Enzymes as diagnostic & therapeutic tool

Prepared by: Shaikh Abusufyan

Objectives

• List the clinically important enzymes and isoenzymes.

• State which enzymes and isoenzymes are found in which tissues

• Use of enzymes as diagnostic & therapeutic tool

Enzymes

Biological catalysisVery efficient –can increase reaction rates

at the order of x 10All are proteins- so liable to denaturation Specific to substratesPartly specific to tissues

Measurement of serum enzymes

Diagnostic enzymology

Enzymes are normally intracellular and LOW concentration

in blood

Enzyme release (leakage)in the blood indicates cell damage

(cell –death, hypoxia, intracellular toxicity)

Quantitative measure of cell/tissue damage

Fairly non invasive possible to do repeated tests

Organ specificity- but not absolute specificity

Information from enzymes measurements in serum

Presence of disease

Organs involved

Aetiology /nature of disease: differential diagnosis

Extent of disease-more damaged cells-more leaked enzymes in blood

Time course of disease

Enzymes routinely measured

NAME OF THE ENZYME PRESENT IN

Aspartate Amino transferase (AST)Serum glutamate-oxaloacetate transaminase (SGOT)

Heart and Liver

Alanine Amino transferase (ALT)Serum glutamate-pyruvate transaminase (SGPT)

Heart and Liver

Alkaline Phosphatase (ALP) Bone, intestine and other tissues

Acid Phosphatase (ACP) Prostate

glutamyl Transferase ( GT)Liver

Creatine kinase (CK) Muscle Including cardiac muscle

Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC

AmylasePancreas

Measurement of enzyme activity

• Enzyme activity is expressed in International unit (IU)

It corresponds to the amount of enzymes that catalyzes the conversion of one micromole (mol) of substrate to product per minute

LACTATE DEHYDROGENASE (LDH)

Pyruvate Lactate (anaerobic glycolysis)

LDH is elevated in myocardial infarction, blood disorders

It is a tetrameric protein and made of two types of subunits namely H = Heart, M = skeletal muscle

It exists as 5 different isoenzymes with various combinations of H and M subunits

Isoenzyme name

Composition Composition Present in Elevated in

LDH1 ( H4) HHHH

Myocardium, RBC

myocardial infarction

LDH2 (H3M1) HHHM Myocardium, RBC

LDH3 (H2M2) HHMM Kidney, Skeletal muscle

LDH4 (H1M3) HMMM Kidney, Skeletal muscle

LDH5 (M4) MMMM Skeletal muscle, Liver

Skeletal muscle and liver diseases

Creatine + ATP phosphocreatine + ADP(Phosphocreatine – serves as energy reserve during muscle

contraction)

Creatine kinase is a dimer made of 2 monomers

Skeletal muscle contains M subunit, Brain contains B subunits

Three different isoenzymes are formed

CREATINE KINASE (CK)

Isoenzyme name Composition Present in Elevated in

CK-1 BB Brain CNS diseases

CK-2 MB Myocardium/ Heart

Acute myocardial infarction

CK-3 MMSkeletal muscle, Myocardium

ALANINE TRANSAMINASE (ALT) or SGPT AND ASPARTATE TRANSAMINASE( AST) or SGOT

Alanine transaminase (ALT) and Aspartate

transaminase (AST) enzymes are the most

abundantly present in the liver

Elevated in blood as a result of leakage from damaged

cells

Measurement of these transaminases is useful for the

diagnosis of liver diseases

ALANINE TRANSAMINASE (ALT) AND ASPARTATE TRANSAMINASE ( AST).......

● In viral hepatitis the enzyme levels are increased 20-50 times

● Alanine transaminase (ALT) increase is specific for liver damage involving hepatocellular damage

● Aspartate transaminase (AST) is moderately increased in Muscular dystrophy and acute myocardial infarction

ENZYMES AS A DRUG TARGET

Renin

Renin:

● Enzyme released by Juxtaglomerular cells of the kidney

● Maintain the BP.

● Role-

Conversion of angiotensinogen to angiotensin- I

Increases the BP

ACE

ACE:

Enzyme relesed by Adrenal cortex of the kidney

● Role:

Conversion of angiotensin- I to angiotensin- II

Increases the BP

Renin- agiotensin system

Monoamine oxidase (MAO)

● It is a membrane-bound mitochondrial enzyme that oxidatively deaminates primary amines to aldehydes.

● Location:

- liver, kidney, intestine and nervous tissue

● Substrates:

- Catecholamines (dopamine, noradrenaline and adrenaline), tyramine, phenylephrine and tryptophan derivatives (5-hydroxytryptamine and tryptamine).

There are 2 type of MAO

1. MAO-A

- It oxidise mainly NA & 5-HT

- Inhibited selectively by v low concentration of

chlorgyline & moclobemide (Antidepressant).

MAO-B:

- It oxidizes DA in the brain

- selectively inhibited by selegiline (Antiparkinson).

