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Clinical Enzymology

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Clinical significance of enzymes

Clinical Enzymology

  1. 1. Biochemistry for Medics www.namrata.co Biochemistry for Medics 7/14/2012 1
  2. 2. 1) Enzymes can act as diagnosticmarkers of underlying diseases .2) Enzymes can also act asreagents for various biochemicalestimations and detections Biochemistry for Medics 7/14/2012 2
  3. 3. 1)Functional plasma enzymes( Plasma derivedenzymes)- Certain enzymes, proenzymes, andtheir substrates are present at all times in thecirculation of normal individuals and perform aphysiologic function in the blood.Examples of these functional plasma enzymesinclude lipoprotein lipase, pseudocholinesterase, and the proenzymes of bloodcoagulation and blood clot dissolution .Themajority of these enzymes are synthesized inand secreted by the liver. Biochemistry for Medics 7/14/2012 3
  4. 4. 2) Nonfunctional plasma enzymes (Cell derivedenzymes)-Plasma also contains numerous otherenzymes that perform no known physiologicfunction in blood.These apparently nonfunctional plasma enzymesarise from the routine normal destruction oferythrocytes, leukocytes, and other cells. Biochemistry for Medics 7/14/2012 4
  5. 5. Tissue damage or necrosis resulting frominjury or disease is generally accompanied byincreases in the levels of severalnonfunctional plasma enzymes. Biochemistry for Medics 7/14/2012 5
  6. 6. A) Increased release i. Necrosis of cell ii. Increased permeability of cell without gross cellular damage iii. Increased production of enzyme within the cell resulting in increase in serum by overflow iv. Increase in tissue source of enzyme as in malignancyB) Impaired disposition i. Increased levels in obstructive jaundice ii. Increased levels in renal failure Biochemistry for Medics 7/14/2012 6
  7. 7. A. Decreased formation which may be i. Genetic ii. AcquiredB. Enzyme inhibitionC. Lack of cofactors Biochemistry for Medics 7/14/2012 7
  8. 8. International unit – One IU is defined as the activity ofthe enzyme which transforms one micro mole ofsubstrate in to products per minute per litre of sampleunder optimal conditions and at defined temperature .It is expressed as IU/LKatal – catalytic unit – One Katal is defined as thenumber of mole of substrate transformed per secondper litre of sample. It is abbreviated as kat or k.60 U=mkat and 1nkat=0.06U Biochemistry for Medics 7/14/2012 8
  9. 9. Single or serial assay of serum activity of a selectedenzyme-1) Helps in making the diagnosis/differential diagnosis/ early detection of a disease2) Helps in ascertaining prognosis of a disease3) Helps in ascertaining the response to drugs in a disease4) Also help in ascertaining the time course of disease. Biochemistry for Medics 7/14/2012 9
  10. 10. Enzyme estimations are helpful in the diagnosisof –1) Myocardial Infarction2) Liver diseases3) Muscle diseases4) Bone diseases5) Cancers6) GI Tract diseases Biochemistry for Medics 7/14/2012 10
  11. 11. •The diagnosis of AMI is usually predicated on theWHO criteria of chest pain, ECG changes, andincreases in biochemical markers of myocardialinjury.•Half of the patients with "typical" symptoms donot have AMI.•The ECG is specific for AMI, but lacks sensitivity.•In contrast, biochemical markers have excellentsensitivity for diagnosing AMI. By combining themost sensitive and the most specific tests,diagnostic accuracy can be enhanced. Biochemistry for Medics 7/14/2012 11
  12. 12. Serum enzymes in AcuteMyocardial InfarctionEnzyme assays routinely carried out for thediagnosis of Acute Myocardial Infarction are-1)Creatine Phospho kinase,2) Aspartate transaminase and3) Lactate dehydrogenaseAcceptable biochemical markers of ischemicheart disease are now considered to includemyoglobin, CK-MB, total CK, and cardiactroponins T and I. Biochemistry for Medics 7/14/2012 12
