A comprehensive presentation on Enzymology Clinical significance of Enzymes & Isoenzymes for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
2. Clinical significance of enzymes
Enzyme units
• International units ( one micromole of substrate conversion / per
minute/l of serum sample ( IU /l )
• Standard international : /SYSTEM INTERNATIONAL /KATAL (catalytic
activity ) number of moles of substrate transformed /second /l of
sample KAT Or K( IU= 60 MICROKATAL )
3. Techniques for estimation of enzymes
• Colorimetry /spectrophotometry
• Fluorometry
• RIA
• ELISA
• Chemiluminescence
4. Factors affecting enzyme estimations
1. Age
2. Sex
3. Pregnancy
4. Time of sampling
5. Temperature
6. p H
7. Substrate concentration
8. Product concentration
9. Presence of drugs in plasma
Therefore strict control on estimation of enzyme is needed
5. Enzyme appear in plasma by 3 ways
1. Functional plasma enzymes
2. Non Functional plasma enzymes
3. Obstruction to secretory pathway
6. Functional plasma enzymes Non Functional plasma enzymes
High concentration in plasma in physiological
conditions
low concentration in plasma in physiological
conditions
Low concentration in tissue in physiological
conditions
High concentration in tissue in physiological
conditions
low concentration in plasma in pathological
conditions
decreased synthesis by damaged liver cells
High concentration in plasma in pathological
conditions( tissue damage )
eg Psuedocholine esterase, ipase SGPT , SGOT, LDH, CPK
7. Obstruction to Secretory Pathway
Physiological conditions balance between synthesis & release
Pathological conditions loss of balance between synthesis & release
8. CONDITIONS RELATED TO INCREASED SERUM ENZYME LEVELS
Significant elevation in serum levels of enzymes is observed under
following conditions :
1. Cellular damaged
2.Increase rate of cell turnover
3. Proliferation of cells
4. Increased synthesis
9. PRINCIPLE OF ESTIMATION OF ENZYMES BY COLORIMETRY /SPECTROMETRY
A:
I. Buffered Substrate + Serum (Enzyme) Product
II. Product + Chemical Reagent Colored Complex
III. Measurement of optical density of colored complex
B :
NADH dependent estimations using UV light as a source : increase or
decrease of Absorbance
10. CLINICAL SIGNIFICANCE OF ENZYMES
Enzyme function Normal range Occurrence Clinical significance
Aldolase F1,6 P Triose
Phosphate
1.5-7.2micromoles /l Myocardium
Skeletal muscles
liver
Sensitive index in muscle
wasting
Muscular dystrophy
Poliomyelitis
Myasthenia Gravis
α-Amylase Starch Maltose Serum – 50-120 IU/L
URINE < 375 IU /L
Salivary gland
Pancreas
placenta
MUMPS > 1000IU/L
Ectopic pregnancy
Acute pancreatitis
Acid phosphatase
( optimum p H)
Hydrolysis of esters of
phosphoric acid
2.5 – 12 IU /L Prostrate
RBC
WBC
Platelet
semen
PROSTRATE CANCER
FORENSIC RAPE CASE
PSA-PROSTRATE
SENTSITIVE
ANTIGEN
(SERINE PROTEASE ) 1 -5 MICROGRAM /L Prostrate
semen
( LIQUIFICATION
OF COAGULUM )
PROSTRATE CANCER
( > 10 MICROGRAM /L )BEFORE
RECTAL EXAMINATION
BENIGN PROSTRATE
ENLARGEMENT (5- 10
MICROGRAM /L )
11. ASPARTATE TRANSAMINASE (AST/SGOT )
PRINCIPLE OF ESTIMATION OF SGOT
α KGA +Aspartate ↔ Glutamate + Oxaloacetate
↓
Pyruvate
Pyruvate +DNPH BROWN COLOR COMPLEX ( Alkaline pH )
Clinical Significance of SGPT
1. Normal range of Serum SGOT = 2-20 IU/L
2. Significant increase observed in Myocardial Infarction
3. Moderate increase observed in liver disease including Hepatoma
4. Isoenzymes –Cytosolic ( Mild Injury )/Mitochondrial (Severe Injury )
12. ALANINE TRANSAMINASE (ALT/SGPT )
PRINCIPLE OF ESTIMATION OF SGPT
α KGA +Alanine ↔ Glutamate +Pyruvate
Pyruvate +DNPH BROWN COLOR COMPLEX (alkaline medium )
Clinical Significance of SGPT
1. Normal range of SGPT =(13-40 IU/L )
2. Significant increase observed in ACUTE HEPITITIS (100-1000 IU/L)
3. Moderate increase observed in liver disease including Hepatoma
4. Increase in Serum ALT>>>Serum AST is observed before clinical
manifestation
5. Chronic Liver Diseases (25-100 Iu/L ) /Cirrhosis /Malignancy
6. Bad prognosis is indicated by SUDDEN FALL in serum levels of SGPT
14. ENZYMES INDICATED IN HEART DISEASES
ENYZYME PATTERN IN HEART DISEASE (AMI )
CPK -MB First enzyme to increase in AMI
Aspartate Amino Transferase increase after CPK, half life 4-5days
Lactate Dehydrogenase (LDH1 ) Last enzyme to get elevated in AMI ,significant half life
15. ENZYMES INDICATED IN MUSCLE DISEASES
ENZYME WHICH SHOW SIGNIFICANT INCREASE IN MUSCLE DISEASES
Creatinine Phosphokinase (CPK -MM )
SGPT
Aldolase (non specific )
16. Enzymes Indicated In Bone Diseases
Serum Alkaline Phosphatase increases Significantly in Paget Disease,
Rickets ,Hyperthyroidism.
