Isoenzymes & Clinical Enzymology

Gandham.Rajeev

Plasma Enzymes

Sources of plasma enzymes: Two types

Plasma derived

Cell derived

Plasma derived enzymes:

These enzymes act on substrates in plasma &

their activity is higher in plasma than cells.

E.g. coagulation enzymes.

Cell derived enzymes

These enzymes have a high activity in cells & overflow

into the plasma.

Further subdivided into

Secretory enzymes:

These are mainly derived from digestive glands &

function in extracellular space.

Metabolic enzymes:

These are concerned with intermediary metabolism &

function in the cells

Classification of Plasma enzymes

Enzymes present in plasma are grouped into

functional & non-functional enzymes

Functional enzymes Non-functional enzymes

Pseudocholinesterase Aspartate transaminase

Lipoprotein lipase Creatinine kinase

Ceruloplasmin Amylase

Blood coagulation enzymes Alkaline phosphatase

Functional enzymes

Few enzymes in plasma are functionally important & they are

involved in blood clotting, lipoprotein metabolism & drug

metabolism.

Synthesized in liver & released into plasma.

E.g. Pseudocholinesterase, Lipoprotein lipase, Ceruloplasmin,

Blood coagulation enzymes

Clinical significance:

These are present in higher concentrations in plasma than cells.

These are clinically significant when the serum level is

decreased below the reference range.

Non-functional enzymes

Most of the enzymes present in plasma serve no function in the

plasma.

Non-functional enzymes are derived from cells of organs &

tissues.

Present in high concentrations within cells

E.g. AST, ALT, LDH,CK,ALP, amylase

Clinical significance:

Non-functional enzymes in plasma are present in low

concentrations.

These are clinically important when the serum level is increased

above the reference range.

Causes for increased level of non-functional enzymes in plasma

Increased level of non-functional enzymes in plasma

include

Hypoxic or infective insults

Disease states of tissues

Excessive synthesis or induction of enzymes by cells with

overflow into plasma

Vigorous exercise

Decreased renal clearance

Isoenzymes

Isoenzymes or isozymes are multiple forms of same

enzyme that catalyze the same chemical reaction

Different chemical and physical properties:

Electrophoretic mobility

Kinetic properties

Amino acid sequence

Amino acid composition

Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is an enzyme present in

a wide variety of organisms

EC 1 = oxidoreductase.

EC 1.1 = acting on the CH-OH group of the donor.

EC 1.1.1 = With NAD or NADP as acceptor.

EC 1.1.1.27 = L-lactate dehydrogenase.

Molecular weight- 32 kD & it is tetramer

M (A) -muscle –chromosome 11(basic)

H (B) -heart – chromosome 12(acidic)

Lactate dehydrogenase, reversibly converts lactate to

pyruvate, in different tissues.

LDH consists of 5 iso-enzymes –

LDH1,LDH2,LDH3,LDH4 & LDH5

These isoenzymes are separated by cellulose acetate

electrophoresis at pH 8.6

Normal values:

Serum -100 -200 U/L

CSF - 7 -30 U/L

Urine - 40 -100 U/L

LDH reaction

LDH isoforms

LDH isoforms

Isoenzyme name

Composition Electrophoretic migration

Present in Elevated in

LDH 1Heat resistant ( H4) Fastest moving

Myocardium, RBC, kidney

myocardial infarction

LDH2Heat resistant (H3M1)

Myocardium, RBC, kidney

Kidney disease,megaloblastic anemia

LDH3 (H2M2) brain Leukemia, malignancy

LDH4Heat labile

(H1M3) Lung, spleen Pulmonary infarction

LDH5Heat labileInhibited by

urea

(M4) Slowest moving Skeletal muscle, Liver

Skeletal muscle and liver diseases

Clinical significance of LDH

In normal serum, LDH2 (H3M) predominant

isoenzyme & LDH5 is rarely seen.

In myocardial infarction, LDH1(H4) levels are greater

than LDH2.

Megaloblastic anemia (50 times upper limit of LDH 1

and LDH 2)

Muscular dystrophy, LDH5 (M4) is increased.

