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Clinical Enzymology

Clinical Enzymology. FE A. BARTOLOME, MD, DPASMAP Dept. of Pathology & Laboratory Diagnosis Our Lady of Fatima University. ENZYMES. protein molecules catalyze chemical reactions without themselves being altered chemically contained primarily within cells

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Clinical Enzymology

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  1. Clinical Enzymology FE A. BARTOLOME, MD, DPASMAP Dept. of Pathology & Laboratory Diagnosis Our Lady of Fatima University

  2. ENZYMES • protein molecules • catalyze chemical reactions without themselves being altered chemically • contained primarily within cells • essential enzymes present in virtually all organs but with slightly different forms in different locations  isoenzymes • classified according to biochemical functions • unit of enzyme activity: • 1 IU = transform 1 mol of substrate/minute • 1 SI (katal) = transform 1 mol substrate/second

  3. Indicators of Cardiac Injury Creatine kinase (CK) • creatine phosphokinase (CPK) • catalyze transfer of a PO4 group between creatine PO4 & ADP to form creatine + ATP • requires magnesium as cofactor • dimeric with pair of two different monomers  M & B • 3 isozymes: CK1 (BB), CK2 (MB), CK3 (MM) • M and B subunits antigenically distinct proteins encoded by different genes

  4. Creatine kinase (CK) • primary tissue sources: • Brain, smooth muscle, prostate, thyroid, gut, lung  CK-BB • Cardiac muscle – MB (20-30%) & MM (70-80%) • Skeletal muscle – MB (1-2%) & MM (98-99%) • Plasma – predom. MM with < 6% MB • relatively small molecular size  allows leakage out of ischemic muscle or brain cells

  5. Creatine kinase (CK) • reference ranges in serum affected by: • Amount of lean muscle mass • Thin, sedentary = 30 – 50 U/L • Muscular, exercising regularly = 500 – 1000 U/L • Age – in neonates, CK-MB 5-10% of total CK • Gender • Race – Africans 30% higher than Europeans • Muscle activity – direct relationship between intensity of exercise and CK level

  6. Creatine kinase (CK) • persons exercising periodically & at usual intensity levels with lower CK than those who do not exercise at all • decrease with severe inactivity • short-term strenuous exercise  10-100 fold increase • marathon runners  up to 2000 U/L as resting value

  7. Diagnostic Applications CM-MM • released from damaged muscles: CK, AST, LD, myoglobin • Myoglobin >> CK >> AST and LD • released during ischemia, injury or inflammation • also increased in: • Chronic myopathies • Chronic renal failure • Acute respiratory exertion – respiratory muscles with more CK than other muscles

  8. Diagnostic Applications CK-BB • Brain trauma or brain surgery • Injury to smooth muscles (e.g. intestinal ischemia) • Patients with malignancies, esp. prostate cancer, small cell lung CA, intestinal malignancies  synthesize B subunit • Transient increase after cardiac arrest  reflect cerebral ischemia

  9. Diagnostic Applications CK-MB • primary clinical use: detection of acute MI • Following MI: • Total CK – 98% sensitive but 68-85% specific; peak value 18-30 hrs; duration 2-5 days; level 5-10x normal • CK-MB – rise proportional to extent of infarction; appears in serum within 6 hrs after AMI; peak value 12-24 hrs; duration 1.5-3 days  persistence indicates extension or infarction or re-infarction

  10. Diagnostic Applications Total Serum CK • Normal: 24 – 170 U/L (women) 24 – 195 U/L (men) • Marked elevation (> 5x normal) • After trauma from electrocution, crush injury, convulsion, tetany, surgical incision or IM injection • Athletic individuals – inc. muscle mass & inc. release during strenuous activity • Muscular dystrophies • Chronic inflammation of muscle (dermatomyositis or polymyositis)

  11. Diagnostic Applications Total Serum CK • Mild or moderate elevation (2 – 4x normal) • Hyper- or hypothermia • Hypothyroidism • After normal vaginal delivery – BB isoenzyme from myometrial contractions • Reye’s syndrome

