3INTRODUCTIONEnzymes are the protein catalysts that increase the rate of specific chemical reaction in the body.Enzymes are found in small amounts mainly within cells ,clotting factors and digestive enzymes function naturally after secretion: Plasma specific – Thrombin Secreted - Lipase, Amylase Intracellular - transaminases, creatine kinaseInjury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream.Elevated enzyme levels are often indicators of tissue problems, and are used in the diagnosis of diseases.Enzyme activities in the body fluids are altered by pathological processes so, its measurement is used for disease investigation.
4Chemical Nature of Enzymes All known enzymes are proteins.They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds.Enzymes can be denatured and precipitated with salts, solvents and other reagents.They have molecular weights ranging from 10,000 to 2,000,000.Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be exerted.This entire active complex is referred to as the HOLOENZYME; i.e., APOENZYME (protein portion) plus the COFACTOR (coenzyme, prosthetic group or metal-ion-activator) .
5Apoenzyme + Cofactor = Holoenzyme Chemical Nature of Enzymes ……contd.Apoenzyme + Cofactor = HoloenzymeThe Cofactor may be:1 • A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely attached to the protein part.2 • A prosthetic group - an organic substance which is dialyzable and thermostable which is firmly attached to the protein or apoenzyme portion.3 • A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++.
7Classification of Enzymes : Enzymes can be classified by the kind of chemical reaction catalyzed.A. Addition or removal of water :1. Hydrolases - these include esterases, carbohydrases, nucleases,deaminases, amidases, and proteases2. Hydrases such as fumarase, enolase, aconitase and carbonicanhydraseB. Transfer of electrons :1. Oxidases2. Dehydrogenases
8Classification of Enzymes …… contd. C. Transfer of a radical: Transglycosidases - of monosaccharides Transphosphorylases and phosphomutases - of a phosphate group Transaminases - of amino group Transmethylases - of a methyl group Transacetylases - of an acetyl group D. Splitting or forming a C-C bond: Desmolases Changing geometry or structure of a molecule 3. Isomerases E. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other tri-phosphate 1. Ligases
9Specificity of Enzymes : One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze.Greater specificity is achieved in three ways:Interpreting investigations in the light of clinical featuresTest pattern recognitionIsoenzyme determination: AST may be due to MI or Hepatitis so, it makesconfusion in diagnosis to be confirmed by LDH levels.- ALP in Cholestasis & bone diseases :- Differentiated by bilirubin & transaminase levels inCholestasis .- Confirmed by GGT in Cholestasis.
10Specificity of Enzymes ………contd. In general, there are four distinct types of specificity:Absolute specificity - the enzyme will catalyze only one reaction.Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.
11Factors affecting serum enzyme activity Rate of synthesisTissue damageMass of enzymeProducing tissueRate of entry into bloodInhibitionSerum enzyme activityRate of removalInactivationClearance
12Factors affecting Serum enzyme activities-1 I- Rate of entry of enzymes into blood is affected by:a – Rate of synthesis of enzyme:- Biliary obstruction hepatobiliary tree enzymes- Drugs :Anticonvulsant drugs ( Phenobarbital & phenytoin) synthesis of enzymes by the hepatocytesb- Mass of enzyme producing cells as in: - alkaline phosphatase: ( in active growth , Paget’s disease. and in 3rd trimester pregnancy). - acid phosphatase ( in cancer prostate).c- Necrosis or Cell damage as in: - Hepatitis transaminases - Myocardial infarction CK - Stored blood LDH
13Factors affecting Serum enzyme activities-2 II- Enzyme inhibitors:- Little effect on enzyme values determined in the lab.- Organophosphorus poisoning irreversible inhibition ofcholinesterase.III- Clearance of enzymes:- Breakdown by Proteases and removal by the reticuloendothelialsystem.- Renal excretion of small molecular enzymes e.g. Amylase
15Schematic diagram showing the effect of temperature on rate of nonenzyme-catalyzed and enzyme catalyzed reactions.
