z

Isoenzymes
Plasma enzymes - 2 classes
 1.Plasma specific enzymes
  * Definite and specific function in plasma       
* plasma their normal site of action
*  present at higher levels than in most tissue
cells.

Eg. * Enzymes of blood coagulation and

clot lysis - plasmin and thrombin

* Ceruloplasmin, cholinesterase
lipoprotein lipase.(Synthesized by the
liver and released at a constant rate)
 2. Non functional plasma enzymes.
* No known physiological function

* Conc. In plasma lower than cells

enzymes of cellular metabolism. Which comes out of cells

due to normal wear and tear.

Located within cells at high conc.

 ENZYME ACTIVITIES MAY BE LOCATED WITHIN CELL
COMPARTMENTS OR ORGANELLES.
cytosol MEMBRANE BORDER
LDH
Alkaline po4 tase
AST
aldolase Invertase
pyruvate kinase Maltase
glucokinase
LAP
NUCLEUS AND NUCLEOLUS
isocitrate DH
Adenyl transferase
Arginase
NAD pyrophosphorylase
NAD(p) nucleosidase
Lysosomes Endoplasmic reticulum

A phosphatase Cholesterol esterase

 Glucuronidase Choline esterase
Cathepsin Glu 6 po4 tase
arylsulphatse Glutathione reductase

Peroxisomes Golgi bodies

Amino oxidase Trypsinogen

Urate oxidase Amylase
catalase
MITOCHONDRIA
Isocitrate DH
Cytochrome oxidase
ATP ase
Succinate Dehydrogenase
Hydroxy butyrate DH
Asp transaminase
Adenylate kinase
Sorbitol DH
NORMAL PLASMA LEVELS:
*  Reflects the balance between synthesis and
release of enzymes during ordinary cell
turnover and their catabolism and excretion

 RAISED PLASMA LEVELS

      * Proliferation of cells
     * cell damage
    * Enzyme induction
NON SPECIFIC CAUSE OF RAISED ENZYME LEVELS
1. PHYSIOLOGICAL
i) NEW BORN: levels of certain enzymes eg. AST raised
ii) childhood: ALP levels are high until after puberty.
Iii) pregnancy: last trimester ALP levels
During and after labor  in AST,ALT AND CK (due to uterine
contractions)
 2. ENZYME INDUCTION BY DRUGS
EG. barbiturates stimulates enzyme
phenytoin, barbiturates gt 
 3. ARTEFACTUAL ELEVATIONS
Haemolysed samples. AST, LD
 DECREASED SERUM ENZYME LEVELS

 GENETIC: eg Wilson's disease in ceruloplasmin

 Acquired: in hepatitis levels of pseudocholine

esterase due to  production
Enzyme inhibition: insecticide poisoning levels of pseudo
cholinesterase

Lack of cofactors:  b6 levels depresses the activity of

transaminase.
SERUM ENZYME ASSAYS IN CLINICAL PRACTICE.
 * Single or serial assay of the serum activity of
a selected enzyme provides
information on nature and extent of disease
 * Few enzyme are specific for any one tissue.
Isoenzyme studies increase specificity.
 
Serum enzymes may be useful in
  * Early detection
* Diagnosis
* Differential diagnosis
* Prognosis
 ISOENZYMES
Moderate elevation (2-3 * normal) in infective hepatitis,
  -Alcoholic hepatitis and hepatocellular carcinoma
DEFINITION: Physically distinct form of a given enzyme.
Different forms of the same enzyme, catalysing the same
chemical reaction. Differs from each other structurally,
electrophoretically and immunologically. Genetically,
determined difference in amino acid sequences.

* Different organs contain diff proportion of

isoenzymes

* it can serve as site detector of tissue

damage
CREATINE KINASE( Normal Male=15-100U/L, F=10-80U/L)
Catalyses ATP generation during muscle contraction.

CK and heart attack

Increases in acute myocardial infarction (5-10 *
normal)
Raises within 4-8hrs of infarction peaks 12-24hrs and
returns to normal 3-4 days.
Useful in detecting early cases
CK is not increased in hemolyses or CCF like LDH
 CK AND MUSCLE DISORDERS

Most sensitive for detecting skeletal muscle involvement

Markedly elevated in muscular dystrophy (50*normal)

Moderately elvated in female carriers of x-Linked muscular

dystrophy

Highly elevated in muscle trauma due to crush Injuries

CREATININE KINASE:
3 isoenzyme each is a dimer of 2 polypeptide chain b and
m in 3 combination

B B
CK1 BRAIN
B M
CK2 MYOCARDIUM
M M
CK3 SKELETAL
MUSCLE
 

CK –MB or CK-2
Estimation of CK-MB is crucial in MYOCARDIAL
INFARCTION

*  Normally : Ck2 (MB) - less than 6 % of total ck.

