Learn about the different types of isoenzymes of LDH & CK.

2. Isoenzymes
 Multiple forms of an enzyme which differ in
physical and chemical properties and catalyze
the same reaction as an enzyme.
 Isoenzymes are produced by a single gene and
some may result from more than one gene.
 Isoenzymes can be seprated by:
1. Heat inactivation
2. Chemical inhibition
3. Electrophoretic techniques(specific)

3. Electrophoresis
• Is technique by which separation of
movement of charged particles through an
electrolyte when subjected to electrical
field.

4. Advantages of isoenzyme
measurement
1. Isoenzyme variants are derived from
different tissue sources.
2. So separation renders increased
specificity to enzyme analysis.
3. Tissue or organ effected can be detected
(where isoenzyme elevation occurs)

5. Types
1. CPK (creatine phospho kinase)
2. LDH (Lactate Dehydogenase)
3. Troponin
4. ALP (Alkaline phosphatase)
5. Aldolase
6. Amylase

6. Introduction (CK)
 Enzyme catalyzing creatine and ATP to
phosphocreatine and ADP
 Action - this enzyme is associated with the
regeneration and storage of high energy phosphate
(ATP)
 It catalyzes the following reversible reaction in the
body.
creatine
 Phospho creatine
ADP ATP
 creatine phophokinase

7.  High quantities are found in
 Skeletal muscle
 Heart muscle
 Brain tissue.
 Smaller quantities are found in
 Kidney
 The bladder
 prostate
 Gestrointestinal tract
 Liver
 Pancreas
 Spleen
 Uretus
 Thyroid
 Lung
 placenta

8. CPK-Creatinine phospho kinase
 CPK test are performed when the total CPK
level is elevated.
 Isoenzyme testing can help differentiate the
source of the damaged tissue.
 CPK is an enzyme found predominantly in the
heart, brain, and skeletal muscle.
 CPK is composed of 3 isoenzymes that differ
slightly in structure:
 CPK is a dimer made up of 2 subunits called
“B” for brain and “M” for muscle.

9. CREATININE KINASE (CK)
 Creatine kinase is a dimer made of 2
monomers occurs in the tissue.
 Skeletal muscle contains M subunit,
brain contains B subunits.
 Three different isoenzymes are formed.

10. Creatine kinase
 Primary tissue sources:
1. Brain, smooth muscle, prostate, thyroid, gut,
lung= CK- BB
2. Cardiac muscle –MB (20-30%) & MM(70-80%)
3. Skeletal muscle – MB (1-2%) & MM(98-99%)
 Relatively small molecular size =allow leakage out
of ischemia muscle or brain cells

11.  Reference range in serum affected by:
1. Amount of lean muscle mass
 Thin, sedentary= 30- 50 U/L
 Muscular, exercising regularly = 500- 1000 U/L
2. Age – in neonated . CK- MB 5- 10% of total CK
3. Gender
4. Race – african 30% higher than europeans
5. muscle activity - direct relationship between
intensity of exercise and CK level.

13. CPK - Isoenzymes
 CPK- l (also called CPK-BB) is concentrated in the
brain and lungs
 CPK- 2 (also called CPK-MB) is found in the heart
and rises when heart muscle is damaged.
 CPK-3 (also called CPK-MM) is found in mostly in
skeletal muscle
 Because the CPK-l isoenzyme is predominately
found in the brain and lung, injury to either of
these organs ( for example, stroke or lung injury
due to a pulmonary embolism) are associated with
elevated level of this isoenzyme.

14.  CPK2 –MB generally rises in response to a heart
attack, inflammation of the heart muscle,
muscular dystrophy, and other problems related to
the heart.
 CPK 3–MM generally rises in response to muscle
damage in your heart, brain, or skeleton after a
crush injury, seizures, muscular dystrophy, muscle
inflammation, or another skeletal muscle disorder.

15. This test may be used to
 Diagnose heart attack
 Evaluate causes of chest pain
 Determine if or how badly a muscle is damaged
 Tell the difference between malignant hypethermia
and postoperative infection.
 Acute renal failure.

16. Lactate dehydrogenase(LDH)
 Lactate dehydrogenase (LDH) is an enzyme present in a wide
variety of organisms
 EC 1 = oxidoreductase.
 EC 1.1 = acting on the CH-OH group of the donor.
 EC 1.1.1 = With NAD or NADP as acceptor.
 EC 1.1.1.27 = L-lactate dehydrogenase.
 Molecular weight- 32 kD & it is tetramer
 M (A) -muscle –chromosome 11(basic)
 H (B) -heart – chromosome 12(acidic)

17.  Lactate dehydrogenase, reversibly converts lactate to
pyruvate, in different tissues.
 LDH consists of 5 iso-enzymes – LDH1,LDH2,LDH3,LDH4 &
LDH5
 These isoenzymes are separated by cellulose acetate
electrophoresis at pH 8.6
 Normal values:
 Serum -100 -200 U/L
 CSF - 7 -30 U/L
 Urine - 40 -100 U/L

18. LDH reaction

19. LDH isoforms

21. Identification……
 The different form can be seperated by electrophoresis.
 The difference in electrophoretic mobilities due to
different electric charges on the isoenzymes due to
difference in content of acidic and basic amino acids.
 The H gene is more strongly negatively charged than
M gene due to higher number of acidic residues
 .
 Exp. LDH-1 has the highest negative charge and hence,
moves fastest during electrophoresis.it contains a higher
proportions of A sp an glutamate than other forms.
 LDH-5 is the lowest moving fraction.

22.  Rate of chemical reaction catalyzed, the different
isoenzymes may catalyzes the same reaction at
different rates.
 Example:
 Rate of oxidation of hydroxy butyrate is greater by
LDH-1 and LDH-2, when compared with rate of
oxidation of LDH-4 and LDH-5.
 This isoenzymes may have different physical
properties also.
 Exp:LDH-4 and LDH-5 are easily destroyed by
heat, whereas LDH-1 and LDH-2 are not, if heated
up to about 60’c(“heat-resistant”)

23.  Myocardial LDH (LDH-1) is found to be move heat
stable than of hepatic LDH.
 Hepatic LDH (LDH-5) is inhibited by urea.
 The isoenzymes have different pH optima and Km
values.
 A pure tetramer if M subunit i.e. M4 has lower Km for
pyruvate and is concentrated in skeleton muscles
which are anaerobic.
 Therefore M4 promotes glycolysis by catalyzing the
production oF lactate from pyruvate quickly and
efficiently.
 On the other hand tetramer of H subunit i.e. H4, has
greater Km for pyruvate and is more concentated in
heart muscles which is aerobic

24. Clinical significance of LDH
 In normal serum, LDH2 (H3M) predominant isoenzyme &
LDH5 is rarely seen.
 In myocardial infarction, LDH1(H4) levels are greater than
LDH2.
 Megaloblastic anemia (50 times upper limit of LDH 1 and LDH
2)
 Muscular dystrophy, LDH5 (M4) is increased.
 Toxic hepatitis with jaundice (10 times more LDH5)

25. Renal disease- tubular necrosis or pyelonephritis, pulmonary
embolism LDH 3 (massive destruction of platelets)
Total LDH is increased in neoplastic diseases.
LDH5 is increased in breast cancer, malignancies of CNS,
prostatic carcinoma.
In leukaemias, LDH2 & LDH3 levels are increased.
In malignant tumors of testis & ovaries, LDH2, LDH3 & LDH 5
levels are increased.