Chemical reaction engineering laboratoryFull description
BioChem
Lab write up for enzyme lab.
Enzyme Histochemistry Introduction
Enzyme Preservation
Enzymes – Enzymes – are are large tertiary proteins that acts as catalyst and are vital in cellular metabolism while maintaining hemostasis in man. Histochemistry – Histochemistry – is is defined as “the identification, localization and qualification in cells and tissue and by chemical or physical test of specific substances, reactive groups and enzyme catalyzed substance.”
Enzyme Histochemistry
This technique produces a final coloured reaction product that is localized to the cells with an intensity proportional to the enzyme activity. Applying on enzyme detection system to tissue section or smears.
Principle of Enzyme Histochemistry
Classification
Classified according to their effect on the substrate: o Oxidoreductases The most important groups to the diagnostic enzyme histochemist. Known as oxidases and dehydrogenases. o Transferases o Hydrolases Oxidative and hydrolytic enzymes o Lyases o Isomerases o Ligases
Activators
Ions of Calcium Magnesium Manganese Sodium Potassium
Fixation o Fixative should be at 4°C for the shortest time. Formol Calcium o Recommended for tissue blocks o Help maintain cell membrane integrity o Used on smears o Cryostat Sections Buffered Formalin or Formal Saline o Satisfactory in most cases (commonly used) Acetone, Formalin Vapour and Formalin-Alcohol Formalin-Alcohol Mixtures
Histochemistry Histochemi stry procedures are based on the simple premise that tissue cells, when placed in a solution chemically react with the solution to produce a colored insoluble product. The amount and location of the endproduct can then be evaluated in the context of the cells or tissue.
Classical histochemical reaction and generally based on one of the FOUR PRINCIPLES: 1. Simple ionic interactions 2. Reactions of aldehydes with schiffs reagent or silver compounds 3. Coupling of aromatic diazonium salts with aromatic residues on protein 4. Conversion acting on a substrate to form colored ppt.
Types of Histochemical Reactions
Simultaneous capture Post incubation coupling Self-coloured substrate Intramolecular Rearrangement
Self-coulered Substrate Technique:
o
Coloured Solube Substrate
Simultaneous Capture Most important technique Principle: o Gomori’s metal ppt. technique o Azp dye method Technique: o Enzyme + Substrate + Primary rxn product + Diazonium salt + Final rxn product (FRP) Disadvantages: o Diffusion of FRP o Rate of hydrolysis of substrate o Diffusion coefficient of the PRP for the buffer o Rate of coupling of the PRP and diazo salt o Enzyme salt and enzyme same PH
Enzyme
Solube Hydrolysis
Grouping remove
Coloured Insolube PRP Avantages: o Diazonium coupling not required
Intramolecular Rearrangement Technique:
o
Soluble substrate
Post Incubation Coupling Technique Hydrolysis
Technique: o Enzyme + Substrate + PRP + Insoluble and remain at site of production for initial incubation -> FRP in substrate medium Advantages: o Incubation Stage o Optimum PH Enzyme Diazonium Salt Disadvantages: o PRP is not completely insoluble o Diffusion
Molecular rearrangement in Spatial structure
Coloured insoluble rxn product PRP
Factors Influence the Reaction
pH Maintained Concentration o Inhibit the enzyme activity Temperature o Can alter the enzyme reaction rate o Some methods requiring incubation at 3°C o Room temperature o
Tetrazolium Salt Method
Demonstration Method 1. 2. 3. 4. 5.
Metal Precipitation Azo-dye Tetrazolium Salt Indigogenic Method Diaminobenzidine (DAB) Cytochrome Oxidase or the peroxidase to form a brown pigment The metabolism of glycogen by phosphorylase to produce a polysaccharide that is colored blue/black with iodine
Routinely used to identify phosphatases o Acid Phosphatase o Alkaline Phosphatase o Adenosine Triphosphate (ATPase)
Azo-dye Method
The azo-dye methods are used to identify acid and alkaline phosphatases, non-specific esterase and chloroacetate esterase. The different substrates contain a napthol group that can be cleaved by one of the above enzymes.
Tetrazolium salts are colourless, watersoluble salts that accept enzymatically-released hydrogen from the substrate to form highly coloured water-insoluble microcrystalline deposits known as Formazans.
Indigogenic Method
Metal Precipitation Method
Diazonium salts or freshly hexazotized pararosaniline are used to react with the napthol group to form a coloured insoluble dye.
The substrate containing indoxyl group, which released as a PRP, which in turn is oxidized by the capture agent potassium ferricyanide to form a turquoise FRP. The incubation solution also includes potassium ferricyanide which prevents over-oxidation of the FRP.
Diagnostic Application of Enzymes Histochemistry 1. Skeletal muscle fiber typing 2. Nerves and ganglia in suspected Hirschsprung’s disease 3. Gastrointestinal tissue to assed malabsorption 4. Demonstration of lymphoid and myeloid cells 5. Identification of early liver degeneration Skeletal Muscle
Diagnosis of neuromuscular disease and congenital myopathies
Skeletal Muscle Biopsy
Application of enzyme histochemical method to cryostat sections of unfixed skeletal muscle shows: o The presence of different fiber types o Changes in the number, size, and relative proportion of different fiber which are valuable in establishing the diagnosis.
Demonstration of Nerves and Ganglia Suspected Hirschsprung’s Disease
In Hirschsprung’s disease in children, a variable segmented of the rectum and colon is devoid of ganglionic cells. In the effected segment peristalsis is impossible and the large bowel become obstructed.
Demonstration of Specific Lactase or Sucrose deficiency in Jejunal Biopsies
An ALP method can be applied, ALP activity resides on the erythrocyte surface, is a sensitive marker of structural and functional integrity of the mucosal absorptive cells. ACP demonstrate some of the inflammatory cells in lamina, propria, also identifies lysososmal activity in villons enterocytes and glandular crypt epithelial cells.
Demonstration of Mast Cells and White Cells of Myeloid Series
Chloroacetate esterase techniques o Formalin-fixed paraffin sections o To assist in the identifications of tissue mast cells and myeloid white cells.
Two methods: o Fast Blue RR Method Which gives a vivid reaction product (Particularly intense in mast cell cytoplasm) o Pararosaniline Method Which gives a pinkish-red reaction product.
Miscellaneous
ACP Method o In the identification of prostate carcinoma o Tumor is infiltrating the colon/bladder wall or o In bone metastases Application of acid and alkaline phosphatases methods to cryostat sections of jejunal mucosal biopsy specimen. ALP Method o In vascular endothelial tumors
