Clinical Enzymology 2

ENZYMES OF CLINICAL INTEREST
DR. APPIAH'S LECTURE SERIES 1

Enzymes of Clinical Interest
• Lactate Dehydrogenase (LDH)
• Creatine Kinase (CK)
• Aspartate Transaminase (AST)/ Glutamate Oxaloacetate Transaminase

(GOT)
• Alanine Transaminase (ALT)/ Glutamate Pyruvate Transaminase (GPT)

Enzymes of Clinical Interest
• Aldolase
• Pseudocholinesterase
• Alkaline phosphatase (ALP)
• Acid phosphatase
• Prostate Specific Antigen (PSA)
• Amylase

Lactate Dehydrogenase (LDH)
• It’s an enzyme (EC 1.1.1.27) present in a wide variety of organisms,

including plants and animals.
• This enzyme catalyzes the reversible inter-conversion of lactate and

pyruvate.
• Lactate + 𝑁𝐴𝐷 + Pyruvate + NADH + 𝐻 +
• It is widely distributed in the body with high concentrations in heart and

skeletal muscle, liver, kidney, brain and erythrocytes.
Lactate Dehydrogenase (LDH)
• Total LDH will begin to rise 2 to 5 days after an MI; the elevation can
last 10 days.
• LDH (molecular weight of about 135,000) exists in body tissues as a

tetramer consisting of four polypeptide chains each with a molecular
weight of 33,000.
• Each chain is formed from one of two distinct monomers; H and M

which can combine in various proportions to give rise to five isomers
according to the chain structure as H4, H3M, H2M2, HM3 and M4.

Isomers of LDH
• LDH-1 (4H) - in the heart and red blood cells.
• LDH-2 (3H1M) - in the reticuloendothelial system.
• LDH-3 (2H2M) - in the lungs
• LDH-4 (1H3M) - in the kidneys, placenta, and pancreas
• LDH-5 (4M) - in the liver and striated muscle

Isomers of LDH
• The isomers are classified into LD1 to LD5 depending on the speed of
migration towards the anode during electrophoresis at pH of 7.4.
• LD1 migrates most rapidly towards the anode whereas LD5 is the
slowest.

Isomers of LDH
• It is possible to divide most tissues into one of three categories.
• Tissues in which the fast moving LD1 and LD2 (anodal isoenzymes)
predominate e.g. heart, red blood cells.
• Tissues in which the slow moving LD5 and LD4 (cathodal isoenzymes)
predominate e.g. liver and most skeletal muscles.
• Tissues in which no single component predominates e.g. lung and

spleen.
Isomers of LDH
• Elevation of LD1 and LD2 (LD1 greater than LD2) occurs in myocardial
infarction, megaloblastic anaemia and renal infarction.
• Elevation of LD2 and LD3 occurs in acute leukaemia.
• LD3 is the main isoenzyme elevated in many other malignancies.
• Elevation of LD5 occurs after damage to liver or skeletal muscle.
• The ‘normal’ level for serum lactate dehydrogenase is 130-500 U/l at

37oC in adults.

LDH
• In MI, LDH can be elevated about five times the normal value.
• It can also be elevated up to twenty (20) times in pernicious anaemia and

leukaemia.
• Lesser increases occur in other states of abnormal erythropoiesis e.g.

thalasemia, myelofibrosis and haemolytic anaemia.
• Moderate increases are observed in viral hepatitis, malignancies, skeletal

muscle disease, pulmonary embolism, and infectious mononucleosis.

Creatine kinase (CK)
• Is an enzyme (EC 2.7.3.2) expressed by various tissues and cell types.
• catalyses the conversion of creatine and consumes adenosine

triphosphate (ATP) to create phosphocreatine (PCr) and adenosine
diphosphate (ADP).

Creatine kinase (CK)
• CK activity is greatest in striated muscle, brain and heart tissues.
• Less activity may be found in tissues like smooth muscle, kidney and
the diaphragm.
• CK is a dimmer formed from different combinations of two types of

polypeptide chains or subunits designated as B or brain and M or
muscle.