● Liver contain equal amount of both MAO enzyme

while brain contain MAO- B.

cholinesterase

2 main types of cholinesterase r

1. Acetylcholinesterase (AchE) or true cholinesterase

2. Butyrocholinesterase (BuChE) or Pseudocholinesterase

Responsible for degradation of Ach

1.Acetylcholinesterase (AchE) or true cholinesterase

Location:

- Neurone, ganglia and myoneural junction.

Function:

- Rapidly hydrolyzes acetylcholine

2. Butyrocholinesterase (BuChE) or Pseudocholinesterase

Location:

Plasma, RBCs, liver, glia and other organs

Function:

- hydrolysis of Ach slowly

The activity of cholinesterase is inhibited by anticholinesterase drugs. (Use in alzheimer disease, glaucoma)

HMG CoA reductase

- It is the rate-limiting enzyme in cholesterol biosynthesis.

- Statins inhibit this enzyme, lowering cytoplasmic cholesterol.

- And used in the treatment of Hyperlipedemia

Cyclo-oxygenase (COX)

There are two main isoforms, COX-1 and COX-2.

- COX-1 is a constitutive enzyme which is present in platelets and other cells.

- COX-2 is an inducible form, which is produced in response to cytokine stimulation in areas of inflammation.

- Produces large amounts of prostaglandins & responsible for inflammation.

Cyclo-oxygenase (COX)...

- CoX inhibitores are used as antiinflammatory

- Eg: Diclofenac, Cilicoxif ect

lanosterol 14-α-demethylase

- It is a fungal cytochrome-haem P450 enzyme,

- Responsible for synthesise of ergosterol from lanosterole.

Inhibition of enzyme

disrupts the acyl chains of fungal membrane phospholipids,

increasing membrane fluidity and causes membrane leakage and dysfunction of membrane-bound enzymes Antifungal activity.

lanosterol 14-α-demethylase

- lanosterol 14-α-demethylase inhibitor: Imidazoles (Antifungal)

Squalene epoxidase

● It is involved in fungal ergosterol biosynthesis.

● ergosterol squaline

- Inhibition of the enzyme lead to accumulation of squaline within the cell

toxic to the organism

● Eg. of squaline epoxidase inhibitor: Terbinafine (Antifungal)

squaline epoxidase

Dihydrofolate reductase

- Responsible for conversion of dihydrofolate to tetrahydrofolate

folic acid synthesis

- Folic acid is required in the synthesis of thymidylate (pyrimidine) and of purine nucleotides and thus for DNA synthesis

Dihydrofolate reductase Inhibitores:

- Methrotrexate (anticancer),

- pyrimethamine (antiprotozoal, antimalarial),

- Trimethoprim (antibacterial)

Thymidylate synthase

● Responsible for synthesis of thymidylate

Inhibitor of Thymidylate synthase

- 5-Fluorouracil & 5-fluorodeoxyuridine (cancer)

Retrovirus

Reverse transcriptase

(Viral RNA dependent DNA polymerase)

DNA copy of the viral RNA

HIV Reverse Transcriptase

HIV Reverse Transcriptase Inhibitors

MOA:

-Competitive inhibition of HIV-1 reverse transcriptase

- Incorporated into the growing viral DNA chain → cause termination

- Most have activity against HIV-2 as well as HIV-1.

- Eg. 3’-azido-2’,3’-dideoxythymidine (AZT)

IV. Enzymes as drug targets

Enzyme Drug/ Inhibitores/Significant

- Mono amine oxidase Ephedrine, Amphetamine

(MAO) (Increases the level of catecholamine)

- Acetyl cholinesterase Neostigmine (Used in glaucoma).

- HMG CoA reductase Lovastatin, Compactin (Inhibit

cholestrol biosynthesis)

- ACE Enalapril, Captopril (Control the BP)

IV. Enzymes as drug targets

Enzyme Drug/ Inhibitores/Significant

- Cyclooxygenase Paracetamol (Non-

(COX) selective NSAIDS), Aspirine

(Antiplateletes)

Cilicoxibe (Selective COX-II

Inhibitores)lanosterol Imidazoles (Antifungal)14-α-demethylase (a fungal cytochrome-haem P450 enzyme)

squalene epoxidase Terbinafine (Antifungal)

IV. Enzymes as drug targets

Enzyme targeting DrugDihydrofolate reductase

Antifolates: methrotrexate (cancer), (Folate metabolism)

pyrimethamine (protozoa, malaria)

trimethoprim (bacteria)

Thymidylate synthase 5-Fluorouracil &

(Pyrimidine metabolism) 5-fluorodeoxyuridine (cancer)

HIV-Reverse transcriptase 3’-azido-2’,3’-dideoxythymidine (AZT)

HIV proteases Ritonavir, saquinavir (clinical trial phase)

SUMMARY

Enzymes are biological catalysts present in every cell of the body.

Isoenzyme patterns can give information about organ-specific

disease.

Important enzymes in the investigation of heart disease are CK, LDH

and AST.

Important enzymes in the investigation of liver disease are AST,

ALT, alkaline phosphatase.

- Creatine kinase has three isoenzymes: CK-MM, CK-MB and

CK-BB.

- LDH has five isoenzymes.

- Increased levels of acid phosphatase are found in prostate cancer.

Thank you