1. CLINICAL ENZYMOLOGY

2. Learning objectives and outcomes
Uses of Enzymes in Diagnosis, prognosis and treatment of diseases.
Enzymes as analytical reagents
Isoenzymes.
Learning Outcomes
List diagnostic applications of enzymes.
Define Isoenzymes.
Describe the role of isoenzymes in diagnosis of diseases.
List therapeutic applications of enzymes
State the use of enzymes as reagents.
Describe the role of enzymes in genetic disorders.

3. Quantitative estimation of enzymes in serum is used to
CLINICAL ENZYMOLOGY
It deals with quantitative estimation of enzymes in body fluids in normal and disease conditions.
Quantitative estimation of enzymes in serum is used to
Confirm the diagnosis
Effectiveness of treatment i.e., prognosis.

4. Plasma Enzymes Functional Enzymes
They are present in plasma at higher level than in most of tissues and they perform functions in plasma.
Pseudocholinesterase. Reacts with succinylcholine, a short-acting muscle relaxant.
Genetic deficiency  extended reaction to the drug.
Cholinesterase levels low  insecticide poisoning 4

5. Plasma Enzymes Non-functional Enzymes :
Present only at minimal concentrations in normals.
No known function in plasma.
Contributed by.
Turnover of tissues: increased tissue breakdown or damage to tissues increased enzymes.
Block in the secretory pathway : plasma level of secretory enzyme is increased.
Increased synthesis increased levels of enzymes

6. Non-functional Enzymes
Enzyme activities differ in different tissues - patterns of release are tissue specific
Amount of enzyme released is proportional to the mass of the affected tissue

7. Examples of causes of altered enzyme activity
Diseased or damaged tissues  “leak” tissue specific enzymes into the blood ….. Myocardial Infarction---CK MB,LDH,AST
Increased synthesis Increased osteoblasts bone isoenzyme of alkaline phosphatase
Microsomal enzyme induction by Alcohol, antiepileptic drugs   γ-glutamyl transferase
Obstruction to secretion – Pancreatitis—Lipase , amylase
Cell proliferation - cancer, metastases
Decreased clearance-- 7

8. Use of Serum Enzymes in Diagnosis
The simple presence of a diagnostic enzyme in serum does not constitute evidence for a specific disease.
What is important is
the level of these serum enzymes
the ratio of component isoenzymes (when applicable)
the timing of their appearance during or following a specific pathologic state.

9. Use of Serum Enzymes in Diagnosis
The normal levels of these enzymes will depend on age, gender, and race.
Monitor more than one enzyme that may be diagnostic for a disease (e.g., the use of creatine kinase and LDH following MI)

10. Criteria for enzyme in a diagnostic test
The enzyme should be stable so that a refrigerated sample may still show the enzyme activity.
The enzyme should be easily and cheaply assayed.
It should have reasonably high activity so that it can be detected at relatively low levels.
It should have a degree of tissue specificity, directing you to a likely pathology in a particular tissue.
The level of the enzyme in serum should also reflect the severity of the pathology.

11. Other considerations for the usefulness of diagnostic enzymes
The time it takes for a serum enzyme to appear during or following a pathologic state. An enzyme that appears early in the course of a disease would obviously allow you to make a therapeutic intervention that much earlier in an effort to arrest or ameliorate the condition.

12. Enzymes used to diagnose disease

13. Serum Enzymes can monitor the course of diseases
Viral hepatitis can result in elevated serum levels of ALT, indicating liver damage.
If the elevated ALT activities remain high over time, it is possible that a chronic form of hepatitis may be present.
Thus, serum enzyme levels can be used in the diagnosis of both acute and chronic diseases.

14. Both α-amylase and lipase are important blood markers to help diagnose acute pancreatitis
The serum level of α-amylase will increase in the first 12 hours following the onset of acute pancreatitis.
During the next 48 to 72 hours, the levels will
usually fall back to normal values.
Serum lipase levels also rise, but they remain elevated after the α-amylase levels have returned to normal and may take 7 to 10 days to normalize.