Isoenzymes of CK
• Three different pairs of subunits
• BB (CK-1), MB (CK-2) and MM (CK-3).
• They have been numbered CK1, CK2 and CK3 on the basis of their electrophoretic
mobility.
• (CK-MM) - The skeletal and cardiac muscles.
• (CK-MB) - Heart muscle (presence in plasma suggests a myocardial infarction).
• CK-BB -brain, thyroid and the smooth muscle of the gastro-intestinal and genital
tracts.
Isoenzymes of CK
• All of them are found in the cell cytosol or are associated with myofibrillar
structures.
• A fourth isoenzyme exist which is designated as CK-Mt.
• Differs from the others both immunologically and electrophorectic mobility.
• located between the inner and outer membranes of mitochondria.
• Constitute about 15% of the total CK activity in the heart.

Creatine Kinase
• Plasma normally contains the MM isoenzyme and increase in total creatine
kinase is always due mainly to MM isoenzyme.
• Marked increases-myocardial infarction, muscular dystrophies and

Rhabdomyolysis, ‘shock’ and circulatory failure.
• Moderate increases-muscle injury, after surgery (for about a week),

hypothyroidism, alcoholism and acute psychotic episodes.
• The ‘normal’ range is:

Female :3-70 U/L Male :5-100 U/L at 37oC

Aspartate transaminase (AST)
• Serum glutamic oxaloacetic transaminase (SGOT).
• A pyridoxal phosphate dependent transaminase enzyme (EC 2.6.1.1).
• Catalyzes the transfer of an amino group from glutamic acid to

oxaloacetic acid to form α-ketoglutaric acid and aspartic acid
respectively.
• Aspartate (Asp) + α-ketoglutarate ⇌ oxaloacetate + glutamate (Glu)

Aspartate transaminase (AST)
• Found in the liver, heart, skeletal muscle, kidneys, brain, and red blood
cells.
• Two major isoenzymes; one cytoplasmic and the other mitochondrial.
• GOT1/cAST, the cytosolic isoenzyme - red blood cells and heart.
• GOT2/mAST, the mitochondrial isoenzyme - liver.

Marked elevations of AST
• Myocardial infarction
• Viral hepatitis
• Toxic liver necrosis (localized death of tissue)
• Circulatory failure with ‘shock’ and hypoxia.

Moderate elevations of AST
• Cirrhosis (2X normal)
• Cholestatic jaundice or obstructive jaundice (10X normal)
• Malignant infiltration of the liver
• Skeletal muscle disease
• Severe haemolytic anaemia
• Infectious mononucleosis
• After trauma (bodily injury, emotional shock) or surgery.

Alanine transaminase (ALT)
• Glutamate pyruvate transaminase (GPT).
• This enzyme catalyzes the transfer of an amino group from glutamic

acid to pyruvic acid to form α-ketoglutaric acid and alanine
respectively.
• glutamate + pyruvate ⇌ α-ketoglutarate + alanine

Marked elevations of ALT
• Viral hepatitis.
• Toxic liver necrosis.
• Circulatory failure with ‘shock’ and hypoxia.

Moderate elevations of ALT
• Cirrhosis (2X normal),
• Cholestatic jaundice (10X normal),
• Liver congestion secondary to cardiac failure
• Infectious mononucleosis with liver involvement
• Extensive trauma and muscle disease (less than AST).

Aldolase
• Catalyzes the splitting of fructose-1,6-diphosphate to
dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.
• Widely distributed.
• Found in the liver, muscle, erythrocytes and kidney.

Marked Elevations of Adolase
• Muscular dystrophy
• Malignant disease
• Hepatocellulary damage
• Haemolytic anaemia
Pseudocholinesterase
• The plasma analogue of acetylcholinesterase, an enzyme which
hydrolyzes acetylcholine.
• Synthesized chiefly in the liver.
• Responsible for the hydrolysis of succinylcholine, an analogue of

acetylcholine.
• The reference range of pseudocholinesterase is 2-5 U/L at 37oC.

Marked elevations of Pseudocholinesterase
• Recovery from liver damage
• Nephrotic syndrome

Decreased levels of Pseudocholinesterase
• Hepatitis
• Cirrhosis
• Anticholinesterases
• Inherited abnormal cholinesterase

Alkaline phosphatase (ALP)
• Alkaline phosphate is a hydrolytic enzyme
• derives its name from working optimally at the alkaline pH of 9-10.5.
• Widely distributed.
• Found in bones, liver, kidney, intestinal wall, lactating mammary and placenta.
• In bone, the enzyme is found in osteoblasts where it has been proposed to be

involved in bone deposition.