* IN MI: CK- MB first enzyme to rise in infraction. CK2 (4 -8h) Following

chest pain. Peaks at 12- 24 hrs. Then falls rapidly.(48-72hrs)

* TOtal MB Level 20 fold above normal.

 z

 CK –MM:

skeletal muscle disorders such as muscular

dystrophy, polymyositis, physical activity,
intra muscular injection, muscle trauma
 z
LACTATE DEHYDROGENASE

LACTATE + NAD LDH PYRUVATE + NADH

-Ubiquitous cytoplasmic enzyme

-Non specific for any disease

-Isoenzyme study is important

LDH ISOENZYME
  * 5 Isoenzymes are present
* 4 polypeptide chains of 2 types h and m
* each isoenzyme contains h and m in different
proportions.
 LD1 HHHH MYOCARDIUM (30%)
LD2 HHHM RBC (35%)
LD3 HHMM BRAIN (20%)
LD4 HMMM LIVER(10%)
LD5 MMMM SKELETAL MUSCLE(5%)
 
 z

- +

LD2 LD1
LD5 LD 4 LD3

PARTIALLY
DESTROYED
DESTROYED STABLE
AT 60 C AT 60 C
z
LDH AND HEART ATTACK

 Begin to rise 12- 24 hrs

 Peaks at 48-72 hrs (10 * normal)

 Remain elevated for 10 days

 FLIPPED PATTERN LD1> LD2

z
OTHER CONDITIONS

 Marked increase in viral hepatitis and  LD5

 Elevateded in megaloblastic anemia, pernicious anaemia &
leukemia – haematological disorders
  In muscular dystrophy,
Physical exercise and crush injuries.
 ALKALANE PHOSPHATASE
hydrolysis of wide variety of phospho mono esters at ph 9-10.5
Sources:
Wide distribution,Liver, bone, placenta, intestine
In liver: cells lining bile canaliculi
In bone: osteoblasts
Isoenzymes Tissue of origin percentage

Alpha ALP Biliary canaliculi 10%

Alpha-2 heat labile ALP Hepatocytes 25%
Β2 heat stable ALP Placenta 1%
Pre-β ALP Bone 50%
𝛄-ALP Intestine 10%
Leucocyte ALP Leucocytes 4%
CLINICAL SIGNIFICANCE – FOR DETECTION
z OF BONE AND HEPATO BILIARY
Normal value- 40 -125 u/l DISORDERS
 Biliary tract diseases> hepato cellular injury

 10-12 * normal seen in extra hepatic obstruction (caused by gall stone, carcinoma
of head of pancreas)

 Intrahepatic obstruction due to virus or by drug chlorpromazine

Moderate elevation (2-3 * normal) in infective hepatitis,
-Alcoholic hepatitis and hepatocellular carcinoma

-Bone disorder and alp

 Osteoblastic activity
Alp
Pagets disease (10-25 *normal)
ATYPICAL ALP ISOENZYME

•REAGAN ISOENZYME: Closely identical to placental isoenzyme.

 Stable upto 63c
 in carcinoma lungs
Enzyme Profile in some important Clinical Condition

Measurment of enzyme activities in plasma, serum,

plural fluid, urine – diagnosis & management of diseases

1. MI. 2. Liver disease 3. Muscle disease 4. Bone

disease 5. Prostatic disease
z

SERUM ENZYME IN HEART

DISEASE
 * Sudden and complete occlusion of a coronary artery by thrombosis

* cuts off the blood supply to a zone of myocardial tissue- o2 and  glu.

* Tissue necrosis soon follows. Patient suffers severe chest pain.

• This leads to the sudden release of cardiac enzyme, which are

removed from the plasma at different rates.
 10
CKMB
ENZYME ONESET OF PEAK DURATION
ELEVATION ACTIVITY OF
DEGREE OF ELEVATION ELEVATION
6
CK CK 4–8h 12 – 24 h 3–4d
5

CKMB 4–8 10-24HR 2–3d

AST 8 – 12 24 5d
4

AST LDH 12 – 24 72 10 d
3

LDH
2
1

1 2 3 4 5 6 7 8 9 10
TIME COURSE OF ELEVATION
TROPONIN: complex of 3 proteins TnT,Tn I, Tn C. cardiac
specific
small amounts of cardiaca necrosis releases seignificant
amounts into serum

myoglobin: heme protein found in striated skeletal and

cardiac muscle

PROTEIN HRS AFTER PEAK RETURNS TO

ONSET NORMAL
MYOGLOBIN 1–3 5 – 12 18 – 30 HRS
TROPONIN T 3–4 10 - 24 10d – 14 d
TROPONIN I 3–6 14 - 20 5 – 10 d
 

Flipped pattern LD1>LD2 highly specific test for Mi.