16How does an enzyme work? Enzymes have an active site- a cleft into which substrate molecules fitThe active site contains amino acids that:- Attract the substrate- Assist in the chemical reactions that convertssubstrate to product
19Sequence of events in enzyme catalyzed reaction 1) E + S ® ES.Enzyme and Substrate collide.Substrate binds to active site of enzyme.A transition state forms where the structure of the substrate is altered.2) ES ® EP.Enzyme catalyses the conversion of substrate to Product.Both substrate and product remain in active site.3) EP ® E + P.Product is released from active site.
21ISOENZYMES Catalyze the same reaction Two or more polypeptide chains Different polypeptide chains are products of different genesDiffer in AA sequence and physical propertiesMay be separable on the basis of chargeAre tissue specific
22Isoenzymes and multiple forms Aminotransferases:ALTASTGGTALT and AST :1. Pyridoxal dependent2. Indicates Hepatitis, Myocardial infarction:- Elevations take > 4 hours to develop, last 4 days- ALT elevation lasts longer than AST- AST elevations are higher than ALT- Elevations occur in most definite infarctions3. Skeletal muscle damage4. Hemolysis5. Pancreatitis
23Isoenzymes and multiple forms GGTHepatobiliary enzyme - highly inducible in 75% of the populationHighest increases in intra or posthepatic biliary obstructionHigher and more persistent increases than AlP (Alkaline Phosphatase)
33Acid phosphatase (ACP) Found in prostate, bone, liver, spleen, kidney, RBCs and plateletsPrimarily used to diagnose prostate cancer . In other prostatic conditions e.g. prostatitis, benign prostatic hypertrophy.In other non prostatic conditions e.g. hemolysis, Paget’s disease, metastatic carcinoma of the breast & Gaucher’s disease.Prostate- Specific Antigen(PSA): an enzyme occurs in prostatic tissue and in cases of metastatic carcinomaTartarate inhibits the prostatic ACP enzyme while Formaldehyde inhibits ACP from other sources
34Alanine aminotransferase (ALT) Widely distributed, although the largest amounts found in the liver.Smaller amounts occur in the heart but usually remains normal after MI .Congestive cardiac failure release from the liverMore specific for liver disease than AST.
35Alkaline phosphatase (ALP) Widely distributed, high concentrations in intestines, liver, bone, spleen, placenta and kidney.The main sources of serum ALP are the hepatobiliary tree and bone disorders.Elevated levels during healing of fractures , active growth and during the 3rd trimester of pregnancy. serum ALP activity in liver disease is mainly due to Cholestasis.Decreased levels are found in the inherited condition “ Hypophosphatasia” which is caused by defective bone calcification
36Alkaline phosphatase (ALP) Causes of increased serum alkaline phosphatase enzyme activity:Physiological :Bone disease:Hepatobiliary disease:Others:- Infancy- Puberty- Pregnancy- Intestinal isoenzymes- Hyperparathyroidism- Osteomalacia, rickets- Paget’s disease of bone- Osteomyelitis- Hepatitis- Cholestasis- CirrhosisCarcinoma of the bronchus
39Amylase (AMS)Clinical Significance : Diagnosis and monitoring of pancreatitis1. Acute: - transient increase in activity within hours- returns to normal in days2. Extent of increase (typically fold) related to probability of acute pancreatitis.3. Serum amylase activity may be falsely normal in presence of severe dyslipidaemia - check urinary amylase.4. Serum amylase may be raised in biliary duct obstruction.5. Serum amylase activity may be normal in chronic pancreatitis.6. Amylase may be raised in pleural effusions and pseudocystic fluids secondary to pancreatitis.7. Salivary gland diseases or trauma may raise serum amylase activity.
