MBBS, DCP, M. Phil. PhD (Biochemistry)

Dept. of Biochemistry

King Edward Medical University

Objectives

• List the clinically important enzymes and isoenzymes.

• State which of the enzymes and isoenzymes are found in which tissues

• Describe plasma enzyme changes in myocardial infarction and liver disease

• Outline different ways of measuring plasma enzymes

• protein molecules

• catalyze chemical reactions without themselves being altered chemically

• contained primarily within cells

• essential enzymes present in virtually all organs but with slightly different forms in different locations isoenzymes

ENZYMES

• classified according to biochemical functions

• unit of enzyme activity:

1 IU = transform 1 mol of substrate/minute

1 SI (katal) = transform 1 mol substrate/second

ENZYMES

• Small amounts of intracellular enzymes are present in the blood as a result of normal cell turnover.

• When damage to cells occurs, increased amounts of enzymes will be released and their concentrations in the blood will rise.

• However, such increases are not always due to tissue damage.

• Other possible causes include:

• increased cell turnover • cellular proliferation (e.g. neoplasia) • increased enzyme synthesis (enzyme

induction) • obstruction to secretion

Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseases.

Enzymes: properties and measurements (I)

Nature: catalytic proteins / denaturation property enzyme

Substrate Product cofactor/coenzyme

• 3-dimensional structure: monomer oligomer• Variants:

1) isozymes (different genes)- tissue-specific forms 2) allozymes (different alleles at single genetic locus)

3) post-translational modifications- cell and tissue specific forms,

e.g., liver- and bone-specific alkaline phosphatase (ALP) differ only in carbohydrate contents attached to the ALP proteins.

Enzymes: properties and measurements (I)

ISOENZYMES• Catalyze the same reaction.

• Differ in AA sequence and physical properties

• Separable on the basis of charge.

• Are tissue specific.

Isoenzymes

• Different Isoenzymes may arise from different tissues and

• Their specific detection may give clues to the site of Pathology.

Alloenzymes• Alloenzymes (or also called allozymes) are variant

forms of an enzyme that are coded by different alleles at the same locus.

• These enzymes generally perform very basic functions found commonly throughout all life forms, such as DNA Polymerase

• These are opposed to isozymes, which are enzymes that perform the same function, but which are coded by genes located at different loci.

MEASUREMENT OF ENZYMES

Enzymes are measured End point assay Kinetic assay

Measurement of enzymes are affected by the presence of

inhibitors or activators.

Hence most of the enzymes are measured by coupled assay.

A coupled assay is one in which a second enzyme is used to act on the product of the enzyme of primary interest.

Second enzyme used NADH as coenzyme.

The rate can be followed by measuring oxidation of NADH which can be done conveniently at 340nm.

ENZYMES IN CLINICAL DIAGNOSIS

• Changes in plasma enzyme activities may sometimes help to detect and localize tissue cell damage .

• The small amounts of intracellular enzymes normally present in the plasma are thought to result from turnover of cells or leakage of enzymes from healthy cells.

• These enzymes almost always function intracellularly, and have no physiologic use in the plasma.

• In healthy individuals, the levels of these enzymes are fairly constant, e. g

• the rate of release from damaged cells into the plasma is balanced by an equal rate of removal of the enzyme protein from the plasma.

• increased plasma levels of these enzyme may indicate tissues damage.

• Some enzymes show relatively high activity in only one or a few tissues.

• The presence of increased levels of these

enzymes in plasma thus reflects damage to the corresponding tissue

Alteration of plasma enzyme levels in disease

states Many diseases that cause tissue damage result in

an increased release of intracellular enzymes into the plasma.

Direct attack on the cell membranes by such agents as

viruses or organic chemicals also cause enzymes release,

which is particularly significant in the case of the liver

A reduction in the supply of oxygenated blood to any tissue also promotes enzyme release.

An example of a clinical condition in which such a reduction occurs is that of myocardial infraction.

The cells of the affected region die rapidly, releasing their enzymes content to the systematic circulation; this release causes rapid rise in serum enzyme activity characteristic of a myocardial infraction.

Enzyme induction is a process in which a molecule (e.g. a drug) induces (i.e. initiates or enhances) the expression of an enzyme.

The process of enzyme induction also increase enzyme production.

An example of such induction from liver is the increased activity of ϒ–glutamyltransferase in serum from the intake ethanol

ENZYMES NAME OF THE ENZYME PRESENT IN

Aspartate Amino transferase (AST)Serum glutamate-oxaloacetate transaminase (SGOT)

Heart and Liver

Alanine Amino transferase (ALT)Serum glutamate-pyruvate transaminase (SGPT)

Heart and Liver

Alkaline Phosphatase (ALP) Bone, intestine and other tissues

Acid Phosphatase (ACP) Prostate

glutamyl Transferase ( GT) Liver

Creatine kinase (CK) Muscle Including cardiac muscle

Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC

Amylase Pancreas

Table 3. Enzyme markers of clinical significanceEnzyme (abbreviation) Clinical significanceAcid phosphatase (ACP) Prostatic carcinomaAlkaline phosphatase (ALP) Obstructive liver diseases, bone disordersAlanine transaminase (ALT,GPT) Hepatic disorders, viral hepatitisAspartate transaminase (AST,GOT) Myocardial infarction, hepatic disordersAlpha-amylase (AMS) Acute pancreatitisAldolase (ALS) Skeletal muscle disordersCreatine kinase (CK) Myocardial infarction, muscle disorders Gamma-glutamyl transferase (GGT) Hepatic disordersG-6-PD Drug-induced hemolytic anemiaLactate dehydrogenase (LD) Myocardial infarction, hepatic disorders, carcionomasLipase (LPS) Acute pancreatitisLeucine aminopeptidase (LAP) Hepatobiliary disorders5’-Nucleotidase (5’NT) Hepatobiliary disordersPseudocholineesterase (PChE) Organophosphate poisoning(butyrylcholine as substrate)Ceruloplasmin (Copper-oxidase) Wilson’s disease (abnormal Cu metabolism)

Isoenzymes and diseases of the heart Most isoenzymes (also called isozymes) are

enzymes that catalyze the same reaction. However, they do not necessarily have the same

physical properties because of genetically determined differences in amino acid sequence.(different genes)

For this reason, isoenzymes may contain different numbers of charged amino acids and may, therefore, be separated from each other by electrophoresis

The pattern of isoenzymes found in the plasma may therefore, serve as a means of identifying the site of tissue damage.

