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## YOUR PATIENT * Pale stool and dark urine ### Complaints: * Anorexia, nausea, vomiting * Pruritus * Severe Abdominal pain * Fever & chills ### Examination: * Fat * Fertile * Palpable gallbladder ## * Serum Bilirubin * Conjugated: ++++ * Unconjugated: +++ * Urobilinogen: ↓ * Urinary b...

## YOUR PATIENT * Pale stool and dark urine ### Complaints: * Anorexia, nausea, vomiting * Pruritus * Severe Abdominal pain * Fever & chills ### Examination: * Fat * Fertile * Palpable gallbladder ## * Serum Bilirubin * Conjugated: ++++ * Unconjugated: +++ * Urobilinogen: ↓ * Urinary bilirubin: + * Urinary bile salts: + * Serum ALP: ↑ * Serum GGT: ↑ * Serum 5-nucleotidase: ↑ * Transaminases: Mildly raised ## ### Ultrasound An image of an ultrasound showing the gallbladder and bile ducts. There is a large stone in the common bile duct and a smaller stone in the cystic duct. * Common hepatic duct * Stone impacted in cystic duct or gall bladder neck * Biliary pain * Acute cholecystitis * Stone in common bile duct * Biliary pain * Obstructive jaundice * Cholangitis * Ampulla of Vater * Duodenum The image is labeled with the following information: * TR 7285.7 * TE 782.0 * TI 191 * VIC 5 ## CLINICAL ENZYMOLOGY ### Types of Enzymes in Blood Plasma * **Diagnostic Enzymology** * Myocardial Infarction * Liver disease * Bone disease * Muscle disease * Cancers * **Therapeutic Enzymology** * **Diagnostic Tools** ## ### Types of Enzymes in Blood Plasma 1. **Functional (in plasma)** e.g. Lipoprotein Lipase (attached to endothelial cells in capillaries) * Zymogens → Enzymes for blood coagulation 2. **Non Functional (Functional in Cell)** e.g. Cell turnover/disease The image contains a diagram showing normal cell turnover on the left and cell necrosis as a result of disease or trauma on the right. * **Increased plasma levels due to cell damage. ** The diagram highlights the connection between tissue damage and increased levels of intracellular enzymes in the blood plasma. ## TWO MAJOR TYPES OF PLASMA ENZYMES * **Functional** * Higher amount * Its substrate is present * Definite function * Eg: - Lipoprotein Lipase * **Non Functional** * Normal low levels due to routine cell destruction * High level in Tissue necrosis/cell proliferation/enzyme induction * No function in blood plasma * Substrate absent ## NON-FUNCTIONAL PLASMA ENZYMES * Enzymes not normally found in blood but in cells (i.e. they have no function in the blood). * If found in blood → Cellular abnormality (i.e. cell damage → leaking enzyme, or cellular proliferation e.g. cancer) * ↑ Tissue specific enzyme/isoenzyme → indicates disease of that tissue ## FUNCTIONAL AND NON-FUNCTIONAL PLASMA ENZYMES | Functional plasma enzymes | Non-functional plasma enzymes | | :--------------------------------------------------------------------- | :------------------------------------------------------------------------------------------- | | Concentration in plasma | Normally, present in plasma in very low concentrations in comparison to tissues | | Present in plasma in higher concentrations in comparison to tissues | No known functions | | Have known functions | Their substrates are absent from the blood | | Their substrates are always present in the blood | Different organs e.g. liver, heart, brain and skeletal muscles | | Liver | Different enzymes increase in different organ diseases | | Decrease in liver diseases | ALT, AST, CK, LDH, alkaline phosphatase, acid phosphatase and amylase | | Clotting factors e.g. prothrombin, Lipoprotein lipase and pseudo-choline esterase | | ## CLINICAL IMPORTANCE Measurement of non-functional plasma enzymes is important for: * **Diagnosis**: Disease of different organs cause