Chapter 13 - Enzyme Histochemistry

ATPase go brrrr

What is the typical diameter and maximum length of a skeletal muscle fiber?

• Diameter: 30–80 µm

• Length: Up to 35 cm

Which structural features distinguish skeletal muscle from other muscle types?

• Multiple elongated nuclei located just beneath the sarcolemma

• Regular cross-striations from the arrangement of actin and myosin

The ___ is the muscle cell membrane, beneath which elongated nuclei are located.

Sarcolemma

True or False: Myofibrils in skeletal muscle are randomly arranged.

False
Explanation: Myofibrils are arranged parallel to the sarcolemma, and their cross-striations are in perfect alignment, contributing to the striated appearance of skeletal muscle.

What are myofibrils, and how are they arranged within skeletal muscle fibers?

• Myofibrils are cylindrical structures packed within the muscle fiber.

• They exhibit prominent cross-striations and are arranged parallel to the sarcolemma

What structures are found in the sarcoplasm of skeletal muscle fibers?

• Mitochondria

• Sarcoplasmic reticulum

• Microtubules

• Intermediate filaments

• Free ribosomes

• Golgi apparatus

• Glycogen and lipid droplets

What is the name of the cytoplasm in muscle cells?

Sarcoplasm

Match the connective tissue structure with what it surrounds:

• Perimysium

• Epimysium

• Endomysium

• Perimysium: Surrounds bundles of muscle fibers (fascicles)

• Epimysium: Encloses the entire muscle

• Endomysium: Separates individual muscle fibers

True or False: The endomysium is easily visible under a light microscope in healthy tissue.

False
Explanation: The endomysium is not usually visible in healthy tissue under light microscopy but may become prominent in pathological conditions.

Where are nerves and blood vessels commonly found in skeletal muscle connective tissue?

• In the perimysium and endomysium

• Capillaries may also be present within the endomysium

What type of muscle activity is associated with muscles responsible for posture?

Continuous, low-intensity contraction over long durations

Fill in the blank:
Extraocular muscles are specialized for ___ contractions of ___ duration.

Rapid; brief

True or False: In humans, slow-twitch and fast-twitch muscles can be distinguished based on color alone.

False
Explanation: Unlike in species such as domestic poultry, human muscle types cannot be visually distinguished by color.

In poultry, which muscles are red and which are white? What does this indicate about fiber types?

• Thigh muscles: Red (slow-twitch, oxidative type I fibers)

• Breast muscles: White (fast-twitch, glycolytic type II fibers)

What are the characteristics of type I muscle fibers (slow-twitch)?

• Predominantly aerobic (oxidative) metabolism

• Abundant mitochondria

• High lipid and myoglobin content

• Good blood supply

• Fatigue-resistant

What are the characteristics of type II muscle fibers (fast-twitch)?

• Anaerobic metabolism

• Few mitochondria and little myoglobin

• Poor blood supply

• Rich in glycogen and glycolytic enzymes

• High resistance to fatigue

Which muscle fiber type contains more glycogen: type I or type II?

Type II (fast-twitch) fibers

Which oxygen-storing molecule is abundant in type I fibers?

Myoglobin

Fill in the blank:
Based on the ATPase histochemical reaction, type II muscle fibers are subdivided into types ___, ___, and ___.

IIA, IIB, and IIC

True or False: Type IIC muscle fibers are common in normal adult muscle.

False
Explanation: Type IIC fibers are not frequently seen in normal adult muscle; they are undifferentiated precursors of other fiber types.

What is the approximate distribution of muscle fiber types in normal human muscle?

• Type I: 33%

• Type II: 67%

  • Type IIA: 50% of type II

  • Type IIB: 50% of type II

What pattern is observed in the distribution of muscle fiber types within normal human skeletal muscle?

A checkerboard pattern of type I, IIA, and IIB fibers

Why are charts of normal fiber distribution used in muscle biopsies?

Because different muscles have different normal distributions of fiber types, and referencing these helps with pathological diagnosis.

What common staining method can reveal basic pathologic changes in muscle tissue?

Hematoxylin and eosin (H&E) stain

Which of the following pathologic changes in muscle can be seen using hematoxylin and eosin (H&E) stain?

• A) Fiber atrophy or hypertrophy

• B) Type grouping

• C) Central cores

• D) Target fibers

A) Fiber atrophy or hypertrophy
Explanation: Changes in fiber size, nuclear location, inflammatory cell infiltration, degeneration/regeneration, and connective tissue increase are visible with H&E. Type grouping, central cores, and target fibers require histochemical techniques.

True or False: Hematoxylin and eosin (H&E) stain is sufficient to detect all forms of muscle pathology.

False
Explanation: H&E reveals some changes (e.g., atrophy, hypertrophy, inflammation), but histochemical stains are required for detailed findings like fiber type grouping, central cores, and target fibers.

Changes in the ___ and ___ of nuclei can be detected on H&E stain during evaluation of pathologic muscle.

Location and appearance

What are some visible signs of pathologic muscle changes on H&E stain?

