Enzymology :Clinical significance of Enzymes & Isoenzymes(Diagnostic uses and Therapeutic uses )

Dr. Rohini C Sane

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 )

Techniques for estimation of enzymes

• Colorimetry /spectrophotometry

• Fluorometry

• RIA

• ELISA

• Chemiluminescence

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

Enzyme appear in plasma by 3 ways1. Functional plasma enzymes

2. Non Functional plasma enzymes

3. Obstruction to secretory pathway

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

Obstruction to Secretory Pathway Physiological conditions balance between synthesis & release

Pathological conditions loss of balance between synthesis & release

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

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

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/LURINE < 375 IU /L

Salivary glandPancreasplacenta

MUMPS > 1000IU/LEctopic pregnancyAcute pancreatitis

Acid phosphatase( optimum p H)

Hydrolysis of esters of phosphoric acid

2.5 – 12 IU /L Prostrate RBC WBCPlatelet 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 EXAMINATIONBENIGN PROSTRATE ENLARGEMENT (5- 10 MICROGRAM /L )

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 )

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

Enzymes indicated Liver DiseasesHEPATIC DISEASE Enzyme of choice for diagnosis

Parenchymal diseases SGPT

Liver dysfunction, cholestasis Nucleotide Phosphatase

Obstructive Jaundice Alkaline Phosphatase

Alcoholic liver Gamma Glutamyl Trans peptidase ( γGT )

Hepatitis LDH 5

Alcoholic liver Alcohol Dehydrogenase

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

ENZYMES INDICATED IN MUSCLE DISEASES

ENZYME WHICH SHOW SIGNIFICANT INCREASE IN MUSCLE DISEASES

Creatinine Phosphokinase (CPK -MM )

SGPT

Aldolase (non specific )

Enzymes Indicated In Bone Diseases

Serum Alkaline Phosphatase increases Significantly in Paget Disease, Rickets ,Hyperthyroidism.

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 )

Enzymes indicated in Kidney Diseases

Beta Glucuronidase –for diagnosis of urinary bladder diseases

Therapeutic uses of EnzymesEnzyme 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

Diagnostic uses of Enzymes

ENZYME FOR DIAGNOSTIC PURPOSE ESTIMATION OF

1 UREASE UREA

2 Uricase Uric acid

3 GLUCOSE OXIDASE GLUCOSE

4 PERIOXIDASE GLUCOSE /CHOLESTEROL

5 HEXOKINASE GLUCOSE

6 CHOLESTEROL OXIDASE CHOLESTEROL

7 LIPASE TRIGLYCERIDE

8 HORSE RADDISH PERROXIDASE ELISA

9 ALKALINE PHOSPHATASE ELISA

10 RESTRICTION ENDONUCLEASE SOURTHEN BLOT

11 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

PRINCIPLE : SUBSTRATE (SERUM )+ENZYME PRODUCT -CHEMICAL REAGENT –OD OF COLOR COMPLEX ( α CONC OF SUBSTRATE )

ISOENZYMESDefinition : 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

Iso enzymes of Lactate dehydrogenase

Reaction is reversible & uses NAD⁺ as a coenzyme

Iso enzymes of Lactate dehydrogenase

Subunit composition of LDH isoenzymesIn heart cells conversion of Lactate to Pyruvate favored by LDH1

In muscle cells conversion of Pyruvate to Lactate favored by LDH5

Comparison of Isoenzymes of Lactate dehydrogenase

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

Iso enzymes of Lactate dehydrogenase

Electrophoretic mobility of LDH Isoenzymes

Electrophoretic mobility of Isoenzymes LDH & CPK

Clinical significance of LDH3

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

Clinical significance of Isoenzymes of Lactate dehydrogenase

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 diffirentchromosome

MECHANISM OF ISOMERISATION OF ENZYMESI.GENETIC FACTOR : LDH

-------------------------------------------H gene M gene

gene expression

^^^^^^^^^^^^^ ^^^^^^^^^^^^^^

M POLYPEPTIDE H POLYPEPTIDE

H4 H3 M H2 M2 HM3 M4

• HEART LIVER /MUSCLES

MECHANISM OF ISOMERISATION OF ENZYMESII 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

MECHANISM OF ISOMERISATION OF ENZYMES

MECHANISM OF ISOMERISATION OF ENZYMES

III Conformational isomerismIso 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

MECHANISM OF ISOMERISATION OF ENZYMES

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 )

Catalysis of Alkaline Phosphatase

Optimum p H : 9- 10Function :hydrolysis of phosphoric esters Cofactors – Mg ⁺⁺,Mn ⁺⁺ ,Zn⁺⁺

Comparison of Isoenzymes of Alkaline Phosphatase

Iso enzyme of Alkaline p04 ase

Occurrence % of total Alkaline PO₄⁻ asein 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