• Generally the enzyme is localized at the membrane border of cells and
appears to be significant in transport processes involving phosphate.
• A meal containing fats and oils increases the total ALP activity due to the
release of the intestinal isoenzyme into the plasma.
• Levels in infants rise rapidly within a few days after birth and 2-3 times the
adult value from age 1 year through puberty.
• Additional increases occur during puberty, after which values fall to adult
range.
• Because of its presence in the placenta, ALP is elevated during pregnancy,

especially in the last trimester.
Typical reference values for the various stages of life
are:
• Children (1-12 years): up to 350 U/L
• During puberty: up to 500 U/L
• Adults (up to 60 years) 20-90 U/L

ALP are increased in the following conditions
• Hepatobiliary disease
• Characterised by an elevation of serum ALP which includes extra and intra-hepatic
cholestasis and infectious hepatitis.
• Bone disease associated with increased osteoblastic activity.

• Paget’s disease,
• Primary and secondary hyperthyroidism (moderate)
• Osteomalacia
• Rickets
• Osteogenic bone cancer

Bone diseases
❑Achondroplasia:
• inherited condition characterized by arrested growth of the long bone
resulting in dwarfism.
❑Paget’s disease:
• excessive ALP causes too rapid bone formation
consequently bone is thin.
• There is loss of stature, crippling deformity, abnormally enlarged head,
collapse of vertebrae; and nervous complication can result.

❑Osteomalacia:
• infectious mineralization of the mature skeleton with
softening and bone pain. It is commonly caused by
insufficient dietary intake of vitamin D.
❑Osteomyelitis:
• inflammation commencing in the marrow of bone.
❑Rickets:
• a disorder of calcium and phosphorus metabolism
associated with a deficiency of vitamin D
• beginning most often in infancy and early childhood
between the ages of 6 months and 2 years.

Drugs that increase ALP activity
• Anabolic steroids • Allupurinol
• Androgens • Aspirin
• Testosterone • Coumarin
• Antibiotics (erythromycin, • Methyldopa

sulphonamides and tetracycline).
• Vitamin D.

Decrease in ALP activity
❑Conditions
• Hypophosphatasia
• Achondroplasia
• Cretinism
• Vitamin C deficiency.
.

Decrease in ALP activity
❑Chemicals
• Arsenicals
• Beryllium
• Cyanide
• high levels of lithium, potassium and zinc

Acid phosphatase (ACP)
• Generic name for a group of enzymes which hydrolyze phosphates at a

pH of 5-6.
• Abundant in prostate and seminal fluid.
• Small concentrations are present in liver, spleen, red cells, platelets,

kidney and bone.

Acid phosphatase (ACP)
• The isoenzymes may be distinguished by measuring activity in the presence
of selective inhibitors.
• Also by using a substrate that is preferentially hydrolyzed by one of the

isoenzymes.
• Most laboratories report either ‘tartrate labile’ or ‘formaldehyde-stable’ acid

phosphatase activity in addition to total plasma ACP activity.
• The reference range for total acid phosphatase in adult plasma is 0.5-5.5 U/L

Degree of Inhibition
Source of ACP Tartrate Labile Formaldehyde
Prostate Almost complete Minimal
Red cells Minimal Marked
Other tissues (Liver, Marked Moderate

Spleen)

Significance of ACP
• The demonstration of the presence and extent of invasive or metastatic
prostatic carcinoma.
• The response of prostatic carcinoma to oestrogen therapy.
• In metastasized prostatic carcinoma, plasma ACP levels rise probably

as a result of increase in the number of prostatic cells

Significance of ACP
• Osteoblastic metastasis results in a raise in plasma ACP to about 9 U/l.
• Severe spread levels above 200 U/l may be attained.
• In carcinoma with the capsule still confined within the capsule of the
prostate, plasma tartrate labile ACP (TLACP) activity does not rise
above the upper limit of the reference values.

Significance of ACP
• A patient with prostatic carcinoma on oestrogen therapy would have a dramatic fall
in the activity of TLACP.
• This indicates response to treatment.
• Any subsequent increase in TLACP is an indication of treatment failure.
• N.B. increased plasma TLACP activity is not absolutely reliable as an indication of

the presence of prostatic carcinoma since its occasionally increased in benign
prostatic hyperplasia.
• In 75% or more of cases with metastasing prostatic carcinoma however, there are
marked elevations.

