Isoenzymes are multiple forms of the same enzyme that catalyze the same chemical reaction but have different physical or chemical properties. Lactate dehydrogenase (LDH) and creatine phosphokinase (CK) are examples of enzymes with multiple isoenzyme forms that exhibit tissue-specific expression and properties. Elevated levels of specific isoenzymes of LDH and CK can indicate tissue damage or diseases of the heart, liver, kidney, and other organs.

1. Enzymes-3
Isoenzymes
Clinical enzymology
RITTU CHANDEL
05-02-13

2. ISOENZYMES
 Isoenzymes or isozymes are mutiple
forms of same enzyme that catalyse
the same chemical reaction
 Different chemical and physical
properties:
 Electrophoretic mobility
 Kinetic properties
 Amino acid sequence
 Amino acid composition
2

3. S. Property E.g.
No
1 Electrophoretic Isoenzymes of Lactate dehydrogenase have
mobility different electrophoretic mobility
2 Heat stability Alkaline phosphatase isoenzymes are either
heat labile or stable
3 Inhibitor An inhibitor can inhibit only one isoenzyme
of an enzyme eg. Acid phosphatase
4 Km Glucokinase and hexokinase
5 Cofactors Mitochondrial isocitrate dehydrogenase
requires NAD+ , cytosolic form requires
NADP+
6 Tissue localisation LDH 1 is present in heart, LDH 5 in muscle
7 Antibodies For creatine kinase, each isoenzyme can be
bound only by a specific antibody 3

4. Lactate dehydrogenase (LDH)
 E.C – 1.1.1.27
 L-lactate :NAD+ oxidoreductase:LDH
 Molecular weight- 134 kDa
 tetramer
 M (A) -muscle –chromosome
11(basic)
 H (B) -heart – chromosome 12(acidic)
4

5. Lactate dehydrogenase (LDH)
Normal values
Serum -100 -200 U/L
CSF - 7 -30 U/L
Urine - 40 -100 U/L
5

6. Isoenzyme Composition Electrophore Present in Elevated in
name tic migration
LDH 1 ( H 4) Fastest Myocardium, myocardial
Heat moving RBC,kidney infarction
resistant
LDH2 (H3M1) Myocardium, Kidney
Heat RBC,kidney disease,megalo
resistant blastic anemia
LDH3 (H2M2) brain Leukemia,malig
nancy
LDH4 (H1M3) Lung,spleen Pulmonary
Heat labile infarction
LDH5 (M4) Slowest Skeletal Skeletal muscle
Heat labile moving muscle, Liver and liver
Inhibited by diseases
urea 6

7. Lactate dehydrogenase (LDH)
This is an example in which two duplicated genes have
become specialized to different tissues.
The isozymes are also differentially expressed in
different developmental stages. Before birth the heart
is more anaerobic compared with adulthood. Indeed,
before birth the main isozyme in the heart is the M4,
and with time it switches to HM3 (at birth), to H2M2
and HM3 at 1 year after birth, and to H3M AND H4
after 2 years.
My main LDH is HM3. Great!
My main LDH is HM3
7

8. Atypical forms of LDH
 sixth isoenzyme LDH- X
 Seventh isoenzyme – LDH -6
8

9. Clinical significance of LDH
 Myocardial infarction (LDH 1>LDH2)
 Megaloblastic anemia (50 times upper
limit of LDH 1 and LDH 2)
 Muscular dystrophy (LDH 5)
 Toxic hepatitis with jaundice (10 times
more LDH 5)
 Renal disease- tubular necrosis or
pyelonepheritis
 Pulmonary embolism LDH 3 (massive
destruction of platelets)
9

10.  Leukemia (LDH 2 and LDH 3)
 Malignancy (LDH 3)
 Hodgkins disease
 germ cell tumors
 Urinary LDH-3 to 6 times normal:
chronic glomerulonephritis
Systemic lupus erythematosus
Diabetic nephrosclerosis
Bladder and kidney malignancies
10

11.  In CSF:
 Bacterial meningitis – LDH 4 and LDH
5
 Viral meningitis - LDH 1
 Metastatic tumors - LDH 5
 Neonatal cases of intracranial
haemorrhage associated with seizures
and hydrocephalus
11

