Clinical Chemistry Intern's Learning Guide 4 PDF

JOSE MARIA COLLEGE FOUNDATION,INC. MEDICAL TECHNOLOGY/MEDICAL LABORATORY SCIENCE Philippine-Japan Friendship Highway, Davao City, 8000 Davao del Sur MEDICAL TECHNOLOGY ASSESSMENT PROGRAM CLINICAL CHEMISTRY- INTERN’S LEARN...

JOSE MARIA COLLEGE FOUNDATION,INC. MEDICAL TECHNOLOGY/MEDICAL LABORATORY SCIENCE Philippine-Japan Friendship Highway, Davao City, 8000 Davao del Sur MEDICAL TECHNOLOGY ASSESSMENT PROGRAM CLINICAL CHEMISTRY- INTERN’S LEARNING GUIDE 4 Non-Protein Nitrogenous Compounds and Liver Function, Bilirubin and Enzymes NON-PROTEIN NITROGENOUS COMPOUNDS Compound Approximate Plasma Approximate Urine Concentration (% of Total Concentration (% of Total NPN) NPN) Urea 45-50% 86.0% Amino Acids 25% - Uric Acid 10% 1.7% Creatinine 5% 4.5% Creatine 1-2% - Ammonia 0.2% 2.8% JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  UREA CREATININE/CREATINE 1.) NPN present in the highest concentration in Creatinine is formed from Creatine and creatine blood. Major excretory product of protein phosphate in the muscles - formed as a by-product of metabolism muscle Physiology 2.) Synthesized in the liver, urea is carried in the Creatinine is excreted into the plasma at a constant blood to the kidney.Most of the urea in the glomerular rate related to muscle mass filtrate is excreted in the urine(90%) only less than -Plasma creatinine is inversely related to glomerular (10%) is reabsorbed via passive diffusion filtration rate (GFR) 3.) The concentration of urea in the plasma is -Plasma creatinine - commonly used to assess renal determined by: renal function and perfusion, the filtration function protein content of the diet and rate of protein catabolism Clinical application Clinical application a.evaluate renal function a.Determine sufficiency of kidney function and severity b. assess hydration status aid in the diagnosis of renal of disease disease b.Monitor the progression of kidney disease c.verify frequency of dialysis c.Measure of completeness of 24 hour collections Note: Study Creatinine Clearance Formula Pathophysiology Pathophysiology Azotemia -Elevated urea in the blood. Elevated creatinine concentration is associated with Uremia - high plasma urea concentration abnormal renal function - may signify glomerular accompanied by renal failure damage Plasma concentration of creatinine is inversely Increased plasma urea concentration: proportional to creatinine clearance a.Prerenal:Due to reduced renal blood flow or Renal damage is suspected when plasma creatinine is increase in protein diet elevated, and glomerular filtration rate is decreased b.Renal:Due to decreased renal function or damaged IMPORTANT: Plasma creatine concentration is not kidneys elevated in renal diseases. c.Postrenal: Due to obstruction of urine flow in the Plasma creatine is elevated in: urinary tract Muscular dystrophy Poliomyelitis To differentiate cause of abnormal urea Hyperthyroidism concentration, calculate BUN to creatinine ratio which is normally between 10:1 to 20:1 The normal BUN/CREATININE RATIO is 10:1 to 20:1. -High urea:creatinine ratio with normal creatinine a.In prerenal disease, it rises to well >20:1. levels - prerenal. b. misue real disease, both BUN and creatinine rise -High urea:creatinine ratio with elevated creatinine together, maintaining BUN/Creatinine ratio at 10-20: levels - postrenal. -Low urea:creatinine ratio - conditions associated with decreased urea concentration. NOTE!!! Urea nitrogen is converted to Urea by multiplying 2.14 Reference values (Bishop, 7 edition) Reference values (Bishop, 7th edition) Plasma or serum: 6-20 mg/dL (2.1-7.1 mmol/L) Plasma or serum Urine, 24 hours: 12-20 g/day (0.43-0.71 mol urea/day) i.Adult male: 0.9-1.3 mg/dL (80-115 umol/L) Conversion factor: 0.357 ii. Adult