Dr. Saidunnisa Associate Professor Department of Biochemistry Isoenzymes and Enzymes as diagnostic tool

Learning Objectives At the end of the session student shall be able to: List the sources of plasma enzymes. Interpret the enzyme levels in diagnosis. Define Isoenzymes and explain the various cardiac enzymes useful in diagnosis of Myocardial infarction. List the enzymes used as therapeutic agents and in diagnosis of disease.

Sources of plasma enzymes They can be : Plasma derived enzymes: These act on substrates in plasma, and their activity is higher in plasma than in cells, e.g., coagulation enzymes. Cell derived enzymes: These have a high activity in cells and overflow into plasma which are coming out from cells of various tissues due to normal wear and tear

Their normal levels in blood are very low but are drastically increased during cell death (necrosis) or disease. Therefore assays of these enzymes are useful in diagnosis of diseases.

Clinical significance of enzyme assays Serum enzymes in heart disease CPK, Myoglobin, Troponin, LDH, AST Serum enzymes in Liver disease ALT,GGT,ALP, Serum enzymes in GI tract disease Serum amylase/Lipase Serum enzymes in muscle diseases Serum aldolase/CPK Serum enzymes in bone diseases ALP Serum enzymes in malignancies Acid phosphatase and PSA

Isoenzymes Multiple forms of an enzyme which differ in physical and chemical properties and catalyze the same reaction as an enzyme. Isoenzymes are produced by a single gene and some may result from more than one gene. Isoenzymes can be separated by: Heat inactivation Chemical inhibition Electrophoretic techniques (specific)

Electrophoresis Is a technique by which separation of Movement of charged particles through an electrolyte when subjected to electrical field.

Advantages of Isoenzyme measurement Isoenzyme variants are derived from different tissue sources. So separation renders increased specificity to enzyme analysis. Tissue or organ effected can be detected ( where isoenzyme elevation occurs)

Types CPK (creatinine Phospho Kinase) Troponin LDH (Lactate Dehydrogenase) ALP (Alkaline phosphatase) Aldolase Amylase

Atherosclerosis Is a condition in which arteries are blocked to a greater or lesser extent by deposition of cholesterol plaques , leading most commonly to coronary heart disease by blocking of coronary arteries i.e ( myocardial infarction MI).

CPK (CK) Creatinine Phospho kinase or Creatinine Kinase catalyses the conversion of creatinine to creatinine Phosphate. Creatinine +ATP Creatinine kinase creatinine phosphate +ADP Creatinine Phospho kinase Normal level: 15-100U/L (males) : 10-80 U/L (females) Sample: in serum it is estimated and not increased in hemolysis.

CPK-Creatinine Phospho kinase CPK Isoenzymes are performed when the total CPK level is elevated. Isoenzyme testing can help differentiate the source of the damaged tissue. CPK is an enzyme found predominantly in the heart, brain, and skeletal muscle. CPK is composed of 3 Isoenzymes that differ slightly in structure: CPK is a dimer made up of 2 subunits called B for brain and M for muscle.

CPK- Isoenzymes CPK-1 (also called CPK-BB) is concentrated in the brain and lungs CPK-2 (also called CPK-MB) is found mostly in the heart CPK-3 (also called CPK-MM) is found mostly in skeletal muscle Because the CPK-1 isoenzyme is predominately found in the brain and lungs, injury to either of these organs (for example, stroke or lung injury due to a pulmonary embolism) are associated with elevated levels of this isoenzyme.

CPK-2 levels rise 3 - 6 hours after a heart attack CPK-2 levels rise 3 - 6 hours after a heart attack. If there is no further damage to the heart muscle, the level peaks at 12 - 24 hours and returns to normal 12 - 48 hours after tissue death. The CPK-3 isoenzyme is normally responsible for almost all CPK enzyme activity in healthy people. When this particular isoenzyme is elevated, it usually indicates muscular dystrophies.

Description LDH LDH is found in the cells of almost all body tissues. Because this enzyme is actually composed of five different Isoenzymes, however, analysis of the different LDH isoenzyme levels in the blood can help in the diagnosis of some diseases.

LDH LDH is an oxidoreductase enzyme whose activity is necessary for the reversible reaction in which Pyruvate and lactate are inter converted. It is important in glycolysis. LDH Isoenzyme is a tetramer with 4 subunits. The subunit may be either H (heart) or M (muscle) .

Clinical significance Total LDH activity is increased in: MI (Myocardial infarction) Acute infective hepatitis Muscular dystrophy Leukemia Normal Value: 100-200U/l

LDH-Isoenzymes The LDH has five Isoenzymes which are: LDH-1 (H4) is found mainly in the heart. LDH-2 ( H3M1) Reticuloendothelial system. LDH-3 (H2M2) is found in the lungs. LDH-4 ( H1M3) in the kidney, placenta, and pancreas, and LDH-5 ( M4) in liver and striated (skeletal) muscle. Normally, levels of LDH-2 are higher than those of the other Isoenzymes

LDH and Heart Attack One of the most important diagnostic uses for the LDH Isoenzymes test is in the differential diagnosis of myocardial infarction or heart attack. The total LDH level rises within 24-48 hours after a heart attack, peaks in two to three days, and returns to normal in approximately five to ten days. The LDH-1 isoenzyme level, however, is more sensitive and specific than the total LDH.