Clinical significance of Isoenzymes of Alkaline Phosphatase

Electrophoretic pattern for Isoenzymes Of Alkaline Phosphatase

MECHANISM OF ISOMERIZATION OF ENZYMES

V Differential gene location on same chromosome or different chromosome

1. Salivary & Pancreatic Amylase

2. Cytosolic & Mitochondrial Malate Dehydrogenase

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 )

Genes coding Isoenzymes of Creatinine Phosphokinase (CPK)

Electrophoretic pattern for Isoenzymes Of Creatinine Phosphokinase (CPK)

Creatinine phosphokinase (CPK) as a Cardiac marker

TROPONINS ( MARKER OF MYOCARDIAL INFARCTION )TROPONINS TYPE Property

TROPONINS C Calcium Binding

TROPONINS I ACTINO MYOCIN INHIBITORY ATPase

TROPONINS T Tropomyosin Binding Element

Comparison of Cardiacbiomarkers including CPK-MB ,LDH, Troponin, Myoglobin

Isoenzymes of Alcohol dehydrogenase

Iso enzymes of Alcohol dehydrogenase

Prevalence

α β 1 Americans

α β 2 (HIGH AFFINITY FOR ALCOHOL )

Chinese ,Japanese

Alcohol Aldehyde (Aldehyde responsible for increase heart rate---Tachycardia ,facial flashings )

Alcohol Aldehyde (Aldehyde responsible for increase in heart rate= Tachycardia, facial flashings )

Toxic effects of Ethanol

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

Industrial Applications of Enzymes

Genetic mutations of enzymes & diseases Disease Defective enzyme

Albinism Tyrosinase

Alkaptonuria Homogenitisate Dioxygenase

Phenylketonuria Phenylalanine mono-oxygenase

Homocystinuria Cysthathione beta synthtase

Albinism is associated with the mutation of Tyrosinase

Phenylketonuria :Genetic mutations in enzymes Phenylalanine Mono oxygenase

PKU : PHENYLKETONURIA HISTORY ,SIGNS,SYMPTOMS & TREATMENT

Alkaptonuria: signs, symptoms & genetic mutation in a gene coding for enzyme Homogenistic acid oxidase

Alkaptonuria: signs, symptoms & genetic mutation in a gene coding for enzyme Homogenistic acid oxidase

Laboratory Tests for Diagnosis of Alkaptonuria

Altered Metabolic Pathway in Alkaptonuria

Genetic mutations in Tyrosine Metabolism: AlkaptonuriaandTyrosinemiaType I

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.

Ochronosis & Alkaptonuria

Deposition of Homogentistic acid in skin & connective tissue .Bluish hue in sclera & ear cartilage

Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

HMP SHUNT ( the first step is catalyzed by G6PD)

Glucose 6 phosphate +NADP 6 Phospho Gluconolactone + NADPH **

measure in increase in absorbance at 340nm .

Glutathione (oxidized )+ NADPH ** Glutathione (reduced )+ NADP ⁺

HMP SHUNT ( the first step is catalyzed by G6PD)

Glucose 6 phosphate dehydrogenase (G6PD ) deficiencyFunctions of Glutathione (reduced )

1. Detoxify peroxides To prevent hemolytic anaemia ( peroxidation of fatty acids of cell membrane prevented )

2. To protect sulphhydryl group from oxidation

SH-SH

↓oxidation

S-S

3.to prevent meth hemoglobin formation (oxidation of ferrous into ferric inhibited

4.To impart resistance to Malaria

Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Laboratory Tests for diagnosis of Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Laboratory Tests for diagnosis of Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Meth haemoglobinemia in Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Meth haemoglobinemia & hemolytic anemia induced when doses of reducing drugs administerd

Meth haemoglobinemia

Meth Hb ( oxidized form ) + NADPH

Meth Hb reductase

Hemoglobin (reduced form )+ NADP⁺

Inheritance of Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

• Hemizygous male (1 normal gene ,1 abnormal gene , Individuals normal)

• Homozygous female

Treatment of Glucose 6 phosphate dehydrogenase (G6PD ) deficiency

Clinical applications of Immobilized enzymes

1. Immobilized enzymes :used for detection of abnormal substances in urine

Paper coated with GOD -POD for Glucose in urine

Glucose + O2 + H2O Glucuronic acid + H2O2

H2O2 H20 + (O)

(O) + O- Toluidine ( colorless ) blue color complex( oxidized O- Toluidine )

2.GOD POD /Urease/ Amylase /Hexokinase For Diagnostic Purpose

3.Chromatography columns with activated Sepharose ( Cyanogen Bromide )& Immobilized Enzymes ( preservation of enzymes without loss of activity )

4. Immobilized enzymes + substrate PRODUCT