For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction.

LIVER,CARDIAC AND SKELETAL ENZYMES

Enzymes in this category include the Aminotransferases, Creatine kinase, Alkaline Pohosphatase Lactate dehydrogenase. Gamma-glutamyl transferase(GTT)

Aminotransferases Aspartate aminotransferase and Alanine aminotransferase are examples of

aminotransferases of clinical interest. Aspartate aminotransferase (AST) was known

formerly as glutamate oxaloacetate transaminase (GOT).

Alanine aminotransferase is also known as alanine transaminase, L-alanine : 2-oxoglutarate aminotransfersae, ALT or AlaAT.

It was known formerly as glutamate pyruvate transaminase (GPT).

They need the cofactor, pyridoxal phosphate. Both enzymes are widely distributed in body

tissues, but ALT is present only in small amounts except in liver.

The aminotransferases are a group of enzymes that catalyze the inter conversion of amino acids to 2-oxo-acids by transfer of amino groups.

Or in other words; These catalyze the exchange of -NH₂ group

between an amino acid and keto acid.

Clinical Significance Transaminases

Both AST and ALT normally are present in Human plasma, Bile, Cerebrospinal fluid (CSF) and Salvia, but none is found in urine. With viral hepatitis and other forms of liver

disease associated with hepatic necrosis, serum AST and ALT levels are elevated even before the clinical signs and symptoms of disease (such as jaundice) appear.

Levels for both enzymes may reach values as high as 100 times the upper limit of the reference interval.

In case of infectious hepatitis and other inflammatory conditions affecting the liver , ALT is characteristically high.

Although serum levels of both AST and ALT becomes elevated whenever disease processes affect liver cell integrity, ALT is the more liver-specific enzyme.

After a myocardial infraction, increased AST activity appears in serum, as might be expected from the relatively high AST concentration in heart muscle.

Aspartate Transaminase (AST) AST (glutamate oxaloacetate transaminase,

GOT) is present in high concentrations in cells of

Cardiac and skeletal muscle, Liver, Kidney and Erythrocytes. Damage to any of these tissues may increase

plasma AST levels.

Causes of raised plasma AST activities

Artefactual Due to in vitro release from erythrocytes if there

is haemolysis or if separation of plasma from cells is delayed.

Physiological: During the neonatal period (about 1.5 times the

upper adult reference limit).

Causes of raised plasma AST activities

Marked increase (10 to 100 times the upper adult reference limit):

Circulatory failure with shock and hypoxia; Myocardial infraction; Acute viral or toxic hepatitis

Moderate Increase: Cirrhosis (may be normal, but may rise to twice the

upper adult reference limit); Infectious mononucleosis (due to liver

involvement); Cholestatic Jaundice (up to 10 times the upper adult

reference limit); Malignant infiltration of the liver.

Skeletal muscle disease: After trauma or surgery (especially after cardiac

surgery) Severe haemolytic episodes (of erythrocyte origin)

L-matate + NAD+

Malate dehydrogenase MDH

Principle involved in AST estimation

Aspartate aminotransferas

e AST

- Oxoglutarate + L-aspartate

L- glutamate + oxaloacetate

NADH + H+

+

Alanine Transaminase (ALT)

ALT (glutamate pyruvate transaminase, GPT) is present in high concentration in

•Liver

•Rich amounts in hepatocytes with high specificity for liver damage Moderate amount Skeletal muscle, Kidney and

Heart.

Alanine Transaminase (ALT)

Small amount:

pancreas,

spleen,

lung,

red blood cells

Causes of Raised Plasma ALT Activities

Marked Increase (10 to 100 times the upper limit of the adult reference range):

Circulatory failure with ‘shock’ and hypoxia; Actual viral or toxic hepatitis.

Causes of Raised Plasma ALT Activities

Moderate Increase: Cirrhosis (may be normal or up to twice the upper

adult reference limit); Infectious mononucleosis (due to liver involvement); Liver congestion secondary to congestive cardiac

failure; Cholestatic jaundice (up to 10 times the upper

reference limits in the adults); Surgery or extensive trauma and skeletal muscle

disease (much less affected than AST)

• primary tissue sources:

1. Brain, smooth muscle, prostate, thyroid, gut, lung CK-BB

2. Cardiac muscle – MB (20-30%) & MM (70-80%)

3. Skeletal muscle – MB (1-2%) & MM (98-99%)

4. Plasma – predom. MM with < 6% MB

• relatively small molecular size allows leakage out of ischemic muscle or brain cells

Creatine kinase (CK)

• reference ranges in serum affected by:

1. Amount of lean muscle mass

Thin, sedentary = 30 – 50 U/L

Muscular, exercising regularly = 500 – 1000 U/L

2. Age – in neonates, CK-MB 5-10% of total CK

3. Gender

4. Race – Africans 30% higher than Europeans

Creatine kinase (CK)

5. Muscle activity – direct relationship between intensity of exercise and CK level

• Short-term strenuous exercise 10-100 fold increase

• Marathon runners up to 2000 U/L as resting value

Creatine kinase (CK)

Creatine Kinase CK is most abundant in cells of cardiac muscle skeletal muscle and in brain, but also occurs in other tissues such as smooth

muscle. Creatine kinase is a dimeric enzyme that

catalyses the reversible phosphorylation of creatine (Cr) by ATP

Increase in plasma CK activity are usually the result of cardiac muscle damage.

