CLINICAL CLINICAL ENZYMOLOGYENZYMOLOGY

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

Small amounts of intracellular Small amounts of intracellular enzymes are present in the enzymes are present in the blood as a result of normal cell blood as a result of normal cell turnover. When damage to cells turnover. When damage to cells occurs, increased amounts of occurs, increased amounts of enzymes will be released and enzymes will be released and their concentrations in the their concentrations in the blood will rise.blood will rise.

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

Other possible causes include:Other possible causes include:

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

induction) induction) obstruction to secretionobstruction to secretion

Enzyme activityEnzyme activity

Enzyme assays usually depend Enzyme assays usually depend on the measurement the catalytic on the measurement the catalytic activity of the enzyme, rather than activity of the enzyme, rather than the concentration of the enzyme the concentration of the enzyme protein itself. protein itself.

One One international unitinternational unit is the is the amount of enzyme that will amount of enzyme that will convert one micromole of convert one micromole of substrate per minute per litre of substrate per minute per litre of sample and is abbreviated as sample and is abbreviated as U/L. U/L.

Katal (catalytic activity)Katal (catalytic activity) is is defined as the number of mole of defined as the number of mole of substrate transformed per substrate transformed per second per litre of sample. second per litre of sample.

Disadvantages of enzyme Disadvantages of enzyme assaysassays

A major disadvantage in the A major disadvantage in the use of enzymes for the use of enzymes for the diagnosis of tissue damage is diagnosis of tissue damage is their lack of specificity to a their lack of specificity to a particular tissue or cell type. particular tissue or cell type. Many enzymes are common to Many enzymes are common to more than one tissuemore than one tissue. .

This problem may be This problem may be obviated to some extent in obviated to some extent in two ways: two ways:

first, different tissues may first, different tissues may contain (and thus release contain (and thus release when they are damaged) two when they are damaged) two or more enzymes in different or more enzymes in different proportionsproportions

SSecond, some enzymes exist in econd, some enzymes exist in different forms (isoforms)different forms (isoforms)

Individual isoforms are often Individual isoforms are often characteristic of a particular characteristic of a particular tissue: although they may have tissue: although they may have similar catalytic activitiessimilar catalytic activities

Alkaline phosphatase (ALP)Alkaline phosphatase (ALP)

Normal serum level of ALP is 40-125 U/L or Normal serum level of ALP is 40-125 U/L or 00..5-15-1..3 3 mmolmmol/(/(hourhour LL).).

This enzyme present in high concentrations in theThis enzyme present in high concentrations in the liverliver bone (osteoblasts)bone (osteoblasts) placenta placenta intestinal epithelium.intestinal epithelium.

The causes of an increase in The causes of an increase in plasma ALP activityplasma ALP activity

Physiological increases are been Physiological increases are been in pregnancy, due to the placental in pregnancy, due to the placental isoenzyme, and in childhood (when isoenzyme, and in childhood (when bones are growing), due to the bones are growing), due to the bone isoenzyme. bone isoenzyme.

Pathological: often >5 x ULNPathological: often >5 x ULN Paget's disease or bone osteomalaciaPaget's disease or bone osteomalacia ricketsrickets cholestasis (intra- and extrahepatic) cholestasis (intra- and extrahepatic) cirrhosis cirrhosis usually <5 x ULNusually <5 x ULN bone tumours (primary and secondary), bone tumours (primary and secondary), primary hyperparathyroidism with bone primary hyperparathyroidism with bone

involvement, healing fractures, involvement, healing fractures, OsteomyelitisOsteomyelitis hepatic space-occupying lesions (tumour, hepatic space-occupying lesions (tumour,

abscess)abscess)

ACID PHOSPHATASE (ACP)ACID PHOSPHATASE (ACP)

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

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

Normal serum value for ACP is Normal serum value for ACP is 2.5-12 U/L or 2.5-12 U/L or 00..025-0025-0..12 12 mmolmmol/(/(hourhour LL).).

ACP total value is increased in prostate ACP total value is increased in prostate cancer and highly elevated in bone cancer and highly elevated in bone metastasis of prostate cancer. metastasis of prostate cancer.

ACP is therefore an important tumour ACP is therefore an important tumour marker.marker.

ACP is present in high concentration in ACP is present in high concentration in semen, a finding which is used in semen, a finding which is used in forensic medicine in investigation of forensic medicine in investigation of rape.rape.

AminotransferasesAminotransferases

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

and and

alanine aminotransferase (ALT). alanine aminotransferase (ALT).

