1. Enzymes as diagnostic & therapeutic tool Prepared by: Shaikh Abusufyan

2. 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

3. Enzymes  Biological catalysis  Very efficient –can increase reaction rates at the order of x 10  All are proteins- so liable to denaturation  Specific to substrates  Partly specific to tissues

4. 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

5. 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

6. Enzymes routinely measured NAME OF THE ENZYMEPRESENT 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  Amylase Pancreas

7. 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

8. 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

9. Isoenzyme name Composition Present inElevated in LDH1( H 4 ) HHHH Myocardium, RBC myocardial infarction LDH2(H 3 M 1 ) HHHM Myocardium, RBC LDH3 (H 2 M 2 ) HHMM Kidney, Skeletal muscle LDH4 (H 1 M 3 )HMMM Kidney, Skeletal muscle LDH5(M 4 )MMMMSkeletal muscle, Liver Skeletal muscle and liver diseases

10. 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)

11. Isoenzyme name CompositionPresent inElevated in CK-1BBBrainCNS diseases CK-2MB Myocardium / Heart Acute myocardial infarction CK-3MM Skeletal muscle, Myocardium

12. 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

13. 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

14. ENZYMES AS A DRUG TARGET

15. Renin Renin: ● Enzyme released by Juxtaglomerular cells of the kidney ● Maintain the BP. ● Role- Conversion of angiotensinogen to angiotensin- I Increases the BP

16. ACE ACE: Enzyme relesed by Adrenal cortex of the kidney ● Role: Conversion of angiotensin- I to angiotensin- II Increases the BP

17. Renin- agiotensin system

18. 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).

19. 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).

20. 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.

21. cholinesterase 2 main types of cholinesterase r 1. Acetylcholinesterase (AchE) or true cholinesterase 2. Butyrocholinesterase (BuChE) or Pseudocholinesterase Responsible for degradation of Ach

22. 1.Acetylcholinesterase (AchE) or true cholinesterase Location: - Neurone, ganglia and myoneural junction. Function: - Rapidly hydrolyzes acetylcholine

23. 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)

24. 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

25. 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.

26. Cyclo-oxygenase (COX)... - CoX inhibitores are used as antiinflammatory - Eg: Diclofenac, Cilicoxif ect

27. 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.

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

29. 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

30. 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

31. Dihydrofolate reductase Inhibitores: - Methrotrexate (anticancer), - pyrimethamine (antiprotozoal, antimalarial), - Trimethoprim (antibacterial)

32. Thymidylate synthase ● Responsible for synthesis of thymidylate Inhibitor of Thymidylate synthase - 5-Fluorouracil & 5-fluorodeoxyuridine (cancer)

33. Retrovirus Reverse transcriptase (Viral RNA dependent DNA polymerase) DNA copy of the viral RNA HIV Reverse Transcriptase

34. 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)

35. 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)

36. 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)

37. IV. Enzymes as drug targets Enzyme targeting Drug Dihydrofolate 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)

38. 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.

39. - 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.

40. Thank you