  12. Atypical Isoenzymes • Forms that migrate electrophoretically in positions different from standard ones • Adenylate kinase • Catalyze formation of ATP & AMP from ADP • Released from erythrocytes • Macro CK type 1 – complex of CK (BB) with antibody (IgG)  mistaken for CK-MB; no known clinical significance • Macro CK type 2 – oligomeric variant of CK; mitochondrial • If (+) in serum – poor prognostic sign • (+) in patients with malignancies & moribund patients

  13. Lactate dehydrogenase (LD) • Zinc-containing; part of glycolytic pathway • Catalyze conversion of lactate to pyruvate using NAD+ as cofactor • CH3 CH3 • HCOH + NAD+ C = O + NADH + H+ • COOH COOH • Tissue source: present virtually in all tissues  cytoplasm of all cells and tissues in the body • Tetramers with 4 subunits of 2 possible forms: H (heart) and M (muscle)

  14. Lactate dehydrogenase (LD) • Five isoenzymes: • LD1 & LD2 – high in heart muscle, erythrocytes, kidney • LD4 & LD5 – high in skeletal muscle & in liver • Normal pattern in serum: • LD2 > LD1 > LD3 > LD4 > LD5 • Highest in newborns and infants; values do not change with age in adults • No gender difference

  15. DIAGNOSTIC APPLICATIONS: • Total LD activity: increase in any disease state where there is cell damage or destruction  non-specific  correlate with AST, ALT and CK • Markedly inc. LD with normal or minimally inc. AST, ALT & CK  damage to biochemically simple cells (e.g. rbc, wbc), kidney, lung, LN or tumors • Inc. LD & CK; inc. AST > ALT  cardiac or skeletal muscle injury • AST & ALT inc. > LD  transiently in liver disease (toxic or ischemic liver injury)

  16. Myocardial damage – • Myocardium normally with LD1 > LD2  similar to rbc • Acute myocardial infarction: • Inversion or flipped LD1/LD2 ratio to a value > 1.0 in serum  stay flipped for several days • Levels inc. after 12-24 hrs, peak (2-10x normal) at 48-72 hrs, return to normal after 8-10 days • used to confirm diagnosis of MI when CK isoenzyme analysis equivocal or after total CK & CK-MB release has returned to normal

  17. Other Applications • Total LD used to estimate tumor mass including metastases • LD1 or LD2 inc. in germ cell tumors (seminoma & dysgerminoma) – serve as tumor marker • Flip LD1/LD2 ratio: • Extreme exercise • Acute myocardial infarction • Hemolytic anemia • Megaloblastic anemia • Renal cortical disease (renal infarct, renal cell CA)

  18. Other Applications • Inc. LD2, 3 and 4 – malignancy & large tumor burden • Inc. LD3 & 4, dec. LD1 & 2 – WBC tumors (leukemia, lymphoma, MM), pulmonary disease • Inc. LD4 & 5 – skeletal muscle injury, ischemic or toxic hepatic injury • Isomorphic pattern – inc. total LD, normal isoenzymes with “tombstone” pattern (relative amounts of isoenzymes the same)  diffuse tissue damage accompanied by shock or hypoxemia

  19. Myoglobin • Small; functions in storage and transfer of O2 from Hgb in the circulation to intracellular respiratory enzymes of contractile cells • With greater affinity for oxygen than Hgb • Only one molecular form • One of the first to diffuse out of ischemic muscle cells, even before CK • Cleared from circulation by kidneys • Measurement in serum with high sensitivity for muscle injury, including acute MI  measure by immunoassay

  20. Myoglobin • In normal individuals, • Levels related to muscle mass and activity • Males > females • Africans > Europeans • Increase with increasing age due to decreasing GFR

  21. Troponins • Bind tropomyosin and govern excitation-contraction coupling • Three subunits • Troponin C (TnC) – calcium-binding subunit • Troponin I (TnI) – bind to actin  inhibitory • Troponin T (TnT) – bind to tropomyosin • TnI and TnT with unique forms expressed in myocardial cells but not in other muscle types  presence of cTnI or cTnT in serum highly specific for myocardial injury

