Electrophoresis of LDH Isoenzymes and Activity Staining

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!