In cardiac muscle, up to 30% is the MB isoenzyme

CK activity is greatest in Striated muscle, Brain and Heart tissue. The liver and erythrocytes are essentially devoid

of activity.

CK consists of two protein subunits, (dimer) the products of two different genes (Loci), M and B, which combine to form three isoenzymes,

BB (CK-1), MB (CK-2) MM (CK-3).

CK-MM is the predominant isoenzyme in skeletal and cardiac muscle and is detectable in the plasma of normal subjects.

CK-MB accounts for about 30% of the total CK activity in cardiac muscle, plasma activity is always high after myocardial infraction.

CK-BB is present in high concentrations in the brain and in the smooth muscle of the gastrointestinal and genital tracts.

Isoenzyme name Composition Present in Elevated in

CK-1 BB Brain CNS diseases

CK-2 MB Myocardium/ Heart

Acute myocardial infarction

CK-3 MMSkeletal muscle, Myocardium

released from damaged muscles: CK, AST, LD, myoglobin

Myoglobin >> CK >> AST and LD

released during ischemia, injury or inflammation

also increased in:

1. Chronic myopathies

2. Chronic renal failure

3. Acute respiratory exertion – respiratory muscles with more CK than other muscles

Diagnostic ApplicationsCM-MM

• Brain trauma or brain surgery

1.Injury to smooth muscles (e.g. intestinal ischemia)

2.Patients with malignancies, esp. prostate cancer, small cell lung CA, intestinal malignancies synthesize B subunit

3.Transient increase after cardiac arrest reflect cerebral ischemia

CK-BB

Diagnostic Applications

• primary clinical use: detection of acute MI

Following MI:

Total CK – 98% sensitive

but 68-85% specific;

peak value 18-30 hrs;

duration 2-5 days;

level 5-10x normal

CK-MB

Diagnostic Applications

• primary clinical use: detection of acute MI

Following MI:

CK-MB – rise proportional to extent of infarction;

appears in serum within 6 hrs after AMI;

peak value 12-24 hrs;

duration 1.5-3 days persistence indicates extension of infarction or re-infarction

CK-MB

Diagnostic Applications

Normal: 24 – 170 U/L (women) 24 – 195 U/L (men)

• Marked elevation (> 5x normal)

1. After trauma from electrocution,

2. crush injury,

3. convulsion,

4. tetany,

5. surgical incision or

6. IM injection

Total Serum CKDiagnostic Applications

Normal: 24 – 170 U/L (women) 24 – 195 U/L (men)

• Marked elevation (> 5x normal)

6. Athletic individuals – inc. muscle mass & inc. release during strenuous activity

7. Muscular dystrophies

8. Chronic inflammation of muscle (dermatomyositis or polymyositis).

Total Serum CKDiagnostic Applications

• Mild or moderate elevation (2 – 4x normal)

1.Hyper- or hypothermia

2.Hypothyroidism

3.After normal vaginal delivery – BB isoenzyme from myometrial contractions

4.Reye’s syndrome

Diagnostic ApplicationsTotal Serum CK

Clinical Significance

CK activity is elevated in many diseases, including those involving

Skeletal muscle, Heart muscles, The central nervous system and The thyroid. The serum enzyme changes after a myocardial

infraction.

Causes of Raised Plasma CK Activities

Physiological: Neonatal period (slightly raised above the adult

reference range); During and for a few days after parturition; Marked Increase: Shock and circulatory failure; Myocardial Infraction; Muscular dystrophies Rhabdomyolysis (breakdown of skeletal muscle)

Moderate Increase:

Muscle Injury; After surgery (for about a week); Physical Exertion. There may be a significant

rise in plasma activity after only moderate exercise, muscle cramp;

Moderate Increase:

Following an epileptic fit; After an intramuscular injection; Hypothyroidism (throxine may influence the

catabolism of the enzyme); Alcoholism (possibly partly due to alcoholic

myositis); Some cases of cerebrovascular accident and head

injury;

Lactate Dehydrogenase (LDH) LDH catalyzes the removal of hydrogen from

the substrate, but is not able to use oxygen as hydrogen acceptor.

The removed hydrogen atoms are taken up by special hydrogen acceptor such as NAD . ⁺

LDH catalyzes the reversible inter-convention of lactate and pyruvate.

Zinc-containing; part of glycolytic pathway

Catalyze conversion of lactate to pyruvate using NAD+ as cofactor

CH3 CH3

HCOH + NAD+ C = O + NADH + H+

COOH COOH

Tissue source: present virtually in all tissues cytoplasm of all cells and tissues in the body

Tetramers with 4 subunits of 2 possible forms: H (heart) and M (muscle)

Lactate dehydrogenase (LD)

• Five isoenzymes:

LD1 & LD2 – high in heart muscle, erythrocytes, kidney

LD4 & LD5 – high in skeletal muscle & in liver

Lactate dehydrogenase (LD)

Normal pattern in serum:

LD2 > LD1 > LD3 > LD4 > LD5

Highest in newborns and infants; values do not change with age in adults

No gender difference

Lactate dehydrogenase (LD)

Lactate Dehydrogenase (LDH) The enzyme is widely distributed in the body,

with high concentrations in the cells of the Cardiac muscle Skeletal muscle Liver Kidney Brain and Erythrocytes So the measurement of plasma total LD activity

is therefore is non specific marker of cell damage.

