Major Clinical Enzymes PDF

MAJOR CLINICAL 2 ENZYMES PHOSPHATASES Class III: hydrolases 1. Alkaline Phosphatase *Alkaline Orthophosphoric Monoester Phosphohydrolase" 2. acid Phosphatase "'Acid Orthophosphoric Monoester Phosphohydrolase" PHOSPHATASES 1. Alkaline Phosphate A NON-SPECIFIC ENZYME CAPABLE OF REACTING WITH MANY DIFF...

MAJOR CLINICAL 2 ENZYMES PHOSPHATASES Class III: hydrolases 1. Alkaline Phosphatase *Alkaline Orthophosphoric Monoester Phosphohydrolase" 2. acid Phosphatase "'Acid Orthophosphoric Monoester Phosphohydrolase" PHOSPHATASES 1. Alkaline Phosphate A NON-SPECIFIC ENZYME CAPABLE OF REACTING WITH MANY DIFFERENT SUBSTRATES. IT FUNCTIONS TO LIBERATE INORGANIC PHOSPHATE FROM AN ORGANIC PHOSPHATE ESTER WITH CONCOMITANT PRODUCTION OF AN ALCOHOL IN HEALTHY SERA, ALP LEVELS ARE DERIVED FROM LIVER AND BONE OSTEOCLAST BONE ISOENZYME INCREASED DUE TO OSTEOBLASTIC ACTIVITY AND IS NORMALLY elevated in children during periods of growth and in adults older than age 50 years (geriatric). Major tissue sources: liver bone placenta intestinal renal Reference values: 30-90 U/L 30-90 U/L Diagnostic Significance: OBSTRUCTIVE JAUNDICE When total ALP levels are increased, it is the major liver fraction that is most frequently elevated PAGET’S DISEASE (OSTEITIS DEFORMANS) highest elevations FOR BONE DISORDERS Carcinoplacental ALP: 1. Regan ALP lung. breast, ovarian and GYNECOLOGICAL CANCERS bone ALP co-migrator most HEAT STABLE ALP (65°C for 30 minutes) inhibited by phenylalanine reagent. 2. NAGAO ALP adenocarcinoma of the pancreas AND BILE DUCT, pleural cancer VARIANT OF REGAN inhibited by L-leucine and phenylalanine. METHODS: 1. Electrophoresis Liver and bone ALPs are the most anodal isoenzymes intestinal ALP is the least anodal. improves separation of bone and liver ALPs. WHEAT GERM LECTIN NEURAMINIDASE 2. Heat Fractionation/Stability Test It is performed at 56°C for 10-15 minutes. Placental ALP is the most heat stable; bone ALP is the most heat labile. Decreasing order of ALP heat stability; placental, intestinal, liver and bone PLACENTAL intestinal LIVER BONE Chemical Inhibition Test uses different concentrations of phenylalanine, synthetic urea and levamisole solutions. PHENYLALANINE: PLACENTAL & INTESTINAL LEVAMISOLE: LIVER AND BONE Bowers and Mc Comb Is considered as the most specific method It is a continous-monitoring technique which requires a pH environment of 10.15 and should be read at 405nm. ALP P-NITROPHENYLPHOSPHATE P- NITROPHENOL + PHOSPHATE ION INCREASED ALP Osteitis deformans Obstructive jaundice Osteomalacia Rickets Osteoblastic bone tumors Sprue Hyperparathyroidism Hepatitis and cirrHosis - slight increased Bone cancer 2. Acid Phosphatase It catalyzes the same reaction made by ALP, except that it is active at pH 5.0. Diagnostic Significance: detection of prostatic carcinoma Tissue sources: prostate (major source) RBC, platelets and bone Reference Values: 2.3-11.7 U/L (total ACP) - male 0-3.5 ng/mL (Prostatic ACP) Useful in forensic clinical chemistry, in the investigation of rape cases - vaginal washings arE examined for seminal fluid- acid phosphatase (ACP) activity, which can persists for up to 4 days. METHOD: Roy and Hillman SUBSTRATE: Thymolphthalein MonoPHOSPHATE END PRODUCT: Free thymolpthalein ACP THYMOLPHTHALEIN MONOPHOSPHATE FREE THYMOLPTHALEIN INHIBITORS: PROSTATIC ACP: L-TARTRATE RED CELL ACP: CUPRIC SULFATE AND FORMALDEHYDE TARTRATE RESISTANT ACP (TRAP) present in certain chronic leukemias and some lymphomas, most notably in hairy cell leukemia. Increased ACP (Metastatic Bone Involvement) Prostatic carcinoma Breast, Lung and Thyroid carcinoma Gaucher's disease Niemann Pick Disease After surgical treatment of prostate cancer, ACP levels falls faster than PSA, and plasma levels are expected to be undetectable following complete