ISOENZYMES

• Iso - Same
• Isoezymes are enzymes that differ in their structure but
catalyses the same reaction.
• They are also called as Isoezymes or Isozymes.
• Differ in their structure, properties and function.
• Isoenzymes have been found in individual cells, these
isoforms of the enzyme are unequally distributed in the
various cells of an organism.
• From the clinical standpoint they have been found to be
selectively activated and inhibited, an observation which
has led to their use in therapy.
ISOENZYMES
 ISOENZYMES
• Isoenzymes are enzymes that differ in amino acid
sequence and amino acid sequence but catalyze the
same chemical reaction.
• Isoenzymes differ in their multimeric quaternary
structure
• These enzymes usually display different kinetic
parameters.
• Isoenzymes displays different regulatory properties.
• Generally exist as multiple forms of enzymes.
• Isoenzymes differ in their Electrophoretic mobility.
• Isoenzymes differ in their Molecular Weight.
• Isoenzymes displays different Physio chemical properties.
 ISOENZYMES
• Isoenzymes are syntesized from different locus of single
gene.
• Different forms of Isoenzymes will be exist in different
tissues.
 ISOENZYMES
• In naming isozymes (isoenzymes), the normal
enzyme name (either systematic or trivial)
• Name of the isozymes - indicated by the short form of
enzyme – as CAPITAL alphabet.
Ex: Lactate Dehydrogenase - LDH
• Naming should be used, followed by a number.
Isoforms of LDH – LDH1, LDH2, LDH3, LDH4, LDH5……….
• The numbers should be allotted consecutively.
• Preferably on the basis of electrophoretic mobility under
defined conditions.
• In Elecetrophoresis fast moving – Lower number, Slow
moving – Higher number.
• Sub classes can be indicated as small alphabets –
 ISOENZYMES
• Two main groups of procedures are available for
the separation of isoenzymes, namely electrophoresis
and ion-exchange chromatography.
• Both depend primarily upon the nature and extent of the
resultant charge on the protein fractions in the buffer
solution used.
• Other methods includes heat inactivation, Chemical
methods.
• Activity of the enzyme can be assayed by varying pH
temperature and ionic concentration.
 ELECTROPHORETIC SEPARATION -
ISOENZYMES
• Electrophoresis – Is a technique in which separation of
movement of charged particles through an electrolyte
when subjected to electrical field.
• Electrophoresis - Protein, Enzymes, DNA
 ISOENZYMES
• Isoenzymes – Clinically Important .
• Detection of Diseases – Cancer, Liver disease, heart
diseases, Thyroid diseases
• Organ Functioning – Liver, Heart, Bone, Brain, Muscle etc.
• Increased and decreased levels of Isoenzymes indicates
clinical complications.
• Isoenzymes are good biomarkers for the identification of
various diseases.
• Distributed in Blood and tissues.
• During tissue damage the levels of Iso enzymes will be
released in blood.
 ISOENZYMES
Clinically Important isoenzymes

1. Lactate Dehydrogenase - LDH

2. Creatine Kinase – CK
3. Alkaline Phosphotase – ALP
4. Acid Phosphotase - ACP
5. Glucokinase – GK
6. Cytochrome P450 – CTYP450
 - LDH
• Lactate dehydrogenase (LDH) is an enzyme required
during the process of turning sugar into energy for your
cells.
• LDH is present in many kinds of organs and tissues
throughout the body, including the liver, heart, pancreas,
kidneys, skeletal muscles, lymph tissue, and blood cells.
 LDH - STRUCTURE
• LDH – 4 Sub units (4 Protein units) – Tetrameric Structure
• Tetrameric Structure – 2 Protein chains – LDH-M, LDH-H

M Chain H Chain
• Predominantly • Predominantly
present in present in
Muscles. Heart.
• M subunit rich • M subunit rich
in Alanine. in Glutamine.
• No change in • No change in
the amino acids the amino acids
present in the present in the
active site. active site.
LDH - ISOFORMS
 LDH - ISOFORMS
• There are five different forms of LDH that are called
isoenzymes. They are distinguished by slight differences in
their structure.
• The isoenzymes of LDH are LDH-1, LDH-2, LDH-3, LDH-4,
and LDH-5.
• Different LDH isoenzymes are found in different body
tissues. The areas of highest concentration for each type of
isoenzyme are:
• H4 - LDH-1: Heart, Red blood cells, Brain
H3M - LDH-2: Heart, Red blood cells
H2M2 - LDH-3: Lymph tissue, Lungs, Platelets,
Pancreas
HM3 - LDH-4: Kidney, Placenta, Pancreas
M4 - LDH-5: Liver and Skeletal muscle
 LDH - ISOFORMS
• Lactate dehydrogenase is composed of four subunits
(tetramer).
• The two most common subunits are the LDH-M and LDH-
H protein, encoded by the LDHA and LDHB genes,
respectively.
• These two subunits can form five possible tetramers
(isoenzymes): 4H, 4M, and the three mixed tetramers
(3H1M, 2H2M, 1H3M).
• These five isoforms are enzymatically similar but show
different tissue distribution: The major isoenzymes of
skeletal muscle and liver, M4, has four muscle (M)
subunits, while H4 is the main isoenzymes for heart
muscle in most species, containing four heart (H)
subunits.
LDH - DISTRIBUTION
SEPARATION
 LDH – NORMAL RANGE
• Normal LDH levels – 140 (U/L) to 280 U/L
• Normal results for isoenzymes are listed below.
LDH-1: 17% to 27%
LDH-2: 27% to 37%
LDH-3: 18% to 25%
LDH-4: 3% to 8%
LDH-5: 0% to 5%
• Normal ratios are:
LDH-1 less than LDH-2
LDH-5 less than LDH-4
 IMPORTANCE