  13. 13. • It is an enzyme found primarily in the heartand skeletal muscles, and to a lesser extentin the brain but not found at all in liver andkidney•Catalyzes the transfer of phosphate betweencreatine and ATP/ADP•Provides rapid regeneration of ATP when ATPis low•Creatine phosphate is regenerated when ATPis abundant Biochemistry for Medics 7/14/2012 13
  14. 14. •After myocardial infarction- serum value is foundto increase within 3-6 hours, reaches a peak levelin 24- 30 hours and returns to normal level in 2-4days (usually in 72 hours).•Normal Value- serum activity varies from 10-50IU/L at 30°C.•CK is a sensitive indicator in the early stages ofmyocardial ischemia.•No increase in activity is found in heart failure andcoronary insufficiency.•In acute MI, CPK usually rises faster than SGOTand returns to normal faster than the SGOT. Biochemistry for Medics 7/14/2012 14
  15. 15. CK/CPK Isoenzymes•There are three Isoenzymes.•Measuring them is of value in the presenceof elevated levels of CK or CPK to determinethe source of the elevation.•Each iso enzyme is a dimer composed of twoprotomers ‘M’ (for muscles) and ‘B’( forBrain).•These isoenzymes can be separated by,Electrophoresis or by Ion exchangeChromatography. Biochemistry for Medics 7/14/2012 15
  16. 16. Isoenzyme Electrophoreti Tissue of Mean c mobility origin percentage in bloodMM(CK3) Least Skeletal 97-100% muscle Heart muscleMB(CK2) Intermediate Heart muscle 0-3%BB(CK1) Maximum Brain 0% Biochemistry for Medics 7/14/2012 16
  17. 17. •Normal levels of CK/CPK are almost entirely MM,from skeletal muscle.•Elevated levels of CK/CPK resulting from acutemyocardial infarction are about half MM and half MB.•Myocardial muscle is the only tissue that containsmore than five percent of the total CK activity as theCK2 (MB) isoenzyme.•Following an attack of acute myocardial infarction,this isoenzyme appears within 4 hours followingonset of chest pain, reaches a peak of activity atapproximately 24 hours and falls rapidly.• MB accounts for 4.5- 20 % of the total CK activity inthe plasma of the patients with recent myocardialinfarction and the total isoenzyme is elevated up to20-folds above the normal. Biochemistry for Medics 7/14/2012 17
  18. 18. •Itis also called as Serum Glutamate Oxaloacetate Transaminase (SGOT).•The level is significantly elevated in AcuteMI.•Normal Value- 0-41 IU/L at 37°C•In acute MI- Serum activity rises sharplywithin the first 12 hours, with a peak level at24 hours or over and returns to normalwithin 3-5 days.•The rise depends on the extent of infarction.Re- infarction results in secondary rise ofSGOT. Biochemistry for Medics 7/14/2012 18
  19. 19. Prognostic significance- Levels> 350 IU/L aredue to massive infarction (Fatal), > 150 IU/L areassociated with high mortality and levels < 50IU/L are associated with low mortality.Other diseases- The rise in activity is alsoobserved in muscle and hepatic diseases. Thesecan be well differentiated from simultaneousestimations of other enzyme activities like SGPTetc, which do not show and rise in activity inAcute MI. Biochemistry for Medics 7/14/2012 19
  20. 20. •Lactate dehydrogenase catalyzes thereversible conversion of pyruvate and lactate.•Normal level- 55-140 IU/L at 30°C. Thelevels in the upper range are generally seenin children.•LDH level is 100 times more inside the RBCsthan in plasma, and therefore minor amountof hemolysis results in false positive result. Biochemistry for Medics 7/14/2012 20
  21. 21. •In Acute MI-The serum activity rises within12 to 24 hours, attains a peak at 48 hours (2to 4 days) reaching about 1000 IU/L and thenreturns gradually to normal from 8 th to 14th day.•The magnitude of rise is proportional to theextent of myocardial infarction.• Serum LDH elevation may persist for morethan a week after CPK and SGOT levels havereturned to normal levels. Biochemistry for Medics 7/14/2012 21