elevation of different plasma enzymes. * **Prognosis**: Effect of treatment, by measuring plasma enzymes before & after the treatment. ## MEDICALLY IMPORTANT NON-FUNCTIONAL PLASMA ENZYMES * AMYLASE → increased in acute pancreatitis. * CREATINE KINASE → increased in heart, brain & skeletal muscle diseases. * LACTATE DEHYDROGENASE → increased in heart, liver & blood diseases. * ALANINE TRANSAMINASE → increased in liver & heart diseases. * ASPARTATETRANSAMINASE → increases in liver & heart diseases. * ACID PHOSPHATASE → increases in prostate cancer. * ALKALINE PHOSPHATASE → increases in obstructive liver diseases & bone diseases. ## ENZYMES AS DIAGNOSTIC MARKERS | NAME OF THE ENZYME | Conditions in which level of activity in serum is elevated | | :----------------------------------------------------------------------- | :------------------------------------------------------------------------------------------------------------------------------------------------ | | Aspartate Amino transferase (AST) | Myocardial infarction, Liver disease especially with liver cell damage | | Serum glutamate-oxaloacetate transaminase (SGOT) | | | Alanine Amino transferase (ALT) | Liver disease especially with liver cell damage | | Serum glutamate-pyruvate transaminase (SGPT) | | | Alkaline Phosphatase (ALP) | Liver disease- biliary obstruction, Osteoblastic bone disease-rickets | | Acid Phosphatase (ACP) | Prostatic carcinoma | | y glutamyl Transferase (y GT) | Liver disorder like liver cirrhosis and alcoholism | | Creatine kinase (CK) | Myocardial infarction and skeletal muscle disease(muscular dystrophy | | Lactate Dehydrogenase (LDH) | Myocardial infarction, other diseases like liver diseases, some blood diseases | ## DIAGNOSTIC ENZYMES * Enzyme activity, and not concentration, is estimated. * Estimated as IU/ml * 1 IU/ml = Activity of enzyme which transforms 1 μmol of substrate/min * Level of Enzyme activity * Correlates with level of tissue damage * Predicts prognosis * Tissue specific enzymes/isoenzymes reflect damage to particular tissue ## MYOCARDIAL INFARCTION An image of a man with a pained expression clutching his chest. * Nausea, or vomiting * Sweating * Pallor (pale skin) * Anxiety * "Crushing" chest pain * Difficulty breathing ## INVESTIGATIONS ### CARDIAC MARKERS & TIME COURSE AFTER ONSET OF MI | Marker | Abnormal activity detectable (hr) | Peak value of abnormality (hr) | Duration of abnormality (days) | | :-------- | :---------------------------- | :------------------------------- | :------------------------------- | | CK-MB | 3-8 | 10-24 | 2-3 | | LD | 8-12 | 72-144 | 8-14 | | AST | 6-12 | 24-48 | 4-6 | | MYOGLOBIN | 1-3 | 6-9 | 1 | | TROPONINI | 3-8 | 24-48 | 3-5 | | TROPONINT | 3-8 | 72-100 | 5-10 | ### Cardiac Enzymes * Cardiac enzymes are markers found in the blood. * They are tested when Myocardial Infarction (MI) is suspected. * The markers are normally present at all times, however, they are significantly elevated during a damage of the heart muscle. ## SERUM ENZYMES IN MYOCARDIAL INFARCTION | Name of enzyme | Starts to rise (hours) | Peak elevation (hours) | Duration of rise (days) | | :------------------------------------------- | :----------------------- | :------------------------- | :----------------------- | | Creatinine kinase (CK) | 4-8 | 24-28 | 3-5 | | Aspartate transaminase (AST) | 6-8 | 24-48 | 4-6 | | Lactate dehydrogenase (LDH) | 12-24 | 48-72 | 7-8 | | Troponins T & troponin I | 4-6 | 24-72 | 5-7 | ## Types of Biochemical Markers for Diagnosis of Myocardial Infarction * **Cardiac enzymes (isoenzymes):** * Total CK * CK-MB activity * CK-MB mass * LDH * AST * **Cardiac proteins:** * Myoglobin * Troponins ## CARDIAC MARKERS A diagram shows the levels of