Toxic hepatitis with jaundice (10 times more LDH5)

Renal disease- tubular necrosis or pyelonephritis, pulmonary

embolism LDH 3 (massive destruction of platelets)

Total LDH is increased in neoplastic diseases.

LDH5 is increased in breast cancer, malignancies of CNS,

prostatic carcinoma.

In leukaemias, LDH2 & LDH3 levels are increased.

In malignant tumors of testis & ovaries, LDH2, LDH3 & LDH

5 levels are increased.

In CSF:

Bacterial meningitis – LDH4 and LDH5

Viral meningitis - LDH1

Metastatic tumors - LDH5

Neonatal cases of intracranial haemorrhage

associated with seizures and hydrocephalus

Creatinine phosphokinase (CPK)

It catalyses creatine to creatine phosphate. Normal serum value: 15-100 U/L for males & 10-80 U/L for females.

CPK consists of 3 isoenzymes.

Each isoenzyme of CK is a dimer;

Molecular weight of 40 kD.

The subunits are called B for brain (chromosome -14)

& M for muscle (chromosome -19)

Creatine Phosphokinase (CPK)

It is an important enzyme in energy

metabolism.

Immediate source of ATP in contracting muscle.

Three Iso-enzymes are separated by electrophoresis.

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

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.

Creatine phosphokinase isoenzymes

ISOENZYMES

SUB-UNIT TISSUE % IN SERUM

CK1 Fast moving

BB Brain 1

CK2 2% of total

MB Heart 5

CK3 Slow moving MM

Skeletal muscle 80

Clinical significance of CK

CPK & heart attack:

CPK2 isoenzymes is very small, (2% of total CPK

activity) & undetectable in plasma.

In myocardial infarction (MI), CPK2 levels are

increased within 4 hrs, then falls rapidly.

Total CPK level is elevated upto 20-folds in MI.

CPK & Muscle diseases

CPK level is elevated in muscular dystrophy (500-

1500U/L)

CPK level is highly elevated in crush injury, fracture &

acute cerebrovascular accidents.

Estimation of total CPK is employed in muscular

dystrophies & CPK-MB isoenzyme is estimated in

myocardial infarction.

Atypical forms of CK

Two atypical isoforms.

1. Macro-CK (CK-macro)

Formation:

Formed by aggregation CK-MB with IgG, sometimes IgA.

Also formed by complexing CK-MM with lipoproteins.

Electrophoretically migrates between CK-MB & CK-MM.

Occurs frequently in women above 50 years.

CK-Mi (Mitochondrial CK-Isoenzyme)

Formation:

It is present bound to the exterior surface of inner

mitochondrial membrane of muscle, liver & brain.

It exist in dimeric form & oligomeric aggregates &

molecular weight 35,000

Electrophoretically, migrates towards cathode & is

behind CK-MM band.

It is not present in normal serum.

Clinical significance

It is present in serum when there is extensive tissue

damage causing breakdown of mitochondrial & cell wall.

Its presence in serum indicates cellular damage, seen in

malignancies.

Alkaline Phosphatase (ALP)

ALP is nonspecific enzyme. It hydrolyses aliphatic, aromatic or heterocyclic

compounds. Optimum pH-9 &10 & it is activated by Mg2+ &Mn. Zn is a constituent of ALP. It is produced by osteoblasts of bone, and is associated

with the calcification process. It is localised in cell membranes -ecto-enzyme. It is associated with transport mechanisms in liver,

kidney & intestinal mucosa.

Normal range-40-125 U/L.

In children, Increased levels are seen, due to increased

osteoblastic activity.

Moderate (2-3) increase in ALP level is seen in hepatic diseases

such as infective hepatitis, alcoholic hepatitis or hepatocellular

carcinoma.

Very high levels of ALP (10-12 times of upper limit) may be

noticed in extrahepatic obstruction (obstructive jaundice) caused

by gallstones or by pressure on bile duct by carcinoma of head of

pancreas

Intrahepatic cholestasis may be due to virus (infective

hepatitis) or by drugs (chlorpromazine).

ALP is produced by epithelial cells of biliary canaliculi &

obstruction of bile with consequent irritation of epithelial

cells leads to secretion of ALP into serum.