  22. 22. Other diseases-The increase in serum activityof LDH is also seen in hemolytic anemias,hepatocellular damage, musculardystrophies, carcinoma, Leukemias, and anycondition which causes necrosis of the bodycells.Since the total LDH is increased in manydiseases, so the study of Iso enzymes of LDHis of more significance. Biochemistry for Medics 7/14/2012 22
  23. 23. •LDH enzyme is tetramer with 4 subunits.•The subunit may be either H(Heart) or M(Muscle)polypeptide chains.•These two chains are the product of 2 differentgenes.•Although both of them have the same molecularweight, there are minor amino acid variations.•There can be 5 possible combinations;H4, H3M1, H2M2, H1M3. M4, these are 5different types of isoenzymes seen in allindividuals. Biochemistry for Medics 7/14/2012 23
  24. 24. Biochemistry for Medics 7/14/2012 24
  25. 25. No. of Subunit Electrophore Activity at Tissue origin PercentageIsoenzyme make up of tic mobility 60°for 30 in human isoenzyme at pH8.6 minutes serum (Mean)LDH-1 H4 Fastest Not Heart 30% destroyed muscleLDH-2 H3M1 Faster Not RBC 35% destroyed.LDH-3 H2M2 Fast Partly Brain 20% destroyedLDH-4 H1M3 Slow Destroyed Liver 10%LDH-5 M4 Slowest Destroyed Skeletal 5% Muscles Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed in myocardial infarction, this is called ‘flipped pattern’. Biochemistry for Medics 7/14/2012 25
  26. 26. •They are not enzymes; however they are accepted asmarkers of myocardial infarction.•The Troponin complex consists of 3 components;Troponin C (Calcium binding), Troponin I(ActomyosinATPase inhibitory element), and Troponin T(Tropomyosinbinding element).•Troponin I is released in to the circulation within 4 hoursof the onset of cardiac manifestations, peak is observed at14-24 hours and remains elevated for 3-5 days postinfarction.•Serum level of TnT increases within 6 hous of myocardialinfarction, peaks at 72 hours and then remains elevatedup to 7-10 days. The TnT2 estimation is 100% sensitiveindex for myocardial infarction. Biochemistry for Medics 7/14/2012 26
  27. 27. One of earliest markers is myoglobin, whichis very sensitive but, in certain clinicalsettings, lacks specificity. Its level rises within 4 hours of infarctionFalsely high levels may be observed inpatients of Renal failure or patients havingmuscle injuries. Biochemistry for Medics 7/14/2012 27
  28. 28. Biochemistry for Medics 7/14/2012 28
  29. 29. Serum enzyme tests can be grouped into twocategories:(1) enzymes whose elevation in serum reflects damage to hepatocytes(2) enzymes whose elevation in serum reflects cholestasis. Biochemistry for Medics 7/14/2012 29
  30. 30. The aminotransferases (transaminases) aresensitive indicators of liver cell injury and aremost helpful in recognizing acutehepatocellular diseases such as hepatitis.These include-1) Aspartate aminotransferase (AST) and2) Alanine aminotransferase (ALT). Biochemistry for Medics 7/14/2012 30
  31. 31. •AST is found in the liver, cardiac muscle,skeletal muscle, kidneys, brain, pancreas,lungs, leukocytes, and erythrocytes indecreasing order of concentration.•Normal level- 0-41 IU/L•ALT is found primarily in the liver.•Normal level-0-45 IU/L•The aminotransferases are normally presentin the serum in low concentrations. Theseenzymes are released into the blood ingreater amounts when there is damage to theliver cell membrane resulting in increasedpermeability. Biochemistry for Medics 7/14/2012 31
