Enzymes and Histochemistry in 316Z Course

Enzymes in Histochemistry

• Enzymes are proteins that catalyze chemical reactions, essential for metabolic changes in living cells and tissues.
• They are named after their biochemical action with the suffix "(ase)" and have a high degree of specificity, but this degree differs from one enzyme to another.
• Enzymes are sensitive to temperature, pH, and ionic concentration.

Types of Enzymes

• Hydrolases (e.g., phosphatases and esterases)
• Oxidoreductases (e.g., oxidases and dehydrogenases)
• Transferases (e.g., phosphorylases)
• Proteolytic enzymes (e.g., leucine aminopeptidases)

Histochemistry of Enzymes

• Enzymes can be demonstrated histochemically only in fresh tissue.
• They are localized by using their specific substrates, which produce a colored or opaque product.
• Enzymes are highly labile and quickly disappear from tissue, so special precautions must be taken to preserve them in their locality at their optimum activity.
• Cryostat and freeze-drying methods have revolutionized enzyme histochemistry.
• Enzyme histochemistry helps illustrate certain cellular structures, such as:
  • Acid phosphatase for lysosomes
  • Alkaline phosphatase for membranes
  • Dehydrogenase and ATPase for mitochondria
  • Esterase for microsomes

Importance of Enzyme Histochemistry

• Enzyme histochemical methods indicate metabolic activities in cells.
• They help compare chemical activities in normal and abnormal cells (e.g., tumors).
• They help identify structures related to certain enzymes when used on ultrathin sections prepared for electron microscopy.
• Enzyme histochemistry is important as a diagnostic tool from a pathological point of view.

Special Precautions for Enzyme Histochemistry

• Tissues must be prepared to preserve enzymes as completely as possible and retain them in their original sites.
• Fixation should not allow diffusion or deterioration of the enzyme.
• Classical embedding techniques are not recommended for most enzymes.
• Control sections are essential to determine specificity and validity of results.
• Ice-cold acetone or alcohol can be used on fresh refrigerated tissues to preserve about 50% of the enzyme.

Enzymatic Reactions

• Enzymatic reactions result in the formation of a Primary Reaction Product (PRP) followed by the production of an opaque or colored Final Reaction Product (FRP).
• Types of enzyme histochemical reactions include:
  • Simultaneous capture
  • Post-incubation coupling (or post-coupling)
  • Self-colored substrate

Types of Techniques Used in Enzyme Histochemistry

• Four basic techniques are available for the demonstration of enzymes:
  • Metal precipitation technique
  • Simultaneous coupling using diazonium salts
  • Post-incubation coupling using diazonium salts
  • Self-colored substrate

Conditions for Histochemical Demonstration of Enzymes

• The quantity of the enzyme in a tissue section must be adequate to exceed the threshold of the technique sensitivity.
• The enzyme must not be lost during tissue preparations (fixation, processing, or cutting).
• PRP must be formed without serious diffusion or loss, and FRP must be formed in the accurate site of the original enzyme activity.
• False positive reactions (non-enzymatic) must be eliminated by the use of controls.