Drastically high levels of ALP (10-25 times of upper limit) are

seen in bone diseases where osteoblastic activity is

enhanced such as Paget's disease (osteitis deformatis),

rickets, osteomalacia, osteoblastoma, metastatic carcinoma

of bone and hyperparathyroidism.

Isoenzymes of ALP

Alpha-1 ALP moves in alpha-1 position, it is synthesized by

epithelial cells of biliary canaliculi.

It is about 10% of total activity and is increased in obstructive

jaundice.

Alpha-2 heat labile ALP is stable at 56oC; but loses its activity

when kept at 65oC for 30 minutes.

It is produced by hepatic cells.

This liver iso-enzyme forms about 25% of total ALP.

Alpha-2 heat stable ALP will not be destroyed at

65oC, but is inhibited by phenylalanine.

It is of placental origin, which is found in blood in

normal pregnancy.

An isoenzyme closely resembling the placental form

is characteristically seen in circulation in about 15%

cases of carcinoma of lung, liver and gut and named

as Regan iso-enzyme or carcinoplacental iso-

enzyme.

Normal level is only 1% of the total ALP.

Pre-beta ALP is of bone origin and elevated levels are seen in bone

diseases.

This is heat labile (destroyed at 56°C, 10 min).

This constitutes about 50% of normal ALP activity.

Heat labile bone iso-enzyme of alkaline phosphatase (BAP) is a marker of

bone disease.

Gamma-ALP is inhibited by phenylalanine and originates from intestinal

cells.

It is increased in ulcerative colitis.

About 10% of plasma ALP are of intestinal origin.

The leukocyte alkaline phosphatase (LAP) is significantly decreased in

chronic myeloid leukemia & it is increased in lymphomas.

Clinical significance of enzymes & iso-enzymes in different disease

conditions

Clinical significance of enzymes & isoenzymes

Serum enzymes in heart diseases/cardiac biomarkers

Biomarker is useful in detecting dysfunction of an organ.

Cardiac biomarkers are used to detect cardiac diseases, include

Any chest pain

Unstable angina

Suspicious ECG changes

History suggestive of myocardial infarction

Following surgical coronary revascularization

Patients with hypotension and dyspnea

Markers for cardiac diseases

Creatine kinase (CK-MB)

Cardiac troponin I (CTI) & Cardiac troponin (CTT)

CTI & CTT are not true enzymes

Brain natriuretic peptide (BNP)

BNP is a reliable marker of ventricular function

Lactate dehydrogenase

Aspartate transferase

Creatinine phosphokinase (CPK)

It catalyses creatine to creatine phosphate. Normal serum value 15-100 U/L for males & 10-80 U/L for females.

CPK consists of 3 isoenzymes.

Each isoenzyme of CK is a dimer;

Molecular weight of 40 kD.

The subunits are called B for brain (chromosome -14)

& M for muscle (chromosome -19)

Creatine Phosphokinase (CPK)

It is an important enzyme in energy

metabolism.

Immediate source of ATP in contracting muscle.

Three Iso-enzymes are separated by electrophoresis.

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

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.

Creatine phosphokinase isoenzymes

ISOENZYMES

SUB-UNIT TISSUE % IN SERUM

CK1 Fast moving

BB Brain 1

CK2 2% of total

MB Heart 5

CK3 Slow moving MM

Skeletal muscle 80

Clinical significance of CK

CPK & heart attack:

CPK2 isoenzymes is very small, (2% of total CPK

activity) & undectable in plasma.

In myocardial infarction (MI), CPK2 levels are

increased within 4 hrs, then falls rapidly.

Total CPK level is elevated upto 20-folds in MI.

CPK & Muscle diseases

CPK level is elevated in muscular dystrophy (500-

1500U/L)

CPK level is highly elevated in crush injury, fracture &

acute cerebrovascular accidents.

Estimation of total CPK is employed in muscular

dystrophies & CPK-MB isoenzyme is estimated in

myocardial infarction.

Cardiac troponins (CTI/CTT)

They are not enzymes.