removal of tumor. TRANSAMINASES/TRANSFERASES 1. Aspartate Aminotransferase (AST) Involved in the transfer of an amino group between aspartate and a-keto acids with the formation of oxaloacetate and glutamate. It has 2 isoenzyme fractions cytOplasm AST - predominant form in serum mitochondrial AST Major tissue sources: cardiac tissue liveR skeletal muscle Reference values: 5-37 U/L kidney pancreas RBC Diagnostic Significance: In the evaluation of myocardial infarction, hepatocellular disorders and skeletal muscle involvement. acute myocardial infarction (AMI) AST levels begin to rise 6-8 hours, peak at 24 hours and normalize within 5 days. It is released to a greater degree in chronic disorders of the liver with progressive damage. METHOD: Karmen Method pH 7.5; 340nm absorbance at 340 nm. Uses malate dehydrogenase (MD) and monitors the change AST Aspartate + a-ketoglutarate oxaloacctate + glutamatE MD OXALOACETATE + NADH + H malate + NAD+ 2. Alanine Aminotransferase (ALT) It has enzymatic activity similar to AST. It catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the formation of glutamate and pyruvate. The highest concentration is in the liver) more liver-specific than AST. Tissue sources: MAJOR: liver MINOR: kidney, pancreas, RBC, heart, skeletal muscles, lungs Reference Values: 6-37 U/L Diagnostic Significance: Significant in the evaluation of hepatic disorders - markedly increased concentration in acute inflammatory conditions than AST. It also monitors the course of hepatitis treatment and the effects of drug therapy. ALT measurement is a more sensitive and specific screening test for posttransfusion hepatitis or occupational toxic exposure. ALT levels are also used to screen blood donors. METHOD: Coupled Enzymatic Reaction: pH 7.5; 340nm ALT ALANINE + a-ketoglutarate PYRUVATE + glutamatE LD PYRUVATE + NADH + H LACTATE + NAD+ SGOT/AST SGPT/ALT Major Organ affected HEART LIVER Substrate Aspartic Alpha Ketoglutaric Acid ALANINE Alpha Ketoglutaric Acid End products Glutamic Acid + OxaloAcetic Acid Glutamic Acid + Pyruvic Acid Color developer 2,4 DNPH 2,4 DNPH Color intensifier 0.4N NaOH 0.4N NaOH Methods Reitman and Frankel Reitman and Frankel AMYLASE It catalyzes the breakdown of starch and glycogen - an important enzyme in the physiologic digestion of starch It is the smallest enzyme in size (with a MW of 50,000 to 55,000 daltons) normally filtered by the renal glomerulus and also appears in the urine. It is the Earliest pancreatic marker. P3 is the most predominant pancreatic amylase isonzyme in AP. Isoenzymes: S-type (ptyalin) P-type/(amylopsiN) Tissue sources MAJOR: acinar cells of the pancreas and the salivary glands MINOR: adipose tissue, fallopian tubes, small intestine and skeletal muscles Reference values: 60-180 SU/dL (somogyo units) 95-290 U/L Increased Amylase: Acute pancreatitis Ectopic pregnancy Peptic ulcers Alcoholism Mumps Diagnostic Significance: Increased AMS blood levels are accompanied by increased urinary excretion acute pancreatitis. (EARLIEST MARKER) In acute pancreatitis (AP), AMS levels rise 2-12 hours after onset of attack, peak at 24 hours, and normalize within 3-5 days. AMS in urine (AP) remains elevated for up to 7 days. In renal failure, increased blood levels are accompanied by decreased urine concentration. (renal cicarance) Salivary gland inflammation (parotitis) due to mumps can also release AMS into the circulation. METHOD: 1. Saccharogenic measures the amount of reducing sugars produced bY the hydrolysis of starch by the usual glucose methods. classic reference method expressed in Somogyi units. 2. Amyloclastic measures amylase activity by following the decreases in substrate concentration (degradation of starch). 3. Chromogenic measures amylase activity by the increase in color intensity of the soluble dye-substrate solution produced in the reaction. 