Clinical enzymology

Dr Smitha Madhavan BHMS,MD(Hom)
This branch of medical science deals with the usage of enzymes for diagnosis & prognosis of various diseases.
Enzymes – a brief description
They are the catalysts of biological reactions in body, by reducing energy for activation,and are colloidal, thermolabile, proteins in nature, characterized by its specificity in action

Substrate :
Substances on which enzymes act to convert them into products.
Enzymes of conjugated protein has 2 parts 1) Protein part – apoenzyme 2) Non protein part-co-enzyme or prosthetic group. Complete structure of prosthetic group and apoenzymes is called the holoenzyme. Enzyme with only one polypeptide chain is called monomeric enzyme eg : ribonuclease, and enzymes with more than one polypeptide chain are called oligomeric enzymes eg: LDH. Co-enzymes are thermostable, low molecular weight non protein part. Reactions involving oxidoreductions, group transfers, isomerisation, covalent bond formation require co-enzyme but lytic reaction do not require (eg: hydrolytic reaction)

Many of the co-enzymes are derived from vit. B complex

• Pantothenic acid – Co. ASH
• Vit. B12 – Conamide
• Folic acid – Tetrahydrofolate (FH4)
• Niacin – NAD, NADP
• Riboflavin – FMN, FAD
• Pyridoxin – Pyridoxal phosphate
• Thiamine – TPP
• Biotin, lipoic acid & even heme can act as coenzymes

Co-enzymes
1) For transfer of groups other than H+ eg : COASH, Biotin, Cobamide
2) For transfer of H + eg : NAD, NADP, FMN, FAD, Lipoic acid etc.
Activity of many enzymes depend on presence of metal ions like Mg2+, K+, Ca2+, ZN2+, Cu2+. Accordingly enzymes can be divided into metal activated enzyme – metal forms a loose bond with enzyme and metalloenzyme – metal bounds in a non dissociated tight way.

Factors affecting enzyme action
1. Temperature : Most active at optimum temperature (37-550C)
2. pH of the medium : Maximum activity at optimum pH (5-9)
3. Effect of enzyme concentration : Initial rate of reaction is directly proportional
4. Effect of product concentration : Excess of product may lower enzymatic reaction or reverse reaction may take place.
5. Substrate concentration : Directly proportional until the enzyme is get saturated with the substrate.
6. Depends on metal ions and co-enzymes
7. Effect of enzyme inhibitors.

Enzyme inhibition

• Competitive inhibition :
• Catalytic site of an enzyme is occupied by a substance other than substrate Eg : physostigmin : inhibit acetylcholine esterase
• Non competitive inhibition :
• Enzyme is acted upon by both inhibitor and substrate.

Sources of enzymes
1 Plasma derived
2 Cell derived

Plasma derived enzyme
Are very little. They act on substrates in plasma (eg: coagulation enzymes.. Coagulation is mediated by a series of proteolytic zymogen activators of serum enzymes. Activated form of one clotting factor catalyses the activation of the next factor, which inturn activate a subsequent factor through extrinsic & intrinsic pathways.) They are called functional plasma enzymes.

Cell derived enzymes
Are non functional plasma enzymes. Present in tissue in higher concentration. The serum levels of these enzyme may increase by diseases that provoke a) an increase in its rate of release
b) decrease in rate of excretion.
It depends on extend of organ damage, location of enzyme in the cell, changes in cell membrane permeability etc. Increased permeability may be with the necrosis of the cell or without necrosis of the cell eg: in early stage of viral hepatitis before jaundice appears, ballooning degeneration of liver cell occur, leading to elevated levels of transaminases. Increase in number of cells or cell mass as in malignancies may cause increased serum enzyme activity eg: Alkaline phosphatase increased in patients with osteoblastic bone lesion.
Decreased serum levels : Due to dicreased formation of enzyme
a) Genetic eg: Wilson’s disease with decreased serum cerulo plasmin (N= 25-43mg /dl)
b) Acquired eg ; Hepatitis- decreased serum pseudocholinesterase
c) Enzyme inhibition
d) Lack of co factors

Classification of enzymes
Can be classified into 6 groups according to the type of reaction catalyses
1. Oxidoreductases : Involved in oxidation and reduction of substrate eg : LDH, glucose 6- phosphate dehydrogenases
2. Transferase : Help in transfer of a particular group from one substrate to another. eg: Transaminases, phosphotransferases (ck, Pyruvate kinase)
3. Hydrolases : Bring about hydrolysis eg: Digestive enzymes, phosphatases
4. Lyases : Facilitate removal of small molecule from a large substrate eg : aldolase
5. Isomerases : Isomerisation of substrate, eg: isomerase, phosphohexose isomerase
6. Ligases : Involved in joining together of 2 substrates eg : synthetases.

