1. Clinical (Diagnostic & Therapeutic ) importance of Enzymes and isoenzymes

2. Objectives List the clinically important enzymes and isoenzymes.
State which of the enzymes and isoenzymes are found in which tissues
Outline different ways of measuring plasma enzyme
Describe plasma enzyme changes in different diseases.

3. Circulatory system enzymes serum enzymes
Functional serum enzyme
responsible for reaction taking place in blood e.g; clotting of blood
Non functional serum enzymes
do not have their function in blood but they are present because of wear and tear of the tissue IN CONSTANT level
Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseasesSmall amounts of intracellular enzymes are present in the blood as a result of normal cell turnover. When damage to cells occurs, increased amounts of enzymes will be released and their concentrations in the blood will rise.

4. INTRODUCTION
Injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream.
Elevated enzyme levels are often indicators of tissue problems, and are used in the diagnosis of diseases.
Enzyme activities in the body fluids are altered by pathological processes so, its measurement is used for disease investigation.
Serum enzyme level may be increased by disease that cause increase in its rate of release or decrease in their rate of excretion.

5. Measurement of serum enzymes
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
Organ specificity- but not absolute specificity in spite of same gene content.
Most enzymes are present in most cells-differing amounts
Time course of disease

6. 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)

7. Enzymes routinely measured
NAME OF THE ENZYME
PRESENT IN
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

8. Myocardial Infarction

9. Enzyme Assays that are Carried out in Mayocardial Infarction
Commonly done:
Creatine kinase (CK)
Aspartate Amino transferase (AST)
Lactate Dehydrogenase (LDH)

10. Myocardial Infarction ( MI )
Necrosis of the myocardium, but not angina pectoris release of CK, AST and LDH into the circulation.
CK is the first to rise (activity within 6 h of MI ).
Total CK reaches a peak at h.
In uncomplicated cases, CK returns to normal within 3 days.

11. Myocardial Infarction ( MI )
Serum AST  more slowly ( maximum activity within 48 h) and returns to normal in 4-5 days.
No significant elevation in LDH seen for the 1st 24 h (reaches maximum at about 3 days & remain  for up to 8 days).
The enzyme is relatively non specific to myocardial tissue.

12. Liver Diseases Hepatic Necrosis Hepatitis Cholestasis Jaundice
Hepatocellular Damage

13. Liver Enzymes ( ALT, AST, GGT, ALP, LDH)
Measurement of serum enzyme activities for :
a - Differential Diagnosis of Jaundice.
b - Monitoring of drug toxicity.
ALT is more specific than AST.
Hepatocellular disease has only modest effect on ALP & GGT (up to 3 times the upper limit of normal)
In Cholestasis, Higher values of ALP & GGT due to  synthesis

14. Alanine aminotransferase (ALT)
Widely distributed, although the largest amounts found in the liver.
Smaller amounts occur in the heart but usually remains normal after MI .
Congestive cardiac failure release from the liver
More specific for liver disease than AST.

15. Aspartate aminotransferase (AST)
This enzyme is widely distributed in the body.
Main sources: Heart, liver, skeletal muscle, and kidney.
Useful in the diagnosis of MI, liver disorders and muscle damage.
Causes of serum AST levels:.
Liver diseases: Hepatitis, hepatic necrosis , cholestasis
Cardiac disease: Myocardial Infarction.
Diseases of skeletal muscle: Crush injury,trauma,myopathy
From Erythrocytes: Hemolysis

16. Alkaline phosphatase (ALP)
Widely distributed, high concentrations in intestines, liver, bone, spleen, placenta and kidney.
The main sources of serum ALP are the hepatobiliary tree and bone disorders.
Elevated levels during healing of fractures , active growth and during the 3rd trimester of pregnancy.
 serum ALP activity in liver disease is mainly due to Cholestasis.
Decreased levels are found in the inherited condition

17. Bone Enzymes - ( Alkaline Phosphatase) ALP
ALP enzyme is usually normal in Osteoporosis as osteoblastic activity is not increased
Modest  of ALP in Osteomalacia and Rickets
Healing fractures  Transient  of ALP
1ry & 2ry Hyperparathyroidism   of ALP
In Paget’s disease of bone    of ALP (10 times)
1ry & 2ry bone tumors   of ALP (5 times normal)

18. Alkaline phosphatase (ALP)
Causes of increased serum alkaline phosphatase enzyme activity:
Physiological :
Bone disease:
Hepatobiliary disease:
Others:
- Infancy
- Puberty
- Pregnancy
- Intestinal isoenzymes
- Hyperparathyroidism
- Osteomalacia, rickets
- Paget’s disease of bone
- Osteomyelitis
- Hepatitis
- Cholestasis
- Cirrhosis
Carcinoma of the bronchus

19. Acid phosphatase (ACP)
Found in prostate, bone, liver, spleen, kidney, RBCs and platelets
Primarily used to diagnose prostate cancer .
 In other prostatic conditions e.g. prostatitis, benign prostatic hypertrophy.
In other non prostatic conditions e.g. hemolysis, Paget’s disease, metastatic carcinoma of the breast & Gaucher’s disease.
Prostate- Specific Antigen(PSA): an enzyme occurs in prostatic tissue and  in cases of metastatic carcinoma

20. Amylase (AMS) HYDROLASES THAT SPLIT COMPLEX POLYSACCHARIDES.
- CA+2 REQUIRING METALLOENZYME
NORMAL LEVEL: U/L INCREASES DRASTICALLY IN ACUTE PANCREATITIS ALSO IN MUMPS
SOURCES :
1.    PANCREAS (P-TYPE)
2.    SALIVARY GLANDS (S-TYPE)
3.    INTESTINAL MALIGNANCY   
CLINICAL SIGNIFICANCE : DIAGNOSIS AND MONITORING OF PANCREATITIS

21. Lipase (LPS)
Breaks down fat into monoacylglycerol and free fatty acids.
Primarily from the pancreas.
Used to diagnose acute pancreatitis.
Pancreatic lipases :
Almost exclusively used clinically in the investigation of pancreatitis.
- Increase within hours of acute attack.
- May remain elevated for many days .
- More specific to acute pancreatitis than amylase.

