KONGUNADU ARTS AND SCIENCE COLLEGE (AUTONOMOUS) …PG)-3.pdfSerum enzymes in GI tract diseases Serum...
Transcript of KONGUNADU ARTS AND SCIENCE COLLEGE (AUTONOMOUS) …PG)-3.pdfSerum enzymes in GI tract diseases Serum...
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KONGUNADU ARTS AND SCIENCE COLLEGE (AUTONOMOUS)
Coimbatore – 641 029 DEPARTMENT OF BIOCHEMISTRY (PG)
C.P.3 Enzymes and Enzyme Technology
Dr.Nithya devi
Associate professor
Department of Biochemistry (PG)
Isoenzymes
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ISOENZYMES
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Isoenzymes or isozymes are mutipleforms of same enzyme that catalysethe same chemical reaction
Different chemical and physical properties:
Electrophoretic mobility
Kinetic properties
Amino acid sequence
Amino acid composition
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S.
No
Property E.g.
1 Electrophoretic
mobility
Isoenzymes of Lactate dehydrogenase have
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
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Lactate dehydrogenase (LDH)
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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)
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Lactate dehydrogenase (LDH)
Normal values
Serum -100 -200 U/L
CSF - 7 -30 U/L
Urine - 40 -100 U/L5
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Isoenzyme
name
Composition Electrophore
tic migration
Present in Elevated in
LDH 1
Heat
resistant
( H4) Fastest
moving
Myocardium,
RBC,kidney
myocardial
infarction
LDH2
Heat
resistant
(H3M1) Myocardium,
RBC,kidney
Kidney
disease,megalo
blastic anemia
LDH3 (H2M2) brain Leukemia,malig
nancy
LDH4
Heat labile
(H1M3) Lung,spleen Pulmonary
infarction
LDH5
Heat labile
Inhibited by
urea
(M4) Slowest
moving
Skeletal
muscle, Liver
Skeletal muscle
and liver
diseases
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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
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Atypical forms of LDH
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sixth isoenzyme LDH- X
Seventh isoenzyme – LDH -6
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Clinical significance of LDH
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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)
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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
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In CSF:
Bacterial meningitis – LDH 4 and LDH 5
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Viral meningitis
Metastatic tumors
- LDH 1
- LDH 5
Neonatal cases of intracranial
haemorrhage associated with seizures
and hydrocephalus
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LDH in starch gelThe H(B) monomer
is very negatively charged
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CREATINE PHOSPHOKINASE
Adenosine triphosphate:creatine N-
phosphotransferase
E.C-2.7.3.2
Dimeric enzyme (82 kDa)
4 -60 IU/L
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14
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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
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Isoenzy
me
name
Compo
sitionPresent in Elevated in
CK-1
Fast
moving
BB
Brain,prostate,GI
tract,lung,bladder,uteru
s,placenta
CNS diseases
CK-2
2% of
total
MB Myocardium/ HeartAcute myocardial
infarction
CK-3
Slow
moving
MMSkeletal muscle,
Myocardium
All 3 in cytosol 16
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atypical forms of CK
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Fourth form - CK-Mt (chromosome 15)
severe illness Malignant tumors
macroCK
type 1- CK BB complexed with
IgG
type 2-oligomeric CK-Mt
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Clinical significance of CK
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CK 1 elevated:
very low birth weight newborns brain
damage in neonates neurological
injury –CK 1 rise inCSF
>200 U/L –die100 – 200 U/L – survive with
neurological defecits
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Elevated CK 1
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Adenocarcinomas of GI tract
Carcinoma lung
Ca
prostate,bladder,testes,kidneys,breast,
ovaries,uterus,CNS,leukemia,lympho
ma and sarcoma
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Elevated CK 2:
myocardial infarction head injuriessubarachnoid haemorrhage exerciseElevated CK 3:muscular dystrophies(DMD- 10000 IU/L)myopathies
hypothyroidism (5 fold more than normal value,also CK 2 is elevated)
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Alkaline phosphatase (ALP)
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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
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Isoenzymes of ALP
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Alpha 1 ALP-epithelial cells of biliary canaliculi
Alpha 2 heat labile ALP- hepatic cells
Alpha 2 heat stable ALP-not destroyed at65˚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
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Clinical significance
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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
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Acid phospatases
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acid phosphatses E.C -3.1.3.2.Hydrolyse phosphoric acid ester at pH 5 -6 In lysosomesExtalysosomal-prostate,bone,spleen,platelet, liver,kidney (pH - 5)RBC (pH – 6)
0 – 0.6 U/LExtremly heat labile
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Isoenzyme of ACP
prostatic
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Erythrocytic
inhibitor
dextrorotatory
tartarate ions
formaldehyde
cupric ions
Majorly the serum contains tartarate
resistant ACP (originating in
osteoclasts)
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Clinical significance
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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
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SERUM AMYLASE(calcium
metalloenzyme)
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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
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Serum aldolase
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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
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Clinical significance of serum
aldolase
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Elevated in:
Progressive muscular dystrophy
particularly high in DMD
Viral hepatitis
Advanced cancer of prostate
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SOURCES
PLASMA
DERIVED/PLASM
A SPECIFIC
CELL
DERIVED/PLASM
A NON SPECIFIC
SECRETOR
YMETABOLIC
BLOOD COAGULATION
ENZYMES
FERROXIDASE
LIPOPROTEIN LIPASE
PSEUDOCHOLINESTERA
SE
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Mechanisms responsible for
abnormal levels
Increased serum
level
decreased serum
level
Increased
release
Impaired
excretion
Decreased
formation
Enzyme
inhibition
genetic acquiredCell
necrosi
s
Increased
permeabilit
y33
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Enzymes in blood
Cell
death
Defects in cellular membrane
Release of cytoplasmic
enzymes initially
In infarctions
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Elevation of enzymes in blood
No.of cellinjured
Gradient ofcell/plasma
Rate ofenzymeentry inplasma
Rate of clearance
from plasma
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Serum enzyme assay in clinical
practice
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In diagnosis
In differential diagnosis
In prognosis
Early detection of disease
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Serum transaminases
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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
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Gamma glutamyl
transpeptidases (GGT)
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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
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cholinesterase
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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
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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)
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Serum lipase
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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
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Serum enzymes in cardiac
diseasesWhy enzyme diagnosis?