Troponins are now accepted as reliable markers for MI

Cardiac troponins have become one of the main tests in early

detection of an ischemic episode and in monitoring the

patient

The troponin complex consists of 3 components

Troponin C (calcium binding subunit),

Troponin I (actomyosin ATPase inhibitory subunit) &

Troponin T (tropomyosin binding subunit)

Troponin I (TnI) is encoded by 3 different genes, giving rise to 3

isoforms; the "slow" and "fast" moving forms are skeletal

variety.

Cardiac isoform is specific for cardiac muscle; the amino acid

sequence is different in skeletal muscle isoform.

Cardiac isoform of CTnT and CTnI are mainly (95%) located in

myofibrils and the remaining 5% is cytoplasmic.

They are identified and quantitated by immunological (ELISA or

immuno turbidimetric) reactions.

Troponins are seen in skeletal and cardiac muscles, but not in

smooth muscles.

Troponin I is released into the blood within 4 hours after the onset of

symptoms of myocardial ischemia; peaks at 14-24 hours and remains

elevated for 3-5 days post-infarction.

CTI is very useful as a marker at any time interval after the heart

attack.

It is not increased in muscle injury; whereas CK2 may be elevated in

some muscle injury.

The initial increase is due to liberation of the cytoplasmic fraction and

sustained elevation is due to the release from myofibrils.

Serum level of Troponin T (TnT) increases within 6 hrs of myocardial

infarction, peaks at 72 hours and then remains elevated up to 7-14

days.

Brain Natriuretic Peptide (BNP)

The natriuretic peptide family consists of three

peptides:

Atrial natriuretic peptide (ANP),

Brain natriuretic peptide (BNP)

C-type natriuretic peptide (CNP).

The clinical significance of CNP is not clear.

ANP is produced primarily in the cardiac atria.

BNP is present in human brain, but more in the cardiac

ventricles.

Human pro–BNP contains 108 amino acids.

It is cleaved by enzymes within cardiac myocytes into the

active C-terminal BNP (32 amino acids) and an inactive

peptide (proBNP 1–76).

Both are seen in circulation.

The active BNP is secreted by the ventricles of the heart

in response to excessive stretching of heart muscle cells

(cardiomyocytes).

Clinical Significance

Patients with congestive heart failure have high plasma

concentrations of ANP and BNP.

The concentrations are correlated with the extent of

ventricular dysfunction.

High concentrations of BNP predict poor long-term survival.

In breathlessness, BNP test helps in the differentiation of the

cause as heart failure or obstructive lung disease.

The best marker of ventricular dysfunction is pro-BNP.

Lactate Dehydrogenase

Lactate dehydrogenase (LDH) is an enzyme present in

a wide variety of organisms, including plants and

animals"

EC 1 = oxidoreductase.

EC 1.1 = acting on the CH-OH group of the donor.

EC 1.1.1 = With NAD or NADP as acceptor.

EC 1.1.1.27 = L-lactate dehydrogenase.

Molecular weight- 32 kD & it is tetramer

M (A) -muscle –chromosome 11(basic)

H (B) -heart – chromosome 12(acidic)

Lactate dehydrogenase, reversibly converts lactate to

pyruvate, in different tissues.

LDH consists of 5 iso-enzymes –

LDH1,LDH2,LDH3,LDH4 & LDH5

These isoenzymes are separated by cellulose acetate

electrophoresis at pH 8.6

Normal values:

Serum -100 -200 U/L

CSF - 7 -30 U/L

Urine - 40 -100 U/L

LDH reaction

LDH isoforms

LDH isoforms

Isoenzyme name

Composition Electrophoretic migration

Present in Elevated in

LDH 1Heat resistant ( H4) Fastest moving

Myocardium, RBC, kidney

myocardial infarction

LDH2Heat resistant (H3M1)

Myocardium, RBC, kidney

Kidney disease,megaloblastic anemia

LDH3 (H2M2) brain Leukemia, malignancy

LDH4Heat labile

(H1M3) Lung, spleen Pulmonary infarction

LDH5Heat labileInhibited by

urea

(M4) Slowest moving Skeletal muscle, Liver

Skeletal muscle and liver diseases

Clinical significance of LDH

In normal serum, LDH2 (H3M) predominant

isoenzyme & LDH5 is rarely seen.