ASPARTATE AMINO ASPARTATE AMINO TRANSFERASE (AST)TRANSFERASE (AST)

It is also called as serum glutamate-It is also called as serum glutamate-oxaloacetate transaminase (SGOT).oxaloacetate transaminase (SGOT).

Normal serum level of AST is 8- 40 U/LNormal serum level of AST is 8- 40 U/L or or ((00..1-01-0..45 45 mmolmmol/(/(hourhourLL))))

It is significantly elevated in myocardial It is significantly elevated in myocardial infarction. It if moderately elevated in liver infarction. It if moderately elevated in liver diseases. diseases.

Measurement of cardiac Measurement of cardiac enzyme levelsenzyme levels

Measure cardiac enzyme levels at regular Measure cardiac enzyme levels at regular intervals, starting on admission and intervals, starting on admission and continuing until the peak is reached or until continuing until the peak is reached or until 3 sets of results are negative. Biochemical 3 sets of results are negative. Biochemical biomarkers are useful for both diagnosis biomarkers are useful for both diagnosis and prognostication and prognostication

Laboratory Diagnosis of Laboratory Diagnosis of Myocardial InfarctionMyocardial Infarction

ALANINE AMINO ALANINE AMINO TRANSFERASE (ALT)TRANSFERASE (ALT)

It is also called as serum It is also called as serum glutamate-pyruvate transaminase glutamate-pyruvate transaminase (SGPT). (SGPT).

Normal serum level of ALT is 5-30 Normal serum level of ALT is 5-30 U/L or (U/L or (00..1-1- 00.68 mmol.68 mmol/(/(hourhourLL))))

Very high values (100 to 1000 U/L) Very high values (100 to 1000 U/L) are seen in acute hepatitis, either are seen in acute hepatitis, either toxic or viral in origin. toxic or viral in origin.

Both ALT and AST are increased in Both ALT and AST are increased in liver diseases, but ALT >AST. liver diseases, but ALT >AST.

Moderate increase (25 to 100 U/L) Moderate increase (25 to 100 U/L) may be seen in chronic liver disease may be seen in chronic liver disease such as cirrhosis, and malignancy in such as cirrhosis, and malignancy in liver.liver.

Ritis coefficient (AST/ALT) in Ritis coefficient (AST/ALT) in normal conditions isnormal conditions is

11..3333 ±± 0,42.0,42.

LACTATE DEHYDROGENASE LACTATE DEHYDROGENASE (LDH) (LD)(LDH) (LD)

Normal value of LDH in serum is 100-200 Normal value of LDH in serum is 100-200 U/L. U/L.

Values the upper range are generally seen in Values the upper range are generally seen in children. Strenuous exercise will slightly children. Strenuous exercise will slightly increase the value. LDH level is 100 times increase the value. LDH level is 100 times more inside the RBC than in plasma, and more inside the RBC than in plasma, and therefore minor amount of hemolysis will therefore minor amount of hemolysis will result in a false-positive test.result in a false-positive test.

This enzyme exists in body This enzyme exists in body tissues as a tetramer. Two tissues as a tetramer. Two monomers, H and M, can monomers, H and M, can combine in various proportions combine in various proportions with the result that five with the result that five isoenymes of LD are known.isoenymes of LD are known.

So five combinations of H and So five combinations of H and M chains are possible; H4, M chains are possible; H4, H3M, H2M2, M3H and M4 H3M, H2M2, M3H and M4

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

Lactate dehydrogenase isoenzymes (as Lactate dehydrogenase isoenzymes (as percentage of total):percentage of total):

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

Increase in total LDH level is seen inIncrease in total LDH level is seen in hemolytic anemias, hemolytic anemias, hepatocellular damage,hepatocellular damage, muscular dystrophy, muscular dystrophy, carcinomas,carcinomas, leukemias, leukemias, and any condition which causes and any condition which causes

necrosis of body cells.necrosis of body cells.

Creatine kinase (CK)Creatine kinase (CK)

Normal serum value for CK is 15-100 U/L Normal serum value for CK is 15-100 U/L for males and 10-80 U/L for females.for males and 10-80 U/L for females.

CK is a dimer; each subunit has a CK is a dimer; each subunit has a molecular weight of 40,000. The subunits molecular weight of 40,000. The subunits are called B for brain and M for muscle.are called B for brain and M for muscle. Three isoenzymes, BB, MM and MB, occur. Three isoenzymes, BB, MM and MB, occur.

Normally CK2 (MB) iso-enzyme is only 5% Normally CK2 (MB) iso-enzyme is only 5% of the total activity. of the total activity.

