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Clinical enzymologyByDr. Saadia Anjum

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Several ways!

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Enzymes as toolsEnzymes have been used in many fields.

1. As drugs and therapeutic agents

2. as targets for therapy

3. As a diagnostic tool

4. As a prognostic tool

5. In forensic science

6. genetic investigations

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Enzymes as toolsEnzymes have been used in many fields.

7. Enzybiotics

8. Food industry

9. Textile industry

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Enzymes may be:

1.Plasma enzymes2.Cellular enzymes

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enzymes

Plasmaenzymes

found inplasma

cellularenzymes

intra-cellular

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Plasma enzymes

The enzymes which aredetectable in serum

No functionin plasma

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Plasma enzymes

Plasma specificenzymes

Non plasmaspecificenzymes

No function inplasma

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Plasma enzymes

Plasma specificenzymes

Meant for &function in

plasma

Non plasmaspecificenzymes

No function inplasma

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enzymes

Plasmaenzymes

found inplasma

cellularenzymes

intra-cellular

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1. Serum enzymes The enzymes which are detectable in

serum are derived from tissue cells and

are of two types

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are of two types

1. Physiologically important

2. Physiologically un-important

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are of two types

1. Those whose primary physiological

function and site of action is in the

plasma2. Those whose presence in serum does

not appear physiologically important

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are of two types





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are of two types





Primary Serum enzymes

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are of two types






Secondary Serum enzymes

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are of two types






Secondary Serum enzymes

Funcional enzymes

Non-Funcional enzymes

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enzymes

Plasmaenzymes

found inplasma

cellularenzymes

intra-cellular

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2. cellular enzymes The enzymes which are found intra-

cellularly and perform within cells

1. They may be produced by the same cell2. Or may have been prodced elsewhere

and transported to their workplace

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1. Those produced by the same cell2. Or may have been prodced elsewhere

and transported to their workplace

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1. Those produced by the same cell

2. Those that have been produced

elsewhere and have been transported

to their workplace

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Small amount ofintracellular

enzymes are detectable in theblood due to:

1. Transportation from site ofsynthesis to site of action

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1. Transportation from site ofsynthesis to site of action

2. as a result ofnormal cellturnover.

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3. It is now accepted that all cellsleak some of their contents

including proteins, without anysign of cell damage

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THEREFORE:

Small amount ofintracellularenzymes are normallypresent in the blood

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Enzymes in plasma

functional Non-functional

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Enzymes in plasma


E in transit

Normal cell turnover

leakage

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Enzymes in plasma


E in transit

Normal cell turnover

leakageE released by

injured cells

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E released by

injured cells

This group forms the bases of

clinical enzymology

(H 56 57)

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(Harper: pg 56, 57)

1.functional plasma enzymes

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(H 56 57)

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(Harper: pg 56, 57)

(H 56 57)

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(Harper: pg 56, 57)

(H 56 57)

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(Harper: pg 56, 57)

Enzymes in plasma

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Enzymes in plasma

(Harper)


Enzymes in plasma

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Enzymes in plasma

(Harper)


present at all times in thecirculation

Enzymes in plasma

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Enzymes in plasma(Harper)


present at all times in thecirculation

perform a physiologic functionin the blood.

En mes in plasma

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Enzymes in plasma

(Harper)

1. functional plasma enzymes

Examples:

1. lipoprotein lipase,

2. pseudo-cholinesterase,

3. Pro-enzymes of blood coagulation

4. ceruloplasmin

En mes in plasma

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Enzymes in plasma

(Harper)2. Non-functional plasma enzymes

Enzymes in plasma

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Enzymes in plasma


perform no known physiological

functions in blood

Enzymes in plasma

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Enzymes in plasma


perform no known physiological

functions in blood Arise from the routine normal

destruction of RBC, WBC, and other

cells.

Enzymes in plasma

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Enzymes in plasma


In healthy individuals, the blood

levels of these enzymes areconstant, as the rate of releasefrom aging cells into blood isequal to the rate of removal ofenzymes from blood.