In myocardial infarction, LDH1(H4) levels are greater

than LDH2.

Megaloblastic anemia (50 times upper limit of LDH 1

and LDH 2)

Muscular dystrophy, LDH5 (M4) is increased.

Toxic hepatitis with jaundice (10 times more LDH5)

Renal disease- tubular necrosis or pyelonephritis

Pulmonary embolism LDH 3 (massive destruction

of platelets)

Total LDH is increased in neoplastic diseases.

LDH5 is increased in breast cancer, malignancies

of CNS, prostatic carcinoma.

In leukaemias, LDH2 & LDH3 levels are increased.

In malignant tumors of testis & ovaries, LDH2,

LDH3 & LDH 5 levels are increased.

In CSF:

Bacterial meningitis – LDH4 and LDH5

Viral meningitis - LDH1

Metastatic tumors - LDH5

Neonatal cases of intracranial haemorrhage

associated with seizures and hydrocephalus

Aspartate aminotransferase (AST)

It was also called as serum glutamate oxaloacetate transaminase

(SGOT).

AST needs pyridoxal phosphate (vitamin B6) as co-enzyme.

Normal serum level: 8 to 20 U/L.

It is a marker of liver injury and shows moderate to drastic increase

in parenchymal liver diseases like hepatitis and malignancies of liver.

AST was used as a marker of myocardial ischemia in olden days.

The level is significantly elevated in myocardial infarction.

But troponins have replaced AST as a diagnostic marker in IHD

Alanine aminotransferase (AST)

It was called as serum glutamate pyruvate transaminase (SGPT) The enzyme needs pyridoxal phosphate as coenzyme.Normal serum level: male is 13-35 U/L & female is 10-30 U/L.Very high values (300 to 1000 U/L) are seen in acute hepatitis,

either toxic or viral in origin.Both ALT and AST levels are increased in liver disease, but ALT >

AST. Rise in ALT levels may be noticed several days before clinical signs

such as jaundice are manifested.Moderate increase (50 to 100 U/L) of ALT may be seen in chronic

liver diseases such as cirrhosis, hepatitis C and non-alcoholic steatohepatitis (NASH).

Alkaline Phosphatase (ALP)

ALP is nonspecific enzyme.

It hydrolyses aliphatic, aromatic or heterocyclic compounds.

Optimum pH-9 &10 & it is activated by Mg2+ &Mn

Zn is a constituent of ALP.

It is produced by osteoblasts of bone and is associated with the

calcification process.

It is localised in cell membranes -ecto-enzyme

Associated with transport mechanisms in liver, kidney & intestinal

mucosa.

Normal range-40-125 U/L.

In children, Increased levels are seen, due to increased

osteoblastic activity.

Moderate (2-3) increase in ALP level is seen in hepatic diseases

such as infective hepatitis, alcoholic hepatitis or hepatocellular

carcinoma.

Very high levels of ALP (10-12 times of upper limit) may be

noticed in extrahepatic obstruction (obstructive jaundice)

caused by gallstones or by pressure on bile duct by carcinoma

of head of pancreas

Intrahepatic cholestasis may be due to virus (infective hepatitis)

or by drugs (chlorpromazine).

ALP is produced by epithelial cells of biliary canaliculi &

obstruction of bile with consequent irritation of epithelial cells

leads to secretion of ALP into serum.

Drastically high levels of ALP (10-25 times of upper limit) are seen

in bone diseases where osteoblastic activity is enhanced such as

Paget's disease (osteitis deformans), rickets, osteomalacia,

osteoblastoma, metastatic carcinoma of bone and

hyperparathyroidism.

Enzyme profiles in liver diseases

Enzymes commonly studied for diagnosis of liver diseases

are:

Alanine amino transaminase (ALT)

Alkaline phosphatase (ALP)

Nucleotide phosphatase (NTP)

Gamma glutamyl transferase (GGT)

Nucleotide Phosphatase (NTP)

It is also known as 5' nucleotidase.