Causes of an increased plasma creatine kinase activity.Causes of an increased plasma creatine kinase activity. often >10often >10 x ULNx ULN polymyositis polymyositis rhabdomyolysis (e.g. trauma, malignant hyperpyrexia) rhabdomyolysis (e.g. trauma, malignant hyperpyrexia) Duchenne muscular dystrophy Duchenne muscular dystrophy myocardial infarction myocardial infarction 5-10 x ULN5-10 x ULN following surgery following surgery skeletal muscle trauma skeletal muscle trauma severe exercise severe exercise myositis myositis carriers of Duchenne muscular dystrophy carriers of Duchenne muscular dystrophy usually <5 x ULNusually <5 x ULN physiological (Afro-Caribbeans) physiological (Afro-Caribbeans) hypothyroidism hypothyroidism drug (statin) treatment drug (statin) treatment

NUCLEOTIDE PHOSPHATASE NUCLEOTIDE PHOSPHATASE (NTP)(NTP)

It is a marker enzyme for plasma It is a marker enzyme for plasma membranes and is seen as an ecto-membranes and is seen as an ecto-enzyme (enzyme present on the cell enzyme (enzyme present on the cell membrane).membrane).

Normal NTP level in serum is 2-10 Normal NTP level in serum is 2-10 U/LU/L. It is moderately increased in . It is moderately increased in hepatitis and highly elevated in biliary hepatitis and highly elevated in biliary obstruction. obstruction.

GAMMA GLUTAMYL GAMMA GLUTAMYL TRANSFERASE (GGT)TRANSFERASE (GGT)

It is seen in liver, kidney, pancreas, It is seen in liver, kidney, pancreas, intestinal cells and prostate gland. intestinal cells and prostate gland.

In the body it is used in the synthesis In the body it is used in the synthesis of glutathione. of glutathione.

Normal serum value of GGT is 6-Normal serum value of GGT is 6-45 U/L in male and 5-30 U/L in 45 U/L in male and 5-30 U/L in female.female.

This value is moderately This value is moderately increased in infective hepatitis increased in infective hepatitis and prostate cancers. The GGT and prostate cancers. The GGT level is highly elevated in level is highly elevated in alcoholism, obstructive alcoholism, obstructive jaundice and neoplasm's of jaundice and neoplasm's of liver. liver.

PROSTATE SPECIFIC PROSTATE SPECIFIC ANTIGEN (PSA)ANTIGEN (PSA)

It is produced from the secretory It is produced from the secretory epithelium of prostal gland. It is epithelium of prostal gland. It is normally secreted into seminal fluidnormally secreted into seminal fluid

Normal value is 1 -5 µg/L. It is very Normal value is 1 -5 µg/L. It is very specific for prostate activity. Values specific for prostate activity. Values between 4-10 µg/L is seen in benign between 4-10 µg/L is seen in benign prostate enlargement; but values prostate enlargement; but values above 10 µg/L is indicative of prostate above 10 µg/L is indicative of prostate cancer.cancer.

CHOLINESTERASE (ChE)CHOLINESTERASE (ChE)

This enzyme is secreted by the liver This enzyme is secreted by the liver into the blood- stream and low plasma into the blood- stream and low plasma activities occur in chronic hepatic activities occur in chronic hepatic dysfunction. Low activities occur dysfunction. Low activities occur physiologically during pregnancy. physiologically during pregnancy.

Interest in this enzyme derives largely Interest in this enzyme derives largely from the fact that it hydrolyzes a muscle-from the fact that it hydrolyzes a muscle-relaxant drug, widely used in relaxant drug, widely used in anaesthesia, called succinylcholine anaesthesia, called succinylcholine (scoline). Occasionally, patients are (scoline). Occasionally, patients are found in whom the effect of this drug, found in whom the effect of this drug, which paralyzes respiration, persists for which paralyzes respiration, persists for several hours after it has been several hours after it has been administered (scoline apnoea). Many of administered (scoline apnoea). Many of these patients have an abnormal these patients have an abnormal cholinesterase activity. cholinesterase activity.

CHOLINESTERASE (ChE)CHOLINESTERASE (ChE)

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

Normal serum range is 2-12 U/mlNormal serum range is 2-12 U/ml It is present in nerve endings and in RBCs. It is present in nerve endings and in RBCs. Plasma cholinesterase activity also falls in Plasma cholinesterase activity also falls in

organophosphate poisoning.organophosphate poisoning. Organophosphorus insecticides (Parathione) Organophosphorus insecticides (Parathione)

irreversibly inhibit ChE in RBCs. Measurement irreversibly inhibit ChE in RBCs. Measurement of ChE level in RBCs is useful to determine the of ChE level in RBCs is useful to determine the amount of exposure in persons working with amount of exposure in persons working with these insecticides.these insecticides.

