Enzyme Histochemistry

Question 1

What do individual skeletal muscle fibers (cells) normally range from?

What are the characteristics of skeletal muscle?

Answer 1

◦ Individual skeletal muscle fibers (cells) normally range from 30 to 80m in diameter.

◦ Characteristics of skeletal muscle are:
1.) Presence of multiple elongated nuclei located at fairly regular intervals.
2.) Presence of regular cross-striations

Question 2

Each muscle fiber is packed with cylindrical structures called what?

What separates muscle fibers from each other?

Bundles of muscle fibers (fascicles) surrounded by a dense layer of collagen called is called the what?

◦ The entire muscle is enclosed in a collagenous connective tissue sheath called the what?

Answer 2

◦ Each muscle fiber is packed with myofibrils (cylindrical structures).

◦ Muscle fibers are separated from each other by a network of fine collagen fibers called the endomysium.

◦ Bundles of muscle fibers (fascicles) are surrounded by a dense layer of collagen called the perimysium.

◦ The entire muscle is enclosed in a collagenous connective tissue sheath called the epimysium.

Question 3

What are the various functions of muscles? hint: there’s 2

How can muscle tissue be classified?

Answer 3

1.) Some are required to contract almost continuously.
2.) Others make rapid contractions of brief durations.

◦ Muscle tissue can be classified into two types:
1).Type I
2.) Type II

Question 4

Type 1 makes up approximately what percent of muscle?

What is type 1 muscle known as?

What type of metabolism does Type 1 muscle utilize?

What organelles/structures are abundant in Type 1 muscle tissue?

Is Type 1 muscle tissue vascularized?

Answer 4

◦ Type I (appx 33% of muscle)

• Type 1 is known as the “slow-twitch” muscle
• Utilizes a predominantly aerobic (oxidative) metabolism
• Contain abundant mitochondria, lipid & myoglobin (oxygen storing molecule)
• Yes it is vascularized, so type 1 has good blood supply.

Question 5

Type 2 makes up approximately what percent of muscle?

What is type 2 muscle known as?

What type of metabolism does Type 2 muscle utilize?

What organelles are in Type 2 muscle tissue? Does it have a poor blood supply?

What molecules are rich in Type 2 muscle tissue?

What does Type 2 muscle tissue have a high resistance to?

Answer 5

Type 2 (appx 67% of muscle)

“fast-twitch” muscle

Have anaerobic metabolism

Contain few mitochondria, myoglobin. Yes, it has poor blood supply

Rich in glycogen and glycolytic enzymes

High resistance to fatigue.

Question 6

Type 2 Muscle tissue can be further divided into specific types, what are they? What are their percentages? hint: there’s 3 types

What is the normal distribution pattern of Type 2 muscle tissue in humans?

Answer 6

1.) IIA (appx 50%)
2.) IIB (appx 50%)
3.) IIC (not frequent in adults)

◦ Normal distribution of types in humans is a “checkerboard pattern”.

Question 7

Are the methods for demonstrating enzymes limited in the present day?

What is the primary use for enzyme histochemistry in the histopathology laboratory?

Some of the same techniques can also be used in hematopathology for what?

Answer 7

◦ No, there’s more than 1,000 methods for demonstrating enzymes today.

◦ Primary use for enzyme histochemistry in the histopathology laboratory is in the diagnosis of muscle diseases.

◦ Some of the same techniques can also be used in hematopathology for differentiation of leukemias.

Question 8

What are catalysts?

What are enzymes?

Enzymes combine with what to initiate the reaction?

After the product is formed, what happens to the enzyme?

Answer 8

◦ Catalysts are substances that change the rate of a chemical reaction (speed up or slow down).

◦ Enzymes are proteins that catalyze chemical reactions occurring in biological systems.

◦ Enzymes combine with their specific substrate to initiate the reaction.

After the product is formed, the enzyme is released to react with another substrate.

Answer 9

Introduction

◦E + S E S P + E

      Enzyme + Substrate             ES Complex               Product + Enzyme Enzymes act alone or in combination with nonprotein structures called cofactors to help speed up the reaction.

