[F] Week 13: Enzyme Histochemistry and Diagnostic Application Flashcards
Detect early metabolic changes in biopsy and autopsy tissue before manifestation on H&E staining or immunohistochemistry
Enzyme histochemistry
- Essential not to inactivate an appreciable proportion of the enzyme
- Precision varies inversely with the length of time required in the incubation medium
Enzyme histochemistry
TOF
Enzyme histochemistry is commonly requested
False
- May be bound to specific cell components or may be free and soluble in the cytoplasm and body fluids
- Speeds up a biochemical process
Enzyme
generally required for histochemical demonstration of enzymes
Frozen Sections
Tissues frozen to -70°C or below are usually well preserved, with little loss of enzyme activity
Several destructive factors
causeing false negative, and disadvantages of unfixed frozen sections:
- Mechanical disruption by freezing and thawing
- Uneven section thickness
- Diffusion of soluble enzymes
- Co-factors leading to loss of reproducibility
- False localization
The best fixative for all enzymes is?
`
chilled acetone
What are the disadvantage of using chilled acetone as the fixative for enzyme
Even the cytological details are not so good, you can still preserve a lot of enzymes
Chilled acetone as fixative is especially recommended when staining for?
Acid Phosphatase
For other enzymes (other than acid phosphatase) this is the prefarable fixative because it gives a better cytological fixation and the tissue is easier to handle.
COLD 90-100% ETHYL ALCOHOL
Most hydrolytic enzymes are reasonably resistant to formalin and can be fixed in?
10% Formalin
(preferably adjusted to pH 6-6.5 with a small amount of phosphate buffer)
entirely unsuitable as a fixative, because it destroys most enzymes
Methyl alcohol
most common technique
for histochemical demonstration of enzymes
METAL PRECIPITATION
Enzymes for which histochemical techniques are known belong in one of the two groups:
- Oxidative enzymes
- Hydrolytic enzymes
catalyze the reaction
between substrate and atmospheric oxygen
Oxidative Enzymes
Transferring of electron from one
substrate to another substrate - the hydrogen
Oxidative Enzymes
Oxidative enzyme can be demonstrated by?
simultaneous coupling
method
simultaneous coupling method, which involves oxidation of the substrate and subsequent reduction of a tetrazolium salt, resulting in
the formation of a?
relatively insoluble formazan deposit at the site of enzyme activity
The two tetrazolium salts commonly used as hydrogen acceptors:
monotetrazolium (MTT) and ditetrazolium chloride-nitro (NBT)
The oxidative enzymes fall into three groups:
- dehydrogenases
- oxidases
- peroxidases
Catalyze the transfer of hydrogen to immediate acceptors other than oxygen and peroxides
Dehydrogenases
other elements can be used such as peroxides.
What are the required coenzymes for dehydrogenases?
linked to the diaphorase or cytochrome system
seen in respiratory/krebs cycle
Delicate enzymes which are largely destroyed by any sort of fixation and
destroyed by embedding
Dehydrogenases
One disadvantage of dehydrogenases, making the procedures HARD
Dehydrogenase
Demonstrates mitochondria and the fine detail of the sarcoplasmic reticulum of the fiber
Nicotinamide Adenine Dinucleotide (NADH) diaphorase
Dehydrogenase
used to detect very minor or early
structural abnormality in the sarcoplasmic reticulum network of the fiber, as well as mitochondrial abnormalities
NADH diaphorase
Dehydrogenases
The three main types of compounds used are
- Methylene blue (not recommended)
- Tetrazolium method (most sensitive; reduced to bright red)
- Tellurite( reduced to black; less sensitive)
What are the guidlines for the specimen when using dehydrogenase?
- tissue need not be absolutely fresh
- Refrigeration for 4 hours at 4°C does not cause any noticeable loss of activity
- Fixation in chilled acetone for 4 hours causes only 40% inactivation of the enzyme
Catalyzing the oxidation of various substrates, mainly phenols
and amines
Oxidase
What are the enzymes under oxidase
- Polyphenol oxidases (PPOs)
- Indophenol Oxidase (Cytochrome Oxidase)
- Tyrosinase
- Dopa oxidase
Oxidase
- belong to copper-containing metalloproteins.