cardiac markers over time, starting from the onset of infarction. * Myoglobin * Total CK * CK-MB * LDH * Troponin I ## SERUM CARDIAC MARKERS A diagram showing a graph of the rise and fall of cardiac markers over time. * Cardiac troponin after “classic” acute MI * CK - MB after acute MI * Cardiac troponin after “microinfarction” (unstable angina) * Upper reference limit ## ECG An image of an ECG displaying a significant ST elevation indicative of myocardial infarction. ## Criteria of Ideal Markers For Myocardial Infarction 1. **Specific:** to myocardial muscle cells (no false positive) 2. **Sensitive:** * Rapid release on onset of attack (diagnose early cases) * So, can detect minor damage * No miss of positive cases (no false negative) 3. **Prognostic:** relation between plasma level & extent of damage 4. **Persists longer:** so, can diagnose delayed admission 6. **Reliable:** procedure depends on evidenced principle 5. **Simple, inexpensive:** * Can be performed anywhere by low costs * No need for highly qualified personnel 7. **Quick:** low turnaround time ## Cardiac Enzymes * **Total CK** (sum of CK-MM, CK-MB & CK-BB) * Non specific to cardiac tissue (available also in skeletal muscles) * **CK-MB (CK-2) activity** * More specific than total CK * BUT: less specific than cardiac troponin I (as CK-MB is also available in skeletal muscles) * Appears in blood: within 4-6 hours of onset of attack (used for early cases) * Reaches maximum peak within: 12-24 hours * Returns to normal: after 2-3 days of onset (no long stay in blood, so not for delayed admissions) * Advantages; * Useful for early diagnosis of MI * Useful for diagnosis reinfarction * Disadvantages: * Not used for delayed admission (more than 2 days) * Not 100% specific (elevated skeletal muscle damage) ## CREATINE KINASE (CK) * It has three isozymes: * CK-MM→ skeletal muscles. * CK-BB → brain. * CK-MB → heart & skeletal muscles. * It was very useful in the diagnosis of myocardial infarction (MI) previously. ## Cardiac Enzymes cont. * **Lactate dehydrogenase (LDH)** * LDH is a tetramer, each chain may be one of two types (H & M) where: * LDH1 is (H4) while LD5 is (M4) * 5 isomers are available, but, each predominates in a certain organ. * LD1 & LD2 predominate in heart * Detected in blood: 18-16 hours after onset of MI attacks (not for early cases) * Reaches a maximum peak level: in 48 h * Remains elevated for: 5-6 days after MI (may remain elevated up to 14 days) * **Disadvantages:** * A non-specific marker of as it is also elevated in diseases of liver, lung, kidney, RBCs etc ## LACTATE DEHYDROGENASE (LDH) * It is a tetrameric enzyme consisting of two monomer types: H (for heart) & M (for muscles) that combine to yield five LDH isozymes: * HHHH → predominates in heart tissue. * HHHM → in the reticuloendothelial system * HHMM → in the lungs * HMMM → in the kidneys, placenta, and pancreas * MMMM → predominates in liver. ## LACTATE DEHYDROGENASE | LDH | Isozyme | Description | | :--- | :------ | :----------------------------------------------------------------- | | 1 | HHHH | Occurs in myocardium (aerobic tissues) | | 2 | HHHM | In acute leukemia | | 3 | HHHM | In acute leukemia | | 4 | HMMM | Occurs in muscle and liver (anaerobic tissues) | | 5 | MMMM | Occurs in muscle and liver (anaerobic tissues) in liver disease | ## SERUM APARTATE AMINO TRANSFERASE (AST) ## SERUM GLUTAMATE OXALO ACETATE (SGOT) 1. NORMAL RANGE: 6-25 IU/L 2. STARTS INCREASING WITHIN 6-12 HRS AFTER CHEST PAIN 3. PEAK HRS → 24-48 HRS 4. Less specific indication of myocardial infarction as it occurrence liver, muscle, hemolytic diseases 5. Prolonged Myocardial Ischemic, Congestive Heart Failure → increase in SGOT (AST) 6. Increase in SGOT (AST) < Increase