Unit of enzyme activity
The amount of enzyme in a sample is measured by the rate of reaction
Catalyzed by the enzyme.This rate is directly proportioned to the amount of enzyme and is expressed in enzyme unit, IU/L Isoenzyme
Physically distinct and separable form of the given enzyme present in different cell types. It is of diagnostic value. Can be separated by electrophorasis. Eg; isoenzymes of lactate dehydrogenase are LD1, LD2 , LD3, LD4and LD5

Value of serum enzyme assay in clinical practice
1. Diagnostic purpose
2. Differential diagnosis eg : MI – SGOT Increased LDH Increased
Pulmonary embolism – SGOT normal and LDH increased
3. Assessing prognosis
4. Early detection of disease eg:SGPTis increased in viral hepatitis before the occurance of jaundice

Lysosome –
Are cell organelles contain packets of enzyme (except in erythrocytes) surrounded by lipoprotein membrane. Acid phosphatase is used as a marker enzyme for this organelle.

Enzymes in lysosomes are
1) proteolytic enzymes
2) Nucleic acid hydrolyzing enzymes
3) Lipid hydrolyzing enzymes
4) Carbohydrate splitting enzymes
5) Acid phosphatase
6) Catalase etc.
When the membrane is ruptured, the enzymes are released into the cytoplasm and can hydrolyses the external substrate. Lysosomal enzyme action occurs in many types of allergic responses and arthritic conditions like gout.

Serum enzymes in heart diseases
Enzyme assays mainly carried out in myocardial infarction
Common enzymes assayed are]
Creatine kinase
Serum glutamate oxaloacetate transaminase(SGOT)
Lactatedehydrogenase (LDH)
I. Creatine kinase
Greater amount in heart muscle and skeletal muscles. None in RBC Kidney and liver. So its level is not affected by haemolysis.Normal value is 10-50 IU/ L More sensitive indicator in early stage of MI, More useful in sub endocardial infraction. No increase in activity in heart failure and coronary insufficiency.Iso enzymes of CK – MM. MB, BB (M-Muscle type, B – Brain type)

Increase in CK seen in
1. Myocardial infarction :MB-CK is specific.It rises by 6-8 hrs of infarction,peak in  24 hrs, down to normal by 3-4 days.
2. Progressive muscular atrophy (1-100w/ml)
3. Smaller increase can be seen in dermatomyositis.
4. Normal persons severely exercised may produce a higher value.
5.Duchenne muscular dystrophy –( autosomal recessive disorder, Proximal arms & legs are affected)

2. Transaminases
Clinically important forms are :
1. SGOT (Serum Glutamate oxaloacetate transaminase
2. SGPT (Serum glutamate pyruvate transaminase)
Normal values
SGPT – 0-45 IU/L
SGOT – 0-41 IU/L
In newborns value upto 120units for SGOT and 90units for SGPT must be considered normal
SGOT > 350 – Massive infarction
>150-High mortality rate
<50 – Low mortality rate

SGOT increase in
1.MI – rises after 8-12 hrs, reach peak levels in 18-36hrs & fall by 3-4 days
2.Trauma to skeletal muscles
3. Neuromuscular dystrophies
4. Normal persons a small excess of SGOT is seen (1.3 to 1)
Normal values in heart conditions without infarction such as angina, pericarditis, pulm embolism and with a/c abdominal conditions ( except in a/c pancreatitis – rise to 600 units within 24 hrs from onset).

SGPT increases in
1.In liver diseases SGPT increases higher than SGOT especially in infective hepatitis. It is the most sensitive diagnostic index. In infective hepatits, the increase begins in the prodromal period.
2. Jaundice due to obstruction of large bile duct, but usually do not exceed 200-300 units
3.In glandular fevers.

3.Lactate dehydrogenase :
Plentiful in cardiac & skeletal muscles, liver, kidney & RBC
Normal value : 60-200 IU/L .It has 5 isoenzymes : LD1 to LD5 Cardiac muscle & Redcells are richest in LD1 and LD5 is more in Liver & skeletal muscles. Normal serum is riche in LD1 & LD2 .So LD1 increases in MI & LD5 in Liver disease .Inhibitors of LD are oxulate & urea

Increased LD level in
1. MI (rises by 12-24hrs, highest 2-6 days, comes down by 2 weeks)
2. Liver diseases : infective hepatitis (not more than transaminases)
3. Leukemia, pernicious anaemia, megaloblastic & haemolytic anaemia
4. Renal diseases,
5. Cancer metastasis.

4.Gamma Glutamyl transpeptidase (G-GTP)
Catalyses transfer of the gamma-glutamyl group from one peptide to another or to an amino acid .Normal value – < 30 units/L at 300 .Highest activity in kidney, then in liver, lungs, pancrease etc. little in heart.