  32. 32. •Levels of up to 300 U/L are nonspecific and may be found inany type of liver disorder.•Striking elevations—i.e., aminotransferases > 1000 U/L—occur almost exclusively in disorders associated withextensive hepatocellular injury such as (1) viral hepatitis, (2)ischemic liver injury (prolonged hypotension or acute heartfailure), or (3) toxin- or drug-induced liver injury.•In most acute hepatocellular disorders, the ALT is higher thanor equal to the AST.•An AST:ALT ratio > 2:1 is suggestive while a ratio > 3:1 ishighly suggestive of alcoholic liver disease.•The AST in alcoholic liver disease is rarely >300 U/L and theALT is often normal.•A low level of ALT in the serum is due to an alcohol-induceddeficiency of Pyridoxal phosphate.•In obstructive jaundice the aminotransferases are usually not Biochemistry for Medics 7/14/2012 32
  33. 33. The activities of three enzymes—1)Alkaline phosphatase,2) 5-nucleotidase, and3) γ-Glutamyl transpeptidase (GGT)—are usuallyelevated in cholestasis.Alkaline phosphatase and 5-nucleotidase arefound in or near the bile canalicular membraneof hepatocytes, while GGT is located in theendoplasmic reticulum and in bile duct epithelialcells. Biochemistry for Medics 7/14/2012 33
  34. 34. The normal serum alkaline phosphatase consists of manydistinct isoenzymes found in theliver, bone, placenta, and, less commonly, small intestine.Physiological Variations-•Patients over age 60 can have a mildly elevated alkalinephosphatase.•Individuals with blood types O and B can have anelevation of the serum alkaline phosphatase after eating afatty meal due to the influx of intestinal alkalinephosphatase into the blood.•It is also nonpathologically elevated in children andadolescents undergoing rapid bone growth, because ofbone alkaline phosphatase.•Itis also high late in normal pregnancy due to the influxof placental alkaline phosphatase. Biochemistry for Medics 7/14/2012 34
  35. 35. Pathological variations-•Elevation of liver-derived alkalinephosphatase is not totally specific forcholestasis, and a less than threefoldelevation can be seen in almost any type ofliver disease.•Alkaline phosphatase elevations greater thanfour times normal occur primarily in patientswith cholestatic liver disorders, infiltrativeliver diseases such as cancer andamyloidosis. Biochemistry for Medics 7/14/2012 35
  36. 36. In liver diseases, the elevation is almost always due to increased•amounts of the liver isoenzyme.•Inthe absence of jaundice or elevated aminotransferases, anelevated alkaline phosphatase of liver origin often, but not always,suggests early cholestasis and, less often, hepatic infiltration bytumor or granulomata.•Intrahepatic cholestasis- Values are increased in drug-inducedhepatitis, primary biliary cirrhosis, rejection of transplanted liver,and, rarely, alcohol-induced steatonecrosis.•Extrahepatic cholestasis- Very high values are found inobstructive jaundice due to cancer, common duct stone, sclerosingcholangitis, or bile duct stricture•The level of serum alkaline phosphatase elevation is not helpful indistinguishing between intrahepatic and extrahepatic cholestasis. Biochemistry for Medics 7/14/2012 36
  37. 37. 1. Hepatic Isoenzyme – Travels fastest towards the anode and occupies the same position as Alpha 2 globulin. Its level rises in extra hepatic biliary obstruction.2. Bone Isoenzyme-Increases die to osteoblastic activity and is normally elevated in children during periods of active growth .3. Placental Isoenzyme - Rises during last 6 weeks of pregnancy.4. Intestinal Isoenzyme- Rise occurs after a fatty meal. May increase during various GI disorders. Biochemistry for Medics 7/14/2012 37
  38. 38. Regan isoenzyme –•Present in plasma of about 15% of patients withcarcinoma of lung, liver or gut•Also seen in chronic smokers•Structurally resembles placental ALPNagao IsoenzymeVariantof Regan isoenzymeDetected in metastatic carcinoma of pleural surfacesand adenocarcinoma of pancreas and bile duct. Biochemistry for Medics 7/14/2012 38