• Atrophy or hypertrophy of individual or groups of fibers

• Abnormal nuclear positioning or morphology

• Inflammatory cell infiltration

• Degenerating or regenerating fibers

• Increased connective tissue

What pathologic muscle features require histochemical techniques to be demonstrated?

• Fiber type grouping

• Fiber type predominance

• Central cores

• Target fibers

Define "type grouping" as it applies to muscle histopathology.

Type grouping refers to the clustering of muscle fibers of the same type (type I or type II), typically due to reinnervation after nerve damage, rather than the normal checkerboard distribution.

Define "fiber type predominance."

Fiber type predominance occurs when there is a marked imbalance in the ratio of type I to type II fibers in a muscle, often seen in neuropathic or myopathic conditions.

___ and ___ fibers are abnormalities of muscle fibers that are visible only with histochemical stains and are associated with certain myopathies.

Central cores; target fibers

True or False: The combination of hematoxylin and eosin (H&E) staining and histochemical stains improves diagnostic accuracy in muscle biopsy.

True
Explanation: Using both H&E and a panel of histochemical stains allows for comprehensive evaluation, distinguishing between primary muscle disease (myopathy) and nerve-related muscle changes (neuropathy).

What two broad categories of disease can be identified by interpreting muscle biopsies using H&E and histochemical stains?

• Primary muscle disease (myopathy)

• Secondary muscle changes due to denervation (neuropathy)

What is the ATPase (pH 9.4 - 10.4) reaction for muscle fiber type I?

Light

What is the ATPase (pH 9.4 - 10.4) reaction for muscle fiber types IIA, IIB, IIC?

Dark for all three

What is the ATPase (pH 4.2 - 4.3) reaction for muscle fiber type I?

Dark

What is the ATPase (pH 4.2 - 4.3) reaction for fiber types IIA, IIB, and IIC?

Light (IIA & IIB), intermediate (IIC)

What is the ATPase (pH 4.6) reaction for types IIA, IIB, and IIC?

• Type IIA: Light

• Type IIB: Intermediate

• Type IIC: Dark

What is the NADH reaction for type I muscle fibers?

• Dark

What is the NADH reaction for types IIA, IIB, and IIC?

• Light for all

What is the SDH reaction for type I muscle fibers?

Dark

What is the SDH reaction fpr types IIA, IIB, IIC?

Light for all

What is the Phosphorylase reaction for type I muscle fibers?

Light

What is the Phosphorylase reaction for types IIA, IIB, and IIC?

Dark for all

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

Diagnosis of muscle disease

True or False: Enzyme histochemistry techniques are used exclusively for muscle pathology.

False
Explanation: While muscle disease diagnosis is the primary application, some techniques are also used in hematopathology (e.g., for leukemia differentiation).

Why is enzyme histochemistry often performed in histopathology labs even for hematologic cases?

Because there is overlap in techniques, so histopathology labs commonly perform all enzyme histochemistry on both tissue sections and smears.

Approximately how many enzyme demonstration methods exist today?

More than 1,000

What are two ways to increase the rate of a chemical reaction?

• Apply heat

• Use a catalyst

Define “catalyst” in the context of chemical reactions.

A substance that changes the rate of a chemical reaction without being consumed in the reaction.

What is the difference between a positive and a negative catalyst?

• Positive catalyst: Increases reaction rate

• Negative catalyst: Decreases reaction rate

A catalyst is released ___ the reaction and can combine again with other chemicals.

After

True or False: Catalysts are permanently changed by the reactions they facilitate.

False
Explanation: Catalysts (including enzymes) are not consumed or altered permanently by the reaction—they are reusable.

What are enzymes, and what do they do?

• Enzymes are proteins that catalyze chemical reactions in biological systems.

• They do so by temporarily combining with their specific substrate and are released unchanged afterward.

Why are enzymes necessary in biological systems?

Because many essential reactions would proceed too slowly at biological temperatures without them.

True or False: Enzymes are consumed by the reactions they catalyze.

False
Explanation: Enzymes act reversibly and remain unchanged, ready to catalyze additional reactions.

What are cofactors in enzyme function?

• Nonprotein components that help enzymes catalyze reactions

• May be metal ions or organic molecules (coenzymes)

Define coenzyme.

A complex organic molecule, often a vitamin, that acts as a cofactor in enzymatic reactions

Coenzymes often act as a ___, meaning they undergo complementary chemical changes to the substrate

Cosubstrate

True or False: Coenzymes are always permanently bound to enzymes.

False
Explanation: Coenzymes bind with varying degrees of affinity and may be loosely or tightly bound depending on the enzyme and function.

List the three ways biological oxidation can occur.

1. Addition of oxygen

2. Loss of hydrogen

3. Loss of electrons

List the three ways biological reduction can occur.

1. Loss of oxygen

2. Gain of hydrogen

3. Gain of electrons

True or False: Oxidation always involves oxygen.

False
Explanation: Oxidation may involve oxygen, but in biological systems, it can also occur via loss of hydrogen or electrons.

True or False: Enzymes can be reactivated after being inactivated by heat.

False
Explanation: Heat causes irreversible denaturation of enzymes, permanently altering their structure and rendering them inactive.