12. LDH in starch gel
The H(B) monomer
is very negatively charged
12

13. CREATINE
PHOSPHOKINASE
 Adenosine triphosphate:creatine N-
phosphotransferase
 E.C-2.7.3.2
 Dimeric enzyme (82 kDa)
 4 -60 IU/L
13

14. 14

15.  Enzyme unstable in serum
 Activity lost due to sulfhydryl group
oxidation at active site
 Dimer (each of 41000 Da)
 B (brain) – chromosome 14
 M (muscle) –chromosome 19
15

16. Isoenzy
Compo
me Present in Elevated in
sition
name
CK-1 Brain,prostate,GI
Fast BB tract,lung,bladder,uteru CNS diseases
moving s,placenta
CK-2
Acute myocardial
2% of MB Myocardium/ Heart
infarction
total
CK-3
Skeletal muscle,
Slow MM
Myocardium
moving
All 3 in cytosol 16

17. 17

18. atypical forms of CK
 Fourth form - CK-Mt (chromosome 15)
severe illness
Malignant tumors
 macroCK
 type 1- CK BB complexed with
IgG
 type 2-oligomeric CK-Mt
18

19. Clinical significance of CK
 CK 1 elevated:
very low birth weight newborns
brain damage in neonates
neurological injury –CK 1 rise in
CSF
>200 U/L –die
100 – 200 U/L – survive with
neurological defecits
<100 U/L – good chance of
recovery
19

20. Elevated CK 1
 Adenocarcinomas of GI tract
 Carcinoma lung
 Ca
prostate,bladder,testes,kidneys,breast,
ovaries,uterus,CNS,leukemia,lympho
ma and sarcoma
20

21. Elevated CK 2:
myocardial infarction
head injuries
subarachnoid haemorrhage
exercise
Elevated CK 3:
muscular dystrophies(DMD-
10000 IU/L)
myopathies
hypothyroidism (5 fold more than
normal value,also CK 2 is elevated)
21

22. Alkaline phosphatase (ALP)
 E.C -3.1.3.1.
 Orthophosphoric monoester
phosphohydrolase
 In mucosa of small intestine, proximal
convoluted tubule, bone, liver,
placenta
 Catalyses alkaline hydrolysis of
naturally occuring and synthetic
substrates
22

23. Isoenzymes of ALP
 Alpha 1 ALP-epithelial cells of biliary canaliculi
 Alpha 2 heat labile ALP- hepatic cells
 Alpha 2 heat stable ALP-not destroyed at 65˚C
 inhibited by phenylalanine
 placental
 Pre beta ALP – bone,heat labile
 Gamma ALP – intestinal cells
 inhibited by phenylalanine
 Leukocyte alkaline phosphatase –decreased in CML
 increase in lymphoma
ATYPICAL ISOENZYMES
 Regan isoenzyme-heat stable,inhibited by L-phenylalanine
 Nagao isoenzyme- variant of regan
 inhibited by L-leucine
23

24. Clinical significance
 Hepatobiliary disease
 Hepatic carcinoma
 Hepatic metastases
 Pagets disease (10 – 25 times)
 Bone cancer
 Healing of bone fracture
 Osteomalacia and rickets
 Hyperparathyroidism
 Ca of ovary,uterus-regan isoenzyme
 Metastatic Ca of pleural surfaces –Nagao
isoenzyme
24

25. Acid phospatases
acid phosphatses
E.C -3.1.3.2.
Hydrolyse phosphoric acid ester at pH 5 -6
In lysosomes
Extalysosomal-
prostate,bone,spleen,platelet,
liver,kidney (pH - 5)
RBC (pH – 6)
0 – 0.6 U/L
Extremly heat labile
25

26.  Isoenzyme of ACP inhibitor
prostatic dextrorotatory
tartarate ions
Erythrocytic formaldehyde
cupric ions
Majorly the serum contains tartarate
resistant ACP (originating in
osteoclasts)
26

27. Clinical significance
 To detect, monitor Ca prostate
 Tartarate resistant ACP increase in
pagets disease and bone cancer
 Marker of bone disease-increases in:
giant cell tumor of bone
normal growing children
Gauchers disease
In high concentrations in semen
27