female: 0.6-1.1 mg/dL (53-97 umol/L) lii. Child: 0.3-0.7 mg/dL (27-62 umol/L) Urine, 24 hours i.Adult male: 800-2000 mg/day (7.1-17.7 mmol/day) ii.Adult female: 600-1800 mg/day (5.3-15.9 mmol/day) Conversion factor: 88.4 JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  URIC ACID AMMONIA 1. Product of the catabolism of purines (guanine 1. Formed through the deamination of amino and adenosine) acids during protein metabolism 2. Filtered by the glomerulus and secreted by the 2. Removed from the circulation and converted distal tubules into the urine, but mostly to urea in the liver reabsorbed in the proximal tubules and reused. 3. Free ammonia is toxic; however ammonia is 3. Relatively insoluble in plasma, and at high present in the plasma in low concentrations concentrations, can be deposited in the joints 4. Marker for DETOXIFICATION and tissue, causing painful inflammation; mostly 5.LEAST NPN present as monosodium urate in plasma, wherein it is insoluble at around pH 7 Clinical application Clinical application a. Assess inherited disorders of purine a. Provide information on clinical conditions such metabolism as hepatic failure, Reye’s syndrome and b. Confirm diagnosis and monitor the treatment of inherited deficiencies of the urea cycle enzymes gout c. Detect kidney dysfunction d. Assist in the diagnosis of renal calculi Pathophysiology Biochemistry Abnormally increased uric acid concentration is -Ammonia (NHs) is consumed by the found in: parenchymal cells of the liver in the production Gout - uric acid deposition in joints of urea Tophi - uric acid deposition in tissues -If liver is damaged, ammonia levels in the blood Disorders in purine metabolism (enzyme is increased deficiencies) -At normal physiologic pH, ammonia exists as i.Lesch-Nyhan syndrome - hypoxanthine ammonium ion, NH4+ guanine phosphoribosyl transferase Pathophysiology deficiency Normally, liver clears ammonia from the ii.Phosphoribosy|pyrophosphate synthetase circulation deficiency In severe liver disease, ammonia is not removed iii.Glycogen storage disease type I - glucose-6- and increases in concentration High phosphatase deficiency concentrations of ammonia are neurotoxic and iv. Fructose intolerance - fructose-1-phosphate often associated with encephalopathy aldolase deficiency Reference values (Bishop, 7th edition) Reference values (Bishop, 7th edition) Plasma or serum (uricase method) Plasma or serum i.Adult male: 3.5-7.2 mg/dL (0.21-0.43 mmol/L) Adult: 19-60 ug/dL (11-35 umol/L) ii.Adult female: 2.6-6.0 mg/dL (0.16-0.36 Child, 10 days to 2 years: 68-136 ug/dL (40-80 mmol/L) umol/L) Child: 2.0-5.5 mg/dL (0.12-0.33 mmol/L) Conversion factor: 0.59 Urine, 24 hours i. Adult: 250-750 mg/day (1.5-4.4 mmol/L) Conversion factor: 0.059 JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  ANALYTICAL METHODS REFERENCE ENZYMATIC OTHER METHODS METHOD METHOD UREA Urease a.Direct method: Also known as Rosenthal method Method - directly measures urea measured at b. Indirect method: Also known as 340 nm Microkjeldahl method - measures nitrogen instead of urea CREATININE/ Isotope -Creatininase Jaffe reaction: CREATINE dilution mass -Creatinase Creatinine + Picric acid=red-orange complex spectrometry -Sarcosine Jaffe-Kinetic:avoid interferences and uses: (IDMS) oxidase -Fuller’s earth reagent (aluminum magnesium -Peroxidase silicate) -Lloyd’s Reagent (Sodium aluminum silicate) URIC ACID Uricase Chemical Method (Caraway method or method phosphotungstic acid method) - It uses measured at phosphotungetic acid to produce allantoin and 293 nm a tungsten blue complex AMMONIA Enzymatic method (most Titration method (Conway method) - uses commonly used) -Uses volatility of ammonia to separate it from the glutamate dehydrogenase sample using a microdiffusion chamber (GLDH) LIVER