Increased level in
1. A/c MI : late occurance, peak value between : 7-11 days, last for a month. So useful in MI in later stages
2.Obstructive jaundice, cholangitis, cholecystitis,Ca. liver
3. Pancreatic diseases (not increased in bone disease).
4. Alcoholics

5.Histaminase
More in human heart muscle .Normal value – .12 to .76 p u/ml
Increased level in :
MI- rises within 6 hrs of infarction, persists for whole of first week. So used in early diagnosis of MI
Higher S. histaminase value associated with poor prognosis.

6.Cholinesterases.
Enzyme which hydrolyse esters of choline to give choline and the acid
Two types 1) True cholinesterase (found in nerve tissue & RBC)
2) Pseudo cholinesterase – Found in Liver heart, intestine,and plasma
Normal value : 2.17 – 5.17 IU/ml
Decreased value in :
Shock, uraemia, anaemia, TB, cancer, mal nutrition, Liver cell damage, (but normal in obstructive jaundice) poisoning by organophosphorus compounds.
Elevation of plasma pseudocholinesterase in a/c MI,Rise within 12 hrs. It is a sensitive index for determination of cellular necrosis in myocardium.
Serum enzymes in liver diseases

Routinely employed tests are
1. Serum transaminases
2. Serum Alkalinephosphatase.
1. SGPT :
Liver is rich in SGPT. Value is increased in infective hepatitis and in serum hepatitis. Increased in early stage of disease. So it is helpful in early detection and prevention.

2. Serum alkaline phosphatase (ALP)
More in bones and Liver, then in small intestine Kidney and in placenta
Normal value 3-13 KA units/100ml. It is used in differential diagnosis of jaundice. Normal in prehepatic jaundice, Increased in hepatic jaundice (30 KA units/100 ml) and Markedly increased in obstructive jaundice.
Increased level in:
Viral hepatitis
Liver abscess
Ca liver, Lymphoma

Enzymes	Haemolytic Jaundice	Hepatic Jaundice	Obstructive Jaundice
1. SGPT	Normal	Markedly    increased (500-1500 IU/L)	Increased (100-300 IU/L)
2. ALP	Normal	Increased slightly < 30 KA	Markedly   Increased More than 35 KA
3. 5- neucleotidase	Normal	Increased (slightly)	Markedly   Increased

3. Serum iso citrate dehydrogenase
Catalyses the reversible oxidation of isocitrate to oxoglutarate in the presence of manganous ions. Rich in Liver, heart, skeletal muscles
Normal value – 9 – 4 IU/L
Increased activity in :
1.Liver diseases (infective hepatitis – usually returns to normal by
the 3rd week after the onset of jaundice)
2.cirrhosis
3.Kwashiorkor
Normal in obstructive jaundice and MI

4. Serum cholinesterase
5. Serum ornithine Carbamoyl tranferase
Normal value – 8-20m – IU
Markedly increased in – Viral hepatitis .Slight elevation in obstructive jaundice, cirrhosis, metastatic cancer. specific and sensitive measure for hepatocellular injury.

6. Serum leucine amino peptidase
Normal value – 15-56 m IU/100ml
Markedly increased in Obstructive jaundice and cirrhosis with hepatomic Mild increase in viral hepatitis,

7. Serum alpha hydroxy butyrate dehydrogenase (SHBD)
Normal value 56-125 IU/L
More sensitive index of MI : Used to differentiate Liver disease and acute MI By taking the ration LDH/SHBD
Normal ratio – 1.18 to 1.6
< 1.18 in MI
1.16< in Liver diseases
In Liver diseases , SHBD is sometimes normal or small increase

8. Serum aldolase
Normal value : 1.5 – 7 IU/L
Increased value in :

• A/c infective hepatitis
• (normal in portal cirrhosis & obstructive jaundice)
• Progressive muscular dystrophy
• Myoglobinuria
• A/c pancreatitis
• Severe haemolytic anaemia
• After MI

9. Serum amylase – Decreased in liver diseases

10. Serum sorbitol dehydrogenase –
It is hepatospecific
Normal value : 0-.2 mIU
Increased in a/c viral hepatitis, carbon tetrachloride poisoning
Normal in C/c hepatitis & obstructive jaundice

