Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Separation and Detection
of LDH Isoenzymes
Ashikh Seethy
Senior Resident & PhD Scholar
Dept of Biochemistry
AIIMS, New Del...
Overview
 Isoenzymes
 Lactate Dehydrogenase
 Tissue Distribution
 Clinical utility
 Separation of LDH Isoenzymes
 Ac...
MULTIPLE FORMS OF AN ENZYME
3
Allelic Variants
 Pseudocholine esterase
Succinyl choline  Succinate + Choline
 G-6-PD
 Allozymes/ Allo-enzymes
 Phar...
Multiple Proteins
From a Single
Polypeptide Chain
5
Genetically Independent Proteins
6
Genetically
Independent Proteins
Hybrid Enzymes:
7
Other Enzyme Modifications:
8
Which of the Following are Isoenzymes?
9
Isoenzymes/ Isozymes
The term “isoenzyme” or “isozyme” should apply only to
those multiple forms of enzymes arising from g...
Isoforms
Post-translational modifications of enzyme molecules
give rise to multiple forms known as isoforms
Tietz Textbook...
Identify the Isoenzymes and the Isoforms
12
Why should there be Isoenzymes?
Evolutionary advantage
13
Clinical Utility of Isoenzymes:
 Functional vs Non-functional plasma enzymes
 Differential tissue distribution
14
LACTATE DEHYDROGENASE
15
Reactions Catalysed:
16
Fate of D-Lactate? 17
LDH Genes 18
Gene Protein
LDHA M-subunit
LDHB
H-subunit
LDHBx subunit
LDHC LDHC subunit
LDHB  H subunit & LDHBx
19
LDHB
H-subunit
LDHBx
PTS1
Translational
Readthrough
Isoenzymes of LDH
20
Isoenzyme
Subunit
Composition
% in Normal
Serum
LD1 HHHH 14–26
LD2 HHHM 29-39
LD3 HHMM 20-26
LD4 HMMM...
Isoenzymes of LDH
21
Isoenzyme
Subunit
Composition
% in Normal
Serum
LD1 HHHH 14–26
LD2 HHHM 29-39
LD3 HHMM 20-26
LD4 HMMM...
Questions:
1. What is the basis of the numbering of iso-enzymes?
 In naming isozymes, the normal enzyme name should be us...
23
Identify:
24
Rossmann Fold
Tissue Distribution of LDH Isoenzymes
25
Isoenzyme Tissue Distribution Elevated in:
LD1
Heart
RBC
Acute MI
Hemolytic anemi...
Tissue Distribution of LDH Isoenzymes
26
Isoenzyme Tissue Distribution Elevated in:
LD1
Heart
RBC
Acute MI
Hemolytic anemi...
Functions of LDH Isoenzymes
27
Lactate is considered to be
a “metabolic dead end”.
Then why convert pyruvate
to lactate?
Functions of LDH Isoenzymes
28
SEPARATION OF LDH ISOENZYMES
29
LDH Isozymes are Separated by:
 Electrophoresis
 Selective inhibitors
 Sodium perchlorate inhibits all LDH isozymes exc...
Electrophoresis of LDH Isozymes:
31
What will be the approximate molecular weight of each subunit?
Electrophoresis
of LDH
Isoenzymes:
32
Electrophoresis of LDH Isozymes:
 Sample: Serum
 Why NOT plasma?
 Stored serum?
 How will you visualize the separated ...
Visualization of LDH Isoenzymes:
34
ACTIVITY STAINING/ ZYMOGRAPHY
35
Zymography: Gross and Lapière (1962)
36
Vandooren et al. 2013
37
Vandooren et al. 2013
Activity Staining for LDH Isoenzymes:
 How will you proceed after electrophoresis?
 Can you use IFCC method of Serum LDH...
Lactate + NAD Pyruvate + NADH
NAD+ + PMS[reduced]NADH + H+ + PMS
NBT + PMS[reduced] + PMS
LDH
Phenazine methosulfate (PMS)...
Nitroblue Tetrazolium Test
40
Chronic granulomatous disease
↓
Defective NADPH oxidase
↓
NBT is not reduced
Densitometry:
42
Reference interval for Serum LDH: 115–221 U/L
Activity Staining:
 Pre-requisites:
 Protein in native state
 Ability to form an insoluble product
 Electrophoresis un...
Electrophoretic Patterns
NORMAL LIVER DAMAGE
MYOCARDIAL INFARCTION MUSCLE DAMAGE
HBD Activity of LDH
 Assay for H-subunit
45
Precautions:
 Avoid EDTA vial for sample collection
 Hemolysis should be avoided
 Proper temperature should be maintain...
Thank you!
Upcoming SlideShare
Loading in …5
×

