Electrophoresis of LDH Isoenzymes and Activity Staining
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The slides prepared for MD(Biochemistry) and MSc (Biochemistry) teaching comprehensively covers isoenzymes, isoforms, clinical utility of Lactate Dehydrogenase (LDH), LDH isoenzymes and basics of zymography. Download and view in presenter mode for better visual experience.
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Electrophoresis of LDH Isoenzymes and Activity Staining
- 1. Separation and Detection of LDH Isoenzymes Ashikh Seethy Senior Resident & PhD Scholar Dept of Biochemistry AIIMS, New Delhi
- 2. Overview Isoenzymes Lactate Dehydrogenase Tissue Distribution Clinical utility Separation of LDH Isoenzymes Activity Staining 2
- 3. MULTIPLE FORMS OF AN ENZYME 3
- 4. Allelic Variants Pseudocholine esterase Succinyl choline Succinate + Choline G-6-PD Allozymes/ Allo-enzymes Pharmacogenomics Evolution 4 Eu Ea Ef Es EuEu EaEa EfEf EsEs Other Combinations
- 5. Multiple Proteins From a Single Polypeptide Chain 5
- 9. Which of the Following are Isoenzymes? 9
- 10. Isoenzymes/ Isozymes The term “isoenzyme” or “isozyme” should apply only to those multiple forms of enzymes arising from genetically determined differences in primary structure, and not to those derived by modification of the same primary sequence. The Nomenclature of Multiple Forms of Enzymes: IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Recommendations (1971) True isoenzymes result from the existence of more than one gene locus coding for the structure of the enzyme protein. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed (2012) 10
- 11. Isoforms Post-translational modifications of enzyme molecules give rise to multiple forms known as isoforms Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed (2012) Isoforms can differ in their activity and regulatory properties 11
- 12. Identify the Isoenzymes and the Isoforms 12
- 13. Why should there be Isoenzymes? Evolutionary advantage 13
- 14. Clinical Utility of Isoenzymes: Functional vs Non-functional plasma enzymes Differential tissue distribution 14
- 17. Fate of D-Lactate? 17
- 18. LDH Genes 18 Gene Protein LDHA M-subunit LDHB H-subunit LDHBx subunit LDHC LDHC subunit
- 19. LDHB H subunit & LDHBx 19 LDHB H-subunit LDHBx PTS1 Translational Readthrough
- 20. Isoenzymes of LDH 20 Isoenzyme Subunit Composition % in Normal Serum LD1 HHHH 14–26 LD2 HHHM 29-39 LD3 HHMM 20-26 LD4 HMMM 8-16 LD5 MMMM 6-16 LD6 ?? LDX/LDHc XXXX or CCCC Macro-LDH Ab-LDH
- 21. Isoenzymes of LDH 21 Isoenzyme Subunit Composition % in Normal Serum LD1 HHHH 14–26 LD2 HHHM 29-39 LD3 HHMM 20-26 LD4 HMMM 8-16 LD5 MMMM 6-16 LD6 ?? LDX/LDHc XXXX or CCCC Macro-LDH Ab-LDH DANNEHOVER ET AL CLINICAL CHEMISTRY, Vol. 30, No. 9, 1984
- 22. Questions: 1. What is the basis of the numbering of iso-enzymes? In naming isozymes, the normal enzyme name should be used, followed by a number. The numbers should be allotted preferably on the basis of electrophoretic mobility under defined conditions- the lower numbers given to the forms with the higher mobility towards the anode. In photographs or diagrams of electrophoretic results, the anode should be oriented to the top or right-hand side of the page. 2. How many E.C numbers will LDH have? 22
- 23. 23
- 25. Tissue Distribution of LDH Isoenzymes 25 Isoenzyme Tissue Distribution Elevated in: LD1 Heart RBC Acute MI Hemolytic anemia/ hemolysis Testicular tumors LD2 RBC Heart Hemolytic anemia/ hemolysis Acute MI LD3 Lung Lymphocytes Spleen Metastasis Pneumonia Lymphomas, ALL LD4 Liver Skeletal muscle Hepatic disorders Muscular dystrophyLD5 LDX/LDHc Testes Various cancers
- 26. Tissue Distribution of LDH Isoenzymes 26 Isoenzyme Tissue Distribution Elevated in: LD1 Heart RBC Acute MI Hemolytic anemia/ hemolysis Testicular tumors LD2 RBC Heart Hemolytic anemia/ hemolysis Acute MI LD3 Lung Lymphocytes Spleen Metastasis Pneumonia Lymphomas, ALL LD4 Liver Skeletal muscle Hepatic disorders Muscular dystrophyLD5 LDX/LDHc Testes Various cancers
- 27. Functions of LDH Isoenzymes 27 Lactate is considered to be a “metabolic dead end”. Then why convert pyruvate to lactate?