- They also belong to oxidoreductases that catalyze the oxidation of a wide range of phenolic compounds by utilizing molecular oxygen.
- The concept involves the transfer of energy from one side to another. They are useful for various biotechnological applications.
Polyphenol oxidases (PPOs)
- A copper-yielding cytochrome complex that catalyzes the oxidation of ferrocytochrome C to produce ferricytochrome C and 2H2O
Indophenol Oxidase (Cytochrome Oxidase)
Indophenol Oxidase (Cytochrome Oxidase)
To stain for cytochrome oxidase, a mixture of
solutions of a
phenol or naphthol and an aromatic diamine is slowly oxidized on exposure to air
intensely blue indophenol dyes
Oxidase
Copper containing monooxygenases that catalyze the production of melanin and other pigments from tyrosine by oxidation.
Tyrosinase
- Increased tyrosinase activity:
- Decreased tyrosinase activity:
- Increased tyrosinase activity: increased melanin
synthesis thereby causing melanoma - Decreased tyrosinase activity: hyperpigmentation (albinism)
Melanin pigment is formed from the amino acid, dihydroxy-phenylalanine (DOPA), by the action of a specific oxidative enzyme that is responsible for the oxidation of tyrosine to dopa and dopa quinone
Dopa oxidase
what are the samples for DOPA OXIDASE?
Adrenal Glands
To demonstrate dopa oxidase, use frozen sections of fresh material or of tissue fixed for only a few hours in?
5% formalin
What are the optimal temerature used to when using Dopa oxidase
20o and 37oC
TOF
Longer fixation may cause partial inactivation of the enzyme dopa oxidase
True
The sections are rinsed very briefly in distilled water and transferred a 0.1 per cent solution of dihydroxyphenylalanine, buffered with a phosphate buffer to pH ____ in an open dish for 4-5 hours
7.3-7.5
Dopa oxidase
It is advisable to change the incubating solution
once or twice
Color reaction for dopa oxidase
dark brown -gray or color black
increased pH = more melanin precipitate
Heme-containing enzymes that use hydrogen peroxide as the electron acceptor to catalyze a few oxidative reactions
Peroxidases
Peroxidase is seen in ____ or ____ wherein positive reaction produces blue color. In this test, it uses
PSEUDOPEROXIDASE.
Guaiac Test or FOBT
Quite resistant to various chemical and physical agents, especially to acids and heat
Peroxidases
a method for demonstrating peroxidase granules in some neutrophils and in eosinophils
Peroxidase stain
What are the dyes used for peroxidase stain
- Benzidine: if oxidized, it will produce blue or brown dye
- Naphthol: if oxidized, it will produce purple-black color
- Leuco-dyes are re-colorized to their original shades (If it does not turn back to its original color, it means it did not react)
Perosxidase smears are fixed with
acetone, alcohol, or formalin alcohol (1:10)
Peroxidases
For tissues, the same fixatives or
formalin-saline
what is the best specimen for the use of peroxidase
Frozen Section
can either use paraffin or celloidin embedding material
- Complex catalytic proteins that use water to break down protein, carbohydrate, nucleic acids, starch, fats, phosphate esters and other molecules
- Group of esterase
- Reactions have been devised for the demonstration of either the
acid or the alcoholic moiety.
Hydrolytic Enzyme
2 HYDROLYTIC ENZYMES
Acids and alcohol combined with?
Acids: metal ions: cobalt, iron, and copper
Alcoholic: (thio?)alcohol or naphtol
What are the hydrolytic enzymes
- Phosphatase (Alkaline phosphatase, Acid phosphatase)
- 5- nucleotidase
- Adenosine Triphosphatase (ATPase)
- Nonspecific esterase
- Chloroacetate esterase
- Acetylcholinesterase
- Phosphorylase
- Aldolase
- Sulfatase