in CPK (TOTAL) 7. HALF LIFE is of small duration & returns to normal within 4-6 days ## Cardiac Proteins * **Cardiac Troponins** * Protein complex located on the thin filament of striated muscles consists of 3 subunits: cTn T, cTn I & cTn C with different structures & functions * cTnI & cTnT are used as biomarkers for MI diagnosis * Cardiac troponins (cTn) are different from skeletal muscle troponins * So, more specific for MI diagnosis ## Cardiac Proteins cont. * **Cardiac Troponin I (cTn I)** * 100% cardiac specific * With greater sensitivity for diagnosing minor damage of MI * Appears in blood: within 6 hours after onset of infarction * Reaches maximum peak: around 24 hours * Disappears from blood: after about 10 days (stays longer) * So, useful for diagnosis of delayed admission * **Prognostic marker:** Matching relation between level in blood & extent of cardiac damage ## Cardiac Proteins * **Cardiac Troponins** * Protein complex located on the thin filament of striated muscles consists of 3 subunits: cTn T, cTn I & cTn C with different structures & functions * cTnI & cTnT are used as biomarkers for MI diagnosis * Cardiac troponins (cTn) are different from skeletal muscle troponins * So, more specific for MI diagnosis ## MYOGLOBIN (MB)as a cardiac marker ## CLINICAL INTERPRETATION of Serum Myoglobin 1. Increase in serum levels after myocardial infarction as early as 1 hr (necrosis) 2. Peak values → 4-12hrs 3. Myoglobin remain high 0-4hrs 4. Time period :in which CPK -2 & CARDIAC TROPONIN IS VERY SHORT. **Disadvantages of Myoglobin as cardiac marker:** 1. non specific eg myoglobin increases in any form of muscle damage 2. Methods not tissue specific (muscle/cardiac) 3. Muscle injury → increase in myoglobin → misdiagnosis of AMI ## HEPATIC ENZYMES An image of a child with jaundice. * **Viral Hepatitis** * Nausea and vomiting * Jaundice (yellowing of the skin & sclera) * Fatigue * Weakness ## SERUM ENZYMES IN HEPATIC DISEASES * Alkaline phosphatase → cholelithiasis (obstructive jaundice). * Aspartate aminotransferase → cell damage/liver cirrhosis. * Alanine aminotransferase → viral hepatitis. * y - glutamyltransferase → alcoholics. ## Serum enzymes in liver diseases Serum enzyme tests can be grouped into two categories: 1. Enzymes whose elevation in serum reflects damage to hepatocytes 2. Enzymes whose elevation in serum reflects cholestasis. ## (1) Enzymes that Reflect Damage to Hepatocytes The aminotransferases (transaminases) are sensitive indicators of liver cell injury and are most helpful in recognizing acute hepatocellular diseases such as hepatitis. These include: 1. Aspartate aminotransferase (AST) and 2. Alanine aminotransferase (ALT). ## (2) Enzymes that Reflect Cholestasis The activities of three enzymes: 1. Alkaline phosphatase, 2. 5'-nucleotidase, and 3. y-Glutamyl transpeptidase (GGT) - are usually elevated in cholestasis. Alkaline phosphatase and 5'-nucleotidase are found in or near the bile canalicular membrane of hepatocytes, while GGT is located in the endoplasmic reticulum and in bile duct epithelial cells. ## LEVELS OF ENZYMES IN DISEASES INVOLVING LIVER DAMAGE A diagram showing levels of ALT, AST and Bilirubin in viral hepatitis over 25 weeks. * **ALT (alanine transaminase)** * **AST (aspartate transaminase)** * **Bilirubin** In viral hepatitis, a rapid rise in transaminases (AST & ALT) in serum occurs even before bilirubin rise is seen. ## Liver Function Tests (LFTs) | LFT Components | Hepatitis Pattern | Cirrhosis Pattern | | :--------------- | :-------------------- | :------------------ | | Total Protein | Normal | ↓ | | Albumin | Normal | ↓ | | Globulin | Normal | ↑ | | A/G ratio | >1 | <1 | | Alkaline Phosphatase | Elevated 1-2 times normal | Elevated 1-2 times normal | | ALT (SGPT) | Values increased into the thousands | ALT, AST are increased up to a maximum of 300 IU | | AST (SGOT) | Values increased into the thousands, but ALT is always > than AST | Never greater than 300 IU AST is always > than ALT | ## STANDARD LIVER PANEL * Albumin * Transaminases * Alkaline phosphatase * Total bilirubin * Direct bilirubin * Gamma glutamyl transpeptidase * INR (international normalised ratio = (prothrombintest/ prothrombincontrol)ISI * 5' Nucleotidase * Lactate dehydrogenase ## SERUM TOTAL PROTEIN & A:G RATIO * **SERUM TOTAL PROTEIN ** * Made up of albumin and globulin. * Globulin is made up of a1, a2, β, and y globulins. * **THE ALBUMIN TO GLOBULIN (A/G) RATIO** * Is an index of disease state * But it is not a specific marker for disease because it does not indicate which specific proteins altered. * The normal A/G ratio is 0.8-2.0. * The A/G ratio can be decreased in response to a low albumin or to elevated globulins. ## SERUM TOTAL PROTEIN & A:G RATIO * **SERUM TOTAL PROTEIN** * Made up of albumin and globulin. * Globulin is made up of a1, a2, β, and y globulins. * **THE ALBUMIN TO GLOBULIN (A/G) RATIO** * Is an index of disease state * But it is not a specific marker for disease because it does not indicate which specific proteins are altered. * The normal A/G ratio is 0.8-2.0. * The A/G ratio can be decreased in response to a low albumin or to elevated globulins. ## HEPATIC ENZYMES | NAME OF THE ENZYME | Conditions in which level of activity in serum is elevated | | :------------------------------------------------ | :------------------------------------------------------------------------------------------------------------------------------------------------ | | Aspartate Amino transferase (AST) | Myocardial infarction, Liver disease especially with liver cell damage | | Serum glutamate-oxaloacetate transaminase (SGOT) | | | Alanine Amino transferase (ALT) | Liver disease especially with liver cell damage | | Serum glutamate-pyruvate transaminase (SGPT) | | | Alkaline Phosphatase (ALP) | Liver disease- biliary obstruction, Osteoblastic bone disease-rickets | ## LACTATE DEHYDROGENASE * Converts lactate to pyruvic acid and back, as it converts NAD+ to NADH and back * LDH is expressed extensively in body tissues, such as blood cells and heart muscle. Because it is released during tissue damage, it is a marker of common injuries and disease such as heart failure. * Many cancers can raise LDH levels, so LDH may be used as a tumor marker, but it is not useful in identifying a specific kind of cancer. Measuring LDH levels can be helpful in monitoring treatment for cancer. Noncancerous conditions that can raise LDH levels include heart failure, hypothyroidism, anemia, pre-eclampsia, meningitis, encephalitis, acute pancreatitis, HIV and lung or liver disease ## GAMMA-GLUTAMYL TRANSFERASE * Gamma-glutamyl transferase is a transferase that catalyzes the transfer of gamma-glutamyl functional groups from molecules such as glutathione to an acceptor that may be an amino acid, a peptide or water * GGT test may be used to determine the cause of elevated alkaline phosphatase (ALP). Both ALP and GGT are elevated in disease of the bile ducts and in some liver diseases, but only ALP will be elevated in bone disease. ## GAMMA-GLUTAMYL TRANSFERASE * Gamma-glutamyl transferase is a transferase that catalyzes the transfer of gamma-glutamyl functional groups from molecules such as glutathione to an acceptor that may be an amino acid, a peptide or water * GGT test may be used to determine the cause of elevated alkaline phosphatase (ALP). Both ALP and GGT are elevated in disease of the bile ducts and in some liver diseases, but only ALP will be