1. Less Commonly Used Cardiac Markers : Lactate

dehydrogenase isoenzymes (LDH) were used widely in
the past for diagnosis of myocardial infarction. (Heart
Injury).
LDH
LDH 1 > LDH 2
2. Hepatic (Liver) Disesaes : lactate dedehydrogenase assay
has been mostly used in evaluating certain hepatic
disorders.
LDH
 IMPORTANCE
3. Celluar Damage : When illness or injury damages in cells,
LDH may be released into the bloodstream, causing the
level of LDH in your blood to rise. High levels of LDH in
the blood point to acute or chronic cell damage. LDH –
4. Other Clinical Complications : LDH is present in so many
types of cells, high levels of LDH may indicate a number
of conditions. Elevated levels of LDH can include LDH –
• blood flow deficiency
• Cerebrovascular Disease (Stroke)
• Certain cancer
• Heart Attack
• Haemolytic anaemia
• Infectious mononucleosis
• Viral Hepatitis & Jaundice – LDH 5
 LDH – CLINICAL
IMPORTANCE
• Muscular dystropy (Muscle injury) – LDH 5
• Pancreatitis
• Tissue Death
• Higher alcohol Intake
• Renal Necrosis (Renal Disease) LDH
• Pulmonary Embolism (Destruction of
Platelets) LDH 3
• Megaloblastic Anaemia LDH 2 & LDH 3
 CREATINE KINASE - CK
• Creatine kinase (CK), also known as Creatine Phospho
Kinase (CPK) or Phospho Creatine Kinase.
• Creatine kinase (CK) is an enzyme found in the heart,
brain, skeletal muscle, and other tissues.
• CK catalyses the conversion of Creatine and uses
adenosine triphosphate and Mg2+ to create Phospho
Creatine and adenosine diphosphate
 CK- STRUCTURE
• CK – 2 Sub units (2 Protein units) – Dimeric Structure
• Dimeric Structure – 2 Protein chains – CK-M, CK-B

M Chain B Chain
• Predominantly • Predominantly
present in present in
Muscles. Brain.
• No change in • No change in
the amino acids the amino acids
present in the present in the
active site. active site.
CK - ISOFORMS
 CK - ISOFORMS
• There are Three different forms of CK that are called
isoenzymes. They are distinguished by slight differences
in their structure.
• The isoenzymes of CK are CK-1, CK-2, and CK-3.
• Different CK isoenzymes are found in different body
tissues. Isoenzyme patterns differ in tissues. The areas of
highest concentration for each type of isoenzyme are:
• CK-MM (98%) - Skeletal muscle
CK-MB (1%) - Skeletal muscle
CK-MM (70%) - Myocardium (Heart muscle)
CK-MB (25–30%) - Myocardium
CK-BB - Brain, Smooth muscle - vascular and
uterine tissue, Placenta, Thyroid, Lung
 CK - ISOFORMS
• Creatine Kinase isoenzyme is composed of four subunits
(Dimer).
• The two most common subunits are the CK-M and CK-B
protein, encoded by the CKM and CKB genes,
respectively.
• These two subunits can form three dimers (isoenzymes):
2M, 2B, and one mixed dimers (MB).
• These three isoforms are enzymatically similar but show
difference in their structure. The major isoenzymes of
skeletal muscle M2, while B2 is the main isoenzymes for
Brain tissue.
CK - DISTRIBUTION
SEPARATION
 CK – NORMAL RANGE
• Total CK levels – 15 (U/L) to 105 U/L – Men
10 (U/L) to 80 U/L – Women
• CK- MB - 9 (U/L)
CK – CLINICAL IMPORTANCE
1. Muscle Damage: The muscle cells in our body need CK to
function. Levels of CK can rise after a heart attack,
skeletal muscle injury, strenuous exercise, or
drinking too much alcohol, and from taking certain
medicines or supplements. If this test shows that your CK
levels are high, you may have muscle or heart damage.
CK
2. Serum CK concentration, mainly the CK-MM subtype, is
the most sensitive indicator of damage to muscles.
Serum CK begins to rise approximately 2 to 12
hours after the onset of muscle injury, peaks within 24 to
72 hours, and then declines gradually in 7–10 days.
 IMPORTANCE
3. Other Clinical Complications : CK is present in so many
types of cells, at high levels may indicate a number of
conditions. Elevated levels of CK can include
• Recent crush and compression muscle injuries,
trauma, burns, and electrocution.
• Inherited myopathies, such as muscular dystrophy.
• Hormonal (endocrine) disorders, such as thyroid
disorders, Addison disease or Cushing disease.
• Strenuous exercise.
• Prolonged surgeries.
• Seizures.
• Confusion or loss of consciousness, even for a brief
moment.
CK – CLINICAL IMPORTANCE

• Garbled or slurred speech.

• Loss of vision or vision changes.
• Muscle aches and pains.
• Muscle stiffness.
• Paralysis.
• Sudden weakness or numbness on one side of
the body.
• Weakness (loss of strength)
OVERVIEW