Enzyme Histochemistry Flashcards
What is the name for a protein that acts as a catalyst, for instance in metabolism.
Enzyme.
True or False: Substrates are chemical reactants that bind with enzymes to facilitate a chemical reaction.
True. The substrate is used or changed by the reaction. The enzyme will be reused.
What are cofactors?
Cofactors or coenzymes are vitamins or minerals that help the enzyme bind with a substrate to form an enzyme-substrate complex.
Why do we need enzyme histochemistry for neuromuscular disorders?
These diseases are best detected by visualizing enzyme activity. Muscle fibers can be typed this way.
Why is enzyme histochemistry performed on frozen tissue?
The heat and pH changes in FFPE processing would denature enzymes. Thawing and refreezing can also affect enzymes. So, snap-freezing and storing at -70° or transporting on dry ice is best.
True or False: All enzymes require a neutral pH.
False: Many do, but each enzyme has an optimal temperature and pH.
Name some enzyme histochemical stains.
ATPase, Esterase, Acid Phosphotase, Cytochrome Oxidase, Succinic Dehydrogenase, NADH
Why is the ATPAse stain used?
ATPase is used for typing fibers. Type 1 fibers stain dark at a low pH and light at a high pH, as pictured. Using the Gomori Metal Precipitate Reaction results in an insoluble black precipitate called Cobalt Sulfide.
What is the difference between mitochondrial ATP and muscle ATP?
The enzyme ATPase in mitochondria is activated by magnesium and inhibited by calcium. When it is attached to the myosin in muscle it is activated by calcium (cofactor) and inhibited by magnesium. Pictured:Type 1 fibers- dark, type 2 fibers- light, type 2C fibers-arrow.
What is the reason for performing the Cytochrome Oxidase, or COX stain?
To detect disorders of the mitochondria.
What is the substrate and reaction product for the Cytochrome Oxidase stain?
The enzyme Cytochrome Oxidase uses the substrate hydrogen peroxide (H₂O₂) with a manganese chloride cofactor (activator) and the resulting color is brown from DAB.
What is the substrate and reaction product for the Succinic Dehydrogenase (SDH) stain?
When the substrate succinate is incubated with nitroblue tetrazolium, the result is a blue color. The stain identifies SDH deficiency in mitochondria. As shown, type 1 fibers stain darker than type 2 fibers, because they have more mitochondria.
What is the substrate and reaction product for the NADH stain?
Used to detect mitochondrial disorders and differentiate between type 1and type 2 fibers, the NADH (Nicotinamide Adenine Dinucleotide) stain uses NADH as a substrate and, when incubated with nitroblue tretrazolium, gives a purple blue color. The image on the right shows demonstrates a loss of myofibullar architecture.
What is the substrate and reaction product for the Esterase stain?
This stain is for denervated muscle fibers, motor end plates, lysosomes, and macrophages. The substrate is alpha-nathyl acetate, and when incubated with azotized pararosanaline produces a dark red-brown color. Normal muscle fibers stsin yellow-brown.
What is the substrate and reaction product for the Acid Phosphotase? Normal muscle shown.
This demonstrates muscle fibers with Acid Maltose Deficiency and inflammatory cells. The substrate is Napthol AS-BL Phosphoric Acid it is incubated with azotized pararosanoline resulting in a red color. Sometimes uses a light green counterstain. AMD shown.