Cofactors can be metal ions or complex organic molecules called CoEnzymes.

Question 10

What are the properties of enzymes?

Answer 10

1.) Enzymes are sensitive to pH & Temperature
2.) Enzymes in environments that are outside their norm will become inactive.

Example:
* Increase in heat can in activate enzymes.
* Enzymes that work in acid pH will not in alkaline pH.

Question 11

What factors influence enzyme demonstration? hint: there’s 5

Answer 11

1.)Treatment of tissue before & during procedure.
2.) Nonoptimal subtrate.
3.) Nonoptimal temperature.
4.) Nonoptimal pH.
5.) Inhibitors (excess of diazonium salts in substrates, fixatives, heat, and some metallic ions).

Question 12

Why should tissue for enzyme demonstration be fixed?

With fixation, certain soluble enzymes are _____, some enzymes will be _______.

What is the recommended fixative for tissue recommended for enzyme studies?

Answer 12

For the preservation of Enzymes. Tissue for enzyme demonstration should be fixed due to considerable diffusion artifact seen in unfixed frozen sections.

◦With fixation, certain soluble enzymes are preserved, some enzymes will be inactivated.

◦ Cold formalin (3℃ to 4℃), cold calcium formalin and cold acetone have been recommended on tissue recommended for enzyme studies.

Question 13

What are enzymes are specific as to?

How are enzymes named?

What 2 older enzymes don’t end in -ase?

Answer 13

◦ Enzymes are specific as to the type of reaction catalyzed.

Enzymes are named by adding “ase” to:
* the specific substrate or molecule on which the enzyme acts, or A stem indication the type of reaction.

◦ Older known enzymes Trypsin & Pepsin,

Question 14

What are the three basic classifications that are of major importance in histopathology?

Answer 14

1.) Hydrolases
2.) Oxidoreductases
3.) Transferases

Question 15

How do Hydrolases behave?

What does Hydro mean?

What are the subgroups of Hydrolases?

What do Esterases do?
What do Phosphatases do?
What do Peptidases do?

Answer 15

◦ Hydrolases act on various substrates through the addition or sometimes the removal of water.

• (Hydro = water)

◦ This group includes subgroups:
1.) Esterases – catalyze the hydrolyses of ester linkages.
2.) Phosphatases – hydrolyze esters of phosphoric acid.
3.) Peptidases – attack peptide bonds.

Question 16

What is the large group of enzymes that deals with Oxidation & Reduction reactions called?

What is oxidation?

What is reduction?

What are the subgroups of Oxidoreductases?

What do Oxidases do?

What do Peroxidases do?

What do Dehydrogenases do?

Answer 16

Oxidoreductases

• Oxidation: Addition of oxygen, loss of hydrogen & electrons
• Reduction: Loss of oxygen, gain of hydrogen & electrons

◦ Includes these subgroups:
1.) Oxidases
2.) Peroxidases
3.) Dehydrogenases

Oxidases: can utilize molecular oxygen as the hydrogen acceptor resulting in water

Peroxidases: catalyze the oxidation of substrates by hydrogen peroxide

Dehydrogenases: remove hydrogen atoms from an organic substrate (Diaphorases).

Question 17

Enzymes that transfer a functional group from one compound to another are called?

Name the only Transferase of concern in the histopathology laboratory

Answer 17

Transferases

• Phosphorylase

Question 18

Muscle tissue is extremely subject to _____, which in turn can interfere with staining.

What is necessary to decrease artifact in muscle tissue?

What is the preferred method of rapid freezing muscle tissue?

What is the recommended temp of Isopentane prior to muscle freezing?

What can be used as an alternative to Isopentane?

Answer 18

◦ Muscle tissue is extremely subject to ice crystal artifact, which in turn can interfere with staining.

◦ Rapid freezing methods are necessary to decrease artifact.

◦ Preferred method is isopentane suspended in liquid nitrogen.

• Isopentane recommended temp is -150℃ prior to muscle freezing.
• Alternative: Dust muscle tissue with talc then immerse directly into liquid nitrogen.