elevated in bone disease. ## LACTATE DEHYDROGENASE * Converts lactate to pyruvic acid and back, as it converts NAD+ to NADH and back * LDH is expressed extensively in body tissues, such as blood cells and heart muscle. Because it is released during tissue damage, it is a marker of common injuries and disease such as heart failure. * Many cancers can raise LDH levels, so LDH may be used as a tumor marker, but it is not useful in identifying a specific kind of cancer. Measuring LDH levels can be helpful in monitoring treatment for cancer. Noncancerous conditions that can raise LDH levels include heart failure, hypothyroidism, anemia, pre-eclampsia, meningitis, encephalitis, acute pancreatitis, HIV and lung or liver disease ## INR * International normalized ratio * The INR is the ratio of a patient's prothrombin time to a normal (control) sample ## SERUM ENZYMES IN MUSCLE DISEASES An image showing an anatomical diagram of the back and shoulder muscles. * Creatine kinase * Aspartate transferase ## MUSCLE DISEASE An image showing an illustrative comparison between healthy and damaged muscles in the lower leg * **Rhabdomyolysis** * Healthy muscles * Damaged muscles ## MARKERS OF MUSCLE DAMAGE * Creatine kinase (CK) This enzyme is released by damaged muscle. CK concentrations rise steadily for 12 hours after the original muscle injury, remain elevated for 1-3 days and then fall * Myoglobin has a short half-life, and is therefore less useful as a diagnostic test in the later stages * Elevated concentrations of the enzyme lactate dehydrogenase (LDH) * Other markers of muscle damage e.g. aldolase, troponin, carbonic anhydrase transaminases, enzymes abundant in both liver and muscle tissue, are also usually increased ## 5) Serum enzymes in Gl tract diseases * **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. ## SERUM ENZYMES IN BONE DISEASES * **Alkaline phosphatase** * Pagets disease (10 – 25 times) * Bone cancer * Healing of bone fracture * Osteomalacia and rickets ## 6) Enzymes as tumor markers | Enzyme | Disease | | :------------------------------ | :---------------------------------------------------------------------- | | Serum acid phosphatase | Cancer prostate | | Serum Alkaline phosphatase | Metastasis in liver, jaundice due to carcinoma head of pancreas, osteoblastic metastasis in bones | | Serum LDH | Advanced malignancies and Leukemias | | B- Glucuronidase | Cancer of urinary bladder | | Leucine Amino Peptidase (LAP) | Liver cell carcinoma | | Neuron specific Enolase | Malignancies of nervous tissue and brain | ## THERAPEUTIC USES OF ENZYMES * **A. Enzymes used systemically** * Streptokinase (proteolytic) - Acute myocardial infarction * Urokinase /"plasmin" -Acute thrombosis of arteries / precursor "plasminogen Pulmonary embolism. Plasmin acts directly on "fibrin" breaking it down to achieve thrombolysis * L-Asparaginase Certain tumours. Acute leukaemia* Malignant lymphomas L-Asparaginase hydrolysesL-Asparagine and growth of tumour cells suffer * Digestive enzymes Available as tablets and syrup Replacement therapy in pancreatic Cystic fibrosis. Chronic pancreatitis/ Following pancreatectomy * In management of inflammatory oedema due to injury, postsurgical infection: and dental procedures * **B. Enzymes used locally** * Hyaluronidase Available as "Hyalase" depolymerisation of Promotes diffusion of fluids * Intra-articular injection of Hyaluronic acid in joints to alleviate pain in osteoarthritis ## DIAGNOSTIC TOOLS FOR ESTIMATION * GLUCOSE OXIDASE – glucose * UREASE - Urea * URICASE – Uric acid * PEROXIDASE - Cholesterol / Glucose * CHOLESTEROL OXIDASE - Cholesterol * ALKALINE PHOSPHATASE – ELISA * REVERSE TRANSCRIPTASE – PCR * RESTRICTION ENDONUCLEASE – Southern blot