Enzyme assays
Creatine phosphokinase (CPK)
Aspartate transaminases (SGOT OR AST)
Lactate dehydrogenase (LDH)
γ-Glutamyl transpeptidase (GGTP)
Histaminase
Pseudocholinesterase
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Cardiac biomarkers in
myocardial infarction
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Onset peak
durationCK-MB
Troponins
AST
Myoglobin
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3-6 hrs 18-24 hrs 36-72
hrs
4-10hrs days 18-24 8-14
6-12hrs 24-48 hrs 6-8 days
24-36 hrs d 4-5days 10-12
1-4hrs 6-7hrs 24hrs
4.5-20% of total
LDHFlipped pattern
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Serum enzymes in GI tract
diseasesSerum 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
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Serum lipase
Acute pancreatitis:
2 -50 times in 4 -8hrs,peaks at 24hrs,decreases in 8 -14 days
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Serum enzymes in liver
disease
Serum transaminases
Severe toxic hepatitis
Extrahepatic cholestasis
Cirrhosis (AST>ALT)
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Hepatic carcinoma (5 -10 fold rise)
Hepatobiliary disease
Extrahepatic obstruction (10- 15 times)
Serum alkaline phosphatases
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Serum LDH
• Toxic hepatitis with jaundice (10 times more LDH 5)
5’nucleotidase
Gamma glutamyl transferase
Extrahepatic and intrahepatic causes (2 -6 fold incr
Early infectious hepatitis
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Alcoholic cirrhosis and alcoholics
Hepatic carcinoma
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Serum enzymes in muscle
diseases
CPK
SGOT/SGPT
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Serum aldolaseProgressive muscular dystrophy
muscular dystrophies
myopathies
hypothyroidism (5 fold more than
normal value,also CK 2 is elevated)
Muscular dystrophy and dermatomyositis
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Serum enzymes in bone
diseases
Alkaline phosphatase
• Pagets disease (10 – 25 times)• Bone cancer• Healing of bone fracture• Osteomalacia and rickets
Acidphosphatase
• Marker of bone disease-increases in:• giant cell tumor of bone• normal growing children
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As tumor markers
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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
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Prostate specific antigen (PSA
or semenogelase)
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From secretory epithelium of prostate gland
32 kDa glycoprotein
Mild Serine protease activity
1- 5 μg/LLevels between 4 -10 μg/L –increased risk
of prostate cancer
>10 μg/L - suggestive of Ca prostate>20 μg/L - Ca prostate with metastases
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Enzymes-therapeutic agent
1.Streptokinase
plasminogen
streptokinase plasmin
fibrinsoluble product 2.urokinase
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3. Bacterial asparginase in leukemia4. α- chymotrypsin-extraction of lens
5.Chymotrypsin,papain –antiinflammatory
6. Collagenase – debridment of dermal ulcers
7. Fibrinolysin – venous thrombosis, pulmonary embolism
8. Hyaluronidase – rapid absorption of drugs injected subcutaneously
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9.Lysosome –antibacterial (eye infections)
10.Trypsin – clean wounds treatment of acute
thrombophlebitis
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Analytical use of enzymes
as reagents
as labels
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Alcohol dehydrogenase
Lactate dehydrogenase
Glucose oxidase and peroxidase
Uricase
Urease
Cholesteroloxidase andperoxidase
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Ethanol
Lactate
Glucose
Uric acid
Urea
cholesterol
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As labels
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In immunoassays
for determining concentration of drugs,
hormones
Glucose -6- phosphate dehydrogenase
Alkaline phosphatase
Beta galactosidase
peroxidase