  22. Troponins • cTnT • 84% sensitivity for MI 8 hrs after onset of symptoms • 81% specificity for MI; 22% specificity for unstable angina • cTnI • 90% sensitivity for MI 8 hrs after onset of symptoms • 95% specificity for MI; 36% specificity for unstable angina

  23. Troponins • Cardiac troponins released in two phases: • Initial damage (acute MI) – leave myocardial cells  enter circulation the same time that CK-MB does  peak at 4-8 hrs • Sustained release from intracellular contractile apparatus – occurs up to days after acute event • First appear in circulation ff. myocardial injury slightly later than when myoglobin enters the blood  rises after 3-6 hrs  peaks at ~ 20 hrs

  24. Troponins • General advantages: • cTnT and cTnI are released only following cardiac damage. • Unlike CK & CK-MB, cTnT and cTnI are present , and remain elevated, for a long time  cTnI detectable up to 5 days & cTnT for 7-10 days following MI • cTnT and cTnI are very sensitive.

  25. Troponins • General disadvantages: • Elevation can occur as a result of causes other than MI  myocarditis, severe cardiac failure, cardiac trauma, pulmonary embolus with cardiac damage • Failure to show a rise in cTnT or cTnI does not exclude the diagnosis of ischemic heart disease. • Both may be elevated in patients with chronic renal failure with sustained levels of elevation.

  26. Troponins • Measured in serum by immunoassay • Ideal time to check is between 6 and 9 hours from onset of symptoms • If onset of symptoms indistinct – take sample on admission, 6 – 9 hrs after and at 12 – 24 hrs after admission

  27. Other enzymes useful in clinical diagnosis Acid phosphatase (ACP) • Optimal activity: pH 5.0 • Tissue source: • Common to many tissues, esp. prostate • Small amounts in rbc, platelets (during clot formation), liver and spleen • Human milk and seminal fluid (very concentrated)

  28. Acid phosphatase (ACP) • Prostatic ACP distinguished from others using thymolphthalein monophosphate highly specific for prostatic ACP • Major applications: • Evaluation of prostatic CA (metastatic & local growth) • Not elevated in CA confined within prostate, BPH, prostatitis or ischemia of prostate • Medicolegal evaluation of rape – vagina with little or no ACP • Measured by radioimmunoassay  acidify serum with citric acid to stabilize ACP activity

  29. Alkaline phosphatase (ALP) • Widely distributed along surface membranes of metabolically active cells • Encoded for by four different genes expressed in: • Placenta • Intestines • Germ cell and lung • Tissues including bone, liver, kidney & granulocyte • Very high activity in bone, liver, intestine, kidney, wbc and placenta

  30. Alkaline phosphatase (ALP) • Methods for distinguishing ALP isoenzymes: • Heat fractionation – easiest & most common; heat serum sample at 56oC x 15 min. then compare with unheated sample • Bone ALP extremely labile  retain 10-20% of original activity • Liver & placental ALP heat stable  liver ALP 30-50% retained, placental ALP with all retained • Chemical inhibition • Urea – block placental ALP • Phenylalanine – block liver & bone ALP • Electrophoresis - definitive

  31. DIAGNOSTIC APPLICATIONS Liver ALP • Derived from epithelial cells of biliary tract  excreted by bile into intestine • Used for establishing diagnosis in jaundice • Pronounced increase (> 5x) • Intra- or extrahepatic bile duct obstruction • Biliary cirrhosis • Moderate increase (3-5x normal) : granulomatous or infiltrative liver disease • Slight increase (up to 3x normal) : viral hepatitis, cirrhosis

  32. Bone ALP • Elevation part of osteoblastic growth • Pronounced increase: • Paget’s disease • Osteogenic sarcoma • Hyperparathyroidism • Moderate increase: metastatic tumors in bone; metastatic bone disease (rickets, osteomalacia) • Slight increase: healing fractures; normal growth patterns in children

  33. Placental ALP • With oncofetal form  turned on and expressed by tumor cells in adults  called Regan isoenzyme • Slight increase in pregnancy • Intestinal ALP • Inc. in inflammatory bowel disease (ulcerative colitis & regional enteritis) • Secreted into the circulation after a meal in persons with blood type “O” and “B”  inc. total ALP in non-fasting specimens