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Plasma contains:

About 70 gms of proteins

Only 1 gm is enzymes

Out of this 99% is functional

enzyme!

h h d

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There are two methods to measure or

detect plasma enzymes

Th h d

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Measure the

enzyme activity

Th h d

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Measure the

enzyme activity

Measure the

enzyme

concentration

Th h d

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Measure the

enzyme activity

Less expensive

Less accurate

Measure the

enzyme

concentration

Th t th d t

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Measure the

enzyme activity

Less expensive

Less accurate

Measure the

enzyme

concentration

Expensive

accurate

Th t th d t

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Measure the

enzyme activity

Less expensive

Less accurate

Measure the

enzyme

concentration

Expensive

accurate

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The measurement of theserum levels of numerous

enzymes has been shownto be ofdiagnostic

significance.

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This is because the presence ofthese enzymes in the serum

indicates that tissue or cellulardamage has occurred resulting

in the release ofintracellularcomponents into the blood.

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Hence, when a physician

indicates that he/she isgoing to assay for liver

enzymes, the purpose is to

ascertain the potential forliver cell damage.

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It was Discovered 50 yrs ago that:

Concentration of non-functional enzymescan be used as markers for clinical diagnosis

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Changes in concentration of these can help

to

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to

1. Detect cell and tissue damage

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to


2. Localize cell and tissue damage

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to


2. Localize cell and tissue damage3. Monitor treatment efficacy

Definitions

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Definitions

A biomarker is a substance used as an

indicator of a biological state.

Cardiac markers are substances

released from heart muscle when it is

damaged as a result of myocardialinfarction.

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DISORDERS DIAGNOSED BY

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70

DISORDERS DIAGNOSED BYENZYMES

1) CardiacDisorders.

2) HepaticDisorders.

3)Skeletal MuscleDisorders.

4) Bone Disorders.

5) Pancreatic

Disorders.

6) Salivary glanddiseae (Mumps)

7) Malignancies

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71

Intracellular Distribution of DiagnosticEnzymes

Liver Heart Pancreas SalivaryGlands

Bone Muscle BiliaryTract

Prostate

LD5ALTAST

LD1ASTCK

MgTpn

LPSAMS

AMS ALP CK ALPGGT

ACP

1 Cardiac Disorders:

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72

1. Cardiac Disorders:

e.g. Acute Myocardial Infarction (AMI).

1) The myocardium becomes ischemic andundergoes necrosis.

2) Cellular contents are released into thecirculation. Blood levels of the followingenzymes increase:

AST LDH1 CK

Tpn Mg

2 H i Di d

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73

2. Hepatic Disorders

a) Hepatocellular Disorders:(1) Viral hepatitis: Hepatitis B & Hepatitis C.

(2) Toxic hepatitis: caused by chemicals &

ToxinsIncreased levels of the following enzymes :

ALT AST LDH5

2 H ti Di d

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74

b) Biliary tract disorders:

The plasma levels of the following

enzymes increase:

ALP GGT

2. Hepatic Disorders

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75

3. Skeletal Muscle Disorders

Muscle dystrophy. Muscle trauma. Muscle hypoxia. Frequent I.M Injections. The plasma levels of thefollowing enzymes increase:

CK AST

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76

4. Bone Disorders:

1)Pagets Bone Disease: caused byincreased osteoclastic activity.

2) Rickets

3) Osteomalacia:The plasma levels of the following

enzyme increase:

ALP

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77

5. Acute Pancreatitis

The plasma levels of the followingenzymes increase:

Lipase AMS

6 Salivary Gland

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78

6. Salivary GlandInflammation:

In Mumps:

The levels of -Amylase (AMS)is significantly increased

l

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79

7. Malignancies

a) Plasma (Acid phosphatase) ACPlevels increase in:

Prostatic carcinoma. Bone metastatic carcinoma

M l

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80

b) Plasma levels of Alkalinephosphatase (ALP) increase in:

Pancreatic carcinoma.

Bile duct carcinoma.

Liver metastasis.

7. Malignancies

7 M li i

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81

c) Plasma levels of Total Lactatedehydrogenase (LDH) increase

in: Leukemia Lymphomas. Liver metastasis.