FUNCTION AND BILIRUBIN Liver -Complex organ that supports all the other body systems One of the most important organs the human body has, and its absence would mean disruption of the body systems Capable of regenerating cells that have been destroyed or damaged due to a short-term disease If repeatedly damaged, it may undergo irreversible changes that affects its vital functions Death may occur within approximately 24 hours due to hypoglycemia if the liver becomes completely nonfunctional I. Basic Anatomy of the Liver Weight of liver = 1.2-1.5 kg Divided unequally into two lobes by a falciform ligament - right lobe is approx. 6 times larger than the left lobe; division of lobes do not affect its functions Blood supply in the liver is from: (1) hepatic artery - which carries oxygen-rich blood from the heart, (2) portal vein - which carries nutrient-rich blood from the digestive tract Lobule - functional unit of the liver; responsible for all metabolic and excretory functions of the liver Two major cell types in the liver: hepatocytes and Kupffer cells Hepatocytes - responsible for performing major functions and for regeneration Kupffer cells - macrophages of the liver; engulfs bacteria, debris, toxins, etc. JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  Biochemical Functions of the Liver Bilirubin It is the end product of hemoglobin metabolism and the principal pigment in bile. It is also formed from destruction of heme-containing proteins such as myoglobin, catalase and cytochrome oxidase. A.EXCRETORY AND SECRETORY JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  Comparison Between Conjugated Bilitrubin and Unconjugated Bilirubin Unconjugated Bilirubin Conjugated Bilirubin Bilirubin 1 Bilirubin 2 Water insoluble Water soluble Non-polar bilirubin Polar bilirubin Indirect (reacting) bilirubin Direct (reacting) bilirubin Hemobilirubin Cholebilirubin Slow-reacting Fast-reacting ↑↑↑ Prehepatic jaundice ↑↑↑ Post hepatic jaundice Note: Delta bilirubin – aka biliprotein; a bilirubin that is covalently bound to albumin thus contributes to the direct bilirubin value. Total bilirubin = unconjugated + conjugated + delta bilirubin B. METABOLISM/SYNTHESIS- All proteins are synthesized by the liver except immunoglobulins and adut hemoglobin, which are produced by B-cells and red blood cells respectively. C.DETOXIFICATION AND DRUG METABOLISM-The liver is capable of detoxifying harmful substances such as toxins and prevent it from reaching the systemic circulation. D. STORAGE-The liver is the storage site for all fat-soluble and water-soluble vitamins. The liver also serves as the storage site for glycogen. Alterations in Liver Function during Diseases Jaundice Used to describe the yellow discoloration of the skin, eyes, and mucous membranes which is most often caused by retention of bilirubin. French word “jaune” which means yellow Jaundice noticeable if bilirubin levels reach at least 3.0-5.0 mg/dL. Icterus is a serum or plasma sample with a yellow discoloration due to bilirubin. Jaundice can be categorized into three: pre-hepatic, hepatic, and post hepatic. In pre-hepatic and post hepatic jaundice, abnormalities are usually not because of liver impairment. Thus, liver function is generally normal. In hepatic jaundice, abnormalities are due to liver problems. a. Pre-hepatic jaundice: ▪ Mild type of jaundice that occurs prior to liver metabolism ▪May also be referred to as unconjugated hyperbilirubinemia - B1 is usually increased ▪ Most common cause are Hemolytic anemia and Ineffective erythropoiesis b. Hepatic jaundice: ▪ Results from intrinsic liver disease due to defects in bilirubin metabolism and transport (i.e. inherited disorders of bilirubin metabolism and jaundice of the newborn) ▪ Due to diseases resulting to hepatocellular injury