Precautions to be observed during ACP determination
• ACP determination should not be done patients who have undergone rectal
examination within 48 hours.
• This causes release of prostatic ACP into circulation.
• The prostatic isoenzyme is unstable hence assay should be done immediately

after sample collection.
• If this is impossible, plasma or serum should be obtained and frozen.

Precautions to be observed during ACP
determination
• Straining at stool in patients with a tendency to constipate can cause the
release of prostatic ACP into the circulation.
• Enquires must thus be made about bowel habit and constipation treated if
necessary
• Total plasma ACP activity is also most often raised due to the presence of
haemolysis.
• It may also be increased in some diseases affecting liver and bone to such
an extent that plasma ACP activity is greatly elevated.

The prostate specific antigen (PSA)
• A serine protease that is normally confined to the epithelial tissue of the prostate
gland.
• It has a molecular weight of 33-34 kDa.
• The molecule consists of approximately 92% peptides and 8% carbohydrates.
• Exists in at least 5 isomers with isoelectric points (pI) ranging from 6.8 to 7.5.
• The major isoform having a pI of 6.9.

The prostate specific antigen (PSA)
• Like other proteases, PSA in serum consists mostly of complexes with
inhibitors.
• The predominant complex is with alpha-1 anti-chymotrypsin.
• Smaller amounts of PSA are also bound with α-1-trypsin inhibitor, α-2-
macroglobulin and inter-α-trypsin inhibitor.
• A minor amount of PSA also exists in the free form but whether the free
form is biologically active remains to be determined

• Most of the commonly available immunoassay kits for PSA appear to detect both
the free PSA and the ACT complex.
• The sum of both fractions contributes to the total PSA.
• As a serine protease, PSA can catalyze the hydrolysis of a number of proteinicious

substances.
• Measurement of serum of PSA provides a very sensitive index of prostate cancer.
• Being increased in over 90% of cases when first diagnosed with about 50% of cases
having increased serum prostatic acid phosphatase (PAP) activity at this stage.

• However serum PSA is increased in most patients with benign prostate
hypertrophy, so the measurement lacks specificity as a test for cancer.
• One potential approach to reducing the mortality from prostate

malignancy is to screen asymptomatic men for early disease
employing the PSA Kit.
• Using, this approach prostate cancer could be detected when it is

confined to the prostate and therefore potentially curable.

Amylase
• Group of hydrolases that split large polysaccharides consisting of α-D-
glucose units.
• Both linear polyglucans such as amylose and branched polyglucans

such as amylopectin and glycogen can be hydrolyzed.
• Two types of amylases have been identified.

• β-amylases
• α –amylases

Types of amylases
• β-amylase
• (e.g. plant and bacterial exoamylase) which acts only at the terminal reducing end of
the polyglucan chain, splitting off 2 glucose units at a time.
• α –amylases
• also called endoamylases are the human amylases.
• They attack the α-1, 4- linkage in a random manner anywhere along the polyglucan
chain.
• Big polysaccharides can thus be broken down into small units (dextrins, maltose and
some glucose).
• The optimum pH for amylase activity is 6.9-7.0. it is usually assayed at 37oC although
it is active at 50oC.

α- amylase
• α- amylase has several isoenzymes.
• The most abundant is derived from the pancreas .
• A potent α –amylase is secreted by the salivary glands.
• This isoenzyme initiates hydrolysis in the mouth and esophagus.

Action of amylase
• The action of the salivary isoenzyme is terminated by acid in the
stomach.
• The pancreatic and intestinal isoenzymes of amylase breakdown the

polyglucans into maltose.
• Intestinal amylase breaks the maltose to glucose which is then

absorbed into the circulation.

Acute Pancreatitis
• Serum levels of the enzyme rise 2-12 hours after the onset of acute pancreatitis in
many patients and within 24 hours in about 85% of cases.
• In most patients the serum amylase level reaches a peak by 24hours and returns to
normal in 48-72 hours.
• The administration of glucose causes a decrease in serum amylase level.
• Thus blood for amylase determination should not be taken during an intravenous
infusion containing glucose.
• Blood for serum amylase measurement should be collected at least 1 hour and
preferably 2 hours after the patient has eaten.