What effect does pH have on enzyme activity?

• Enzymes are sensitive to pH changes

• Some work best at acidic pH, others at alkaline pH

• Most function optimally near neutral pH (~7.0)

• An enzyme active at acidic pH becomes inactive in alkaline conditions, and vice versa

Enzymes are highly specific to their ___, which they recognize by specific chemical groups and spatial arrangement.

Substrate

What are five key factors that can influence enzyme demonstration in histochemistry?

• Tissue treatment before/during the procedure

• Nonoptimal substrate

• Nonoptimal temperature

• Nonoptimal pH

• Inhibitors

True or False: Fixation always preserves enzymes for histochemical analysis.

False
Explanation: Many enzymes are removed or destroyed by fixation; some are also sensitive to freezing and thawing, requiring compromise between enzyme activity and localization.

What trade-off often occurs when preparing tissue for enzyme histochemistry?

• A sacrifice of enzyme activity may be made for better localization, or

• Maximum activity may be preserved at the expense of increased diffusion

Why might a nonoptimal substrate be used in enzyme histochemistry?

• The optimal substrate may be insoluble

• The substitute may be hydrolyzed more slowly than natural or other available substrates

What is the typical temperature threshold above which enzyme activity is destroyed?

>56°C

Some methods, such as ___, may require a nonoptimal temperature for reaction, which can reduce enzyme activity.

Azo dye simultaneous coupling techniques

True or False: All enzymes are best demonstrated at neutral pH.

False
Explanation: Although most enzymes are best demonstrated near pH 7.0, some (e.g., acid and alkaline phosphatases) have nonneutral pH optima.

Why might a histochemical method intentionally use a nonoptimal pH?

Certain techniques (e.g., azo dye simultaneous coupling methods) require it, even though it may lower enzyme activity.

List potential inhibitors that can reduce or abolish enzyme activity in tissue samples.

• Excess diazonium salts

• Fixatives

• Heat

• Certain metallic ions

True or False: Diazonium salts always enhance enzyme histochemistry.

False
Explanation: Excess diazonium salts in the substrate can act as inhibitors, reducing or abolishing enzyme activity.

True or False: Fixation is always avoided when preserving enzymes for histochemical demonstration.

False
Explanation: While some enzymes are inactivated by fixation, others require some fixation to prevent diffusion and retain enzymatic activity. The decision depends on the enzyme and technique used.

Why is fixation often desirable before freezing tissue for enzyme demonstration?

• To prevent diffusion artifacts

• To retain soluble enzymes that might otherwise be lost in unfixed frozen sections

Postfixation of sections does ___ prevent enzyme diffusion if tissue was unfixed prior to freezing.

Not

What potential problem occurs when frozen sections are cut from unfixed tissue?

Considerable diffusion artifact — enzymes may spread from their original location, distorting localization results.

What fixatives are commonly used cold (3–4°C) for enzyme studies?

• Cold formalin

• Cold calcium formalin

• Cold acetone

What is the benefit of using cold fixatives for enzyme preservation?

Cold temperatures help preserve enzyme activity while still minimizing diffusion and tissue degradation.

What solution can be used to store fixed tissue for enzyme studies? At what temperature?

Gum sucrose solution (30% sucrose with 1% gum acacia), stored at 4°C

How long can fixed tissue be stored in gum sucrose solution at 4°C?

Several weeks

True or False: Blood smears should be fixed immediately before drying.

False
Explanation: Blood smears must be fixed as soon as they are dry — fixing before drying would cause morphological distortion and loss of enzyme activity.

Why must blood smears be fixed as soon as they are dry for enzyme analysis?

Delays in fixation cause loss of enzyme activity in smears.

How are muscle biopsies typically prepared for enzyme histochemistry?

• Frozen unfixed

• Sections are postfixed if the technique allows

Why is rapid freezing essential for muscle biopsies?

Muscle is especially prone to ice crystal artifact, which can distort tissue morphology and enzyme localization.

What is the preferred method for freezing muscle tissue to preserve enzyme activity?

• Use isopentane cooled in liquid nitrogen

• Let isopentane reach −150°C before immersing the muscle

True or False: The cryostat freezer plate is the best method for freezing muscle for enzyme studies.

False
Explanation: The cryostat plate is slower and may not prevent ice crystal artifact, making it less suitable than rapid freezing in −150°C isopentane.

What precaution should be taken if muscle must be frozen directly in liquid nitrogen?

• Dust the specimen with talc

• This prevents gaseous nitrogen from forming a barrier that impedes rapid freezing

If immersed directly in liquid nitrogen, the formation of ___ nitrogen around the specimen can impede freezing.

Gaseous

What are the four histochemical techniques used to demonstrate hydrolytic enzymes?

1. Simultaneous capture or coupling

2. Postincubation coupling

3. Self-colored substrate

4. Intramolecular rearrangement

In the simultaneous coupling method for hydrolase demonstration, what compound is commonly used with azo-dye methods?

A diazonium salt.

What is the function of esterase enzymes?

They hydrolyze bonds between carboxylic acid and alcohols, phenols, or naphthols.