c.Post-hepatic Jaundice: Results from biliary obstructive disease.Most common causes – gall stones or tumors in the biliary tree. Since bilirubin does not reach the intestines, stool becomes clay-colored. JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  HEPATIC JAUNDICE Inherited disorders of bilirubin metabolism Physiologic Jaundice of the Newborn Gilbert's Syndrome -Usually occurs in Inherited, autosomal recessive mild form of unconjugated premature infants due to hyperbilirubinemia absence of UDPGT ↑ unconjugated bilirubin in the blood enzyme - UDPGT is one of the last liver functions to be → Serum bilirubin: 1.5 – 3 mg/dL activated in prenatal life -Infants suffer from Crigler-Naiiar Syndrome increased levels of Characterized by impaired conjugation due to absence of unconjugated bilirubin in UDPGT enzyme their blood which can be ↑ unconjugated bilirubin in the blood fatal because B1 may deposit in the brain. Type I Crigler- Type Il Crigler-Najjar -Kernicterus caused by the Najjar Syndrome Syndrome build-up of Unconjugated bilirubin in the brain and Complete absence Partial deficiency of nerve cells resulting in cell of UDPGT enzyme UDPGT enzyme damage and death in newborns. Total absence of B2 Small amounts of B2 production are produced Conjugated hyperbilirubinemia Serum bilirubin: >20 Serum bilirubin: 5 – 20 mg/dL mg/dL → conjugated bilirubin is >1.5 mg/dL Kernicterus No kernicterus → most important causes – Therapy: Liver Px responds to UV transplant therapy idiopathic neonatal hepatitis and biliary atresia Rotor Syndrome Clinically similar to Dubin-Johnson syndrome because of its Unconjugated inability to excrete conjugated bilirubin to the bile hyperbilirubinemia (a) physiologic jaundice of (↑Conjugated) the newborn (b) hemolytic disease Dubin-Johnson Syndrome (c) breastmilk Characterized by liver's failure to excrete conjugated hyperbilirubinemia bilirubin from the liver cell to the bile. In this condition B2 that circulates in the blood is bound to albumin. Delta bilirubin - conjugated bilirubin bound to albumin JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  Liver Function Tests Van den Bergh reaction → bilirubin + diazotized sulfanilic acid → azobilirubin → done using serum samples Jendrassik-Grof method → diazotized sulfanilic acid + accelerator/solubilizer (caffeine sodium benzoate) → strong alkaline tartrate is added to convert original purple color into blue (measured spectrophotometrically at 600nm) → Advantages: Insensitive to sample pH changes and variation in protein concentration of sample Not affected by hemoglobin up to 750 mg/dL Has adequate optical sensitivity even for low bilirubin concentrations Evelyn-Malloy method → accelerator:50% methanol → azobilirubin: red to reddish purple color in acid pH (measured at 560nm →pH:11.2 Ammonia Test for detoxification function Useful in detecting hepatic failure, hepatic coma, and Reye syndrome E. Enzyme Tests Liver enzymes play a vital role in assessing liver function and in differentiating hepatocellular from obstructive liver diseases. Any injury to the liver resulting in cytolysis or necrosis will cause the release of liver enzymes in the circulation. High levels of liver enzymes in the circulation = liver damage Enzymes secreted by the liver: i. Alkaline phosphatase (ALP) ii. Alanine aminotransferase (ALT) iii. Aspartate aminotransferase (AST) iv. 5'-Nucleotidase V. y-Glutamyltransferase (GGT) vi. Lactate dehydrogenase (LDH) CLINICAL ENZYMOLOGY A. General Characteristics of Enzymes: 1. Active site: Has binding and catalytic site; It is where substrate interacts with the enzyme 2. Allosteric site: site other than the active site; binds to regulator molecules 3. Cofactors: non-protein substances needed for maximal activity of the enzyme a. Inorganic cofactors: activators (anions and cations) b. Organic cofactors: coenzymes; acts as co-substrate in enzyme reactions 4. Holoenzyme: complete active enzyme system with full catalytic activity; apoenzyme + prosthetic group 5. Proenzyme/Zymogen: inactive form of an enzyme 6. Isoform: multiple forms of serum proteins that are functionally related; results from post translational modifications 7. Isoenzymes: multiple forms of an enzyme that catalyzes the same biochemical reaction 8.Activator: Inorganic cofactor, metal ions such as magnesium and chloride 9.Coenzyme: Organic cofactor, such as heme, biotin, FAD, NAD, or coenzyme A 10. Apoenzyme:Complete and active enzyme with its cofactor JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi) ENZYME NOMENCLATURE Enzymes are biological catalysts that increase the rate of chemical reactions. Enzyme Nomenclature: Enzymes are named based on their substrate and end with the suffix "-ase." They are also assigned numerical designations by the International Union of Biochemistry (e.g., EC 3.1.3.1 for Alkaline Phosphatase). First number - classification Second two numbers - subclass & sub-subclass Last number - serial number Michaelis-Menten Equation: Describes the relationship between reaction velocity (V) and substrate concentration ([S]). The equation V = Vmax [S] / (Km + [S]) demonstrates that: At low [S], V increases linearly with [S]. At high [S], V approaches Vmax (maximum velocity), becoming independent of [S]. Km (Michaelis-Menten constant) represents the [S] at which V is half of Vmax. Zero-order kinetics-reaction is directly proportional to substrate concentration First-order kinetics-reaction is directly proportional to enzyme concentration JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi) Factors Affecting Enzyme Activity: Temperature: Enzymes have optimal temperatures; higher temperatures can denature them. pH: Enzymes have optimal pH ranges; outside this range, activity decreases. Enzyme Concentration: V is directly proportional to enzyme concentration. Inhibitors: Molecules that bind to enzymes and decrease their activity. Competitive inhibitors compete with substrate for the active site. Non-competitive inhibitors bind to allosteric sites, altering enzyme conformation. Enzyme Specificity: Enzymes are highly specific for their substrates due to the complementary shape and chemical properties of the active site. This ensures that enzymes catalyze only intended reactions. Enzyme Theory 1. Emil Fisher's/Lock and Key Theory t is based on the premise that the shape of the key (substrate) must fit into the lock (enzyme) 2. Kochland's/Induced Fit Theory It is based on the substrate binding to the active site of the enzyme Units for Expressing Enzymatic Activity: 1. International Unit (IU or U) -1 micromole of substrate/minute 2. Katal Unit (KU) - 1 mole of substrate/second HEPATIC ENZYME PROFILE Purpose: Assess liver function and differentiate between hepatocellular injury and cholestasis. Key Enzymes: Alanine Aminotransferase (ALT): Most specific for liver damage, found primarily in hepatocytes. Elevated in hepatitis, cirrhosis, and liver tumors. Aspartate Aminotransferase (AST): Less specific for liver damage, found in many tissues including heart and skeletal muscle. Elevated in liver diseases, but also in cardiac and muscle disorders. Alkaline Phosphatase (ALP): Found in liver, bone, and other tissues. Elevated in both hepatocellular and obstructive liver diseases, as well as bone disorders. Gamma-Glutamyl Transferase (GGT): More sensitive than ALP for detecting liver disease, especially cholestasis and alcohol-related liver damage. 