17. Enzymes indicated in Prostrate Diseases
• Acid phosphatase (Tartaric acid labile ) - Prostrate Cancer ( Malignant /
Benign )
• Diagnosis conformed by estimation of PROSTRATE SPECIFIC ANTIGEN
(PSA )–Prostrate Cancer ( Malignant / Benign )
18. Enzymes indicated in Kidney Diseases
Beta Glucuronidase –for diagnosis of urinary bladder diseases
19. Therapeutic uses of Enzymes
Enzyme Therapeutic use
1 Asparginase Acute Lymphatic Leukemia (cells need Asparagine for its
growth )
2 Streptokinase LYSE INTRACELLULAR CLOT
3 Uro kinase Lyse Intracellular Clot
4 Plasminogen PLASMIN /CLOT LYSIS
5 Streptokinase DNA ase applied locally
6 Hyaluronidase Enhance local anesthesia
7 Pancreatic (Lipase & Trypsin ) Pancreatic insufficiency – oral administration
8. Papain Anti-inflammatory
9. Alpha Anti Trypsin Emphysema
21. ISOENZYMES
Definition : Enzymes occurring in different molecular forms which differ in their
physiochemical prosperities but catalyze the same reaction
Physio-chemical properties of Isoenzymes
1. differential mobility on electrophoresis
2. differential mobility in column chromatography
3. differential kinetic properties
a. Km
b. V max
c. Optimum temperature
d. Optimum p H
e. Relative sensitivity to inhibitors
f. Degree of denaturation
22. Iso enzymes of Lactate dehydrogenase
Reaction is reversible & uses NAD⁺ as a coenzyme
23. Iso enzymes of Lactate dehydrogenase
Subunit composition of LDH isoenzymes
In heart cells conversion of Lactate to
Pyruvate favored by LDH1
In muscle cells conversion of Pyruvate
to Lactate favored by LDH5
25. Comparison of Isoenzymes of Lactate dehydrogenase
LDH1 LDH5
Optimum
condition
AEROBIC ANAEROBIC
Km high low
Affinity for
pyruvate
low high
Synthesis of
lactate
Not favored Favored
26. Iso enzymes of Lactate dehydrogenase
Electrophoretic mobility of LDH Isoenzymes
28. Iso enzymes of Lactate dehydrogenase in AMI
ENZYME HALF LIFE
LDH 1 8 DAYS
LDH 6- 8 DAYS
CPK -MB 2 DAYS
SGOT 4 DAYS
AREA UNDER CURVE ,SLOPE OF INITIAL RISE α INFARCT
30. MECHANISMS OF ISOMERISATION OF ENZYMES
1. Genetic factors
2. Polymerization
3. Conformational isomerization
4. Presence of charged group
5. Differential gene location on same chromosome or diffirent
chromosome
31. MECHANISM OF ISOMERISATION OF ENZYMES
I.GENETIC FACTOR : LDH
-------------------------------------------
H gene M gene
gene expression
^^^^^^^^^^^^^ ^^^^^^^^^^^^^^
M POLYPEPTIDE H POLYPEPTIDE
H4 H3 M H2 M2 HM3 M4
• HEART LIVER /MUSCLES
32. MECHANISM OF ISOMERISATION OF ENZYMES
II Polymerization: eg Cholinesterase ( Type 1-5 )
Differ in surface charges differential electrophoretic mobility
Cholinesterase 5 ---(dilution ) yields 5 polypeptide chains
CHE 1 CHE2 CHE3 CHE4 CHE5
Separation of isoenzymes of Cholinesterase ( Type 1-5 ) by starch gel
electrophoresis of serum
34. MECHANISM OF ISOMERISATION OF ENZYMES
III Conformational isomerism
Iso enzymes have similar
1. Amino acid sequence
2. Active site
3. Enzymatic property
Dissimilar
1. Tertiary structure (folding of chain )
2. Electrophoretic mobility
eg Cytoplasmic Aspartate Transaminase
Microsomal Aspartate Transaminase are Conformational Isomers
36. MECHANISM OF ISOMERISATION OF ENZYMES
• IV PRESENCE OF CHARGED GROUPS
Isomers OF Alkaline Phosphatase differ in the number of Sialic acid