Causes of Raised Plasma total LD Activity Artefactual: Due to in vitro haemolysis or delayed separation of

plasma from whole blood. Marked Increase : Circulatory failure with ‘shock’ and hypoxia; Myocardial infraction; Some haematological disorders, such as megaloblastic anaemia, acute leukaemias and lymphomas.

Smaller increases occur in other disorders of erythropoiesis such as

thalasemia, and haemolytic anaemias Renal infraction Moderate Increase: Viral hepatitis Malignancy of any tissue Skeletal muscle disease Pulmonary embolism Infectious mononucleosis

Isoenzyme of Lactate Dehydrogenase Five isoenzymes can be detected by electrophoresis

and are referred to as LD1 to LD 5. Predominates in cells of cardiac muscle,

erythrocytes and kidneys. The slowest moving isoenzymes, LD5 is the most

abundant form in the liver and in skeletal muscle. Predominant elevation of LD1 occurs after

myocardial infraction, in megaloblastic anaemia after renal infraction.

Isoenzyme name

Composition Composition Present in Elevated in

LDH1 ( H4) HHHH

Myocardium, RBC

myocardial infarction

LDH2 (H3M1) HHHM Myocardium, RBC

LDH3 (H2M2) HHMM Kidney, Skeletal muscle

LDH4 (H1M3) HMMM Kidney, Skeletal muscle

LDH5 (M4) MMMM Skeletal muscle, Liver

Skeletal muscle and liver diseases

LACTATE DEHYDROGENASE IN MI

The iso-enzymes are usually separated by cellulose acetate electrophoresis at pH 8.6.

Lactate dehydrogenase isoenzymes (as percentage of total):

LDH1 14-26 % LDH2 29-39 % LDH3 20-26 % LDH4 8-16% LDH5 6-16 %

Acute Myocardial Infarction Acute myocardial infarction is

the rapid development of myocardial necrosis caused by a critical imbalance between the oxygen supply

and demand of the myocardium. It is an irreversible myocardial

injury from prolonged ischemia. Accurate and early diagnosis is

important in minimizing cellular damage and, consequently, in obtaining a successful outcome for the patient

Cardiac Markers

Markers of cardiac myocyte necrosis:MyoglobinCKCk-MBTroponin I & T

Creatine kinase Cytoplasmic CK is a dimer,

composed of M and/or B subunits, which associate forming CK-MM, CK-MB and CK-BB isoenzymes.

CK-MM is the main isoenzyme found in striated muscle

CK-MB is found mainly in cardiac muscle

CK-BB is the predominant isoenzyme found in brain

Myoglobin

Myoglobin is an iron and oxygen binding protein found in muscle tissue

It is only found in the blood stream when it is released following muscle injury

It is a sensitive marker for muscle injury making it a potential marker for myocardial infarction

However elevated myoglobin has low specificity for the diagnosis of myocardial infarction and therefore is not the preferred test

Serum total CK activity and CK-MB concentration rise in parallel following myocardial injury,

starting to increase 4± 6 h after injury,

reaching peak serum concentrations after 12±24 h and returning to baseline after 48±72 h.

Serum CK-MB is considerably more specific for myocardial damage than is serum total CK, which may be elevated in many conditions where skeletal muscle is damaged.

Consequently, CK should not be used for the diagnosis of myocardial injury unless used in combination with other more specific cardiac markers.

Diagnosis of myocardial infarction Measurements of plasma enzymes have long

been used to assist in the diagnosis of myocardial infarction.

The first enzyme to increase is the MB isoenzyme of creatine kinase (CK-MB), followed by

total CK, aspartate aminotransferase (AST) and hydroxybutyrate dehydrogenase (HBD, the

cardiac isoenzyme of lactate dehydrogenase)

Diagnosis of myocardial infarction

Myocardial muscle is the only tissue that contains more than five percent of the total CK activity as the CK2 (MB) isoenzyme.

Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium.

Following an acute myocardial infarction, this isoenzyme appears approximately four to eight hours following onset of chest pain, and reaches a peak of activity at approximately 24 hours

Enzyme Starts to rise (Hours)

Time after infraction of peak elevation (Hours)

Duration of rise (Days)

CK (Total) 4-6 24-48 3-5

AST 6-8 24-48 4-6

LD H 12-24 48-72 7-12

Cardiac muscle contains about 30% of its CK as CK-MB; the proportion in healthy skeletal muscle is about 1%.

Thus, even if total plasma CK is elevated (for example as a result of trauma or vigorous exercise), the presence of more than about 5% of the total as CK-MB suggests cardiac muscle damage

Newer markers for myocardial infarction: Troponin T and Troponin I These are regulatory proteins involved in

myocardial contractility. Certain subtypes of troponin (cardiac troponin I

and T) are very sensitive and specific indicators of damage to the heart muscle (myocardium).

They are measured in the blood to diagnose myocardial infarction (heart attack) in patients with chest pain.

They are released into the plasma in response to cardiac damage.

Cardiac specific troponin T(cTnT) and troponin I(cTnI) are highly sensitive markers for damage to cardiac tissue.

Increase in troponin T(cTnT) and troponin I(cTnI) levels are seen at 4-8 hours after myocardial infarction.

Remained elevated up to 5-10days respectively.

Troponins

Troponin is a regulatory complex of 3 protein subunits located on the thin filament of the myocardial contractile apparatus.

Its function is the regulation of striated and cardiac muscle contraction.

The complex regulates the calcium-modulated interaction between actin and myosin on the thin filament.

Troponins Troponin C (18 kd) • Calcium-binding subunit • No cardiac specificity • Troponin I (26.5 kd) • Actomyosin-ATP-inhibiting subunit • Cardiac-specific form • Troponin T (39 kd) • Anchors troponin complex to theTropomyosin strand

In the absence of calcium ions, tropomyosin blocks access to the mysosin binding site of actin.