  39. 39. It is involved in amino acid transport across the membranes.Found mainly in biliary ducts of the liver, kidney and pancreas.Enzyme activity is induced by a number of drugs and in particular alcohol. -GT increased in liver diseases especially in obstructive jaundice. -GT levels are used as a marker of alcohol induced liver disease and in liver cirrhosis. Biochemistry for Medics 7/14/2012 39
  40. 40.  Moderately increased in hepatitis and highly elevated in biliary obstruction. Unlike ALP the level is unrelated to osteoblastic activity and is thus unaffected by bone disease. The enzyme hydrolyses 5’ nucleotides to 5’ nucleosides at an optimum p H of7.5 Biochemistry for Medics 7/14/2012 40
  41. 41. In viral hepatitis Rapid rise in transaminases (AST & ALT) in serum occurs even before bilirubin rise is seenBiochemistry for Medics 7/14/2012 41
  42. 42. 1) Alkaline Phosphatase-Rises in Rickets, osteomalacia, hyperparathyroidism and in Paget’s disease. Also rises in primary and secondary malignancies of bones.2) Acid Phosphatase-Highly increased in bony metastasis of carcinoma prostate Biochemistry for Medics 7/14/2012 42
  43. 43. 1. Aspartate Transferase- Not commonly done2. Aldolase-Moderate increase in Dermatomyositis, muscular dystrophies, highest values are seen in Deuchenne type of muscular dystrophies3. CPK- Elevated in neurogenic muscular dystrophies, highest values are seen in Deuchenne type of muscular dystrophies Biochemistry for Medics 7/14/2012 43
  44. 44.  Amylase-Serum activity > 1000 units is seen within 24 hours in acute Pancreatitis, values are diagnostic. A raised serum activity is also seen in perforated peptic ulcer and intestinal obstruction. Lipase-Levels as high as 2800 U/l are seen in acute pancreatitis. Also reported high in perforated duodenal and peptic ulcers and intestinal obstruction. Biochemistry for Medics 7/14/2012 44
  45. 45. Enzyme DiseaseSerum acid phosphatase Cancer prostateSerum Alkaline phosphatase Metastasis in liver, jaundice due to carcinoma head of pancreas, osteoblastic metastasis in bonesSerum LDH Advanced malignancies and LeukemiasΒ- Glucuronidase Cancer of urinary bladderLeucine Amino Peptidase (LAP) Liver cell carcinomaNeuron specific Enolase Malignancies of nervous tissue and brain Biochemistry for Medics 7/14/2012 45
  46. 46. Enzyme Used for testingUrease UreaUricase Uric acidGlucose oxidase GlucoseCholesterol oxidase CholesterolLipase TriglyceridesAlkaline phosphatase ELISAHorse radish Peroxidase ELISARestriction endonuclease Recombinant DNA technologyReverse transcriptase Polymerase chain reaction Biochemistry for Medics 7/14/2012 46
  47. 47. Enzyme Therapeutic ApplicationStreptokinase/Urokinase Acute MI, Pulmonary embolism, DVT(Deep vein thrombosis)Trypsin, lipase and amylase Pancreatic insufficiencyAsparaginase/Glutaminase Acute lymphoblastic leukemiasHyaluronidase Enhanced local anesthesia and for easy diffusion of fluidsPapain Anti inflammatoryChymotrypsin Pain killer and Anti inflammatoryAlpha- 1 Antitrypsin Deficiency and EmphysemaSerratopeptidase Pain killer and Anti inflammatory Biochemistry for Medics 7/14/2012 47
  48. 48. Conditions in which level of activity NAME OF THE ENZYME in serum is elevatedAspartate Amino transferase (AST) Myocardial infarction, Liver diseaseSerum glutamate-oxaloacetate especially with liver cell damagetransaminase (SGOT)Alanine Amino transferase (ALT) Liver disease especially with liver cellSerum glutamate-pyruvate damagetransaminase (SGPT)Alkaline Phosphatase (ALP) Liver disease- biliary obstruction Osteoblastic bone disease-ricketsAcid Phosphatase (ACP) Prostatic carcinoma glutamyl Transferase ( GT) Liver disorder like liver cirrhosis and alcoholismCreatine kinase (CK) Myocardial infarction and skeletal muscle disease(muscular dystrophyLactate Dehydrogenase (LDH) Myocardial infarction,7/14/2012diseases Biochemistry for Medics other 48

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