residues (charged groups ) attached to enzyme –Post transcription
modification
Position of alkaline phosphatase in electrophoresis location
α 2 (ALPHA 2 ) Liver
γ (GAMMA ) Intestine ( No Sialic Acid Residues )
PRE BETA Bone ( Heat Labile )
PRE BETA Placenta ( Heat Stable )
37. Catalysis of Alkaline Phosphatase
Optimum p H : 9- 10
Function :hydrolysis of phosphoric esters
Cofactors – Mg ⁺⁺,Mn ⁺⁺ ,Zn⁺⁺
38. Comparison of Isoenzymes of Alkaline Phosphatase
Iso enzyme of
Alkaline p04 ase
Occurrence % of
total
Alkaline
PO₄⁻ ase
in serum
Clinical significance ( increase in serum levels observed in )
1 α 1 Liver 10 Obstructive Jaundice ,Hepatoma
2 α 2 ( heat labile –
DENATURED BY
BOILING AT 65⁰ C
FOR 30 MINS )
Liver 20 Hepatitis
3 α 2 ( HEAT STABLE
INHIBITED BY Phe )
Placenta 10 Lung /liver/ GIT CARCINOMA
4 Pre beta heat labile Bone 5 Bone carcinoma ,Paget's, Osteitis , Osteomalacia
5 γ ( gamma )
INHIBITED BY Phe
Intestinal cells 10 Ulcerative colitis
6 Leucocyte (LAP ) MYELOID LEUKAMIA ,LYMPHOMAS
39. Clinical significance of Isoenzymes of Alkaline Phosphatase
Electrophoretic pattern for Isoenzymes Of Alkaline Phosphatase
40. MECHANISM OF ISOMERIZATION OF ENZYMES
V Differential gene location on
same chromosome or different
chromosome
1. Salivary & Pancreatic Amylase
2. Cytosolic & Mitochondrial
Malate Dehydrogenase
41. Iso enzymes of Creatinine phosphokinase (CPK)
• Normal range in serum( males) : 15-100 IU
• Normal range in serum (females ): 10-80 IU
Iso enzyme Abbreviation Location Elevated
serum levels
observed in
Electrophoretic
mobility
CPK 1 CPK-BB Brain Maximum
CPK 2 CPK-MB Heart Acute Myocardial
infarction
Intermediate
CPK 3 CPK-MM Muscles Muscular Dystrophy Least
CPK –MT
(MITOCHONDRIAL )
42. Genes coding Isoenzymes of Creatinine Phosphokinase (CPK)
Electrophoretic pattern for Isoenzymes Of Creatinine Phosphokinase (CPK)
44. TROPONINS ( MARKER OF MYOCARDIAL INFARCTION )
TROPONINS TYPE Property
TROPONINS C Calcium Binding
TROPONINS I ACTINO MYOCIN INHIBITORY ATPase
TROPONINS T Tropomyosin Binding Element
49. Genetic mutations in enzymes & diseases
Biochemical changes in Mutation of enzyme
1. Gain in amino acids
2. Loss in amino acids
3. Replacement by another amino acids
Gene mutation defective enzyme
A. Amino acid residues from active site of enzymes altered
B. Amino acid residues from catalytic site of enzymes altered
C. Three dimensional structure of enzymes altered
D. Catalytic activity of enzymes reduced ( different Km,V max ) or lost (inactive )
Defective Enzyme –Lethal Disturbance /Mental Retardation
Remedy : capsule containing normal enzyme enter blood circulation toxic
metabolites metabolized & normal products produced
60. Silent features of Alkaptonuria
Silent features
•Urine: turns black on standing (due to oxidation of homogentisic acid).
•On long standing urea is hydrolysed into ammonia which then reacts with
homogentisic acid in presence of oxygen to form a black pigment similar to
melanin.
•Ochronosis: Occurs due to deposition of homogentisic acid in skin and
connective tissue. Leads to bluish hue especially of the sclera and ear cartilage.
•Joints: Chronic osteoarthritis involving large joints (spine, hip, knee).
•CVS: Aortic/mitral valvulitis, myocardial infarction.