Question 19

What is the recommended protocol for freezing muscle tissue? hint: 7 steps

Answer 19

◦ Recommended Procedure:
1.) Orient the fresh unfixed muscle tissue so that cross sections will be obtained.

2.) Prepare a piece of cork (label with patient info), punch small hole and run a piece of thread.

3.) Mount the oriented tissue on the cork with gum tragacanth.

4.) Suspend a steel beaker of isopentane in a Dewer flask containing liquid nitrogen.

5.) When the temperature reached -150C, place the cork containing the muscle biopsy in the isopentane (appx 15 seconds), then put specimen directly into liquid nitrogen.

6.) Specimen can be left in liquid nitrogen until ready to cut.

7.) After sectioning wrap in plastic wrap, then foil, & store at -70C.

Question 20

What is the purpose of the α-Napthyl Acetate Esterase Stain for Muscle Biopsies

How is the tissue prepared for α-Napthyl Acetate Esterase Stain? What measurement is the tissue cut at?

What color are the motor end plates stained?

What color are the normal muscle fibers stained?

What color are the denervated muscle fibers stained?

What color are the macrophages and lysosomes stained?

Answer 20

• Purpose: differentiating between type II atrophy and neurogenic atrophy, as denervated muscle stains dark and type II do not. Also demonstrates motor end plates & lysosomes in inflammatory cells.
• Frozen, unfixed tissue cut at 10µm.
• Motor end plates: Brick red
• Normal muscle fibers: Very pale yellow
• Denervated muscle fibers: Dark red-brown
• Macrophages & lysosomes: Dark red-brown

Question 21

What are Esterases? What are they capable of doing?

What happens to the hydolyzed alcohlic residue? What is the result?

Answer 21

Esterases are enzymes that are capable of hydrolyzing aliphatic and aromatic ester bonds.

The hydrolyzed alcoholic residue will couple with the hexazotized pararosaniline solution to resulting in an insoluble brightly colored azo-dye as the reaction product at the enzyme site.

Question 22

What is the purpose of the Naphthol AS-D Chloroacetate Esterase Staining Technique?

This technique is rare in that it can be performed on what kind of embedded tissue?

What color are positive cells such as granulocytes and mast cells stained?

What color is the nuclei stained?

Answer 22

• Purpose: Identify granulocytes in the classification of leukemias or chloromas
• This technique is rare in that it can be performed on paraffin embedded tissue.
• Results:
• Positive cells (granulocytes and mast cells): Bright red
• Nuclei: Blue

Question 23

What is the purpose of the ATPase Muscle Tissue Stain?

How is the tissue prepared for the ATPase Stain?

What the results of the ATPase stain at pH 9.4?

What the results of the ATPase stain at pH 4.3?

What the results of the ATPase stain at pH 4.6?

Answer 23

◦ ATPase Stain - With preincubation at various pHs, will differentiate between type I, type IIA, and type IIB fibers

• Frozen sections of unfixed tissue cut at 10µm (3 slides – pH4.3, pH4.6 & pH9.4)
• Results:
• pH 9.4: Type II fibers are dark and type I fibers are light to unstained
• pH 4.3: Type I fibers are dark and type II fibers are light to unstained
• pH 4.6: Type I fibers are dark, type IIA fibers are light, and type IIB fibers are intermediate.

Question 24

ATPase Stain method involves a metal-precipitation of the hydrolyzed orthophosphoric acid with ____.

◦ Complex series of reactions leads to the formation of what?

◦ Further procedures exchange calcium with cobalt, then sulfide, resulting in formation of what?

Answer 24

ATPase Stain method involves a metal-precipitation of the hydrolyzed orthophosphoric acid with calcium.

◦ Complex series of reactions leads to formation of colored metallic sulfide.

◦ GO BACK TO THIS PART IN POWERPOINT TO MAKE ANOTHER QUESTION!!

◦ Further procedures exchange calcium with cobalt, then sulfide, resulting in formation of black, insoluble cobaltus sulfide.

Question 25

What doe the presence of Acid Phosphatase in Muscle Biopsies indicate?

How should the muscle tissue be prepared?

Sites containing acid phosphatase activity will be what color?