5'-Nucleotidase (5'NT): Elevated specifically in obstructive liver diseases, helps differentiate from bone disorders when ALP is elevated. Lactate Dehydrogenase (LDH): Non-specific enzyme found in many tissues, including liver. Elevated in various conditions, including liver disease, but isoenzyme fractionation is needed for interpretation. JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi) A.HEPATIC ENZYMES: TRANSFERASES/TRANSAMINASES Enzyme: Clinical Significance: Methods: Aspartate Aminotransferase (AST) -Less specific for liver -Karmen method: Coupled EC numerical code: 2.6.1.1 damage than ALT, found in enzymatic reaction, measures o Highest Concentrations are found in many tissues. decrease in absorbance at 340 nm CARDIAC TISSUES, LIVER, and -Elevated in liver diseases, due to NAD+ formation. Uses malate SKELETAL MUSCLE; cardiac disorders, and dehydrogenase - pH 7.5, 340 nm. Fewer amount in the KIDNEY, muscle disorders. PANCREAS, and RBCs -Used in conjunction with o Previously known as SGOT (serum ALT to assess liver glutamic-oxaloacetic transaminase) function. Two isoenzyme fractions: (a) Cytoplasmic AST – most predominant in serum (b) Mitochondrial AST – becomes higher than cytoplasmic AST in serum in cellular necrosis. Reference value:5-37 U/L Alanine Aminotransferase (ALT) -Most specific for liver. -Coupled Enzymatic Method EC numerical code: 2.6.1.2 It is significant in the -LD as the indicator enzyme; o Tissue source: highest evaluation of hepatic change in absorbance at 340m concentration in liver and kidney, disorders markedly measured continuously is directly smaller amount in cardiac tissue and increased concentration proportional to ALT activity skeletal muscle in acute inflammatory Optimal pH: 7.3-7.8 o Formerly known as serum conditions than AST. -Reitman Frankel glutamic-pyruvic transaminase -It monitors the course of Reaction: alanine + a ketoglutaric (SGPT) liver (like hepatitis) o More “liver specific” than AST treatment and the → pyruvic acid o Involved in the reversible transfer of effects of drug therapy Addition of DNPH (2,4- an amino group between alanine and dihydrophenythydrazine) to alpha-ketoglutarate pyruvie acid to produce color Reference value: 6-37 U/L AST/SGOT ALT/SGPT Major Organ affected Heart (AMI) Liver (Liver Disease) Substrate Aspartic Alpha Alanine Alpha Ketoglutaric Acid Ketoglutaric Acid End products Glutamic Acid + Glutamic Acid + Oxaloacetic Acid Pyruvic Acid. Color developer 2,4 DNPH 2,4 DNPH Color intensifier 0.4N NaOH 0.4N NaOH ALT/AST RATIO "De Ritis ratio" differentiates viral from non-viral etiology viral etiology: high ALT if ALT/AST is > 1 = viral non-viral: high AST if ALT/AST is Continuation at the table below LACTATE DEHYDROGENASE (LD/LDH) Enzyme that catalyzes the(interconversion of lactic and pyruvic acids Highest concentration: HEART, LIVER, SKELETAL MUSCLE, KIDNEYS and RBCs Lesser amounts in lungs, smooth muscle, and brain Measurement is done either forward (lactate to pyruvate: many labs use this or the reverse (dry slide) 5 isoenzymes (electrophoresis):4D1, LD2, LD3, LD4, LD5 i.LD1 (anodic) fastest ii.LD5 (cathodic) iii.LD1 and LD2: heat stable iv. LD5: most labile v. LD6-ALCOHOL DEHYDROGENASE 4 SUBUNITS in each isoenzyme/tetramer ISOENZYME Subunits Shorthand Subunits High Concentration LD1 HHHH LD H4 Heart/RBC LD2 HHHM LD H3M Heart/RBC LD3 HHMM LD H2M2 Heart/Liver (lung, lymphocyte, spleen, pancreas) LD4 HMMM LD HM3 Liver/Skeletal muscle LD4 MMMM LD M4 Liver/Skeletal muscle JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi) HEPATIC ENZYMES CONT. Enzymes Clinical Significance: Methods: Gamma-Glutamyl Transferase -More sensitive than ALP for -Szasz and Rosalki: IFCC recommended (GGT) detecting liver disease, especially method, uses gamma-L-glutamyl-p- It catalyzes the transfer of glutamyl cholestasis and alcohol-related liver nitroanilide as substrate. groups between peptides or amino damage. -Other methods include Persijn and Van der acids through linkage at -Helps differentiate between liver Slik, and Goldberg methods. a gammy carboxyl group. and bone disorders when ALP is Reference value: 5-30 U/L(F) / 6- elevated. 