15. Serum enzymes can be used to monitor the effects of therapeutic treatments for disease.
A patient with breast cancer may develop metastases to bone, with subsequent bone destruction shown by elevated serum alkaline phosphatase.
Chemotherapy for the patient with breast cancer may result in decreased serum alkaline phosphatase as bone destruction declines or is halted.
Analysis of serum alkaline phosphatase following remission of the cancer would be advisable to rule out the possibility of recurrence of the disease.

16. Isoenzymes

17. What is an isoenzyme ? Isoenzymes are
Physically distinct forms of the same enzyme.
differences in their amino acid sequences or by the presence of different posttranslational modifications in each isozyme.
Catalyze same reaction but differ in physiochemical properties.
They are tissue specific.
The ability to control which isozymes are expressed in a particular cell allows each cell to adjust the enzyme activity based on the specific condition that exist in the cell. = 17

18. An example of isoenzyme
All of the lactate dehydrogenase isoenzymes catalyze the interconversion between lactate and pyruvate

19. Lactate Dehydrogenase is composed of four monomers

20. Separation of Iso-enzymes
The serum lactate dehydrogenase (LDH) iso-enzyme pattern is obtained by subjecting serum to electrophoresis at pH 8.6.
Separates into five bands. {LDH1, LDH2, LDH3, LDH4 and LDH5}
Myocardial infarction endothelial cells rupture release of contents into the bloodstream  increased serum levels of LD1 and LD2

21. Serum LDH isoenzymes and MI
Heart muscle is particularly enriched in the LD1 isoenzyme, followed by the LD2 isoenzyme.
However, in normal blood, the LD2 isoenzyme predominates.
Following MI, there is a shift in the relative values of these two isoenzymes so that there is a predominance of LD1 to LD2 in serum.
Dotted line = normal pattern; solid line = 12–24 hours after MI.
The elevation of LDH in the blood
Develops more slowly following an MI than does the elevation of creatine kinase. As well, the elevated LDH levels persist longer in blood, making LDH a useful tool in the diagnosis of an MI
that occurred a week previously .. 21

22. Creatine Phosphokinase (CK) Isoenzyme
CK is a dimer. It consist of two subunits M and B. CK has three isoenzymic forms They differ in subunit composition. CK1 : BB CK2: MB CK3 : MM Diagnosis of a myocardial infarction can be confirmed by noting an elevation and peaking of creatine kinase (MB)

23. Enzymes as Reagents
Amounts of various compounds are measured using enzymes.

24. Coupled Assay : Hexokinase Method for Estimation of Glucose in Plasma

25. Enzymes in Genetic Disease
A defective gene can result in the lack or deficiency of a specific enzyme activity.
Hexosaminidase A deficiency failure to remove N-acetylgalactosamine from gangliosidesaccumulation of non- degraded lipid byproduct, with lysosomal and cellular swelling and cell death neurologic signs and symptoms(blindness, retardation, and death) can occur by a very early age. Tay-Sachs disease . 25

26. USE OF ENZYMES AS THERAPEUTIC AGENTS
Recombinant enzymes can be used in various replacement therapies.
Recombinant Human DNAse reduces the viscosity of sputum in Cystic fibrosis patients.
Cystic fibrosis (CF) lung disease is characterized by a chronic bacterial airway infection associated with a massive influx of neutrophils.
The rapid turnover of airway neutrophils leads to the accumulation of large amounts of extracellular DNA which is thought to hinder the clearance of respiratory mucus 26

27. USE OF ENZYMES AS THERAPEUTIC AGENTS
Proteolytic enzyme papain that can be used to clear away dead cells from an area of necrosis.
The use of the enzyme streptokinase, urokinase, or tissue plasminogen activating factor as “clot busters” in stroke or MI. These proteins activate the plasma protease plasmin, which can dissolve thrombi blocking the cerebral or coronary vessels, thereby restoring blood flow and ending ischemia.