This enzyme hydrolyses 5' nucleotides to corresponding nucleosides

at an optimum pH of 7.5.

Nickel ions inhibit NTP but not ALP.

It is a marker enzyme for plasma membranes and it is ecto-enzyme

Normal NTP level in serum is 2-10 IU/L.

It is moderately increased in hepatitis and highly elevated in biliary

obstruction.

Unaffected by bone diseases.

Gamma Glutamyl Transferase (GGT)

It can transfer gamma glutamyl residues to substrate. In the body it is used in the synthesis of glutathioneGGT has 11 iso-enzymes. It is seen in liver, kidney, pancreas, intestinal cells & prostate

gland.Normal serum value of GGT is 10-30 U/L.It is moderately increased in infective hepatitis and prostate

cancersGGT is clinically important because of its sensitivity to detect

alcohol abuse. GGT is increased in alcoholics.Increase in GGT level is generally proportional to the amount of

alcohol intake.

Enzyme profiles in muscle diseases

Enzymes commonly studied for diagnosis of muscle diseases are:

AST

CPK

Aldolase

In muscular dystrophies, probably due to increased leakage of

enzymes from damaged cells.

CPK is most reliable indicator of muscular diseases.

Aldolase

It is a tetrameric enzyme with A and B subunits; so there

are 5 iso-enzymes.

It is a glycolytic enzyme.

Normal range of serum is 1.5-7 U/L.

It is drastically elevated in muscle damages such as

progressive muscular dystrophy, poliomyelitis, myasthenia

gravis and multiple sclerosis.

It is a very sensitive early index in muscle wasting

diseases.

Enzyme profiles in bone diseases

Serum alkaline phosphatase remains the only useful

enzyme assay for investigation.

ALP is most valuable index of osteoblastic activity.

Increased ALP activity is seen in rickets, osteomalacia,

hyperparathyroidism & in Paget’s disease.

In malignancies of bone, the level depends on the severity

& degree of new bone formation.

In hypophosphatasia, where there is defective calcification,

low serum ALP activity is observed.

Enzyme profiles in malignancies

Enzymes commonly studied for diagnosis of malignancies are:

Serum acid phosphatse (ACP)

Serum prostate specific antigen (PSA)

Serum enolase

Serum alkaline phosphatase (ALP)

Serum LDH

Serum aldolase

β-Glucuronidase

Serum acid phosphatase (ACP)

It hydrolyses phosphoric acid ester at pH between 4 and 6.

Normal serum value for ACP is 2.5-12 U/L.

ACP is secreted by prostate cells, RBC, platelets and WBC.

The prostate iso-enzyme is inactivated by tartaric acid.

Normal level of tartrate labile fraction of ACP is 1 U/L.

ACP total value is increased in prostate cancer and highly

elevated in bone metastasis of prostate cancer.

In these conditions, the tartrate labile iso-enzyme is elevated.

Very helpful in follow-up of treatment of prostate cancers.

Prostate specific antigen (PSA)

It is produced from the secretory epithelium of prostate

gland.

It is normally secreted into seminal fluid, where it is

necessary for the liquefaction of seminal coagulum.

It is a serine protease, and is a 32 kD glycoprotein.

In blood it is bound to alpha-2-macroglobulin and alpha-1-

antitrypsin; a very small fraction is in the free form also.

Normal value is 1-5 microgram/L.

It is very specific for prostate activity.

Values above 10 microgram/L is indicative of prostate

cancer.

Enolase

It is a glycolytic enzyme.

Neuron-specific enolase (NSE) is an iso-enzyme seen in neural

tissues

NSE is a tumor marker for cancers associated with

neuroendocrine origin, small cell lung cancer, neuroblastoma,

pheochromocytoma, medullary carcinoma of thyroid, etc.

It is measured by RIA or ELISA.

β-Glucuronidase

Not routinely done.

β-Glucuronidase most abundant in liver, spleen,

endometrium, breast & adrenals.

Leukocytes contain high content.

Increased levels are seen in cancer of urinary bladder.

Very high levels are seen in carcinoma of head of pancreas &

in 50% cases of breast cancer.

