By Dr.

Saadia Anjum

Clinical enzymology

Several ways!

Enzymes as tools
Enzymes 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

Enzymes as tools
Enzymes have been used in many fields. 7. Enzybiotics 8. Food industry 9. Textile industry

Enzymes may be:

1.Plasma enzymes 2.Cellular enzymes

enzymes
Plasma enzymes
found in plasma

cellular enzymes
intracellular

Plasma enzymes
The enzymes which are detectable in serum
No function in plasma

Plasma enzymes
Plasma specific enzymes

Non plasma specific enzymes

No function in plasma

Plasma enzymes
Plasma specific enzymes

Non plasma specific enzymes

Meant for & function in plasma

No function in plasma

enzymes
Plasma enzymes
found in plasma

cellular enzymes
intracellular

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Physiologically important 2. Physiologically un-important

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Those whose primary physiological function and site of action is in the plasma 2. Those whose presence in serum does not appear physiologically important

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Those whose primary physiological function and site of action is in the plasma 2. Those whose presence in serum does not appear physiologically important

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Those whose primary physiological Primary Serum enzymes function and site of action is in the plasma 2. Those whose presence in serum does not appear physiologically important

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Those whose primary physiological Primary Serum enzymes function and site of action is in the plasma 2. Those whose presence in serum does Secondary Serum enzymes not appear physiologically important

1. Serum enzymes
The enzymes which are detectable in serum are derived from tissue cells and are of two types 1. Those whose primary physiological Primary Serum enzymes function and site of enzymes action is in the Funcional plasma 2. Those whose presence in serum does Secondary Serum enzymes notNon-Funcional appear physiologically important enzymes

enzymes
Plasma enzymes
found in plasma

cellular enzymes
intracellular

2. cellular enzymes
The enzymes which are found intracellularly and perform within cells 1. They may be produced by the same cell 2. Or may have been prodced elsewhere and transported to their workplace

2. cellular enzymes
The enzymes which are found intracellularly and perform within cells 1. Those produced by the same cell 2. Or may have been prodced elsewhere and transported to their workplace

2. cellular enzymes
The enzymes which are found intracellularly and perform within cells 1. Those produced by the same cell 2. Those that have been produced elsewhere and have been transported to their workplace

 Small amount of intracellular enzymes are detectable in the blood due to: 1. Transportation from site of synthesis to site of action

 Small amount of intracellular enzymes are detectable in the blood due to: 1. Transportation from site of synthesis to site of action 2. as a result of normal cell turnover.

 Small amount of intracellular enzymes are detectable in the blood due to: 3. It is now accepted that all cells leak some of their contents including proteins, without any sign of cell damage

THEREFORE: Small amount of intracellular enzymes are normally present in the blood

Enzymes in plasma
functional Non-functional

Enzymes in plasma
functional Non-functional
E in transit leakage

Normal cell turnover

Enzymes in plasma
functional
E released by injured cells

Non-functional
E in transit leakage

Normal cell turnover

This group forms the bases of clinical enzymology

E released by injured cells

(Harper: pg 56, 57)

1.functional plasma enzymes

(Harper: pg 56, 57)

(Harper: pg 56, 57)

(Harper: pg 56, 57)

Enzymes in plasma (Harper)

1.functional plasma enzymes

Enzymes in plasma (Harper)

1.functional plasma enzymes present at all times in the circulation

Enzymes in plasma (Harper)

1.functional plasma enzymes present at all times in the circulation perform a physiologic function in the blood.

Enzymes in plasma (Harper)

1. functional plasma enzymes Examples:
1. 2. 3. 4. lipoprotein lipase, pseudo-cholinesterase, Pro-enzymes of blood coagulation ceruloplasmin

Enzymes in plasma (Harper)

2. Non-functional plasma enzymes

Enzymes in plasma (Harper)

2. Non-functional plasma enzymes perform no known physiological functions in blood

Enzymes in plasma (Harper)

2. Non-functional plasma enzymes perform no known physiological functions in blood Arise from the routine normal destruction of RBC, WBC, and other cells.

Enzymes in plasma (Harper)

2. Non-functional plasma enzymes In healthy individuals, the blood levels of these enzymes are constant, as the rate of release from aging cells into blood is equal to the rate of removal of enzymes from blood.

Plasma contains:
About 70 gms of proteins Only 1 gm is enzymes Out of this 99% is functional enzyme!

There are two methods to measure or detect plasma enzymes

There are two methods to measure or detect plasma enzymes

Measure the enzyme activity

There are two methods to measure or detect plasma enzymes

Measure the enzyme activity

Measure the enzyme concentration

There are two methods to measure or detect plasma enzymes

Measure the enzyme activity

Less expensive Less accurate

Measure the enzyme concentration

There are two methods to measure or detect plasma enzymes

Measure the enzyme activity

Less expensive Less accurate

Measure the enzyme concentration

Expensive accurate

There are two methods to measure or detect plasma enzymes

Measure the enzyme activity

Less expensive Less accurate

Measure the enzyme concentration

Expensive accurate

The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance.

