ENZYME HISTOCHEMISTRY.................pptx
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2. AST and ALT are aminotransferases that catalyze transfer reactions and are released from hepatocytes during injury. ALP is a hydrolase involved in membrane transport. GGT and 5'NT also indicate cholestasis.
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Enzymes are biological catalysts that are usually proteins which increase the rate of chemical reactions without being consumed. They are very specific and function by lowering the activation energy of reactions. Enzymes work by binding substrates to form enzyme-substrate complexes, and either the "lock and key" or "induced fit" models describe this interaction. Enzyme activity can be regulated by factors like substrate concentration, products, pH and temperature changes. It can also be regulated by allosteric effectors or covalent modification. Inhibitors decrease enzyme activity by competing or non-competitively binding to the active site.
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Enroll Now to secure your spot in this crucial training session and ensure your HIPAA compliance is robust and audit-ready.
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ENZYME HISTOCHEMISTRY.................pptx
- 2. ENZYMES - DEFINITION Enzymes are biological catalysts that accelerates a chemical reaction. ENZYME HISTOCHEMISTRY Utilising histochemical techniques to demonstrate enzyme in tissues.
- 3. PRESERVATION For good demonstration, maximum amount of enzyme activity should be preserved - with accurate localisation and by preventing diffusion of enzymes. Lyo-enzymes: dissolved in cytoplasm; likely to diffuse Desmo-enzymes: attached to cytoplasmic constituents; less likely to diffuse
- 4. SAMPLE PREPARATION FOR ENZYME HISTOCHEMISTRY Fresh preparation or frozen/cryostat/freeze dried preparations are necessary. Autopsy tissue: unsuitable for demonstration of enzymes. Cold fixative should be used to preserve maximum enzyme activity Eg: Ice cold formalin, ice cold alcohol
- 5. FACTORS AFFECTING ENZYME ACTIVITY Temperature: optimal temperature in EHC is 4°C -30 °C PH optimum pH-7 ALP-9.2 ACP-5.0
- 6. Inhibitors Specific Inhibitors :eserine for choline esterase Non-Specific Inhibitors: heat Competitive Inhibitors: malonate ions for succinate dehydrogenase Activators:Mg ions for ALP
- 7. 2 TERMS USED IN ENZYME TECHNIQUE Primary Reaction Product: the product of the reaction of an enzyme on a substrate Final Reaction Product: the product of an insoluble uncolored PRP, which has been made colored or opaque
- 8. METHODS OF DEMONSTRATION 1. Simultaneous coupling 2. Post Incubation coupling 3. Self- Coloured substrate 4. Intra-molecular Rearrangement 5. Metal Precipitation
- 9. SIMULTANEOUS COUPLING Single incubation Mixture: Substrate + Diazonium salts
- 10. POST INCUBATION COUPLING Substrate (for enzyme hydrolysis) and Diazonium salts (Coupling) are in separate solutions
- 11. Advantages: Optimal pH for enzyme substrate reaction and possibly different pH for coupling can be achieved. Deleterious effect of diazonium salts on enzyme substrate reaction can be avoided.
- 14. METAL PRECIPITATION • A type of Simultaneous Coupling but uses Metallic lons instead of Diazonium salts.
- 15. USE OF CONTROLS Substrates, Diazonium salts and other components deteriorate with time leading to false positives and false negative reactions. Therefore, Control sections should be carried together with test sections. Positive Controls: to demonstrate that all reagents are working Negative Controls: prepared by a) destroying the enzyme in the test section by immersing in boiling water for 15 minutes b) by including a specific enzyme inhibitors c) By omitting the substrate from the incubation medium
- 16. APPLICATION OF ENZYME HISTOCHEMISTRY PATHOLOGICAL DIAGNOSIS OF MuSCULAR DISORDERS 1. 5' Nucleotidase 2. Adenosine Tri-Phosphatase 3. Cytochrome oxidase 4. Lactate adehydrogenase 5. Choline Esterase 6. Mono-amine-oxidase 7. Phosphorylase
- 17. DIAGNOSIS OF HEMATO-LYMPHOID MALIGNANCIES 1. Peroxidase 2. Alkaline phosphatase 3. Acid phoshphatase 4. β-glucuronidase 5. Non-specific esterase 6. DOPA-Oxidase 7. Chloroacetate-esterase
- 18. DETERMINATION OF ALKALINE PHOSPHATASES Optimal activity at 9-9.6 pH Activated by Mg, Mn and Co ions Cyanide and Cysteine inhibits ALP-ase activity- thus, incorporated in incubation medium as a control METHODS 1. Gomori’s Calcium Phosphate method (Metal Substitution) 2. Azo Dye Coupling methods – Simultaneous Coupling – Post Coupling
- 19. AZO-DYE SIMULTANEOUS COUPLING METHOD USING SUBSTITUED NAPHTHOL
- 20. Incubation mixture: 1. Tris Buffer pH 9.1 2. Distilled Water 3. Substrate Solution (Naphthol phosphoric acid) 4. Fast Garnet GBC salt
- 21. Procedure: Fix sections in methanol or cold acetone in freezing temperature for 30 sec Rinse in Distilled water. Keep the section in the incubation mixture for 1-2 hrs at RT. Rinse in water. Counter stain with hematoxylin Mount in glycerine.
- 22. RESULT Positive cells – granular dark brown
- 23. THANK YOU