When calcium binds to troponin, the positions of troponin and tropomyosin are altered on the thin filament and myosin then has access to its binding site on actin.

When the calcium level decreases, troponin locks tropomyosin in the blocking position and the thin filament slides back to the resting state.

Tissue Specificity of Troponin Subunits

Troponin C is the same in all muscle tissues

Troponins I and T have cardiac-specific forms, cTnI and cTnT

cTnI and cTnT remain elevated for 10 to 14 days

Troponin Release Kinetics

• Detectable in blood 4-12 h, similar to CKMB • Peaks 12-38 h• Remains elevated for 10-14 days

Troponins Bind tropomyosin and govern excitation-

contraction coupling

Three subunits

1. Troponin C (TnC) – calcium-binding subunit

2. Troponin I (TnI) – bind to actin inhibitory

3. Troponin T (TnT) – bind to tropomyosin

TnI and TnT with unique forms expressed in myocardial cells but not in other muscle types presence of cTnI or cTnT in serum highly specific for myocardial injury

cTnT

84% sensitivity for MI 8 hrs after onset of symptoms

81% specificity for MI; 22% specificity for unstable angina

cTnI

90% sensitivity for MI 8 hrs after onset of symptoms

95% specificity for MI; 36% specificity for unstable angina

Troponins

Cardiac troponins released in two phases:

1.Initial damage (acute MI) – leave myocardial cells enter circulation the same time that CK-MB does peak at 4-8 hrs

2.Sustained release from intracellular contractile apparatus – occurs up to days after acute event

First appear in circulation after myocardial injury slightly later than when myoglobin enters the blood rises after 3-6 hrs peaks at ~ 20 hrs

Troponins

General advantages:

1.cTnT and cTnI are released only following cardiac damage.

2.Unlike CK & CK-MB, cTnT and cTnI are present , and remain elevated, for a long time cTnI detectable up to 5 days & cTnT for 7-10 days following MI

3.cTnT and cTnI are very sensitive.

Troponins

General disadvantages:

1.Elevation can occur as a result of causes other than MI

2.myocarditis,

3. severe cardiac failure,

4.cardiac trauma,

5.pulmonary embolus with cardiac damage

Troponins

General disadvantages:

6. Failure to show a rise in cTnT or cTnI does not exclude the diagnosis of ischemic heart disease.

7. Both may be elevated in patients with chronic renal failure with sustained levels of elevation.

Troponins

Measured in serum by immunoassay

Ideal time to check is between 6 and 9 hours from onset of symptoms

If onset of symptoms indistinct – take sample on admission, 6 – 9 hrs after and at 12 – 24 hrs after admission

Troponins

Other enzymes useful in clinical diagnosisAcid phosphatase (ACP)

• Optimal activity: pH 5.0

• Tissue source:

Common to many tissues, esp. prostate

Small amounts in rbc, platelets (during clot formation), liver and spleen

Human milk and seminal fluid (very concentrated)

Prostatic ACP distinguished from others using thymolphthalein monophosphate highly specific for prostatic ACP

Major applications:

1. Evaluation of prostatic CA (metastatic & local growth)

Not elevated in CA confined within prostate, BPH, prostatitis or ischemia of prostate

2. Medicolegal evaluation of rape – vagina with little or no ACP

Acid phosphatase (ACP)

vaginal acid phosphatase activity in non-coital women is less than 10 U/liter of broth, and in recently

post-coital women is more than 50 U/liter

Measured by radioimmunoassay acidify serum with citric acid to stabilize ACP activity

Alkaline Phosphatase (ALP)

The alkaline phosphatase are a group of enzymes that hydrolyses organic phosphotase at high pH.

The exact metabolic function of ALP is unknown but it is probably important for calcification of bone.

In adults plasma ALP is derived mainly from bone and liver in approximately equal proportions. The proportions due to the bone fraction is

increased when there is increased osteoblastic (new bone formation) activity that may be physiological.

They are present in most tissues but high concentrations are found in

Osteoblasts of bone Cells of the hepatobiliary tracts Intestinal walls Renal tubules and Placenta

Causes of Raised Plasma ALP Activity

Physiological: During the last trimester of pregnancy the

plasma total ALP activity rises due to the contribution of the placental isoenzyme.

In preterm infants plasma total ALP activity is up to five times the upper reference limit in adults and consists predominantly of the bone isoenzyme.

In children the total activity is about 2.5 times and increase up to five times from its upper limit during the pubertal bone growth spurt.

There is a gradual increase in the proportion of the liver ALP with age.

Bone Disease Rickets and osteomalacia Paget’s disease of bone (may be very high) Secondary malignant deposits in bone Osteogenic sarcoma, only if very extensive Primary hyperparathyroidism

These are disorders caused by insufficient levels of vitamin D in the body.