This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood.

Hence, when a physician indicates that he/she is going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell damage.

It was Discovered 50 yrs ago that:

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

It was Discovered 50 yrs ago that:

Concentration of non-functional enzymes can be used as markers for clinical diagnosis Changes in concentration of these can help to

It was Discovered 50 yrs ago that:

Concentration of non-functional enzymes can be used as markers for clinical diagnosis Changes in concentration of these can help to 1. Detect cell and tissue damage

It was Discovered 50 yrs ago that:

Concentration of non-functional enzymes can be used as markers for clinical diagnosis Changes in concentration of these can help to 1. Detect cell and tissue damage 2. Localize cell and tissue damage

It was Discovered 50 yrs ago that:

Concentration of non-functional enzymes can be used as markers for clinical diagnosis Changes in concentration of these can help to 1. Detect cell and tissue damage 2. Localize cell and tissue damage 3. Monitor treatment efficacy

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 myocardial infarction.

DISORDERS DIAGNOSED BY ENZYMES

1) Cardiac
4) Bone Disorders.

Disorders.

2) Hepatic Disorders. 3) Skeletal Muscle Disorders.

5) Pancreatic Disorders. 6) Salivary gland diseae (Mumps) 7) Malignancies

70

Intracellular Distribution of Diagnostic Enzymes

Liver Heart Pancreas Salivary Glands Bone Muscle Biliary Tract Prostate

LD5 ALT AST

LD1 AST CK Mg Tpn

LPS AMS

AMS

ALP

CK

ALP GGT

ACP

71

1. Cardiac Disorders:
e.g. Acute Myocardial Infarction (AMI). 1) The myocardium becomes ischemic and undergoes necrosis.

2) Cellular contents are released into the circulation. Blood levels of the following enzymes increase:

AST LDH1 CK Tpn Mg

72

2. Hepatic Disorders
a) Hepatocellular Disorders: (1) Viral hepatitis: Hepatitis B & Hepatitis C. (2) Toxic hepatitis: caused by chemicals & Toxins Increased levels of the following enzymes :

ALT

AST

LDH5
73

2. Hepatic Disorders
b) Biliary tract disorders: The plasma levels of the following enzymes increase:

ALP

GGT
74

3. Skeletal Muscle Disorders

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

CK

AST
75

4. Bone Disorders:
1) Pagets Bone Disease: caused by increased osteoclastic activity. 2) Rickets 3) Osteomalacia: The plasma levels of the following enzyme increase:

ALP
76

5. Acute Pancreatitis
The plasma levels of the following enzymes increase:

Lipase

AMS
77

6. Salivary Gland Inflammation:

In Mumps:
The levels of -Amylase (AMS) is significantly increased

78

7. Malignancies
a) Plasma (Acid phosphatase) ACP levels increase in:

Prostatic carcinoma. Bone metastatic carcinoma

79

7. Malignancies
b) Plasma levels of Alkaline phosphatase (ALP) increase in:

Pancreatic carcinoma. Bile duct carcinoma. Liver metastasis.

80

7. Malignancies
c) Plasma levels of Total Lactate dehydrogenase (LDH) increase in: Leukemia Lymphomas. Liver metastasis.

81

82

Clinical Applications of Enzymes

Clinical examples and case studies.

 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 to palpation.

Case presented

Hepatitis?
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 to palpation.

 Liver function tests were abnormal; plasma ALT was 1500 IU/L (Alanine aminotransferase 6.0 21 IU/L); AST was 400 IU/L (Aspartate aminotransferase 7.0 20 IU/L).

 Liver function tests were abnormal; plasma ALT was 1500 IU/L (Alanine aminotransferase 6.0 21 U/L); AST was 400 IU/L (Aspartate aminotransferase 7.0 20 U/L). During the next 24 hours the man developed jaundice, and his plasma total bilirubin was 9.0 mg/dL (0.2 1 mg/dL).

 Liver function tests were abnormal; plasma ALT was 1500 IU/L (Alanine aminotransferase 6.0 21 U/L); AST was 400 IU/L (Aspartate aminotransferase 7.0 20 U/L). During the next 24 hours the man developed jaundice, and his plasma total bilirubin was 9.0 mg/dL (0.2 1 mg/dL). A diagnosis of hepatitis was made.

Isoenzymes
found in plasma

Isoenzymes
Multiple forms of same enzyme

Isozymes
Multiple forms of same enzyme Catalyze same reaction Differ in molecular weight ,structure and charge Have different Km for same substrate Important in diagnosis of diseases
109

Isozyme or isoenzymes

Isozyme or isoenzymes
are enzymes that differ in amino acid sequence but catalyze the same chemical reaction

Isozyme or isoenzymes
are enzymes that differ in amino acid sequence but catalyze the same chemical reaction They are differentiated by variations in physical properties.

Isozyme or isoenzymes They catalyze the same reaction but migrate differently on electrophoresis

What is electrophoresis?

