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Histology Spring 2020 > Enzyme Histochemistry (IH) > Flashcards

Enzyme Histochemistry (IH) Flashcards

1
Q

Epimysium

A

Collagenous tissue sheath that covers an entire muscle

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2
Q

Perimysium

A

Dense layer of collagen that covers bundles of muscle fibers to form a fascicle

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3
Q

Endomysium

A

Fine collagen fibers that surround individual muscle fibers

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4
Q

Enzymes

A

Proteins that catalyze (change the rate of) chemical reactions by temporarily combining with their substrate (target)

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5
Q

Oxidation

A

Addition of oxygen
loss of hydrogen
loss of electrons

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6
Q

Reduction

A

Loss of oxygen
gain of hydrogen
gain of electrons

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7
Q

Hydrolase

A

An enzyme that typically adds (or sometimes removes) water

Includes: esterases, phosphatases, peptidases

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8
Q

Simultaneous Coupling

A

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

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9
Q

Esterase

A

Enzymes that break bonds between carboxylic acid and alcohols, phenol, and napthols.

Most are capable of hydrolyzing a-napthyl cetate

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10
Q

Phosphatase

A

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

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11
Q

Phosphorylase

A

A type of transferase that move phosphate groups

Used for synthesis and decomposition of saccharides such as glycogen

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12
Q

What are 3 properties of enzymes?

A

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

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13
Q

What are the 6 basic enzyme reactions?

A

Oxidoreductases
Transferases
Hydrolases

Lyases
Isomerases
Ligases

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14
Q

What are 5 factors that affect enzyme demonstration?

A

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)

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15
Q

What is the artifact that may be seen in frozen sections?

A

freezing artifact, disruption of tissue? Looks like lots of little holes that disrupt morphology

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16
Q

How to preserve tissue for enzyme studies?

A

Need to be fixed before freezing to prevent diffusion artifact

17
Q

What is the preferred method for freeing muscle tissue

A

isopentane suspended in liquid nitrogen, because it causes less ice-crystal freezing artifact

18
Q

Summarize enzyme-substrate chemical reaction

A

Enzyme + Substrate -> temporarily combine-> yields Product + Enzyme

19
Q

α-naphthyl acetate esterase reaction

A

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

20
Q

Naphthol AS-D choloracetate esterase reaction

A

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

21
Q

ATPase reaction

A

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

22
Q

Acid phosphatase reaction

A

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

23
Q

NADH diaphorase reaction

A

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

24
Q

SDH Succinic dehydrogenase reaction

A

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

25
Q

Phosphorylase reaction

A

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.

26
Q

Cytochrome C Oxidase reaction

A

(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

27
Q

α-naphthyl acetate esterase Result

A

Motor end plates: Brick red
Normal Muscle Fibers: Very pale yellow
Denervated Muscle fibers: Dark red-brown
Macrophages and lysosomes: Dark red-brown

28
Q

Naphthol AS-D choloracetate esterase Results

A

Positive cells (granulocytes and mast cells): Bright red
Nuclei: Blue
Background: Unstained

29
Q

ATPase reaction Results

A

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

30
Q

Acid phosphatase Results

A

Sites of acid phosphatase activity: Red

Background: Green

31
Q

NADH diaphorase Results

A

Site of enzyme activity: Dark purple deposits
Type I muscle fibers: Dark purple
Type II muscle fibers: Light

32
Q

SDH Succinic dehydrogenase Results

A

Sites of SDH activity: Blue

Type I are dark and Type II are light

33
Q

Phosphorylase Results

A

Phosphorylase activity: Varying shades of brown, blue, and purple
Darker for longer chains, light for no activity
Total absence of activity: Indicates McArdle disease

34
Q

Acid phosphatase Results

A

Sites of enzyme activity: pink-red to red

Nuclei: Blue

Histology Spring 2020 (14 decks)

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