They are really the same condition: rickets is the name used when it occurs in children whereas osteomalacia is the term used for adults

Liver Disease: Intra- or extrahepatic cholestasis( Bile

obstruction) Hepatic tumuors

• Widely distributed along surface membranes of metabolically active cells

• Encoded for by four different genes expressed in:

1. Placenta

2. Intestines

3. Germ cell and lungs

(A germ cell is any biological cell that gives rise to the gametes of an organism)

Alkaline phosphatase (ALP)

4. Tissues including bone, liver, kidney & granulocyte

•Very high activity in bone, liver, intestine, kidney, wbc and placenta

Methods for distinguishing ALP isoenzymes:

1.Heat fractionation – easiest & most common; heat serum sample at 56oC x 15 min. then compare with unheated sample

Bone ALP extremely labile retain 10-20% of original activity

Liver & placental ALP heat stable liver ALP 30-50% retained, placental ALP with all retained

Alkaline phosphatase (ALP)

1. Chemical inhibition

Urea – block placental ALP

Phenylalanine – block liver & bone ALP

2. Electrophoresis - definitive

Alkaline phosphatase (ALP)

DIAGNOSTIC APPLICATIONS

Derived from epithelial cells of biliary tract excreted by bile into intestine• Used for establishing diagnosis in jaundice

• Pronounced increase (> 5x)

Intra- or extrahepatic bile duct obstruction

Biliary cirrhosis

• Moderate increase (3-5x normal) : granulomatous or infiltrative liver disease

• Slight increase (up to 3x normal) : viral hepatitis, cirrhosis

Liver ALP

Elevation part of osteoblastic growth• Pronounced increase:

Paget’s disease

Osteogenic sarcoma

Hyperparathyroidism

• Moderate increase: metastatic tumors in bone; metastatic bone disease (rickets, osteomalacia)

• Slight increase: healing fractures; normal growth patterns in children

Bone ALP

Placental ALP

• With oncofetal form turned on and expressed by tumor cells in adults called Regan isoenzyme

• Slight increase in pregnancy

• (Oncofetal antigens are substances which are produced by tumors and also by fetal tissues but they are produced in much lower concentration by adult tissues)

Intestinal ALP

•Inc. in inflammatory bowel disease (ulcerative colitis & regional enteritis)

•Secreted into the circulation after a meal inc. total ALP in non-fasting specimens

Renal ALP

• Normally excreted into urine from renal tubular cells

Granulocyte ALP

• Used as marker of granulocyte maturity in leukocytosis

• Lymphocytes infected with HIV release specific ALP fraction (band-10) surrogate marker for HIV infection in children

• Glycolytic enzyme split fructose-1,6-diphosphate into two triose phosphate molecules in glucose metabolism

• Distributed in all tissues

• Elevated in serum following:1. Skeletal muscle disease or injury – reflect severity of

dermatomyositis

2. Metastatic CA to liver 5. Hemolytic anemia

3. Granulocytic leukemia 6. Tissue infarction

4. Megaloblastic anemia

Aldolase

Amino acid + Glutathione -glutamyl amino acid + Cysteinylglycine

It is involved in aminoacid transport across the membranes.

Found mainly in biliary ducts of the liver, kidney and pancreas.

Enzyme activity is induced by a number of drugs and in particular alcohol.

( GT)

glutamyltransferase ( GT)

Glutathione Glutathione is the

tripeptide Gamma-glutamylcysteinylglycine containing a sulfhydryl group. Glutathione has several important role.

serves as a transporter in the gamma-glutamyl cycle for amino acids across cell membranes

protects erythrocytes from oxidative damage

Glutathione cycles (Meister cycle) figure.9-16

The enzyme gamma-glutamyl transpeptidase, located on the cell membrane of kidneys and other tissue cells, catalyzes glutathion (GSH) to transfer its glutamyl group to amino acid, then the gamma-glutamyl-ammino acid is transported inside of the cell.

Glutathione cycles (Meister cycle) figure.9-16

The gamma-glutamyl-amino acid releases amino acid and 5-oxiproline. This is the process for amino acid transportation into the cell.

The 5-oxiproline converts to glutamate under the action of enzyme and uses ATP.

Glutathione cycles (Meister cycle figure.9-16 The 5-oxiproline converts

to glutamate under the action of enzyme and uses ATP.

Glutamate and the other parts of GSH, glycine and cysteine, are regenerated GSH in cytosol and 2 ATPs are used. So 3 ATPs are required for the transportation of each amino acid.

Glutathione cycles (Meister cycle figure.9-16

key enzyme of the gamma-glutamyl cycle is gamma-glutamyl transpeptidase which is found in high levels in the kidneys

Glutathione cycles (Meister cycle) figure.9-16

Glutathion cycles between a reduced form with a sulfhydryl group (GSH) and an oxidized form (GSSG), in which two GSHs are linked by a disulfide bond. GSH is reductant, its sulhydryl group can be used to reduce peroxides formed during oxygen transport.

Glutathione cycles (Meister cycle) figure.9-16 Glutathione plays a key

role in detoxification by acting with hydrogen peroxide and organic peroxide.

Glutathion peroxidase catalyzes this reaction, in which GSH converts to GSSG. Then GSSG is reduced to GSH by glutathione reductase, an enzyme containing NADPH as a cofactor.

Gamma-glutamyl transferase (GTT) Gamma-glutamyl transferase (GTT) present in

cells of Liver Kidney Pancreas Prostate

Causes of Raised Plasma ALP Activity

Induction of enzyme synthesis, with out cell damage, by drugs or alcohol.

Cholestatic Liver Disease Hepato-cellular damage e.g. infectious hepatitis Very high plasma GTT levels Alcoholic hepatitis Induction by chronic alcohol intake

“gamma glutamyltranspeptidase

Catalyze transfer of glutamyl groups between peptides or amino acids through linkage at a -COOH group important in transfer or movement of amino acids across membranes

Large amounts in:Pancreas and renal tubular epithelium

Hepatobiliary cells

Gamma glutamyltransferase (GGT)

increased activity:.