Pseudocholinesterase or type II Pseudocholinesterase or type II ChE is non-specific and can ChE is non-specific and can hydrolyse acyl esters. It is hydrolyse acyl esters. It is produced mainly by liver cells.produced mainly by liver cells.

Normal serum level is 8-18 U/ml. Normal serum level is 8-18 U/ml.

GLUCOSE-6-PHOSPHATE GLUCOSE-6-PHOSPHATE DEHYDROGENASEDEHYDROGENASE

This is an important enzyme in the This is an important enzyme in the hexose monophosphate shunt pathway hexose monophosphate shunt pathway of glucose. of glucose.

Normal value of GPD in RBC is 125-Normal value of GPD in RBC is 125-250 U/10250 U/101212 cells cells. .

It is mainly used for production of It is mainly used for production of NADPH. It has a special role in the RBC NADPH. It has a special role in the RBC metabolism. metabolism.

AMYLASEAMYLASE

This splits starch to maltose. It is This splits starch to maltose. It is activated by calcium, chloride and activated by calcium, chloride and fluoride ions. fluoride ions.

It is produced by pancreas and It is produced by pancreas and salivary glandssalivary glands

Normal serum value is 50-120 U/LNormal serum value is 50-120 U/L, , ((12-32 12-32 gg/(/(hour× Lhour× L))).).

The value is increased about 1000 The value is increased about 1000 times in acute pancreatitis which is a times in acute pancreatitis which is a life-threatening condition. The peak life-threatening condition. The peak values are seen between 5-12 hours values are seen between 5-12 hours after the onset of disease and returns after the onset of disease and returns to normal levels within 2-4 days after to normal levels within 2-4 days after the acute phase has subsided.the acute phase has subsided.

Moderate increase in serum levels Moderate increase in serum levels are seen in chronic pancreatitis, are seen in chronic pancreatitis, mumps (parotitis), obstruction of mumps (parotitis), obstruction of pancreatic duct and in renal disease. pancreatic duct and in renal disease.

Normal urine value is 20-160 Normal urine value is 20-160 gg/(/(hour× Lhour× L)) or (less than 375 U/L) or (less than 375 U/L)..

It is increased in acute pancreatitis. It It is increased in acute pancreatitis. It is increased on the 1 st day and is increased on the 1 st day and remains to be elevated for 7-10 days.remains to be elevated for 7-10 days.

LIPASELIPASE

It will hydrolyse triglyceride to β-It will hydrolyse triglyceride to β-monoglyceride and fatty acid. monoglyceride and fatty acid.

The enzyme is present in pancreatic The enzyme is present in pancreatic secretion.secretion.

Normal serum range is 0.2-1.5 Normal serum range is 0.2-1.5 U/LU/L. .

It is highly elevated in acute It is highly elevated in acute pancreatitis and this persists for pancreatitis and this persists for 7-14 days. Thus, lipase remains 7-14 days. Thus, lipase remains elevated longer than amylase. elevated longer than amylase.

Moreover, lipase is not increased Moreover, lipase is not increased in mumps. in mumps.

Aldolase (ALD)Aldolase (ALD) It is a tetrameric enzyme with A and B It is a tetrameric enzyme with A and B

subunits; so there are 5 iso-enzymes. It subunits; so there are 5 iso-enzymes. It is a glycolytic enzyme. is a glycolytic enzyme.

Normal range of serum is 1.5-7 U/L.Normal range of serum is 1.5-7 U/L. It is drastically elevated in muscle It is drastically elevated in muscle

damages such as progressive muscular damages such as progressive muscular dystrophy, poliomyelitis, myasthenia dystrophy, poliomyelitis, myasthenia gravis and multiple sclerosis. gravis and multiple sclerosis.

EnolaseEnolase

It is a glycolytic enzyme. Neuron-It is a glycolytic enzyme. Neuron-specific enolase (NSE) is an iso-specific enolase (NSE) is an iso-enzyme seen in neural tissues and enzyme seen in neural tissues and Apudomas. NSE is a tumour marker for Apudomas. NSE is a tumour marker for cancers associated with neuro-cancers associated with neuro-endocrine origin, small cell lung endocrine origin, small cell lung cancer,neuroblastoma,pheochromocytcancer,neuroblastoma,pheochromocytom, medullary carcinoma of thyroid, om, medullary carcinoma of thyroid, etc. etc.

Upper limit of NSE is 12 μg/ml.Upper limit of NSE is 12 μg/ml.