40Salivary Enzymes in Normal Function and Abnormal Pathology α-Amylase—normally present in saliva—important in digestion______________________________________________LysozymeHyaluronidaseChondrosulfataseAryl SulfataseNeutral ProteaseCollagenasesSpecific activities are increased in gingivitis and periodontal disease
41Causes of Hyperamylasemia and Hyperamylasuria 1. Pancreatic disease (P-type):Pancreatitis:- Acute- Chronic- Complications:PseudocystAscites and pleural effusionAbcessPancreatic Trauma, including investigative maneuversPancreatic carcinoma
42Causes of Hyperamylasemia and Hyperamylasuria 2. Disorders of non-pancreatic origin(mechanism unknown) :a- Renal insufficiency (mixed)b- Neoplastic hyperamylasemia - usually bronchogenicor ovarian (usually S-type)c- Salivary gland lesions, e.g. mumps, calculus disease(S-type)d- Macroamylasemia (predominantly S-type)
43Causes of Hyperamylasemia and Hyperamylasuria 3. Disorders of complex origin (mechanism unknown or uncertain)Biliary tract diseaseIntra-abdominal disease (other than pancreatic diseases):Perforated peptic ulcer (P-type)Intestinal obstruction (P-type)Mesenteric infarction (P-type)Peritonitis (mixed; depends on cause)Acute appendicitisRuptured ectopic pregnancy (S-type)Aortic aneurysm with dissectionCerebral trauma (type depends on other organ damage)Burns and traumatic shockPostoperative hyperamylasemia (usually S-type)Diabetic ketoacidosis (mixed)Renal transplantation (S-type)Acute alcoholism (mixed)Drugs: - Medicinal opiates (P-type) - Heroin addiction (S-type)
44Aspartate aminotransferase (AST) This enzyme is widely distributed in the body.Main sources: Heart, liver, skeletal muscle, and kidney.Useful in the diagnosis of MI, liver disorders and muscle damage.Causes of serum AST levels:Physiological : Neonates.Liver diseases: Hepatitis, hepatic necrosis , cholestasisCardiac disease: Myocardial Infarction.Diseases of skeletal muscle: Crush injury,trauma,myopathyFrom Erythrocytes: Hemolysis
45Creatine kinase (CK)Creatine kinase is associated with ATP regeneration in muscle and nervous tissue.Elevated blood levels of CK are used as indicators of MI, muscular dystrophy, and stroke.CK occurs as a dimer of 2 different subunits, M and B. - CK-BB: Brain type. - CK-MB: Hybrid type. - CK-MM: Muscle type.These can be separated by electrophoresis.CK-MB is released from cardiac muscle cells after MI.
46Creatine kinase (CK) BB % MB % MM % A dimer - M and B protein strands which are the products of different genes - true isoenzymes.5% cutoff by general agreement2 of 3 - history, ECG, enzymesBB %MB %MM %< 1<20> 80MM - skeletal muscle> 40> 60MB - cardiac muscle> 95< 3BB - brain
47Gamma-glutamyltransferase (GGT) A microsomal enzyme its synthesis induced by ethanol and anticonvulsant drugs.Found mainly in the kidney and significant amounts in liver, brain, prostate, and pancreas.Used primarily for diagnosis of hepatobiliary problems.ALT, AST and GGT are the main liver function tests.Marked elevation of serum GGT level is seen in alcoholic liver disease. serum GGT activity sometimes following MI or congestive cardiac failure.
48Glucose 6-phosphate dehydrogenase (G6PD) First (and control) enzyme for pentose phosphate pathway (P.P.P.).Important in production of NADPH + H+, especially in RBC.NADPH + H+ keeps glutathione reduced.Antimalarial drugs are oxidants, and adversely affect this system in RBCs.Some populations, especially African-Americans, have a high frequency of G6PD deficiency.If given antimalarial drugs, or fava beans, they develop hemolytic anemia.
