ENZYME HISTOCHEMISTRY

KWABENA OWUSU DANQUAH (PhD)

 Roadmap
• Structure-function of Enzymes

• Diagnostic enzymes in pathologies

• Concepts for enzyme demonstration

• Some examples of enzymes

• Take away questions

RECAP OF HISTOCHEMISTRY

The location where chemistry

exposes the nature of tissue
components!!!!
 What are enzymes?
• Are biological catalysts that transform substances,
and also mediate in energy transfer.

• With an exception to Ribozyme, an RNA, all

enzymes are proteins.

• Are specific and enzymatic activities occur in the

active site.

• Require cofactors to work, such as metal ions: Mg

and Mn ions (activators) or small molecules: NAD
and NADP (coenzyme).
Types of Enzymes
 What is enzyme histochemistry?

• It is the application of chemistry to locate

enzymes in pathological cases.

• In enzyme histochemistry, it is all about

LOCATION, LOCATION, LOCATION!!!

• Thus care and caution are needed to ensure

enzymes do not diffuse to trigger false
interpretation.
 What can kill enzymes?
• Enzymes are proteins, thus care should
be taken not to ‘kill’ or denature them.

• Fixation, tissue processing and handling

could ‘kill’ enzymes, making their
detection and localisation impossible.

• A decision should be made on whether

to fix or freeze a tissue for enzyme
demonstrations.
Application of Enzyme Histochemistry

• Skeletal muscle biopsy (PFK & aldolase).

• Diagnosis of Hirschsprung’s disease (via

detection of ganglia and nerves in rectum).

• Lactase/sucrase deficiency in jejunal biopsies.

• Demonstration of chloroacetate esterase in

myeloid series and mast cells.
 Application of Enzyme Histochemistry
More specific examples
• Alkaline phosphatase: prostate carcinoma, when
infiltrating into colon, bladder & bone.

• Acid & alkaline phosphatase: gluten enteropathy

• Alkaline phosphatase: vascular endothelial

tumour.
Methods for enzyme demonstration
 Hydrolytic enzymes
• Catalyse the introduction of water or elements
into specific substrate bonds.

• Or removal of water in some cases.

• Include esterases, lipases, phosphatases,

glycosidases, peptidases and
pyrophosphatases.
Demonstration of hydrolytic enzymes
 Diazonium salts

• Related to tetrazolium salt, and are colourless

salts.

• Few are dyes such as pararosanilin & fast garnet

GBC.

• Reacts with primary products to give coloured &

insoluble reaction products.

• The solubility of the final coloured products is

breached in low pH.
 Controls in Enzyme Histochemistry
Positive control
• Are performed to show that all the chemical solutions are working,
thus it should be carried out throughout the test procedure.
• Omission of substrate
• Inclusion of inhibitors
• Inclusion of competitive inhibitors
• Pre-treatment such as boiling of sections
• Incubation in distil water instead of buffers

Expected result???
No reaction products indicate positive results and activity in negative
control sections means false result.
 Differentiation of Esterases
• None specific esterases are grouped into A, B and C (as
recommended by Pearse).

• To differentiate the types, inhibitors are used.

• Diethyl-p-nitrophenyl phosphate (E600) inhibits B-

esterases (carboxylesterase).

• A-esterases (arylesterases) are differentiated from C-

esterases (acetylesterases) by incubation in p-
chloromercuribenzoate (PCMB). C-esterases are rather
activated by PCMB.

• Cholinesterases are inhibited by Eserine.

 Oxidative enzymes
• Are demonstrated by simultaneous coupling
methods.

• Substrates are oxidised and follow by reduction

of tetrazolium salt to produce insoluble
formazan deposits.

• Oxidation of substrates: reaction between S

and oxygen (Oxidases) or removal of H from S
and transfer it along hydrogen acceptor
(dehydrogenases).
 Tetrazolium salts
• Used as a hydrogen acceptor.

• Two commonly used: monotetrazolium and

ditetrazolium.

• Monotetrazolium such as 3-(4:5-dimethyl thiazolyl-

2)-2:5-diphenyl tetrazolium bromide (MTT)
produces fine lipid-soluble granular formazan.

• Ditetrazolium includes ditetrazolium chloride-nitro

(NBT) & produces highly coloured lipid-insoluble
formazan.
Demonstration of Oxidative enzymes
 Take away Questions

• Is enzyme histochemistry still useful in the era

of immunohistochemistry?

• Which method: enzyme histochemistry or

immunohistochemistry is the best approach of
diagnosing muscle and myeloid samples?
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