The background will stain what color?

Answer 25

Acid Phosphatase in Muscle Biopsies indicates:
1.) The presence of inflammatory cells.
2.) Muscle fibers in acid maltase deficiency also show an increase in acid phosphatase.

• Frozen sections of unfixed tissue cut at 10um.
• Results:
• Sites of acid phosphatase activity – Red
• Background - Green

Question 26

Acid phophatases are phosphomonoesterases that hydrolyze esters of orthophosphoric acid at an acid pH, varying from ___ to ____.

Alcohol residue of reaction reacts with hexazotized pararosaniline to produce what?

Answer 26

Acid phophatases are phosphomonoesterases that hydrolyze esters of orthophosphoric acid at an acid pH, varying from 4.5 to 6.0 pH.

Alcohol residue of reaction reacts with hexazotized pararosaniline to produce a highly colored insoluble azo dye.

Question 27

Alkaline Phosphatase is type of stain used to detect what?

How should the muscle tissue be prepared?

Sites of enzyme activity will be stained what color?

The nuclei will be stained what color?

Answer 27

Detection of regenerating muscle fibers.

Frozen sections of unfixed tissue cut at 10µm.

• Results:
• Sites of enzyme activity – Pink red to red
• Nuclei - Blue

Question 28

Alkaline phophatases are phosphomonoesterases that hydrolyze esters of orthophosphoric acid at an acid pH, varying from ___ to pH ____.

Alcohol residue of reaction reacts with fast red violet to produce what?

Answer 28

Alkaline phophatases are phosphomonoesterases that hydrolyze esters of orthophosphoric acit at an acid pH, varying from 8.6 to 8.8 pH.

Alcohol residue of reaction reacts with fast red violet to produce a highly colored insoluble azo dye.

Question 29

NADH stands for what?

What does the NADH Diaphorase staining method demonstrate?

How is the tissue cut?

What color will the sites of enzyme activity be?

What color will the type 1 muscle be?

what color will the type 2 muscle be?

Answer 29

Nicotinamide Adenine Dinucleotide Reductase

Demonstrates abnormalities in mitochondria, z-band material & sarcoplasmic reticulum.

Frozen sections cut at 10µm.

◦ Results:
*Sites of enzyme activity: Dark purple depostis
*Type I muscle: Dark purple
*Type II muscle: Light purple( double check)

Question 30

What happens in the NADH Diaphorase staining reaction?

Answer 30

In this reaction, a tetrazolium salt acts as a hydrogen acceptor. NADH cannot reduce the tetrazolium salt directly.

Question 31

What does the Succinic Dehydrogenase (SDH) staining method demonstrate?

How is the muscle tissue cut?

What color are the sites of SDH activity stained?

Answer 31

Succinic Dehydrogenase (SDH) further identifies a source of NADH diaphorase activity, as only mitochondria show positive SDH activity

• Frozen sections of unfixed tissue cut at 10µm.

*Results:
*Sites of SDH activity – Blue

Question 32

How does Succinic Dehydrogenase SDH stain demonstrate the enzyme?

Answer 32

Enzyme is demonstrated by incubating the fresh frozen sections with the succinate substrate in the presence of a tetrazolium compound such as nitro blue tetrazolium.

Question 33

What is the Phosphorylase Stain for Muscle useful for?

How is the tissue cut?

What color will the areas with Phosphorylase activity be stained?

A total absence of Phosphorylase activity indicates what?

Answer 33

Useful in the diagnosis of McArdle disease, as in the disease there is a single enzyme defect.

*Frozen sections of unfixed tissue cut at 10µm.

*Results:
*Phosphorylase activity - Varying shades of brown, blue & purple
*Total absence of activity – Indicates McArdle disease

Question 34

In vitro muscle phosphorylase produces an unbranched polysaccharide of what?

Length of chain formed is proportional to the amount of what?

Answer 34

In vitro muscle phosphorylase produces an unbranched polysaccharide of amylose type from glucose-1-phosphate.

Length of chain formed is proportional to the amount of phosphorylase activity present and with stained with iodine, will vary in color depending on length of chain.