49Lactate dehydrogenase (LDH or LD) Converts pyruvate to lactate (and vice versa) during and after anaerobic metabolism.LDH occurs as a tetramer of 2 different subunits: LD-1 (HHHH) from the heart:Elevated after MI. LD-2 (HHHM) from the kidney:Elevated after renal infarction. LD-3 (HHMM) from the lung, spleen and pancreas: Elevated in pulmonary embolism.LD-4 (HMMM) and LD-5 (MMMM), both from the liver and skeletal muscle:Elevated in injury to liver or skeletal muscle.
51Lipase (LPS) Pancreatic lipases : Breaks down fat into monoacylglycerol and free fatty acids.Primarily from the pancreas.Used to diagnose acute pancreatitis.Pancreatic lipases :- A group of enzymes that hydrolyze glycerol esters of long chain fatty acids.- Some substrate specificity e.g. LPL (Lipoprotein Lipase).- Bile salts are necessary for activity.-Almost exclusively used clinically in the investigation of pancreatitis.- Increase within hours of acute attack.- May remain elevated for many days .- More specific to acute pancreatitis than amylase.- Less sensitive to acute exacerbations in chronic pancreatitis.
52Plasma cholinesterase Similar to cholinesterase in nervous system, degradesacetylcholine (neurotransmitter and hormone).Elevated in hepatitis and cirrhosis.Also elevated in organophosphate (pesticide) poisoning.Degrades succinylcholine, a muscle relaxant givenduring general anesthesia in surgery.Some people are deficient in plasma cholinesterase, sothe normal dose of succinylcholine would kill themTherefore, a determination of plasma cholinesterase ismade prior to major surgery.
53Low Plasma Cholinesterase ExamplesCategory of causeInfancy, 3rd Trimester of pregnancyPhysiological reasonsScoline sensitivity(ChE variants)Inherited abnormalityAcquired abnormality:Impaired protein synthesisA) Liver diseaseOrganophosphorus insecticidesB) Industrial poisoningOral contraceptives, MAO inhibitors, Cytotoxic drugsC) Drug effects
54Pancreatic Trypsin A Serine Protease (hydrolyses peptide bonds formed by the carboxyl groups of lysine/arginine).Inactive Zymogens secreted (Type 1 and 2) under the influence of vagus nerve .alpha1-antitrypsin and alpha2-macroglobulin protect serum proteins (consider alpha-1 AT deficiency).Little clinical application in modern practice.
57Myocardial Infarction ( MI ) Necrosis of the myocardium, but not angina pectoris release of CK, AST and LDH (HBD) into the circulation.CK is the first to rise (activity within 6 h of MI ).Total CK reaches a peak at h.In uncomplicated cases, CK returns to normal within 3 days.Serum AST more slowly ( maximum activity within 48 h) and returns to normal in 4-5 days.No significant elevation in HBD seen for the 1st 24 h (reaches maximum at about 3 days & remain for up to 8 days).It is important to consider the timing of sample when interpreting test results.CK & HBD are useful as early and late indicators of MI, and more specific than AST.
58Myocardial Infarction ( MI ) CK from skeletal muscle may be following intramuscular injection, chest compression for resuscitation or electrical defibrillator.CK specificity is by measuring CK-MB.HBD activity may be due to non cardiac factors (hemolysis).Cardiac enzyme measurements are very sensitive indicators of MI because it is in over 95% of cases.They are of particular value in the following conditions:Atypical clinical presentation (absence of chest pain)If the patient presents some time after a suspected event.Difficulty in interpreting ECG (Arrhythmia or previous MI).If further MI is suspected few days of a previous one.
59CK-2 & CK-3 in normal subject and in patient 24 hrs after Myocardial InfarctionCreatine Kinase isoenzymes in blood
60Plasma levels following myocardial infarction CPK---Creatine KinaseLDH---Lactate DehydrogenaseHBDH—α-Hydroxybutyrate dehydrogenase
64TroponinTroponins are a complex of I, T and C subunits uniquely present in striated muscle and regulate the calcium mediated interaction of actin and myosin.Troponin-I has three isoforms:sTnI (for fast twitch muscle);TnI (for slow twitch muscle) andcTnI (for cardiac muscle).cTnI levels are elevated in plasma within 4 hr after infarction.Better test than LDH and CKMB.