28. SERUM AMYLASE(calcium
metalloenzyme)
 E.C -3.2.1.1.
 Molecular weight -54 -62 kDa
 From salivary gland and pancreas
 Enzymes are products of 2 closely
linked loci on chromosome 1
 macroamylases
28

29. 29

30. Serum aldolase
 Tetramer
 Catalyses interconversion of fructose-
1,6-bi-phosphate and triose phosphate
 5 isoenzymes
 Subunits
A
B
1- 7.5 U/L
Skeletal muscle,liver,brain,heart
30

31. Clinical significance of serum
aldolase
 Elevated in:
 Progressive muscular dystrophy
particularly high in DMD
 Viral hepatitis
 Advanced cancer of prostate
31

32. SOURCES
PLASMA CELL
DERIVED/PLASM DERIVED/PLASM
A SPECIFIC A NON SPECIFIC
BLOOD COAGULATION
ENZYMES
FERROXIDASE
LIPOPROTEIN LIPASE
PSEUDOCHOLINESTERA
SE SECRETOR
METABOLIC
Y
32

33. Mechanisms responsible for
abnormal levels
Increased serum decreased serum
level level
Increased Impaired Decreased Enzyme
release excretion formation inhibition
Cell Increased
necrosi permeabilit genetic acquired
s y
33

34. 34

35. Enzymes in blood
Cell
death
Defects in cellular
membrane
Release of cytoplasmic
enzymes initially
In infarctions
35

36. Elevation of enzymes in blood
No.of cell Gradient of
injured cell/plasma
Rate of Rate of
enzyme clearance
entry in from
plasma plasma
36

37. Serum enzyme assay in clinical
practice
 In diagnosis
 In differential diagnosis
 In prognosis
 Early detection of disease
37

38. Serum transaminases
38

39. Serum transaminases
 Catalyse interconversion of
aminoacids to ketoacids by transfer of
amino group
 AST-aspartate
aminotransferases(SGOT)
10-30 U/L
 ALT-alanine aminotransferases
(SGPT)
10-40 U/L
Both present in plasma,bile,CSF,saliva 39

40. Gamma glutamyl
transpeptidases (GGT)
 E.C -2.3.2.2.
 Γ – glutamyl – peptide:amino acid Γ –
glutamyl transferases
 In proximal convoluted
tubule,liver,pancreas,intestine
 Clinical significance
 hepatobiliary disease
 neoplasms
 heavy drinkers
40

41. cholinesterase
 Hydrolyse acetylcholine
 Types-
 acetylcholinestarase -3.1.1.7
 pseudocholinesterase – 3.1.1.8
Clinical significance
insecticide poisoning
atypical form of enzymes who are at
risk to muscle relaxants
sensitive indicator of synthetic capacity
of liver
41

42. Glucose – 6 –phosphate
dehydrogenase
 Dimer with identical subunits
 In HMP - for production of NADPH
 G-6-P + NADP+ 6-PG + NADPH + H+
 hemolytic anemia
 prolonged neonatal jaundice
 conditions are directly related to the inability
of specific cell types to regenerate reduced
nicotinamide adenine dinucleotide phosphate
(NADPH)
42

43. Serum lipase
 E.C – 3.1.1.3.
 Molecular weight -48 kDa
 Hydrolyses glycerol esters of long
chain fatty acids
 Pancreas, intestinal and gastric
mucosa
 0.2 – 1.0 U/L
43

44. Serum enzymes in cardiac
diseases
Why enzyme diagnosis?
Enzyme assays
Creatine phosphokinase (CPK)
Aspartate transaminases (SGOT OR
AST)
Lactate dehydrogenase (LDH)
γ-Glutamyl transpeptidase (GGTP)
Histaminase
Pseudocholinesterase
44

45. Cardiac biomarkers in
myocardial infarction
45

46. Onset peak
duration
3-6 hrs 18-24 hrs 36-72
CK-MB hrs
4.5-20% of total
Troponins 4-10hrs
days
18-24 8-14
LDH 6-12hrs 24-48 hrs 6-8 days
Flipped pattern
24-36 hrs 4-5days 10-12
AST d
Myoglobin 1-4hrs 6-7hrs 24hrs
46

