CLINICAL ENZYMOLOGY

Dr. Michael Appiah

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Enzymes
• Enzymes are proteins and certain class of RNA (ribozymes)
which enhance the rate of a thermodynamically feasible
reaction and are not permanently altered in the process.

• Most enzymes have tertiary and quaternary structures

• Catalysts for biological reactions.

• Does not effect equilibrium

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Enzymes
• Remains unchanged in overall process.

• Reactants bind to enzymes, products are released.

• Activity lost if denatured.

• May contain cofactors such as metal ions or organic (vitamins)

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 Enzymes
• Are specific for what
they will catalyze

• Are Reusable

• End in –ase
-Sucrase
-Lactase
-Maltase
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Characteristics of Enzymes
• Accelerate the rate of making and breaking of covalent catalysis.

• Highly specific: React with one substrate

• Not changed in the reaction.

• Catalyze many cycles (turnovers) of the reaction.

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Characteristics of Enzymes
• Usually proteins, but can be RNA.

• Bind substrates in special regions called ‘active sites’.

• Catalyze the forward and reverse reactions equal extent.

• They do not change the position of the equilibrium.

• Function by stabilizing the transition state.

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Plasma Enzymes
• Its easier to measure enzyme activity in body fluids than its
concentration which is normally low.

• Many enzymes are present in plasma of healthy individuals and more

are found in disease conditions.

• The activity of enzymes in plasma and other body fluids can be affected
through:

• Rate of release from the organs

• Distribution of the enzyme in the ECF
• Rate and route of elimination and inactivation
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❑These factors are influenced by

• Variability in an individual.

• Disease

• Drugs

• Exercise

• There is the need to consider this to ensure a more efficient diagnostic use
of enzymes in clinical practice.
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Uses of Plasma Enzymes in Clinical Pathology
• Mostly aimed at detecting, evaluating and monitoring organ damage
based on the increase in organ-specific enzyme.

• Enzymes are used to:

• to evaluate the synthetic capacity of an organ.
• to diagnose the adverse effects of toxic compound which are enzyme inhibitors
• to monitor the inductive activity of exogenous compounds or enzyme activation
by minerals or vitamins

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Classification of plasma enzymes

❑There are two main categories of plasma enzymes:

➢Functional Enzymes

➢Non functional enzymes

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Functional Enzymes (plasma-active)
• These are enzymes with a clearly defined function in blood. E.g.
coagulation factors such as prothrombin factor V, VII and XI and also
lipoprotein lipase.

• These enzymes originate from the liver and their concentration in

blood is higher than in other tissues.

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❑A reduction in the synthesizing capacity of the target organ may be due
to:

➢A decrease in number of intact cells

➢Cellular damage to organ of origin.

➢All this would lead to a depression of the enzyme activities in the plasma.

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Non-Functional Enzymes.

• These are enzymes with no known function in the blood and which
have been released from cells as a result of ‘leakage’ or of cell death in
the normal process of wear and tear of tissues, or due to diffusion
through undamaged cell membranes.

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Sources of Non Functional Enzymes
• Increase in the rate of enzyme systhesis e.g. Bilirubin increases the rate
of synthesis of alkaline phosphatase in obstructive liver disease.

• Obstruction of normal pathway e.g obstruction of bile ducts increases

alkaline phosphatase.

• Increased permeability of cell membrane as in tissue hypoxia.

• Cell damage with the release of its content of enzymes into blood e.g.
M.I and viral hepatitis

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Medical Importance of Non Functional Enzymes

❑Measurement of non-functional plasma enzymes is important for:

• Diagnosis of diseases as diseases of different organs causes elevation of

different plasma enzymes.

• Prognosis of diseases: following the effects of treatment by measuring

plasma enzymes before and after treatment

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 Functional plasma enzymes Non-functional plasma
enzymes
Concentration in plasma Present in plasma in higher Normally, present in plasma in
concentrations in comparison to very low concentrations in
tissues comparison to tissues

Function Have known functions No known functions

The substrates Their substrates are always Their substrates are absent from
present in the blood the blood
Site of synthesis Liver Different organs e.g. liver, heart,
brain and skeletal muscles

Effect of diseases Decrease in liver diseases Different enzymes increase in

different organ diseases
Examples Clotting factors e.g. ALT, AST, CK, LDH, alkaline
prothrombin, phosphatase, acid phosphatase
Lipoprotein lipase and pseudo- and amylase,
choline esterase

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Alterations in the activity of plasma enzymes are observed in the event
of the following:

• Altered synthesis of the enzymes within the cells.

• Change in the amount of enzyme forming tissue.

• Change in cell permeability.

• Change in the rate of inactivation or disposal of enzyme.

• Obstruction to a normal pathway of enzyme excretion.

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The rate and extent of enzyme release may be affected by the following
factors:

• The amount of the enzyme within the cell.

• The molecular weight of the enzyme; small molecules are released more
readily than large ones.

• The intracellular site of the enzyme; cytoplasmic enzymes are released

more readily than mitochondrial enzymes.

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Enzyme assays provide useful clinical information
by:
• Identifying the presence of disease.

• Distinguishing the specific disease from other diseases.

• Providing estimates of the severity and duration of disease.

• NB: Serum is preferred to plasma in enzyme assays since anti-

coagulation factors may have inhibitory effects on the activity of the
enzyme.
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Factors to consider in selecting an enzyme for
diagnosis
❑The choice of enzymes and technique employed depends on the following
factors:

• Sensitivity
• Specificity
• The Rate of Release
• Duration of Release
• Half-Life of the Enzyme
• Technical Factors
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❑ Sensitivity
• The technique must be capable of detecting small amounts of an enzyme
by its activity measurement.

• This helps overcome the problem of making a clinical diagnosis when

tissue damage is minimal.

❑Specificity
• This is the ability to identify which tissue has been damaged.

• This problem may be overcome by measuring several different enzyme

activities or by the study of its isoenzymes.
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❑The Rate of Release
• Usually increased enzyme activity in plasma occurs three hours after
tissue damage.

• Depending on the pathology of the condition its activity rises more or less
quickly to its maximum.

❑Duration of Release
• The activity of the enzyme should be elevated soon after the onset of the
disease and should persist for appreciable period.

• This depends solely on the nature and extent of pathological process.

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❑Half-Life of the Enzyme
• Some enzymes have very short half-lifes whereas others have long lifes
i.e. 5 days or more.

❑Technical Factors
• The test must be precise and accurate, easy to perform and inexpensive.

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ISOENZYMES

• Are enzymes that differ in amino acid sequence but catalyze the same
chemical reaction.

• Isoenzymes are usually the result of gene duplication.

• Isoenzymes may originate from different organs (e.g. alkaline phosphatase)

or from different cell compartments (e.g. AST) or from the same
compartment (e.g. LDH).

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Distinguishing isoenzymes
• The isoenzymes can be distinguished by their biochemical and
physical properties.

• They may be distinguished by their kinetic properties, i.e. Michaelis

constant (Km and Vmax) or by different sensitivities to temperature
changes.

• The most popular of these detection methods has undoubtedly been

electrophoresis (using cellulose acetate, agar, starch or acrylamide
gel).

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Distinguishing isoenzymes
• They have similar catalytic activity, but are different biochemically or
immunologically and can be demonstrated and differentiated by:

• Electrophoretic mobility (i.e. surface charge)

• Differences in absorption properties

• By their reaction with specific antibody

• Heat

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Distinguishing isoenzymes

• Inhibitors

• Activators

• ion-exchange chromatography

• isoelectric focusing

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THANK YOU