1. In urine – renal tubular damage

2. Hepatocellular & hepatobiliary diseases correlates better with obstruction & cholestasis than with pure hepatocellular damage “obstructive” enzyme

Diagnostic Applications

GGT & alcohol

Alcohol induces microsomal activity inc. GGT synthesis indicator of alcohol use

GGT levels return to normal after 3-6 wks of abstention from alcohol test for compliance in alcohol-reduction programs

Diagnostic Applications

• GGT & drugs

Barbiturates, phenytoin & other drugs (acetaminophen) inc. microsomal activity of GGT

Potentially useful in drug treatment protocols

Diagnostic Applications

digestive enzyme

Acts extracellularly to cleave starch into smaller groups & finally to monosaccharides

Major sources: salivary glands

exocrine pancreas

Amylase (Diastase)

secretion stimulated by pancreozymin (cholecystokinin)

enter duodenum at ampulla of Vater via sphincter of Oddi

Low levels found in:

1.Fallopian tubes 3. Small intestine

2.Adipose tissue 4. Skeletal muscle

readily cleared in urine

Pancreatic amylase

Acute Pancreatitis

• Levels rise within 6-24 hours remain high for a few days return to normal in 2-7 days

• Serum amylase normal but with suspicion of pancreatitis measure 24-hour urine amylase or serum lipase

DIAGNOSTIC APPLICATIONS

Morphine administration

• Constrict pancreatic duct sphincter dec. intestinal excretion & inc. absorption in the circulation

Renal failure

• Failure to clear normally released amylase from the circulation no diagnostic significance

DIAGNOSTIC APPLICATIONS

Malabsorption & liver disease

(+) circulating complexes of amylase with a high MW compound such as Ig’s macroamylasemia prevent renal clearance

no diagnostic significance

DIAGNOSTIC APPLICATIONS

Tumors

1.serous ovarian tumors

• epithelium similar to FT produce cyst fluid with amylase appear in serum & urine

2.Lung CA

• ectopic production of amylase

DIAGNOSTIC APPLICATIONS

Conditions AffectingSerum AmylasePronounced Elevation (> 5x normal)

Acute pancreatitisPancreatic pseudocystMorphine administration

Moderate Elevation (3-5x normal)Pancreatic CA (head of pancreas)MumpsSalivary gland inflammationPerforated peptic ulcerIonizing radiation.

α-Amylase Amylase breaks down starch and glycogen to maltose. It is present at high concentration in pancreatic juice Saliva gonads, fallopian tubes, skeletal muscle and adipose tissue. In normal subjects most plasma amylase is derived from

pancreas and salivary glands. Being of relatively low molecular weight, it is excreted

in the urine.

Causes of Raised Plasma Amylase Activity

Marked Increase Acute pancreatitis Severe glomerular impairment Severe diabetic ketoacidosis Perforated peptic ulcer Moderate Increase

Acute cholycystitis Intestinal obstruction Abdominal trauma Ruptured ectopic pregnancy

Salivary gland disorder: Mumps Salivary calculi Sjogren’s syndrome After injection of contrast medium into salivary ducts for

sialography Morphin administration (spasm of the sphincter of Oddi) Severe glomerular dysfunction (may be markedly raised) Myocardial infraction (occasionally) Acute alocoholic intoxiacation Diabetic ketoacidosis (may be markedly raised) Macroamylasaemia

LipaseAlimentary lipase

• Cleave dietary TG’s into free fatty acid & glycerol

• Secreted by exocrine pancreas into the duodenum

• Found almost exclusively in pancreas highly specific

LipaseAlimentary lipase

Not cleared into the urine remain elevated after amylase has returned to normal

Highest levels in acute pancreatitis

Moderate increase: pancreatic CA

Inc. after administration of morphine or cholinergic drugs (+) constriction of sphincter of Oddi

Lipase

Blood lipase

• Cleaves fatty acids from lipoproteins and clears chylomicrons from the circulation

• Bound to vascular endothelium membrane

• Released into plasma by administration of heparin occurs within minutes of IV heparin dose post-heparin lipolytic activity (PHLA)

Table 1. Half-lives of clinically important enzymes in plasma Enzyme Range (hours) Lactate dehydrogenase (LD) LD-1 (H4) 50-70 LD-5 (M4) 8-14 Alanine transaminase (ALT, GPT) 40-50 Aspartate transaminase (AST, GOT) mitochondrial AST 6-7 cytosolic AST 12-17 Creatine kinase (CK) CK-MM 10-20 CK-MB 7-17 CK-BB 3 Alkaline phosphatase (ALP) liver ALP 190-230 bone ALP 30-50

Table 3. Enzyme markers of clinical significanceEnzyme (abbreviation) Clinical significanceAcid phosphatase (ACP) Prostatic carcinomaAlkaline phosphatase (ALP) Obstructive liver diseases, bone disordersAlanine transaminase (ALT,GPT) Hepatic disorders, viral hepatitisAspartate transaminase (AST,GOT) Myocardial infarction, hepatic disordersAlpha-amylase (AMS) Acute pancreatitisAldolase (ALS) Skeletal muscle disordersCreatine kinase (CK) Myocardial infarction, muscle disorders Gamma-glutamyl transferase (GGT) Hepatic disordersG-6-PD Drug-induced hemolytic anemiaLactate dehydrogenase (LD) Myocardial infarction, hepatic disorders,

carcionomasLipase (LPS) Acute pancreatitisLeucine aminopeptidase (LAP) Hepatobiliary disorders5’-Nucleotidase (5’NT) Hepatobiliary disorders

Pseudocholineesterase (PChE) Organophosphate poisoning (butyrylcholine as substrate)Ceruloplasmin (Copper-oxidase) Wilson’s disease (abnormal Cu metabolism)

Table 2. Serum normal (reference) ranges of clinical enzymes

Enzyme Abbreviation Range Stability (male> female)