Electrophoresis of LDH Isoenzymes and Activity Staining

2,941 views

Published on

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.

Published in: Health & Medicine
  • Be the first to comment

Electrophoresis of LDH Isoenzymes and Activity Staining

  1. 1. Separation and Detection of LDH Isoenzymes Ashikh Seethy Senior Resident & PhD Scholar Dept of Biochemistry AIIMS, New Delhi
  2. 2. Overview  Isoenzymes  Lactate Dehydrogenase  Tissue Distribution  Clinical utility  Separation of LDH Isoenzymes  Activity Staining 2
  3. 3. MULTIPLE FORMS OF AN ENZYME 3
  4. 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. 5. Multiple Proteins From a Single Polypeptide Chain 5
  6. 6. Genetically Independent Proteins 6 Genetically Independent Proteins
  7. 7. Hybrid Enzymes: 7
  8. 8. Other Enzyme Modifications: 8
  9. 9. Which of the Following are Isoenzymes? 9
  10. 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. 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. 12. Identify the Isoenzymes and the Isoforms 12
  13. 13. Why should there be Isoenzymes? Evolutionary advantage 13
  14. 14. Clinical Utility of Isoenzymes:  Functional vs Non-functional plasma enzymes  Differential tissue distribution 14
  15. 15. LACTATE DEHYDROGENASE 15
  16. 16. Reactions Catalysed: 16
  17. 17. Fate of D-Lactate? 17
  18. 18. LDH Genes 18 Gene Protein LDHA M-subunit LDHB H-subunit LDHBx subunit LDHC LDHC subunit
  19. 19. LDHB  H subunit & LDHBx 19 LDHB H-subunit LDHBx PTS1 Translational Readthrough
  20. 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. 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. 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. 23
  24. 24. Identify: 24 Rossmann Fold
  25. 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. 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. 27. Functions of LDH Isoenzymes 27 Lactate is considered to be a “metabolic dead end”. Then why convert pyruvate to lactate?
  28. 28. Functions of LDH Isoenzymes 28
  29. 29. SEPARATION OF LDH ISOENZYMES 29
  30. 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. 31. Electrophoresis of LDH Isozymes: 31 What will be the approximate molecular weight of each subunit?
  32. 32. Electrophoresis of LDH Isoenzymes: 32
  33. 33. Electrophoresis of LDH Isozymes:  Sample: Serum  Why NOT plasma?  Stored serum?  How will you visualize the separated isoenzymes? 33
  34. 34. Visualization of LDH Isoenzymes: 34
  35. 35. ACTIVITY STAINING/ ZYMOGRAPHY 35
  36. 36. Zymography: Gross and Lapière (1962) 36 Vandooren et al. 2013
  37. 37. 37 Vandooren et al. 2013
  38. 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. 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. 40. Nitroblue Tetrazolium Test 40 Chronic granulomatous disease ↓ Defective NADPH oxidase ↓ NBT is not reduced
  41. 41. Densitometry: 42 Reference interval for Serum LDH: 115–221 U/L
  42. 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. 43. Electrophoretic Patterns NORMAL LIVER DAMAGE MYOCARDIAL INFARCTION MUSCLE DAMAGE
  44. 44. HBD Activity of LDH  Assay for H-subunit 45
  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. 46. Thank you!

×