Enzyme profiles in pancreatic diseases

Amylase

The enzyme splits starch to maltose & activated by Ca2+ & Cl-

ions.

It is produced by pancreas and salivary glands.

Normal serum value is 50-120 IU/L.

The value is increased about 1000 times in acute pancreatitis

which is a life-threatening condition.

The peak values are seen between 5-12 hours after the onset of

disease and returns to normal levels within 2-4 days after the

acute phase has subsided.

Moderate increase in serum levels are seen in chronic

pancreatitis, mumps (parotitis) and obstruction of pancreatic

duct.

Normal value of amylase in urine is less than 375 U/L.

It is increased in acute pancreatitis.

It is increased on the 1st day and remains to be elevated for 7-

10 days.

lipase

It will hydrolyse triglyceride to beta-monoglyceride and fatty

acid.

The enzyme is present in pancreatic secretion.

The level in blood is highly elevated in acute pancreatitis and this

persists for 7-14 days.

Lipase remains elevated longer than amylase.

Lipase is not increased in mumps.

Lipase estimation has advantage over amylase.

It is moderately increased in carcinoma of pancreas, biliary

diseases and perforating peptic ulcers.

Cholinesterase (ChE)

Acetyl cholinesterase or true ChE or Type 1 ChE can act mainly on acetyl

choline.

It is present in nerve endings and in RBCs.

Newly formed RBC will have high levels of ChE which is slowly reduced

according to the age of the cell.

ChE level in RBCs will be proportional to the reticulocyte count.

Organophosphorus insecticides (Parathione) irreversibly inhibit ChE in

RBCs.

Measurement of ChE level in RBCs is useful to determine the amount of

exposure in persons working with these insecticides

Pseudo cholinesterase or type II ChE is nonspecific and can

hydrolyse acyl esters.

It is produced mainly by liver cells.

Succinyl choline is a widely used muscle relaxant.

It is a structural analogue of ACh, and competitively fixes on post-

synaptic receptors of ACh.

Succinyl choline is hydrolysed by the liver ChE within 2–4 minutes.

But in certain persons the ChE activity may be absent; this is a

genetically transmitted condition.

In such individuals when succinyl choline is given during surgery,

it may take hours to get the drug metabolized.

Glucose-6-phosphate Dehydrogenase

It is a dimer with identical subunits.

It is an important enzyme in the hexose monophosphate shunt

pathway of glucose

It is mainly used for production of NADPH.

Hydrogen peroxide is continuously formed inside the RBC.

Peroxide will destroy RBC cell membrane.

Glutathione and NADPH will prevent this process.

NADPH is very essential for preserving the RBC integrity.

Drug Induced Hemolytic Anemia

Normal value of GPD in RBC is 6-12 U/g of Hb.

This is reduced in drug induced hemolytic anemias.

In the GPD deficient individuals, RBC life-span may be reduced, but

there will be no disease manifestations.

But when certain drugs (aspirin, mepacrine, primaquine, sulpha) are

taken by such individuals, there will be sudden damage to RBCs.

Primaquin stimulates peroxide formation.

In G6PD deficient cells the level of NADPH is low, leading to

unchecked build up of peroxides resulting in premature cell lysis.

This drug-induced hemolytic anemia is characteristic of GPD

deficiency

Therapeutic use of enzymes

Enzyme Therapeutic application

Asparaginase Acute lymphoblastic leukemia

Streptokinase To lyse intravascular clot

Urokinase To lyse intravascular clot

Pancreatin (trypsin & lipase) Pancreatic insufficiency; oral administration

Papain Anti-inflammatory

Alpha-1-antitrypsin AAT deficiency; emphysema

Enzymes used for diagnostic purpose

Enzyme Used for testing

Urease Urea

Uricase Uric acid

Glucose oxidase Glucose

Peroxidase Glucose, Cholesterol

Hexokinase Glucose

Cholesterol oxidase Cholesterol

Lipase Triglycerides

Alkaline phosphatase ELISA

Horse radish peroxidase ELISA

Restriction endonuclease Southern blot; RFLP

Reverse transcriptase Polymerase chain reaction (RT=PCR)

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