7. Malignancies

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Clinical

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Clinical

Applications ofEnzymes

Clinical examples

and case studies.

Case

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Case

presented A 36-year old man was admitted to a

hospital following episodes of nausea,vomiting, and general malaise.

His urine was darker than usual.

Upon examination it was discovered that

his liver was enlarged and tender topalpation.

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Hepatitis?

A 36-year old man was admitted to ahospital following episodes of nausea,vomiting, and general malaise.

His urine was darker than usual.

Upon examination it was discovered that

his liver was enlarged and tender topalpation.

Li f ti t t b l

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Liver function tests were abnormal;plasma ALT was 1500 IU/L (Alanine

aminotransferase 6.0 21 IU/L); AST was400 IU/L (Aspartate aminotransferase 7.0 20 IU/L).

Li f ti t t b l

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aminotransferase 6.0 21 U/L); AST was400 IU/L (Aspartate aminotransferase 7.0 20 U/L).

During the next 24 hours the mandeveloped jaundice, and his plasmatotal bilirubin was 9.0 mg/dL (0.2 1mg/dL).

Liver function tests were abnormal

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aminotransferase 6.0 21 U/L); AST was400 IU/L (Aspartate aminotransferase 7.0 20 U/L).

During the next 24 hours the mandeveloped jaundice, and his plasmatotal bilirubin was 9.0 mg/dL (0.2 1mg/dL).

A diagnosis of hepatitis was made.

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Isoenzymes

found in

plasma

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Isoenzymes

Multiple forms of same

enzyme

I

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Isozymes

Multiple forms of same enzyme

Catalyze same reaction

Differ in molecular weight ,structureand charge

Have different Km for same substrate Important in diagnosis of diseases

109

Isozyme or isoenzymes

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are enzymes that differ in aminoacid sequence but catalyze the

same chemical reaction


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are enzymes that differ in aminoacid sequence but catalyze the

same chemical reaction

They are differentiated by

variations in physical properties.


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They catalyze the samereaction but migrate

differently on

electrophoresis


What is electrophoresis?

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Electrophoresis is a

separation technique thatis based on the mobility of

ions in an electric field.

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Example of isoenzymes

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Example of isoenzymes:

1. Alkaline Phosphatase

2. Lactate Dehydrogenase Test

3. Creatinine phospho kinase

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halflife

t1/2

What is half life?

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What is half life?

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the time it takes for the blood plasma

concentration of a substance to half ("plasmahalf-life").

What is half life?

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The period of time required for the conc. or

amount of drug in the body to be reduced toexactly one-half of a given conc. or amount.

What is half life?

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usually denoted by the abbreviation t1/2

What is half life?

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usually denoted by the abbreviation t1/2

The half life ofcardiac LD is about three days,

while the half life ofLD released from skeletal

muscle and liver is about ten hours

LDH exists in 5 forms, which differ

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slightly in structure. All of these can

be measured in the blood. LDH-1 heart muscle , RBC

LDH-2 white blood cells

LDH-3 lung

LDH-4 kidney, placenta, pancreas

LDH-5 liver , skeletal muscle

Greater-than-normal LDH levels may

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suggest:

Heart attack

Hemolytic

anemia

Hypotension

Infectious

mononucleosis Liver disease such

as hepatitis

Muscle injuryMuscular dystrophy

Pancreatitis

Lung tissue death

Stroke

Ischemic

cardiomyopathy

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Cardiac Enzyme Studies

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Cardiac enzyme studies measurethe levels of enzymes and

proteins that are linked with

injury of the heart muscle.


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Cardiac enzyme studies measurethe levels of enzymes and

proteins that are linked with

injury of the heart muscle.