  34. Renal ALP • Normally excreted into urine from renal tubular cells • Granulocyte ALP • Used as marker of granulocyte maturity in leukocytosis • Lymphocytes infected with HIV  release specific ALP fraction (band-10)  surrogate marker for HIV infection in children

  35. Aldolase • Glycolytic enzyme  split fructose-1,6-diphosphate into two triose phosphate molecules in glucose metabolism • Distributed in all tissues • Elevated in serum following: • Skeletal muscle disease or injury – reflect severity of dermatomyositis • Metastatic CA to liver 5. Hemolytic anemia • Granulocytic leukemia 6. Tissue infarction • Megaloblastic anemia

  36. Aminotransferase (Transaminase) • Catalyze reversible transfer of an amino group between an amino acid and an alpha-keto acid • R R’ R R’ • HCNH2 + C = O C = O + HCNH2 • COOH COOH COOH COOH • Requires pyridoxal phosphate (vitamin B6) as cofactor

  37. Alanine aminotransferase (ALT) • “glutamate-pyruvate transaminase” (GPT) • Rich amounts in hepatocytes  with high specificity for liver damage • Moderate amount: kidney, heart, skeletal muscle • Small amount: pancreas, spleen, lung, red blood cells

  38. Diagnostic Applications • Inc. AST & ALT – excellent indicators of liver damage • ALT increased in serum ff. acute MI 6 hrs after onset, peaks at 24-48 hrs, returns to normal in 3-4 days • AST inc. in conditions that can be confused with acute MI or that may complicate or co-exist with acute MI: • Shock or circulatory collapse from any cause • Acute pancreatitis • Cardiac arrhythmias or ischemic insult that do not progress to infarction

  39. Gamma glutamyltransferase (GGT) • “gamma glutamyltranspeptidase • Catalyze transfer of glutamyl groups between peptides or amino acids through linkage at a -COOH group  important in transfer or movement of amino acids across membranes • Large amounts in: • Pancreas and renal tubular epithelium • Hepatobiliary cells

  40. Diagnostic Applications • increased activity: • In urine – renal tubular damage • Hepatocellular & hepatobiliary diseases  correlates better with obstruction & cholestasis than with pure hepatocellular damage  “obstructive” enzyme

  41. Diagnostic Applications • GGT & alcohol • Alcohol induces microsomal activity  inc. GGT synthesis  indicator of alcohol use • GGT levels return to normal after 3-6 wks of abstention from alcohol  test for compliance in alcohol-reduction programs

  42. Diagnostic Applications • GGT & drugs • Barbiturates, phenytoin & other drugs (acetaminophen) inc. microsomal activity of GGT • Potentially useful in drug treatment protocols

  43. Amylase (Diastase) • digestive enzyme • Acts extracellularly to cleave starch into smaller groups & finally to monosaccharides • Major sources: salivary glands • exocrine pancreas

  44. Pancreatic amylase • secretion stimulated by pancreozymin (cholecystokinin) • enter duodenum at ampulla of Vater via sphincter of Oddi • Low levels found in: • Fallopian tubes 3. Small intestine • Adipose tissue 4. Skeletal muscle • readily cleared in urine

  45. DIAGNOSTIC APPLICATIONS • Acute Pancreatitis • Levels rise within 6-24 hours remain high for a few days  return to normal in 2-7 days • Serum amylase normal but with suspicion of pancreatitis  measure 24-hour urine amylase or serum lipase

  46. DIAGNOSTIC APPLICATIONS • Morphine administration • Constrict pancreatic duct sphincter  dec. intestinal excretion & inc. absorption in the circulation • Renal failure • Failure to clear normally released amylase from the circulation  no diagnostic significance

  47. DIAGNOSTIC APPLICATIONS • Malabsorption & liver disease • (+) circulating complexes of amylase with a high MW compound such as Ig’s  macroamylasemia  prevent renal clearance • no diagnostic significance

  48. DIAGNOSTIC APPLICATIONS • Tumors • serous ovarian tumors • epithelium similar to FT  produce cyst fluid with amylase  appear in serum & urine • Lung CA • ectopic production of amylase

  49. Conditions Affecting Serum Amylase

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