AST – extent of infarction
Myoglobin - very early indicator
 TROPONIN I - very good indicator of prognosis
z
CARDIAC TROPONINS

 Troponin I – marker for MI

 Released into the blood within 4hrs after the

onset of symptoms of myocardial ischemia

 Peaks at 14-24 hrs and remain elevated for 3-5

days

 Not elevated in muscle injury

 AST, ALT, GGT,ALP, LDH, LD5
z
 To differentiate between hepato cellular from biliary obstruction – ALP,
GGT, 5’NT
 GGT – sensitive marker of occult alcohol abuse, alcoholic cirrhosis

 DIFFERENTIAL DIAGNOSIS
 SKELETAL HEPATIC
 ALP  
 GT N 
z
TRANSAMINASES

 AST /SGOT

 ALT/ SGPT

 CATALYSE THE TRANSFER OF AMINO GROUPS TO KETO

ACID(for the synthesis of non essential amino acid and
degradation)
 z
 AST:aspartate transaminase

 Normal level 5-35 u/l

 Present in liver, myocardial and skeletal muscle
 Markedly elevated in MI(10-100*normal)
 Rise 6-8 hrs, peak at 24 hrs, return n within 5 days
 Used to assess the extent of damage and rule out mi.
 Ed in viral hepatitis and toxic liver necrosis
 Moderate  cirrhosis of liver, obstructive jaundice, muscular
crush injury
 z
ALANINE TRANSAMINASE

 Confined to the evaluation of hepatic disorders

 Viral -100 *normal
 Toxic – 20* normal
 ALT>AST , remain elevated longer in viral hepatitis
 Alcoholic cirrhosis ratio >2
 Both AST AND ALT elevated even before jaundice
appears
 GAMMA GLUTAMYL TRANSFERASE(GGT)
z
 source- cells that line biliary tract and hepatic canaliculi
 use: detection & differential diagnosis of hepato biliary disorders
 5-30 * normal – intrahepatic & extrahepatic biliary obstruction
 More sensitive than alp .

 GGT is not in bone. So indicates hepatobiliary disorders in children and

during pregnancy.

 Induced by alcohol & drugs

 Sensitive indicator of alcohol abuse.

 z NUCLEOTIDE PHOSPHATASE(5’NT/NTP)

 Increase parallels that of ALP &GGT.

 Unaffected by bone diseases.

 Useful in differentiating bone & liver elevations of ALP

MUSCLE DISEASE
 MUSCLE DISEASE
 MUSCLE ENZYMES ELEVATED
* creatinine kinase
•transaminase
•LDH
* CK estimation most valuable.
*

- muscular dystrophy, polymyositis, physical exertion,

crushing muscular injuries muscular injections
 BONE DISEASE
 
ALP - Valuble index of osteoblastic
activity

ALP -
Paget’s disease (osteitis deformans)
Rickets, osteomalacia,
hyperparathyroidism
Bone malignancy
The age of the patient is important in Interpretation of ALP levels.
 GI TRACT DISEASE
 Acute pancreatits life thretening

- s.Amylase (4-6 * normal)

S. Calcium

Serum amylase - mumps,other forms of

parotiditis

  - Perforated peptic ulcer,

intestinal obstruction

Urine amylase and amylase:creatinine

Clearance also useful
 SERUM LIPASE
- More specific not usually done
- Supplement the diagnosis.
- In acute pancreatitis, remain elevated longer.
z
PROSTATE DISORDER

 Acid phosphatase & PSA

 To detect prostate carcinoma

 Total ACP non specific

 Prostate specific acid phosphatase – monitor the

treatment of prostate carcinoma
 SERUM ENZYMES IN MALIGNANCY
ACID POSPHATASE cancer of prostate
ALP metasis of liver
metasis of bone
jaundice due to carcinoma head of
pancreas
LDH wide spread malignancy
ALDOLASE advanced leukemias
PHOSPHOHEXOSE
ISOMERASE
-GLUCURONIDASE cancer of urinary bladder
IN URINE
cancer head of pnacreas
LAP liver cell carcinoma
DIAGNOSTIC SIGNIFICANCE OF CERTAIN ENZYMES

ACID  in carcinoma prostate

PHOSPHATASE
ALKALINE  in obstructive jaundice
PHOSPHATASE (rickets,hyperparathyroidism)
AMYLASE  in acute pancreatitis
 
