Enzymes in Body Fluids Lecture outline Dr. Essam H. Jiffri

Upon completion of this chapter, the following will be covered: - Introduction - Factors affecting serum enzyme Rate of entry of enzymes into blood Enzyme inhibitors Clearance of enzyme

Specificity of serum enzyme measurements Test results and clinical features Test pattern recognition Isoenzymes

Major enzymes of diagnostic interest Phosphatases Transaminases G-Glutamyl transferase Amylase and Lipase Cholinesterase Creatine kinase Lactate dehydrogenase

Serum enzymes in disease Myocardial infarction Muscle disease Liver disease Bone disease Enzymes in urine Haematological disorders Tissue enzymes

- Methods for the determination in serum and urine - Case histories

Introduction -Enzymes are protein catalysts which are found in small amounts, mainly within cells such as clotting factors. -Most enzymes with diagnostic applications function within the cells in which they are synthesized and since they have a large molecular mass, they do not cross cell membranes readily.

Introduction -Normally only small quantities of intracellular enzymes leak from cells into blood or other body fluids. -The amounts are too low for enzyme mass to be measured, their activities can be monitored.

Introduction -Most clinical enzyme measurements using serum, occasionally other fluids, such as urine and gut secretions, are investigated. -In general, increased rather than decreased activities of enzymes are of diagnostic interest in body.

Factors Affecting Serum Enzyme Activities The activity of an enzyme in the circulation depends on a balance between: 1- The rate of release from tissues (rate of entry of enzyme into blood), 2- The presence of inhibitors, and 3- The rate of removal

1.Rate of entry of enzyme into blood The main factors affecting the rate of entry of enzyme into blood are: The rate of synthesis The mass of enzyme producing cells, and Cell damage

Enzyme Synthesis -The rate of enzyme synthesis is increased particularly in conditions affecting the liver. -Biliary obstruction causes increased synthesis of enzyme located in the hepatobiliary tree. -Some agents induce increased synthesis of enzyme by hepatocytes, example ( phenobarbitone and phenyrtoin).

Mass of Enzyme Producing cells -Serum alkaline phosphatase originating from bone reflects osteoblastic activity, this is increased (leading to high serum alkaline phosphatase activity in children who are actively growing or where bone disease is present in which increased osteoblastic activity occurs, e.g. Paget’s disease).

Mass of Enzyme Producing cells -The placenta produces alkaline phosphatase, causing increased levels in the third trimester. -Metastatic carcinoma of the prostate produces increased acid phosphatase levels.

Cell Damage -Increased amounts may leak from tissues that are inflamed, necrotic, or metabolically abnormal, leading to increased serum levels. -Examples include raised transaminase levels in hepatitis, creatine kinase (CK) following myocardial infarction and lactate dehydrogenase (LDH).

2. Enzyme Inhibitors -Organophosphate poisoning which irreversibly inhibits cholinesterase.

3. Clearance of Enzymes -Serum enzyme activity is also affected by the rate of removal of enzymes from the circulation, and understanding of these mechanisms is incomplete. -Possibilities include removal by reticuloendothelial system. -Renal excretion appears unimportant, except for amylase which is small enough to be cleared by the kidney.

FACTORS AFFECTING SERUM ENZYME ACTIVITY Tissue damage Rate of synthesis Mass of enzyme producing tissue Rate of entry into blood Inhibition Serum enzyme activity Rate of removal Clearance Inactivation

Specificity of Serum Enzyme Measurements - Many enzymes which are used diagnostically originate from more than one tissue which potentially limits their specificity.

Main applications Origin Enzyme Metastatic carcinoma of prostate Prostate, erythrocytes Acid phosphatase Hepatocelluar disease Hepatocytes Kidney Alanine aminotransferase (ALT, SGPT) Cholestatic disease Bone disease Hepatobiliary tree Bone GI tract, placenta, kidney Alkaline phosphatase (ALP) Acute pancreatitis Pancrease Pancrease, Salivary glands Lipase Amylase Myocardial infarction Muscle disease Cardiac muscle Skeletal muscle Aspartate aminotransferase (AST, SGOT) Organophosphorous poisoning Liver Cholinesterase Skeletal Muscle Heart Muscle Brain Creatine kinase (CK) Cholestasis Alcohol abuse Kideny y-Glutamyl transferase (GGT) Erythrocytes, liver Lactate dehydrogenase (LDH)

Specificity of Serum Enzyme Measurements - Increased serum CK could be due to myocardial infarction or skeletal muscle disease, and increased LDH occurs through multiple causes. - This would limit the usefulness of enzyme measurements if their specificity was not increased.

Specificity of Serum Enzyme Measurements - Greater specificity is achieved in three ways: Interpreting investigations in the light of clinical features (test results and clinical features). Test pattern recognition, Isoenzyme determination.

Test Results and Clinical Features Serum aspartate aminotransferase (AST) activity may be raised due to myocardial infarction or because of diseases affecting hepatocytes, such as viral hepatitis. - Occasionally, increased AST may originate from the liver because of complications of myocardial infarction, such as congestive cardiac failure.

Test Pattern Recognition - Investigations are rarely done in isolation and recognition of test patterns may aid differential diagnosis. - Alkaline phosphatase is raised in cholestasis and bone disease, in cholestasis, there are often increases in bilirubin and transaminase levels, while these do not occur in bone disease.

Test Pattern Recognition -If an isolated increase in alkaline phosphatase occurs, the estimation of gamma-glutamyl- tranferase may be helpful, as high serum activities of this enzyme occur in cholestasis while levels are normal in bone disease.

Isoenzymes Multiple forms of enzymes (isoenzymes) occur which have similar catalytic activities but different structures. - Different isoenzymes are often organ-specific and their determination may improve the specificity of enzyme tests.

Isoenzymes - The heterogeneity of some isoenzymes is due to different protein subunits which are coded for by separate genes. - Lactate dehydrogenase has four subunits of two different types (H and M), five isoenzymes occur, H4 originating from the heart and M4 from the liver.

Isoenzymes Creatine kinase has two subunits, M and B; three isoenzymes occur, BB from brain, MM from skeletal muscle and MB from the heart.

Isoenzymes - Isoenzymes may be differentiated because of different physicochemical properties by techniques such as: Electrophoresis Immunochemical properties (immunoassay) or Chemical properties (differential activity for some substrates or susceptibility to inhibitors).