Normally, the level of LDH-2 is higher than the level of LDH-1. An LDH-1 level higher than that of LDH-2, a phenomenon known as "flipped LDH," is strongly indicative of a myocardial infarction. The flipped LDH usually appears within 12-24 hours after a heart attack. A normal LDH-1/LDH-2 ratio is considered reliable evidence that a heart attack has not occurred.

Preparation This test requires a blood sample. The patient need not fast (nothing to eat or drink) before the test. LDH level is 100 times more in the RBC than plasma, therefore minor amount of hemolysis will result in a false positive.

Blood is drawn from a vein (venipuncture), usually from the inside of the elbow or the back of the hand. A needle is inserted into the vein, and the blood is collected in an air-tight vial or a syringe. Preparation may vary depending on the specific test.

Cardiac Enzymes

Myoglobin Myoglobin, a low molecular weight Heme protein found in cardiac & skeletal muscles. Although it lacks cardio specificity, yet it is released more rapidly from the infracted myocardium than CK-MB & may be detected as early as 2 hours after MI. Myoglobin is best used in negative prediction of myocardial injury in emergency room.

Cardiac enzymes Enzyme Initial rise Peak Back to Normal Myoglobin <2 h 6-9 h 1 day CK-MB 3-6 h 12-24h 2-3 days Troponin I <4hrs 14-24hrs 3-5days AST Raises after CPK 48hrs 4-5days LDH 24-48 h 5-10 day

Troponin Because the conventional serum cardiac marker, creatine kinase (CK) and its MB isoenzyme (CK-MB) lack sufficient sensitivity and specificity, there is a need for more sensitive and cardiac-specific markers of myocardial necrosis. The Troponin complex consists of 3 subunits:

TROPONINS (contd.) Troponin T binds the troponin complex to tropomyosin, while Troponin I binds to actin and inhibits interactions between actin and myosin. Troponin C is responsive to changes in intracellular calcium concentration.

Amino acid sequences of the skeletal and cardiac isoforms of troponin I and troponin T have sufficient dissimilarity that monoclonal antibody-based immunoassays have been developed to detect, cardiac-specific troponin T (cTnT) and I (cTnI). Because the amino acid sequence of troponin C is the same in cardiac and skeletal muscle, no immunoassays of cTnC have been developed for clinical purposes.

Cardiac specificity cTnI raises <4hrs peak 14-24hrs normal 3-5days. 75% sensitive index for MI. It is not increased in crush injury. cTnT is 100% sensitive index for MI. These are assayed by ELISA or RIA technique.

Alkaline Phosphatase (ALP) Isoenzymes are five: ALP-1 present in liver increased in obstructive jaundice, biliary cirrhosis. ALP-2 in bone increased in rickets and Pagets ALP-3 in placenta increase in 2nd and 3rd trimester of pregnancy and decrease indicates placental insufficiency and foetal death.

4. ALP-4 in intestine increased in intestinal disease and after gastrectomy surgery 5. ALP-5 in kidney increases in kidney disorders. In normal serum liver and bone fractions are present. Abnormal ALP Isoenzymes Regan and nagao are present in carcinomas and metastasis. Normal level: 40-125 U/L

Amylase Two Isoenzymes: Salivary in normal serum 60% increased in Parotitis/ mumps Pancreatic 40% increased in pancreatitis The peak levels are seen between 5-12hours after onset of disease and returns to normal level with in 2-4 days. Normal level: 50-120 IU/L

Aldolase Three Isoenzymes: Aldolase-A present in muscle increased in muscular dystrophies. Aldolase-B in liver increased in hepatitis. Aldolase-C in brain Normal level: 1.5-7 U/L

Acid phosphatase Present in Prostate, RBC and platelets. Increased in Prostate cancer, Gaucher’s disease and thromboembolic disorders Normal level: 2.5-12 U/L

Case-1 A 45 year old male company director, weighing 90kg was a smoker for the past 25 years. For the past 6 months, he was feeling mild chest pain when climbing stair case. One night he felt crushing chest pain which radiated to the left arm. He was sweating profusely, diagnosed as MI. Name the specific blood markers which help to diagnose MI.

Case-2 40 year old female with history of intermittent right upper quadrant pain worsening after greasy meals and now with constant midepigastric pain, nausea and vomiting diagnosed as acute pancreatitis. Name the specific blood markers which help to diagnose acute pancreatitis.

Case-3 A 50 year male complains of bone pain, in thigh bone (femur), and gives a previous history of fracture, and arthritis. The diagnosis of Paget's disease of the bone was made. Name the specific blood markers which help to diagnose Paget's disease .

Case-4 A 52 year old male complains of muscle cramps, stiffness, and spasm associated with myositis a diagnosis of myopathy a muscular disease was made. Name the specific blood markers which help to diagnose Paget's disease

Case -5 A 55 year old male complains of frequent urination at night, painful urination, blood in the urine, and a diagnosis of prostate cancer was made. Name the specific blood markers which help to diagnose Prostate cancer.

Therapeutic agents Enzymes are used as therapeutic agents in treatment of certain disorders: Streptokinase (from streptococcus) can lyse intravascular clots and are used in MI. Pepsin and Trypsin are given to patients with indigestion. Asparaginase is used as an anticancer drug.

Diagnosis Enzymes are used in various biochemical estimations for diagnosing diseases. Glucose oxidase is used to estimate glucose. Urease in urea estimations. Cholesterol oxidase Reverse transcriptase- PCR Restriction endonuclease- Blotting