66Muscular Dystrophy Genetically determined degenerative disorders. Duchenne muscular dystrophy is an X-linked recessive disorder caused by an abnormal dystrophin gene (progressive weakness of muscles).CK activities before the onset of clinical symptoms (values >10 times the normal upper level).Serum CK is in 75 % of female carriers.Becker’s muscular dystrophy is a benign form of Duchenne MD.CK elevated pattern similar to that of Duchenne MD.
67Malignant Hyperpyrexia Toxic MyopathyCauses:- Drugs & chemicals (Alcohol, D-penicillamine, ..etc) generalized myopathy- IM injections ( Trauma & Chemical irritation)CK activity by narcotic analgesics given in MI.Rapid in body temp, shock& convulsions.(in general Anesthesia Serum CK activity during attacks .Pre-operative CK should be measured in patients with a family history of malignant hyperpyrexia.Malignant Hyperpyrexia
68Traumatic MyopathiesMuscle trauma (surgery, I.M. injection, etc..) release of enzymesHigh serum CK values occurs post-operatively .If MI suspected, CK-MB should be measured.Serum CK usually return to normal within 48 h of a single intramuscular injection.Vigorous exercise of short duration and prolonged moderate exercise serum CK
70Liver Enzymes ( ALT, AST, GGT, ALP, LDH) Measurement of serum enzyme activities for :a - Differential Diagnosis of Jaundice.b - Monitoring of drug toxicity.ALT is more specific than AST.Hepatocellular disease has only modest effect on ALP & GGT (up to 3 times the upper limit of normal)In Cholestasis, Higher values of ALP & GGT due to synthesis ( the values are 5-10 times the upper normal level) .
72Bone Enzymes - ( Alkaline Phosphatase) ALP ALP enzyme is usually normal in Osteoporosis as osteoblastic activity is not increasedModest of ALP in Osteomalacia and RicketsHealing fractures Transient of ALP1ry & 2ry Hyperparathyroidism of ALPIn Paget’s disease of bone of ALP (10 times)1ry & 2ry bone tumors of ALP (5 times normal)
73Enzymes in Urine Enzymes appear in Urine from 2 sources: - Filtration of plasma- Leaking from cells lining the urinary tract Amylase is normally detected in urine (small size). Indicators of tubular damage:- Alkaline phosphatase- N-acetyl--glucosaminindase (NAG)
74Enzymes in Hematological Disorders Inherited or acquired diseases - Hemolytic diseases- Spherocytosis- Methemoglobinemia G-6-PD deficiency Hemolysis on exposure to oxidant drugs as antimalarial drugs (Primaquine) or ingestion of fava beans Pyruvate kinase , Glutathione synthetase, Hexokinase Defects Hemolysis
78Enzymes Proteins. Increase reaction rates by lowering activation energy.Increase rates byAllow reactions to occur under much milderconditions (low temperature, atmosphericpressure, around neutral pH).Enzymes do not affect the thermodynamicproperties of a reaction- they do not alter DG.
90Some other Enzymes of Diagnostic Value Plasma AST in:Liver DiseaseAcute Renal DiseaseAcute PancreatitisAlkaline PhosphataseLiver isoenzyme in:Liver cancer and fatty liverBone isoenzyme in:Osteoblastic bone tumorsMaternal plasma AP up in the third trimester of pregnancy
94Functions of Released Enzymes Components of bone—Collagen, Hyaluronic acid, Sulfated glycoproteins, HydroxyapatiteHyaluronic Acid NAG + Glucuronic AcidHUSulfated Glycoproteins NAG sulfate + GlucuronicAS, CS AcidAcids produced breakdown the hydroxyapatite crystal lattice
95Bone Resorption Bone Collagen type I Abnormal Hydroxyapatite Crystal latticeCollagenase type I¾ and ¼ ChainsNeutral ProteinaseAmino acids and peptides