Causes of Increased Serum Amylase
• > 10X upper limit of normal: - acute pancreatitis.
• 5X upper limit of normal

• perforated duodenal ulcer,
• intestinal obstruction,
• acute oliguric renal failure,
• diabetes

Causes of Increased Serum Amylase
• Usually < 5X upper limit of normal:-
• Salivary gland disorders e.g. calculi and inflammation (including mumps)
• Chronic renal failure
• Macroamylasaemia
• Morphine administration (spasm of sphincter of Oddi)

Clinical Enzymology 2

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Clinical Enzymology 2

Uploaded by

Copyright:

Available Formats

ENZYMES OF CLINICAL INTEREST

DR. APPIAH'S LECTURE SERIES 1

• Lactate Dehydrogenase (LDH)

• Creatine Kinase (CK)

• Aspartate Transaminase (AST)/ Glutamate Oxaloacetate Transaminase

• Alanine Transaminase (ALT)/ Glutamate Pyruvate Transaminase (GPT)

DR. APPIAH'S LECTURE SERIES 2

• Alkaline phosphatase (ALP)

• Prostate Specific Antigen (PSA)

DR. APPIAH'S LECTURE SERIES 3

• It’s an enzyme (EC 1.1.1.27) present in a wide variety of organisms,

• This enzyme catalyzes the reversible inter-conversion of lactate and

• Lactate + 𝑁𝐴𝐷 + Pyruvate + NADH + 𝐻 +

• It is widely distributed in the body with high concentrations in heart and

• LDH (molecular weight of about 135,000) exists in body tissues as a

• Each chain is formed from one of two distinct monomers; H and M

DR. APPIAH'S LECTURE SERIES 5

• LDH-2 (3H1M) - in the reticuloendothelial system.

• LDH-3 (2H2M) - in the lungs

• LDH-4 (1H3M) - in the kidneys, placenta, and pancreas

• LDH-5 (4M) - in the liver and striated muscle

DR. APPIAH'S LECTURE SERIES 6

DR. APPIAH'S LECTURE SERIES 7

• Tissues in which no single component predominates e.g. lung and

• Elevation of LD2 and LD3 occurs in acute leukaemia.

• LD3 is the main isoenzyme elevated in many other malignancies.

• Elevation of LD5 occurs after damage to liver or skeletal muscle.

• The ‘normal’ level for serum lactate dehydrogenase is 130-500 U/l at

DR. APPIAH'S LECTURE SERIES 9

• It can also be elevated up to twenty (20) times in pernicious anaemia and

• Lesser increases occur in other states of abnormal erythropoiesis e.g.

• Moderate increases are observed in viral hepatitis, malignancies, skeletal

DR. APPIAH'S LECTURE SERIES 10

• catalyses the conversion of creatine and consumes adenosine

DR. APPIAH'S LECTURE SERIES 11

• CK activity is greatest in striated muscle, brain and heart tissues.

• CK is a dimmer formed from different combinations of two types of

DR. APPIAH'S LECTURE SERIES 12

• BB (CK-1), MB (CK-2) and MM (CK-3).

• (CK-MM) - The skeletal and cardiac muscles.

• (CK-MB) - Heart muscle (presence in plasma suggests a myocardial infarction).

• A fourth isoenzyme exist which is designated as CK-Mt.

• Differs from the others both immunologically and electrophorectic mobility.

• located between the inner and outer membranes of mitochondria.

• Constitute about 15% of the total CK activity in the heart.

DR. APPIAH'S LECTURE SERIES 14

• Marked increases-myocardial infarction, muscular dystrophies and

• Moderate increases-muscle injury, after surgery (for about a week),

• The ‘normal’ range is:

DR. APPIAH'S LECTURE SERIES 15

• Serum glutamic oxaloacetic transaminase (SGOT).

• A pyridoxal phosphate dependent transaminase enzyme (EC 2.6.1.1).

• Catalyzes the transfer of an amino group from glutamic acid to

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

DR. APPIAH'S LECTURE SERIES 16

• Two major isoenzymes; one cytoplasmic and the other mitochondrial.

• GOT1/cAST, the cytosolic isoenzyme - red blood cells and heart.

• GOT2/mAST, the mitochondrial isoenzyme - liver.

DR. APPIAH'S LECTURE SERIES 17

• Toxic liver necrosis (localized death of tissue)

• Circulatory failure with ‘shock’ and hypoxia.

DR. APPIAH'S LECTURE SERIES 18

• Cholestatic jaundice or obstructive jaundice (10X normal)