Acid phosphatase ACP, AP 0.2-5.0 U/L +Alkaline phosphatase ALP 30-95 U/L +++Alanine transaminase ALT, G PT 6-37 U/L ++++Aspartate transaminase AST, GOT 5-30 U/L +++ Alpha-amylase AMS 95-290 U/L ++++ Aldolase ALS 1.5-8.0 U/L ++++ Creatine kinase CK, CPK 15-160 U/L -- Gamma-glutamyl transferase GGT 6-45 U/L ++++ Glucose-6-phosphate dehydrogenase G-6-PD 0-0.2 U/L +++ Lactate dehydrogenase LD, LDH 100-225 U/L + Lipase LPS 0-2 U/ml ++++Leucine aminopeptidase LAP 11-30 U/L +++5’-Nucleotidase 5’NT 2-15 U/L +++ Pseudocholineesterase PChE 5-12 U/ml ++++Ceruloplasmin (Copper-oxidase) 0.2-0.6 g/L

Therapeutic Enzymes

Therapeutic enzymes have a broad variety of specific uses Oncolytics Anticoagulants Thrombolytics Replacements for metabolic deficiencies

Digestive aids Metabolic storage disorders, etc

Miscellaneous enzymes of diverse function

Therapeutic Uses of Enzymes

Name of Enzyme

Mechanism of Action Indication

Enzymes used

systemically

•Streptokinase

and

•Urokinase

Increase amount of

proteolytic enzyme

“plasmin” by either

•Increasing the circulating

level of its precursor

“plasminogen” or

•Increasing the conversion

of plasminogen to plasmin.

Plasmin acts directly on

“fibrin”breaking it down to

achieve thrombolysis.

•Acute myocardial

infraction

•Acute thrombosis of

arteries

•Deep vein

thrombosis (DVT)

•Pulmonary embolism

L-Asparaginase Certain tumor cell require;

L-Asparagine for growth

L-Asparaginase hydrolyzes

L-Asparagine and growth of

tumour cell suffer.

•Acute leukaemia

•Malignant

lymphomas

Digestive

enzymes,

amylase, lipase

and protease

Replacement therapy in

pancreatic insufficiency

•Cystic fibrosis

•Chronic pancreatitis

•Following

pancreatectomy

Enzymes used

locally

Brings about

depolymerization of ground

substance and helps in

absorption of fluids.

•Promotes diffusion of

fluids given

subcutaenously (SC)

Pancreatic Pseudocyst If the plasma amylase activity fails to fall after

an attack of acute pancreatitis there may be leakage of pancreatic fluid into the lesser sac (a pancreatic pseudocyst).

Urinary amylase levels are high, differentiating it from macroamylasamaemia.

This is the one of few indications for estimating urinary amylase activity, which is inappropriately low relative to the plasma activity if there is glomerular impairment or macroamylasaemia.

Leakage of Enzymes from Cells

Enzymes are retained within their cells of origin by the plasma of membrane surrounding the cell.

The plasma membrane is a metabolically active part of the cell, and its integrity depends on the cell’s energy production.

Any process that impairs energy production, either through deprivation of oxidizable substrates or restriction of access of oxygen necessary for energy production, promotes deterioration of the cell membrane.

In such cases the membrane leaks its cellular components and, if cellular injury becomes reversible, the cell dies.

Small molecules are the first to leak from damaged or dying cell, followed by large molecules, such as enzymes; ultimately the entire contents of the necrotic cells are discharged.

Aspartate (Asp) + α-ketoglutarate ⇌ oxaloacetate + glutamate (Glu)

Altered Enzyme Production This contribution of enzymes to the circulating

blood may decrease, either as the result of genetic deficiency of enzyme production or the depression of enzyme production as a result of disease.

However, cases in which enzyme production is increased are for more general interest in diagnostic enzymology.

Two aminotransferases are used in diagnosis and management: aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

Hepatobiliary Disease

The response of the liver to any form of biliary tree obstruction is to induce the synthesis of ALP.

Alkaline Phosphatase

Alkaline phosphatase (ALP) catalyzes the alkaline hydrolysis for a large variety of naturally occurring and synthetic substrates, but the natural substrates on which they act in the body are not known.

ALP is present in practically all tissues of the body, especially at or in the cell membranes and it occurs at particularly high levels in intestinal epithelium, kidney tubules, bone (osteoblasts), liver and placenta.

Bone Disease

Among the bone diseases the highest levels of serum ALP activity are encountered in individuals with paget’s disease as a result of the action of osteoblastic cells.

Lactate Dehdrogenase (LD) Mosby

This enzyme exists in body tissues as a tetramer. Two monomers, H and M, can combine in various

proportion with the result that five isoenzymes of LD are known.

Increase in plasma LD activity are seen in a wide variety of conditions including acute damage of to the liver, skeletal muscle and kidneys, and also in megaloblastic and haemolytic anaemias.

In both cardiac muscle and red blood cells LD1 (H4) is the predominant isoenzyme.

Lactate Dehydrogenase

Lactate Dehydrogenase is a hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate with the mediation of NAD+ as hydrogen acceptor as follows.

The subunits composition of five isoenzymes, in order of decreasing are; LD-1, LD-2, LD-3, LD-4 and LD-5.

Changes in the serum LD activity are after a myocardial infraction.

Biochemistry ALT catalyzes the analogous reaction:

COO- COO- COO- COO-

│ │ │ │ H � C � NH2 + C=O ↔ C=O + H � C � NH2

│ │ │ │ CH3 CH2 CH3 CH2

│ │

CH2 CH2

│ │

COO- COO-

L-Alanine 2-Oxoglutarate Pyruvate L-Glutamate

Biochemistry AST catalyzes the following reaction:

COO- COO- COO- COO-

│ │ │ │ H � C � NH2 + C=O ↔ C=O + H � C � NH2

│ │ │ │ CH2 CH2 CH2 CH2

│ │ │ │

COO CH2 COO CH2

│ │

COO- COO-

L-Aspartate 2-Oxoglutarate Oxaloacetate L-Glutamate