 Hrs 2-3 days

Comparison

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The Importanceof the timing of Enzyme Assays in the Diagnosis

of MI

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>>>Enzyme assays are laboratory methods for

measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

A rapid laboratory turn around time for cardiac markers is essential.

Once a sample is drawn from a patient, structural changes in the enzymes can take place, which can alter the result of a test, therefore, a quick turnaround time must be practiced to avoid these changes.

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NON-ENZYME

PROTEINSignificant to MI diagnosis

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Troponin Troponin - protein involve in muscle contraction in

skeletal and cardiac but not in smooth muscle. - normally it is tightly bounded to

contractile apparatus of muscle protein with minimal amount presence in the blood.

- 0-0.1 ug/LTroponin I - prevents binding of myosin and actinTroponin C- calcium ion accceptorTroponin T- binds Troponin C and Troponin I to

tropomyosin

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TroponinThe most sensitive and specific test for

myocardial damage. Because it has increased specificity compared with CK-MB, troponin is a superior marker for myocardial injury

The troponin T and troponin I of cardiac muscle differ structurally, and therefore antigenically, from their skeletal muscle counterparts.

Skeletal troponin is not detected by cardiac troponin assay.

Troponin C of both cardiac and skeletal muscle have the same structure.

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Troponin

• Troponin I (cTnI) or T (cTnT) are the forms frequently assessed.  – appears in blood 2 - 6– Peaks in 12 - 16 hours – Returns to normal 5-9 days

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MyoglobinMyoglobin is a monomeric protein of red muscle,

stores O2 as a reserve against O2 deprivation.Elevated when muscle tissue is damaged but it

lacks specificity.earliest marker for myocardial injury.Used together with cardiac troponin and CK-MB

assays, the myoglobin assay can give valuable clinical information as to the time of injury and the presence of re-injury.

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MyoglobinA negative test is useful 1-2 hours after the onset of

symptoms to rule out myocardial necrosis. But because the same myoglobin is present in all types of muscle, many conditions can produce a positive myoglobin test.

• Time sequence after myocardial infarction– Rises fast (2 hours) after myocardial infarction– Peaks at 6 - 8 hours– Returns to normal in 20 - 36 hours

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ENZYMES Initial Rise Peak Level Descent

Troponin 4-8 Hrs 12-16 Hrs 5-9 days

Myoglobin 2 Hrs 6-8 Hrs 20-36 Hrs

Comparison

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Thrombolytic Drugs

Used in MI

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Classification of Drugs Mechanism of Action sample

Anticoagulant

It is a substance stops blood from clotting or limits the ability of the

platelets to clot

Heparin, Coumadin,

LMWH

Antiplatelet

They decreases platelet aggregation

and inhibits thrombus formation

Aspirin,Clopidogrel

Thrombolytic used in medicine to dissolve blood clots

Streptokinase, tPA, Urokinase

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Heparin

Heparin is a biological substance, usually made from pig intestines.

It works by activating antithrombin III, which blocks thrombin from clotting

blood.

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Aspirin

Irreversibly inhibits the enzyme COX, resulting in reduced platelet production of

TXA2 (thromboxane - powerful vasoconstrictor which lowers cyclic AMP and initiates the platelet release reaction)

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Streptokinase• a protein secreted by several species of streptococci

(group C, ß-hemolytic streptococci) can bind and activate human plasminogen.

• Streptokinase belongs to a group of medications known as fibrinolytics, and complexes of streptokinase with human plasminogen can hydrolytically activate other unbound plasminogen by activating through bond cleavage to produce plasmin.

• It is used as an effective and inexpensive clot-dissolving medication.

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Tissue Plasminogen Activator tPA PLAT• Derived by recombinant genetics from human DNA

• It s a protein involved in the breakdown of blood clots. It activates plasminogen associated with fibrin directly by enzymatic action.

• As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat only embolic or thrombotic stroke

• Recombinant tissue plasminogen activator (Alteplase, Reteplase, Tenecteplase)

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Thrombolytic Theraphy• It is the drugs use to dissolve clot

• The primary goal of treatment is to quickly open the blocked artery and restore blood flow to the heart muscle, a process called reperfusion

• When the blood clot forms in the blood vessel, it may cut off and reduced the blood flow to the parts of the body that are served by the blood vessels. This can cause a severe damage to that part of the body

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Repurfusion Injury

• Refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia.

• The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.

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END

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