Enzyme Histochemistry (IH) Flashcards
Epimysium
Collagenous tissue sheath that covers an entire muscle
Perimysium
Dense layer of collagen that covers bundles of muscle fibers to form a fascicle
Endomysium
Fine collagen fibers that surround individual muscle fibers
Enzymes
Proteins that catalyze (change the rate of) chemical reactions by temporarily combining with their substrate (target)
Oxidation
Addition of oxygen
loss of hydrogen
loss of electrons
Reduction
Loss of oxygen
gain of hydrogen
gain of electrons
Hydrolase
An enzyme that typically adds (or sometimes removes) water
Includes: esterases, phosphatases, peptidases
Simultaneous Coupling
Enzyme acts on substrate and a reaction product is released
Then the reaction product is rapidly captured or coupled with a diazonium salt to yield an insoluble azo dye, or captured with a metallic ion
Esterase
Enzymes that break bonds between carboxylic acid and alcohols, phenol, and napthols.
Most are capable of hydrolyzing a-napthyl cetate
Phosphatase
hydrolytic enzymes that break the bond between an alcohol and phosphate group
found in lysosomes (acidic and alkaline versions)
ATPase is in mitochondria and bound to myosin for muscle contraction
Phosphorylase
A type of transferase that move phosphate groups
Used for synthesis and decomposition of saccharides such as glycogen
What are 3 properties of enzymes?
Act as catalysts to promote or inhibit a reaction
They are specific to the shape of a substrate based on chemical groups and orientation
They are sensitive to temperature and pH to function
What are the 6 basic enzyme reactions?
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
What are 5 factors that affect enzyme demonstration?
Treatment before and during procedure (fixation/freeing/thawing)
Nonoptimal substrate (poor solubility or hydrolyzation)
Nonoptimal temperature (enzymes denatured at 56C)
Nonoptimal pH (pH7 is optimal wit some exceptions)
Inhibitors (excess diazonium salts, heat, and some metallic ions)
What is the artifact that may be seen in frozen sections?
freezing artifact, disruption of tissue? Looks like lots of little holes that disrupt morphology
How to preserve tissue for enzyme studies?
Need to be fixed before freezing to prevent diffusion artifact
What is the preferred method for freeing muscle tissue
isopentane suspended in liquid nitrogen, because it causes less ice-crystal freezing artifact
Summarize enzyme-substrate chemical reaction
Enzyme + Substrate -> temporarily combine-> yields Product + Enzyme
α-naphthyl acetate esterase reaction
a-napthyl acetate + water in presence of esterases yields -> a-napthol + acetic acid
a-napthol + hexazotized pararosaniline (basic fuchsin) yields -> azo dye for detection
non-specific esterase stain for differentiating type II atrophy and neurogenic atrophy
Naphthol AS-D choloracetate esterase reaction
a-napthyl acetate + water in presence of esterases yields -> a-napthol + acetic acid
a-napthol + hexazotized pararosaniline (basic fuchsin) yields -> azo dye for detection
This is a specific esterase stain, the only one that can be done on paraffin, and it is used to identify granulocytes for classifying leukemia’s
ATPase reaction
differentiate type I, type IIA, type IIB fibers
Gomori-type metal precipitation of the hydrolyzed orthophosphoric acid with calcium. A complex series of reactions leads to the formation of a colored metallic sulfide
(ATP + H2O) in the presence of ATPase yields -> (ADP + orthophosphate + energy)
orthophosphate combines with calcium to form calcium phosphate
calcium is exchanged with cobalt
cobalt is exchanged with sulfide to form visible insoluble cobaltous sulfide
Acid phosphatase reaction
Presence of inflammatory cells, marker for lysosomes
phosphomonoesterases hydrolyze esters of orthophosphoric acid at an acidic pH
Alcohol residue of the napthol AS-BI-phosphate reacts with hexazotized pararosaniline (basic fuchsin) to yield a red azo dye via simultaneous coupling method
NADH diaphorase reaction
Identifies abnormal mitochondria, z-bands, and sarcoplasmic reticulum
NADH (reduced) in the presence of diaphorase yields -> NAD+ (oxidized) + hydrogen
Nitro blue tetrazolium (NBT) + hydrogen yields -> formazan
SDH Succinic dehydrogenase reaction
Identify mitochondria only from NADH diaphorase activity
SDH is mitochondrial and is part of the Krebs cycle
succinate in the presence of succinic dehydrogenase yields -> fumarate + hydrogen
NBT + hydrogen yields -> formazan
Phosphorylase reaction
Diagnosing McArdle disease (glycogen storage disease)
Complete absence of staining indicates disease state
Length of polysaccharide chains is proportional to amount of phosphorylase activity. Stain color varies based on chain length, darker for longer chains.
Cytochrome C Oxidase reaction
(COX) is an enzyme of the inner mitochondrial membrane and part of respiration
DAB acts as an electron donor to COX and forms an insoluble brown polymer when oxidized
Then the reaction is enhanced by cytochrome c
And finally catalase increases specificity by eliminating endogenous hydrogen peroxide
α-naphthyl acetate esterase Result
Motor end plates: Brick red
Normal Muscle Fibers: Very pale yellow
Denervated Muscle fibers: Dark red-brown
Macrophages and lysosomes: Dark red-brown
Naphthol AS-D choloracetate esterase Results
Positive cells (granulocytes and mast cells): Bright red
Nuclei: Blue
Background: Unstained
ATPase reaction Results
pH 9.4 (alkaline) Type I are light to unstained, Type II are dark
pH 4.3 (acidic) Type I are dark, type II are light to unstained
pH 4.6 (acidic) Type I are dark, IIA are light, and IIB are intermediate
Acid phosphatase Results
Sites of acid phosphatase activity: Red
Background: Green
NADH diaphorase Results
Site of enzyme activity: Dark purple deposits
Type I muscle fibers: Dark purple
Type II muscle fibers: Light
SDH Succinic dehydrogenase Results
Sites of SDH activity: Blue
Type I are dark and Type II are light
Phosphorylase Results
Phosphorylase activity: Varying shades of brown, blue, and purple
Darker for longer chains, light for no activity
Total absence of activity: Indicates McArdle disease
Acid phosphatase Results
Sites of enzyme activity: pink-red to red
Nuclei: Blue
