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
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
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
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
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
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
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
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
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
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