- 28. Functions of LDH Isoenzymes 28
- 29. SEPARATION OF LDH ISOENZYMES 29
- 30. LDH Isozymes are Separated by: Electrophoresis Selective inhibitors Sodium perchlorate inhibits all LDH isozymes except LDH1 Ion exchange chromatography Immunoprecipitation Separates LDH1 and LDH 5 from other LDH isoenzymes 30
- 31. Electrophoresis of LDH Isozymes: 31 What will be the approximate molecular weight of each subunit?
- 33. Electrophoresis of LDH Isozymes: Sample: Serum Why NOT plasma? Stored serum? How will you visualize the separated isoenzymes? 33
- 34. Visualization of LDH Isoenzymes: 34
- 36. Zymography: Gross and Lapière (1962) 36 Vandooren et al. 2013
- 37. 37 Vandooren et al. 2013
- 38. Activity Staining for LDH Isoenzymes: How will you proceed after electrophoresis? Can you use IFCC method of Serum LDH estimation for activity staining? 38 Pyruvate + NADH+H+ Lactate + NAD+ Rate of disappearance measured at 340nm pH= 7.4-7.8 LDH No Soluble
- 39. Lactate + NAD Pyruvate + NADH NAD+ + PMS[reduced]NADH + H+ + PMS NBT + PMS[reduced] + PMS LDH Phenazine methosulfate (PMS) acts as electron carrier for reduction of tetrazolium salts to coloured product Activity Staining for LDH Isoenzymes: NBT[reduced] In Insoluble
- 40. Nitroblue Tetrazolium Test 40 Chronic granulomatous disease ↓ Defective NADPH oxidase ↓ NBT is not reduced
- 41. Densitometry: 42 Reference interval for Serum LDH: 115–221 U/L
- 42. Activity Staining: Pre-requisites: Protein in native state Ability to form an insoluble product Electrophoresis under optimum conditions Shortcomings: Artificial environment Semi-quantitative data 43
- 43. Electrophoretic Patterns NORMAL LIVER DAMAGE MYOCARDIAL INFARCTION MUSCLE DAMAGE
- 44. HBD Activity of LDH Assay for H-subunit 45
- 45. Precautions: Avoid EDTA vial for sample collection Hemolysis should be avoided Proper temperature should be maintained during electrophoresis Staining should be done in dark as PMS is light sensitive. Standard precautions while handling serum 46
- 46. Thank you!
Editor's Notes
- Other pathway where MDH1 and MDH2 are invoved?
- M- Chr 11 H- Chr 12
- in vitro differences in kinetic properties of the isoenzyme may be inappropriate to explain actual physiological actions for several reasons: differences in kinetic properties between LDH-1 and LDH-5 are less marked at 37~ (body temperature) than at 25~ high intracellular concentrations of the enzyme are present, and differences in the actual ratio of ketopyruvate to enolpyruvate exist (the enol form may be the more potent inhibitor). Furthermore, the occurrence of similar isoenzyme patterns in widely different tissues with divergent metabolic goals (e.g., LDH-5 in liver and muscle; LDH-1 in heart and erythrocytes) points out our tack of understanding of their precise role.