(intestinal obst, acute parotitis,  in
acute liver
disease)
LIPASE  in acute pancreatitis
pancreatic
carcinoma
 in liver disease
CERULOPLASMIN  in cirrhosis
 
 in wilson’s disease
CHOLINE ESTERASE  nephrotic syndrome
 
 acute liver disease
 organo phosphorous poisoning
CPK  in acute MI
muscular dystrophies
SGOT  in acute mi, toxic
liver disease
SGPT  in viral hepatitis
LDH  in acute MI, hepatitis
 ENZYME ASSAYS IN DIFFERENT DISEASES
DISEASE ENZYME ASSAY

MI CPK,SGOT,LDH

LIVER DISEASE SGOT,SGPT,ALP,GT

G.I DISORDER AMYLASE

MUSCLE DISEASE CK

BONE DISEASE ALP

 MACRO ENZYMES
* Large molecular mass enzyme complex
* usually enzyme bound to IG
*  unexpected plasma enzyme activity
* Unusual isoenzyme electrophoretic pattern

Macro amylase
*  Usually bound to ig a
*  Associated with chronic pancreatitis,
alcoholism, liver disease
*  Diabetes and cancer
 z
AMYLASE

 Hydrolyses of  1-4 glycosidic linkage in starch & glycogen

 Tissue source- salivary glands & acinar cells of pancrease.

 Serum & urine amylase elevated in acute pancreatitis.

 Also elevated in mumps, parotitis, perforated peptic ulcer

 z

 In acute pancreatitis- increase in serum amylase (4-6 fold)

occurs within 2-12 hr of onset

 Peaks in 12-72 hrs

 Returns to normal by day 3 or 4.

 Cholecystitis – increased amylase due to pancreatic

involvement.

 Macroamylasaemia – amylase + IgA/ IgG

 Ratio of amylase clearance to creatinine clearance < 0.02

 z
LIPASES
 Hydrolysis of TGL. Sources : pancreas

 Acute pancreatitis -activity parallels that of amylase

 Lipase more organ specific

 Mumps – amylase is increased but not lipase.

 Obstruction of pancreatic duct by calculus/carcinoma – rise in lipase

 Increase of lipase after acute pancreatitic attack (2- 50 xURL)-

increase within 4-8hrs
 Peaks at 24 hrs. Decrease within 4 – 8 days.

 May be useful in late presenting acute pancreatitis.

 ACID PHOSPHATASE
z

 Hydrolysis of phospho monoesters in acidic ph 4.5

 Noraml value 2.5-12 u/l

 Elevated in prostatic disease, prostate carcinoma, benign prostate hypertrophy

 Prostate isoenzyme is inactivated by tartaric acid.

 Prostate carcinoma : tartrate labile ACP is elevated.

 Prostate specific ACP=total ACP- ACP after tartrate inhibition

 Sample collection
 Blood sample without haemolysis
 Sample collection before rectal examination
 CHOLINESTERASE(ChE)
z

ACETYL CHOLINESTERASE (TYPE1)

HYDROLYSIS ESTERS OF CHOLINE

 Present in nerve endings and RBCs.

- Destroys ach at neuromuscular junction

 Inhibited irreversibly by organo phosphorous insecticide

(Parathione)

 Useful measure of exposure of insecticides and nerve gases

 z

PSEUDOCHOLINESTERASE (TYPE II ChE)

-Non specific

-To detect the presence of abnormal variant of ChE that

hydrolyze succinyl choline - a muscle relaxant within 2-4
min.

-Variant of ChE cause – Drug metabolism prolonged scoline

apnoea- night mare of anaesthetist

-In acute hepatitis

 z
GLUCOSE 6 PHOSPHATE
DEHYDROGENASE
 Important enzyme of HMP pathway

 Production of NADPH- vital for preserving the RBC integrity

Drug induced hemolytic anaemia

- G6PD is deficient

-  Life span of RBC & no disease manifestation

 Aspirin, pamaquine, sulpha induce haemolysis

 Favism- haemolytic anaemia induced by fava beans (star beans , corner beans)
ENZYME ASSAY
NAD DEPENDENT DEHYDROGENASES ASSAYED AT 340
nm
NADH,NADPH – MAXIMUM ABSORPTION AT 340 nm
NADH NAD
DECREASE IN O.D AT 340 nm
NADPH NADP
ANY ENZYME CATALYSING OXIDATION OF NADH BY ITS
OXIDIZED SUBSTRATE
RATE OF DECREASE IN O.D  ENZYME CONCENTRATION
 GLUCOSE
ATP

HEXOKINASE
ADP

GLUCOSE 6 - P
NADP
G6PDH

NADPH + H+
6 P GLUCONOLACTONE
COUPLING TO A DEHYDROGENASE
ENZYME ASSAYS ALSO DONE BY MEASURING
SUBSTRATE OR PRODUCT FORMED OR COUPLE THE
PRODUCT TO A DH REACTION