4. Coupled-enzyme measures amylase activity by a continuous-monitoring technique. AMS Maltopentose maltrotriose + maltose a-glucosidase Maltrotriose + maltose 5-glucose Hexokinase 5-glucose + 5 ATP 5-glucose-6-phosphate + 5 ADP G-6-PD phosphate + 5 NAD 5,6-phosphogluconolactone + 5 NADH LIPASE An enzyme that hydrolyzes the ester linkages of fats to produce alcohol and fatty acid. It catalyzes partial hydrolysis of dietary TAG in the intestine to the 2-monoglyceride intermediate, with the production of long chain fatty acids. Most specific pancreatic marker: secreted exclusively in the pancreas not affected by renal disorders. Concentrations are normal in conditions of salivary gland involvement. Major tissue source: Pancreas Reference values: 0-1.0 U/mL Diagnostic Significance: In acute pancreatitis (AP), LPS levels rise 6 hours after onset of attack, peak at 24 hours, remains elevated for days) and normalize in 8-14 days. In chronic AP, acinar cell/degradation occurs resulting in loss of amylase and lipase PRODUCTION METHODS: It uses olive oil as the substrate because other esterases can hydrolyze TAG and synthetic diglycerides. Addition of colipase (protein secreted by the pancreas) and bile salts will make assay more sensitive and specific for AP detection. Hemoglobin inhibits the activity of LPS leading to falsely low values. Triolein (more pure form of TAG) is used also as a substrate for LPS assay. 1. Cherry Crandal reference method Principle: Hydrolysis of olive oil after incubation for 24 hours at 37°C and titration of fatty acids using NaOH. Substrate: 50% olive oil End product: Fatty acid LPS Triglyceride + 2 H20 2-monoglyceride + 2 fatty acids 2. Peroxidase coupling most commonly used method; does not use 50% olive oil. LACTATE DEHYDROGENASE (LD) It is an enzyme that catalyzes the interconversion of lactic and pyruvic acids. It is a zinc-containing enzyme that is part of the glycolytic pathway and is found in virtually all cells in the body. It is a hydrogen-transfer enzyme that uses the coenzyme nicotinamide dinucleotide (NAD+). It is a tetrameric molecule containing four subunits of two possible forms (H and M). In plasma, the majority of LD comes from breakdown of erythrocytes and platelets, with varying contributions from other organs. Tissue sources: LD-1 AND LD-2 HEART, RBCS AND KIDNEYS LD-3 LUNGS, PANCREAS AND SPLEEN LD-4 AND LD-5 SKELETAL MUSCLES, LIVER, INTESTINE REFERENCE VALUES: Forward reaction 100-225 U/L Reverse reaction 80-280 U/L DIAGNOSTIC SIGNIFICANCE: pernicious anemia and hemolytic disorders Highest serum levels AMI LD levels begin to rise within 12-24 hours, peak levels within 48-72 hours and remains elevated for 10-14 days. Hepatic carcinoma and toxic hepatitis 10-fold increased. LD-5 is MARKEDLY increased Viral hepatitis and cirrhosis slightly increased values (2-3x URL) LD-5 is moderately increased myocardial infarction and hemolytic ANEMIA LD-1 > LD-2 “flipped pattern” acute leukemia, germ cell tumors, breast and lung cancers LD-2, LD-3, LD-4 = LD cancer markers LD Isoenzyme as a Percentage of Total LD: LD-1 17-27% relatively abundant in cardiac muscle not found in the skeletal muscles and liver LD-2 27-37% major isoenzyme in the sera of healthy persons greater than LD-1 is seen in healthy sera. never found in the skeletal muscles LD-3 18-25% LD-4 3-8% LD-5 0-5% LD-6 more abundant in skeletal muscle. undetectable level in the heart, RBCs and renal cortex. represents the alcohol dehydrogenase enzyme 6th band in electrophoresis elevated in drug hepatoxicity and obstructive jaundice METHODS: Lactate is a more specific substrate compared to pyruvate. LD-1 prefers the forward reaction, whereas LD-5 prefers the reverse reaction. LD is stable at room temperature for 48 hours. 