Myocardial

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infarction

Classical definition of myocardial

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y

infarction ---WHO

Presence of at least two of the

following criteria:

1. Typical history

2. Enzyme increase typical of

myocardial necrosis

3. ECG characteristic of MI

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BIOCHEMICAL MAKERS OF

CARDIAC DISEASE

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CARDIAC DISEASE

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CARDIAC DISEASE

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CARDIAC DISEASE

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CARDIAC DISEASE

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diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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Typical history

ECG findings

Cardiac enzymes

diagnosis

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes

diagnosis

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diagnosis

Typical history

ECG findings

Cardiac enzymes / markers

AST LD1

CK

Mgb troponin

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AST LD1 CK

Mgb troponin

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myoglobinl l l i ht h t i f d i

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a low molecular weight heme protein found in

cardiac and skeletal muscle,


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cardiac and skeletal muscle,a low molecular weight hemeprotein found in cardiac and

skeletal muscle,


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cardiac and skeletal muscle,It may appear in the blood inabnormal levels as early as 1

to 3 hours after onset of

myocardial ischemia.


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cardiac and skeletal muscle,is valuable in the earlyevaluation of chest pain

patients


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cardiac and skeletal muscle,Myoglobin is not specific tocardiac muscle


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cardiac and skeletal muscle,Myoglobin is not specific tocardiac muscle

it is useful in the detection of

MI in the absence of skeletal

muscle trauma

LDH

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LDH

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AST

LDH

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LDH in myocardial disease

LDH i f ll i di l

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LDH may rise following myocardial

infarction and in myocarditis.

LDH in myocardial disease

F ll i di l i f i

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Following a myocardial infarction:

levels increase after 12 to 24 hours

peak at 48 to 72 hours

return to normal after 8 to 10 days,providing there are no complications

the peak reached is from two to ten times

the upper reference limitthe normal plasma LD1:LD2 isoenzyme

ratio of is reversed

What is creatine kinase?

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An enzyme


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An enzyme

Can remove ~P from creatineP

and paste it on ADP ATP


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An enzyme


and paste it on ADP ATP Found in tissues that require lots

ofATP


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An enzyme


and paste it on ADP ATP Found in tissues that require lots

ofATP Starts its job when ATP gets

depleted

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CPK levels

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CPK levels

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When the total CPK level is very high,

it usually means there has been injury

or stress to the heart, the brain, or

muscle tissue.

CPK isoenzymes test

Th CPK i t t

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The CPK isoenzymes test measures

the different forms of creatinephosphokinase (CPK) in the blood.

This test is done if a CPK test revealselevated levels.

CPK isoenzyme testing can helppinpoint the exact source of the

damaged tissue.

CPK is made of three slightly different

substances

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substances

CPK-1 (also called CPK-BB) is found mostly inthe brain and lungs

CPK-2 (also called CPK-MB) is found mostly in

the heart CPK-3 (also called CPK-MM) is found mostly in

skeletal muscle

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Troponin I

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in acute MI. Cardiactroponins

have become the goldstandard

Troponin

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Troponin actually refers to threeproteins the body produces that

are classed as I, C and T.

These are present in cardiac

muscles and skeletal muscles, and

most times they are at extremely

low levels.

Troponin I

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Cardiac troponins T and I are

highly sensitive and specific

for cardiac damage. Troponin I

and T are of equal clinical

value

Troponin

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Cardiac troponins elevaterelatively early

have the most favorableprofile with respect to both

sensitivity andspecificity forcardiac necrosis

Troponin

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located at regular intervals along thelength of actin filaments

play a key role in muscle contraction.

Troponin I (TnI) and T (TnT) have

cardiac specific isoforms and are used

for assessing cardiac injury.

Troponin

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located at regular intervals along the

l th f ti fil t

Troponin

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play a key role in muscle contraction.

Troponin I (TnI) and T (TnT) have cardiac

specific isoforms and are used for assessing

cardiac injury

Troponin

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Apart from their proximity to each other in the

troponin complex, troponin T, C and I are

otherwise unrelated proteins

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Troponin

a myocardial

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y

specific myo-filament

component.

Troponin

relatively early

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relatively early

release from dead

cells.

Troponin

The slower release of

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the myofilamentcomponent provides

for detection for aslong as a week

following MI.

Troponinis released as early as

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y

CK-MB and remainselevated for as long as

LDH

Troponin

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has the potential toserve as a sole

marker for MI.

Troponin I & Ta myocardial

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y

specific myo-filament

component.

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Diagnostic enzymes

Cardiac enzymes

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