22. Specificity of Enzymes :
Greater specificity is achieved in three ways:
Interpreting investigations in the light of clinical features
Isoenzyme determination:
 AST may be due to MI or Hepatitis so, it makes
confusion in diagnosis to be confirmed by LDH levels.
-  ALP in Cholestasis & bone diseases :
- Differentiated by  bilirubin & transaminase levels in Cholestasis .
- Confirmed by  GGT in Cholestasis.

23. Conditions in which level of activity in serum is elevated
NAME OF THE ENZYME
Conditions in which level of activity in serum is elevated
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate transaminase (SGOT)
Myocardial infarction, Liver disease especially with liver cell damage
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate transaminase (SGPT)
Liver disease especially with liver cell damage
Alkaline Phosphatase (ALP)
Liver disease- biliary obstruction
Osteoblastic bone disease-rickets

24. Acid Phosphatase (ACP)
Prostatic carcinoma
 glutamyl Transferase ( GT)
Liver disorder like liver cirrhosis
Creatine kinase (CK)
Myocardial infarction and skeletal muscle disease(muscular dystrophy
Lactate Dehydrogenase (LDH)
Myocardial infarction, other diseases like liver disease.some blood diseases
 Amylase
Acute pancreatitis

25. ISOENZYMES Catalyze the same reaction Two or more polypeptide chains
Different polypeptide chains are products of different genes
Differ in AA sequence and physical properties
May be separable on the basis of charge
Are tissue specific

26. Diagram illustrating the origin of Isoenzymes

27. LACTATE DEHYDROGENASE (LDH)
converts pyruvate to lactate (and vice versa)
LDH occurs as a tetramer of 2 different subunits H & M:
(product of 2 diff. gene)
normal LDH2 is high in serum but after MI LDH1 rises
increase of total is seen in
hemolytic anemia,hepatocellular damage,carcinomas,leukemias & any condition of necrosis.

28. , five isoenzymes of LDH that occurs as a dimer of 2 different subunits H &M

29. Isoenzyme name
Composition
Present in
Elevated in
LDH1
( H4)
HHHH
Myocardium, RBC
myocardial infarction
LDH2
(H3M1)
HHHM
LDH3
(H2M2)
HHMM
Kidney, Skeletal muscle
LDH4
(H1M3)
HMMM
LDH5
(M4)
MMMM
Skeletal muscle, Liver
Skeletal muscle and liver diseases

30. Creatine kinase (CK)
Creatine kinase is associated with ATP regeneration in muscle and nervous tissue.
Elevated blood levels of CK are used as indicators of MI, muscular dystrophy, and stroke.
CK occurs as a dimer of 2 different subunits, M and B.
        - CK-BB: Brain .
        - CK-MB: cardiac muscle.
        - CK-MM: Muscle type.
CK-MB is released from cardiac muscle cells after MI.

31. Isoenzyme name
Composition
Present in
Elevated in
CK-1 BB
Brain
CNS diseases
CK-2 MB
Myocardium/ Heart
Acute myocardial infarction
CK-3 MM
Skeletal muscle, Myocardium

32. „ Cardiac enzymes“
The figure is adopted from the book: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, ISBN 0‑471‑15451‑2

33. ENZYMES IN THERAPY
Substitution of missing production of digestive enzymes – digestive enzymes – pepsin trypsin…
Removal of deposits of death tissue or fibrin (e.g. in lungs, eyes), treatment of skin defects – proteinases, nucleases, collagenase
Acceleration of fibrinolysis in lungs embolization (activation of plasmin and plasminogen) –
streptokinase, urokinase

34. Thank you

35. Quiz
1.An activated enzyme consisting of polypeptide chain and a cofactor is called:
A.Apoenzyme B.Holoenzyme
2.Which one forms the raw material for coenzymes?
A.Viitamins B.Carbohydrates
3.A cofactor made of inorganic ion which is detachable is called
A.Prosthetic group B.Coenzyme

36. 4. Enzymes _________ the activation energy of a chemical reaction
A.Increases B.Decreases
5.  Three dimensional dcavity bearing a specific charge by which the enzyme reacts with its substrate is called
A.Active site B.Binding site
6. Which step causes activation of catalytic site of an enzyme?
A.Change in pH of the surroundings.
B.Formation of Enzyme Susstrate complex

37. 7. If the concentration of enzyme is kept constant and amount of substrate is increased a point is reached where increase in substrates concentration does not affect the reaction rate because of
A.Enzymes get denatured at higher substrate conc.
All the active sites on enzyme molecule are occupied.

38. 7. f more substrate to already occurring enzymatic reaction is added and there is no effect on the rate of the reaction what is the form given to this situation:
A.Saturation B.Denaturation
8.Optimal temperature of enzymes present in human body is
A.27C B.37C

39. 9.  A chemical substance which can react (in place of substrate) with the enzyme but is not transformed into product/s and thus blocks the active site temporarily or permanently is called
A.Co-enzyme B.Blocker C.Inhibitor
10 The structure of an enzyme is altered by:
A.Irreversible inhibitor B.Reversible inhibitor