1. Wacker Method (forward/direct reaction) reaction is at ph 8.8 It is the most commonly used method because it produces a positive rate (NADH) and not affected by product inhibition. LD Lactate + NAD Pyruvate + NADH 2. Wrobleuski La Due (reverse/indirect reaction) reaction is at ph 7.2 It is about 2x faster as the forward reaction. It is the preferred method for dry slide technology It uses a less costly cofactor and it has a smaller specimen volume requirement. ld Pyruvate + NADH Lactate + NAD 2. Wrobleuski Cabaud 3. Berger Broida Increased LDH Anemias pernicious, hemolytic, megaloblastic Myocardial infarction Leukemia Renal infarction Hepatitis and hepatic cancer Muscular dystrophy Delirium tremens Malignancy Pneumocystis jerovecii pneumonia CREATINE KINASE It catalyzes the transfer of a phosphate group between creatine phosphate and adenosine diphosphate. It is involved in the storage of high-energy creatine PO4 in the muscles. It is a dimeric molecule with smali molecular size, composed of a pair of two different monomers called M and B. It is found in small amounts throughout the body, but is found in high concentrations only in muscle and brain, although CK from brain virtually never crosses the blood-brain barrier to reach plasma. Major tissue sources: brain tissue, smooth and skeletal muscles and cardiac muscles Reference values: Male 15-160 U/L Female 15-130 U/L CK-MB < 6% of total CK isoenzymes 1. CK-BB (brain type) most anodal and labile isoenzyme dominant isoenzyme of CK found in brain, intestine, and smooth muscle. Serum of adults rarely contains CK-BB of brain origin due to its high molecular size; it may be normally present in neonatal sera. 2. ck-mm (muscle type) abundantly present in the cardiac and skeletal muscles. In the sera of healthy persons, CK-MM is the major isoenzyme (94100%). 3. ck-mb (hybrid type) Cardiac tissues contain significant amount of CK-MB (20%) myocardium is the only tissue from which CK-MB enters the serum in significant quantities. CK-MB in serum of healthy person is < 5 ug/L. diagnostic significance It is a very sensitive indicator of acute myocardial infarction (AMI) and Duchenne disorder. Highest elevation of total CK is seen in Duchenne's muscular dystrophy (50x URL). CK-MB is found mainly in myocardial tissue it is used as a serodiagnostic test for AMI. Demonstration of elevated levels of CK-MB, ≥ 6% of the total CK, is considered the most specific indicator of myocardial damage, particularly AMI. Following AMI, the CK-MB levels begin to rise within 4-8 hours, peak at 12-24 hours and normalize within 48-72 hours. CK-MB is not elevated in angina. Injury to both cardiac and skeletal muscle accounts for the majority of CK-MM elevations. Total CK is markedly elevated after trauma to skeletal muscle from crush injury, convulsions, tetany, surgical incision or intramuscular injections. METHODS: 1. Tanzer-Gilbarg Assay (forward/ direct method) cpk Creatine + ATP Creatine PO4 + ADP pk AD + phosphoenolpyruvate Pyruvate + ATP ld Pyruvate + NADH Lactate + NAD 2. Oliver-Rosalki ( reverse/indirect method) most commonly used method; faster reaction; pH 6.8; 340nm cpk Creatine po4 + AdP Creatine + AtP hk Atp + glucose adp + glucose-6-po4 ld glucose-6-po4 + NADp 6-phosphogluconate + NADPH Increased Creatine kinase Duchenne's muscular dystrophy Myocardial Infarction Hypothyroidism Pulmonary infarction Reye's syndrome Strenous exercise and intramuscular injection Cerebral vascular accident (occasional) Rocky Mountain Spotted Fever - CK-MB Carbon monoxide poisoning ALDOLASE It is a glycolytic enzyme that splits fructose-1,6-diphosphate into two triose phosphate molecules in the metabolism of glucose. Increased: skeletal muscle disease, leukemia, hemolytic anemia and hepatic cancer Isoenzymes: Aldolase A = Skeletal muscles Aldolase B = WBC, liver, kidney Aldolase C = Brain Tissue Nucleotidase (5'N) It is a phosphoric monoester hydrolase; predominantly secreted from the liver. It is a marker for hepatobiliary diseas and infiltrative lesions of the liver. Method: Dixon & Purdon, Campbell, Belfield & Goldberg Reference value: 0-1.6 units Gamma Glutamyl Transamine Peptidase/Transferase (GGT) It catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at a gammy carboxyl group. It is located in the canaliculi of the hepatic cells and particularly in the epithelial cells lining the biliary ductules; also in the kidney, prostate and pancreas. It affects the cell membrane and microsomal fractions - elevated among individuals undergoing warfarin, phenobarbital and phenytoin therapies. Substrate: v-glutamyl-p-nitroanilide Method: Szass, Rosalki & Tarrow, Orlowski Reference value: 5-30 U/L (F) / 6-45 U/L (M) Diagnostic Significance: It is useful in differentiating the source of an increased ALP level. It is elevated in all hepatobiliary disorders - biliary tract obstructions. It is a sensitive indicator of alcoholism (occult alcoholism) - most sensitive marker of acute alcoholic hepatitis. It is useful in monitoring the effects of abstention from alcohol. It is also increased in pancreatitis and prostatic disorders. Pseudocholinesterase (PChE) It is secreted by the liver it reflects synthetic function rather than hepatocyte injury. antixenobiotic enzyme It catalyzes the removal of benzyl group from cocaine is a marker for insecticide/pesticide poisoning (organophosphate poisoning) that causes low serum PChE. Is used to monitor the effect of muscle relaxants (succinylcholine) after surgery. It is involved in the metabolism of anticholinergic drugs. PChE reflects acute toxicity while AChE (true cholinesterase or choline esterase) in the red blood cells better reflect chronic exposure. Tissue source: liver, myocardium and pancreas Decreased:acute hepatitis, cirrhosis, carcinoma metastaticto liver and malnutrition Method: Ellman technique and potentiometric Reference value: 0.5-1.3 pH units (plasma) Angiotensin-Converting Enzyme (ACE) It is also known as peptidyldipeptidase A or kininase ii; a hydrolase enzyme. It converts angiotensin I to angiotensin Ii within the lungs. It is a possible indicator of neuronal dysfunction (Alzheimer's diseaseCSF). Is a critical target for inhibitory drugs designed to lower blood pressure. Tissue source: lungs, testes, macrophages and epitheloid cells Diagnostic significance: for the diagnosis and monitoring of sarcoidosis Increased: sarcoidosis, multiple sclerosis, addison's disease, acute and chronic bronchitis, HIV infection and leprosy Ceruloplasmin It is a copper-carrying protein and also an enzyme. It is a marker for Wilson's disease (hepatolenticular disease). Ornithine Carbamoyl Transferase (OCT) It is a marker for hepatobiliary diseases. Glucose-6-Phosphate Dehydrogenase (G-6-PD) It functions to maintain NADPH in the reduced form in the erythrocytes. It is a newborn screening marker. It is found in adrenal cortex, spleen, RBC and lymph nodes. Deficiency of this enzyme can lead to drug-induced hemolytic anemia after taking primaquine, an antimalarial drug. Increased: myocardial infarction and megaloblastic anemia Specimen: red cell hemolysate and serum Reference value: 10-15 U/g hemoglobin or 1200-2000 mU/mL packed RBC QUIZ 2 1. HIGHEST ELEVATION OF ALP IS SEEN IN WHAT CONDITION? 2. ENZYME USED TO DIAGNOSE RAPE VICTIMS 3. SMALLEST ENZYME 4. MOST SPECIFIC ENZYME AS MARKER OF PANCREATITIS 5. MOST NON-SPECIFIC ENZYME BECAUSE OF ITS PRESENCE IN ALMOST ALL CELLS 6. CREATININE KINASE THAT IS FASTEST 7. LD-1 > LD-2 IS TERMED AS 8. ENZYME USED TO DIAGNOSE ORGANIC PHOSPHATE POISONING 9. MOST LIVER ENZYME SPECIFIC TRANSFERASE 10. MOST SENSITIVE MARKER OF ALCOHOLIC HEPATITIS