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Separation and Detection of LDH Isoenzymes
Ashikh Seethy
Senior Resident & PhD Scholar
Dept of Biochemistry
AIIMS, New Delhi
Overview
Isoenzymes
Lactate Dehydrogenase
Tissue Distribution
Clinical utility
Separation of LDH Isoenzymes
Activity Staining
2
MULTIPLE FORMS OF AN ENZYME
3
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 OtherCombinations
Multiple Proteins From a Single Polypeptide Chain
5
Genetically Independent Proteins6
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 tothose multiple forms of enzymes arising from geneticallydetermined differences in primary structure, and not tothose derived by modification of the same primarysequence.
The Nomenclature of Multiple Forms of Enzymes: IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Recommendations (1971)
True isoenzymes result from the existence of more thanone gene locus coding for the structure of the enzymeprotein.
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed (2012)
10
Isoforms
Post-translational modifications of enzyme moleculesgive 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 regulatoryproperties
11
Identify the Isoenzymes and the Isoforms12
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
LDHBH-subunit
LDHBx subunit
LDHC LDHC subunit
LDHB H subunit & LDHBx
19
LDHBH-subunit
LDHBx
PTS1
Translational Readthrough
Isoenzymes of LDH20
IsoenzymeSubunit
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
Isoenzymes of LDH21
IsoenzymeSubunit
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 ALCLINICAL CHEMISTRY, Vol. 30, No. 9, 1984
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 ofelectrophoretic mobility under defined conditions- the lowernumbers given to the forms with the higher mobility towards theanode.
In photographs or diagrams of electrophoretic results, the anodeshould be oriented to the top or right-hand side of the page.
2. How many E.C numbers will LDH have?
22
23
Identify:24
Rossmann Fold
Tissue Distribution of LDH Isoenzymes25
Isoenzyme Tissue Distribution Elevated in:
LD1HeartRBC
Acute MIHemolytic anemia/ hemolysis
Testicular tumors
LD2RBC
HeartHemolytic anemia/ hemolysis
Acute MI
LD3Lung
LymphocytesSpleen
MetastasisPneumonia
Lymphomas, ALL
LD4 LiverSkeletal muscle
Hepatic disordersMuscular dystrophyLD5
LDX/LDHc Testes Various cancers
Tissue Distribution of LDH Isoenzymes26
Isoenzyme Tissue Distribution Elevated in:
LD1HeartRBC
Acute MIHemolytic anemia/ hemolysis
Testicular tumors
LD2RBC
HeartHemolytic anemia/ hemolysis
Acute MI
LD3Lung
LymphocytesSpleen
MetastasisPneumonia
Lymphomas, ALL
LD4 LiverSkeletal muscle
Hepatic disordersMuscular dystrophyLD5
LDX/LDHc Testes Various cancers
Functions of LDH Isoenzymes27
Lactate is considered to bea “metabolic dead end”.Then why convert pyruvateto lactate?
Functions of LDH Isoenzymes28
SEPARATION OF LDH ISOENZYMES
29
LDH Isozymes are Separated by:
Electrophoresis
Selective inhibitors
Sodium perchlorate inhibits all LDH isozymes exceptLDH1
Ion exchange chromatography
Immunoprecipitation
Separates LDH1 and LDH 5 from other LDH isoenzymes
30
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 isoenzymes?
33
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 estimationfor activity staining?
38
Pyruvate + NADH+H+ Lactate + NAD+
Rate of disappearance measured at 340nm
pH= 7.4-7.8
LDH
No
Soluble
Lactate + NAD Pyruvate + NADH
NAD+ + PMS[reduced]NADH + H+ + PMS
NBT + PMS[reduced] + PMS
LDH
Phenazine methosulfate (PMS) acts as electron carrier forreduction of tetrazolium salts to coloured product
Activity Staining for LDH Isoenzymes:
NBT[reduced]
In
Insoluble
Nitroblue Tetrazolium Test40
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 under optimum conditions
Shortcomings:
Artificial environment
Semi-quantitative data
43
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 maintained duringelectrophoresis
Staining should be done in dark as PMS is lightsensitive.
Standard precautions while handling serum
46
Thank you!