47. Serum enzymes in GI tract
diseases
Serum amylase
A cute pancreatitis-4 -6 fold increase in 2 -12 hrs ,
maximum level 12 -72 hrs , normal in 3 - 4 day
Urinary amylase - increased on 1st day and remains
elevated till 8- 10 day
Ca pancreas- amylase in ascitic and pleural fluid
Cholecystitis – 4 fold elevation
47

48. Serum lipase
Acute pancreatitis:
2 -50 times in 4 -8
hrs,peaks at 24
hrs,decreases in 8 -14 days
48

49. Serum enzymes in liver
disease
Severe toxic hepatitis
Extrahepatic cholestasis
Cirrhosis (AST>ALT)
Serum
transaminases Hepatic carcinoma (5 -10 fold rise)
Hepatobiliary disease
Extrahepatic obstruction (10- 15 times)
Serum alkaline
phosphatases
49

50. • Toxic hepatitis with jaundice (10 times more
Serum LDH
LDH 5)
Extrahepatic and intrahepatic causes (2 -6 fold incre
5’nucleotidase Early infectious hepatitis
Alcoholic cirrhosis and alcoholics
Gamma glutamyl
Hepatic carcinoma
transferase
50

51. Serum enzymes in muscle
diseases
Serum aldolase
Progressive muscular dystrophy
muscular dystrophies
CPK
myopathies
hypothyroidism (5 fold more than
normal value,also CK 2 is
elevated)
SGOT/SGPT Muscular dystrophy and dermatomyositis
51

52. Serum enzymes in bone
diseases
• Pagets disease (10 – 25 times)
• Bone cancer
Alkaline • Healing of bone fracture
phosphatase • Osteomalacia and rickets
• Marker of bone disease-increases in:
Acid • giant cell tumor of bone
phosphatase • normal growing children
52

53. As tumor markers
 Aldolase-liver
 ALP – bone,liver,leukemia,sarcoma
 Placental ALP – ovarian,lung,hodgkins
 Amylase – pancreatic
 CPK BB –
prostate,lung,breast,colon,ovarian
 GGT – liver
 LDH – liver,lymphoma,leukemia
 Neuron specific enolase – tumors of
neuroendocrine origin 53

54. Prostate specific antigen (PSA or
semenogelase)
 From secretory epithelium of prostate
gland
 32 kDa glycoprotein
 Mild Serine protease activity
 1- 5 μg/L
Levels between 4 -10 μg/L –increased risk
of prostate cancer
>10 μg/L - suggestive of Ca prostate
>20 μg/L - Ca prostate with metastases
54

55. Enzymes-therapeutic agent
1.Streptokinase
plasminogen
streptokinase
plasmin
fibrin soluble product
2.urokinase
55

56. 3. Bacterial asparginase
in leukemia
4.α- chymotrypsin-extraction of lens
5.Chymotrypsin,papain –anti
inflammatory
6.Collagenase – debridment of dermal
ulcers
7.Fibrinolysin – venous thrombosis,
pulmonary embolism
8.Hyaluronidase – rapid absorption of
drugs injected subcutaneously
56

57. 9.Lysosome –antibacterial (eye
infections)
10.Trypsin – clean wounds
treatment of acute
thrombophlebitis
57

58.  Analytical use of enzymes
 as reagents
 as labels
58

59. Alcohol
Ethanol
dehydrogenase
Lactate Lactate
dehydrogenase
Glucose Glucose
oxidase and
peroxidase
Uricase Uric acid
Urease
Urea
Cholesterol
oxidase and
peroxidase cholesterol
59

60. As labels
 In immunoassays
 for determining concentration of
drugs, hormones
 Glucose -6- phosphate
dehydrogenase
 Alkaline phosphatase
 Beta galactosidase
 peroxidase
60

61. BIBLOGRAPHY
 T.B. OF BIOCHEMISTRY:
 SATYANARAYAN
 VASUDEVAN
 PANKAJA NAIK
 RANA SHINDE
 TEITZ
 HARRISON-INTERNAL MEDICINE
THANK YOU
61