What is electrophoresis? Electrophoresis is a separation technique that is based on the mobility of ions in an electric field.

Example of isoenzymes:
1. Alkaline Phosphatase 2. Lactate Dehydrogenase Test 3. Creatinine phospho kinase

halflife
t1/2

What is half life?

What is half life?

the time it takes for the blood plasma concentration of a substance to half ("plasma half-life").

What is half life?

the time it takes for the blood plasma concentration of a substance to half ("plasma half-life"). The period of time required for the conc. or amount of drug in the body to be reduced to exactly one-half of a given conc. or amount.

What is half life?

the time it takes for the blood plasma concentration of a substance to half ("plasma half-life"). The period of time required for the conc. or amount of drug in the body to be reduced to exactly one-half of a given conc. or amount. usually denoted by the abbreviation t1/2

What is half life?

the time it takes for the blood plasma concentration of a substance to half ("plasma half-life"). The period of time required for the conc. or amount of drug in the body to be reduced to exactly one-half of a given conc. or amount. usually denoted by the abbreviation t1/2 The half life of cardiac LD is about three days, while the half life of LD released from skeletal muscle and liver is about ten hours.

LDH exists in 5 forms, which differ slightly in structure. All of these can be measured in the blood.
LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 heart muscle , RBC white blood cells lung kidney, placenta, pancreas liver , skeletal muscle

Greater-than-normal LDH levels may suggest:

Heart attack Hemolytic anemia Hypotension Infectious mononucleosis Liver disease such as hepatitis Muscle injury Muscular dystrophy Pancreatitis Lung tissue death Stroke Ischemic cardiomyopathy

Cardiac Enzyme Studies

Cardiac Enzyme Studies

Cardiac enzyme studies measure the levels of enzymes and proteins that are linked with injury of the heart muscle.

Cardiac Enzyme Studies

Cardiac enzyme studies measure the levels of enzymes and proteins that are linked with injury of the heart muscle.

Myocardial infarction

Classical definition of myocardial 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

BIOCHEMICAL MAKERS OF CARDIAC DISEASE

BIOCHEMICAL MAKERS OF CARDIAC DISEASE

BIOCHEMICAL MAKERS OF CARDIAC DISEASE

BIOCHEMICAL MAKERS OF CARDIAC DISEASE

BIOCHEMICAL MAKERS OF CARDIAC DISEASE

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes

diagnosis
Typical history ECG findings Cardiac enzymes / markers

AST

LD1

CK

Mgb

troponin

AST

LD1

CK

Mgb

troponin

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

a low molecular weight heme protein found in cardiac and skeletal muscle,

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

It may appear in the blood in abnormal levels as early as 1 to 3 hours after onset of myocardial ischemia.

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

is valuable in the early evaluation of chest pain patients

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

Myoglobin is not specific to cardiac muscle

myoglobin
a low molecular weight heme protein found in cardiac and skeletal muscle,

Myoglobin is not specific to cardiac muscle it is useful in the detection of MI in the absence of skeletal muscle trauma

LDH

LDH

AST

LDH

LDH in myocardial disease LDH may rise following myocardial infarction and in myocarditis.

LDH in myocardial disease

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 limit the normal plasma LD1:LD2 isoenzyme ratio of is reversed

What is creatine kinase?

What is creatine kinase?

An enzyme

What is creatine kinase?

An enzyme Can remove ~P from creatineP and paste it on ADP ATP

What is creatine kinase?

An enzyme Can remove ~P from creatineP and paste it on ADP ATP Found in tissues that require lots of ATP

What is creatine kinase?

An enzyme Can remove ~P from creatineP and paste it on ADP ATP Found in tissues that require lots of ATP Starts its job when ATP gets depleted

CPK levels

CPK levels

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 The CPK isoenzymes test measures the different forms of creatine phosphokinase (CPK) in the blood. This test is done if a CPK test reveals elevated levels. CPK isoenzyme testing can help pinpoint the exact source of the damaged tissue.

CPK is made of three slightly different substances

CPK-1 (also called CPK-BB) is found mostly in the 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

Troponin I
in acute MI. Cardiac troponins have become the gold standard

Troponin
Troponin actually refers to three proteins 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
Cardiac troponins T and I are highly sensitive and specific for cardiac damage. Troponin I and T are of equal clinical value

Troponin
Cardiac troponins elevate relatively early have the most favorable profile with respect to both sensitivity and specificity for cardiac necrosis

Troponin
located at regular intervals along the length 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

located at regular intervals along the length of actin filaments

Troponin

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

Apart from their proximity to each other in the troponin complex, troponin T, C and I are otherwise unrelated proteins.

Troponin

a myocardial specific myofilament component.

Troponin

relatively early release from dead cells.

Troponin

The slower release of the myofilament component provides for detection for as long as a week following MI.

Troponin

is released as early as CK-MB and remains elevated for as long as LDH

Troponin

has the potential to serve as a sole marker for MI.

Troponin I & T

a myocardial specific myofilament component.

Diagnostic enzymes
Cardiac enzymes