11. Serum gamna – Glutamyl transferase
Serum enzymes in gastrointestinal diseases
1. Serum. amylase
Two methods for detection – 1) Amyloclastic
2) Saccharagenic
Normal value – 80-180 somogy units /100 ml
Increased in :(not exceeding 500 units) in perforated peptic ulcer
Intestinal obstruction
A/c pancrealitis (>1000 units), within 24 hrs. & returned to normal within 3 days
Mumps, other forms of parotitis, salivery duct stone.
Macroamylasemia
2. Serum lipase
Normal value : <150 units /L
Increased in :-
Perforated duodenal ulcer
Intestinal obstruction
A/c pancreatitis, at the time of onset of symptoms elevated level persist for 10 – 14 days, Gradual fall
In a/c pancreatitis duodenal juice amylase value is unaltered ,serum level is altered
Serum enzymes in muscle diseases
1.SGPT/ SGPT
2.Aldolase Increased in muscular dystrophy
3.CPK
Serum enzymes in bone diseases
Serum alkaline phosphatase
Normal value: 5-13 KA
Valuable in diseases of osteoblastic activity
Increased in –
Ricket
Osteomalacia
Hyperparathyroidism
Bone malignancies
Normal – in bone destruction
Decreased in – hypophosphatasia (defective calcification)

Serum enzymes in malignancies
1. Acid phosphatase :
2 types depending on activity in different PH.
1.A type of acid phosphatase : (PH – 5) found in prostate epithelium,
spleen, kidney, plasma, liver, pancrease.
2. A type of acid phosphatase: ( PH – 6 )found in erythrocyte
Normal value – 1-5 KA units/ 100ml
Increased in : –
Prostate cancer
Gaucher`s disease
Thrombocytosis
C/c granulocytic leukemia
Myeloproliferative disorders
No rise in lymphocytic leukemia or lymphomas

2. Beta glucoronidase
Most abundant in liver, spleen, endometrium, breast, adrenals
Normal value : 210 – 550 mIU in males: 90 – 400 mIU in females
Increased in :
Ca.urinary bladder
Ca head of pancrease. (very high)
Ca cervix without liver metastasis (very high)
Last trimester of pregnancy, falls to normal by about 5th post partum day.

Enzyme	Disease
S. A P A L PLD B Glucoronidase	Ca. prostate Metastasis in liver, osteoblastic metastasis bone, Jaundice due to Ca head of pancreaseWide spread malignancies Advanced leukemias Ca. urinary bladder

Glucose – 6 – Phosphate dehydrogenase (G-6- PD)

• Rich in red cells
• Normal value : 12-18 units /ml
• Increase in – MI (less marked than transaminases & LD)
• Main use of determination is to recognise an inherited deficiency of the enzyme in the red cells which causes haemolytic anaemia. There are 400 isoenzymes for G-6-PD.

G-6-PD deficiency
Common in Mediterranean population. It is sex linked, recessive, transmitted from mother to son. Deficiency gives partial protection against fulciparum malaria.

Verities of deficiency :

• Class I – enzyme activity is below 10% results in c/c haemolysis
• Class II – activity is below 10%, no chronic haemolysis
• Class III – Activity10-60%, haemolysis only after enposure to drugs
• Class IV – Normal functional activity
• Class V – Increased activity.

Causes :
a) Drugs. 1) Antimalarial – Quinine, primaquine, chloroquine.
2)Sulfagroup -Sulfanilamide,sulfapyridine,acetylsulfanilamide
3) Analgesics : Acetanilide, phenacetin, phenazone
4) Other drugs- Furozolidone, nitrofurantoin ,chloramphenicol paraaminosalicylic acid, naphthalene, vit. k
Drug induced haemolysis starts 24-72 hrs after administration of drugs.
b) Food – Favabeans
Lab. findings – Severe anaemia
– Red cells show Heinz bodies
– Haemoglobinuria
– Reticulocytosis
Reticuloaytes are generally rich in G6PD, the enzyme activity may be normal in the presence of active reticuloytosis. In such cases, the enzyme activity test should be repeated after reticulocytosis has subscribed.
Therapy – withdrawal of drugs
– Blood transfusion in sever cases

Recombinant dna technology :
It is a specific means of studying segments of DNA & how they work.
Help in :
1. Understanding molecular basic of number of diseases (eg : Thalassemia, familial hypercholesterolemia, sickle cell disease) enzyme mapping of DNA derived from fetal cells in amniotic fluid
2. Human proteins can be produced for therapy (eg : insulin, GH etc)
3. Produce proteins for vaccine
4. Diagnosis and prognosis of disease

Certain endonucleases, enzymes that cut DNA at specific DNA sequences within the molecule are important in DNA technology. These enzymes are called restriction endonucleases. By this we get a clear cut DNA mapping. The fragments produced can be isolated by electrophoresis & the neucleotide sequences can be studied. There by we can detect the point mutation.

References :
1.Harper’s Biochemistry
2.Practical clinical biochemistry by Harold Vurley
3.Practical clinical biochemictry by Ranjna Chawla
4.Text book of biochemistry by West
5.Hawk’s physiological chemistry
6.Biochemistry by Victor . L. Davidson
7.Text book of medical biochemistry by MN Chatterjea and Rana Shinde