45 U/L (M) MOST sensitive marker for: Chronic Alcoholism!!! Lactate Dehydrogenase (LDH) -Non-specific enzyme -Elevated in -Wacker method (pH8.8): FORWARD It is an enzyme that catalyzes the various conditions, including liver METHOD and It is the most commonly used interconversion of lactic and pyruvic disease,and MI. method.Measures conversion of lactate to acids. -Isoenzyme fractionation is pyruvate, either kinetically at 340 nm or It is a tetrameric molecule necessary for interpretation. colorimetrically. containing four subunits of two -Normal: LD2>LD1 -Wroblewski and La Due(pH:7.2): possible forms (H and M). -LD-1 > LD-2 also known as the REVERSE METHOD and It is about 2x faster Reference value: "flipped pattern" is seen in as the forward reaction. 100-225 U/L (Forward reaction) myocardial infarction and HIGHEST It is the preferred method for dry slide 80-280 U/L (Reverse reaction) IN :hemolytic anemia/ pernicious technology. It uses a less costly cofactor and anemia it has a smaller specimen. Measures conversion of pyruvate to lactate, kinetically at 340 nm. 5'-Nucleotidase (5'NT -Elevated specifically in obstructive -Usually measured by kinetic assays using It is a phosphoric monoester liver diseases. adenosine monophosphate as substrate. hydrolase; predominantly secreted -Helps differentiate from bone from the liver.) disorders when ALP is elevated. Reference value: 0-1.6 units Alkaline Phosphatase (ALP) -Elevated in both hepatocellular and -Bowers and McComb (Szasz Isoenzymes: obstructive liver diseases. modification): (MOST SPECIFIC MERHOD o Normal ALP isoenzymes – -Also elevated in bone disorders FOR ALP) intestinal, placental, bone, and liver (Paget's disease, osteosarcoma) IFCC recommended method, uses p- o Abnormal ALP isoenzymes – aka and during pregnancy. nitrophenyl phosphate as substrate.-Other carcinoplacental ALPs Highest elevations (5-10x) seen in methods include Bessey-Lowry-Brock, (a) Regan ALP – highest incidences biliary obstruction and bone Huggins-Talalay, and Klein-Babson-Read is found in ovarian and disorders. methods, using different substrates. gynecological cancers -Moderate elevations (up to 3x) in (b) Nagao ALP – observed in hepatocellular diseases and other pleural cancer and pancreatic and conditions. bile duct carcinomas (c) Kasahara ALP – observed in hepatoma and GIT tumors ALP GGT 5’-NT Pregnancy Increased Normal Normal Hepatobiliary disorders Increased Increased Increased Bone disorders Increased Normal Normal JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  CARDIAC DISORDER PROFILE Purpose: Diagnose and monitor myocardial infarction (MI) & other heart conditions. Key Enzymes: Creatine Kinase (CK): Found in cardiac and skeletal muscle, brain. Total CK levels rise within hours of MI, peak at 24 hours, and return to normal within 3- 4 days. CK-MB isoenzyme: More specific for cardiac muscle damage. Elevated levels confirm MI diagnosis. Troponin: Most specific and sensitive marker for MI. Remains elevated for up to 10 days, allowing detection of even minor cardiac injury. Myoglobin: Early marker for MI, rises within 2 hours. Less specific than troponin, but useful for early rule-out. Clinical Significance: Elevated levels of these biomarkers, especially troponin, are indicative of myocardial damage. The cardiac disorder profile is crucial for early diagnosis and risk stratification in patients with suspected MI, as well as monitoring the effectiveness of treatment and predicting long-term outcomes. CREATINE KINASE EC numerical code: 2.7.3.2 o Catalyzes the transfer of phosphate to creatine o Requires Magnesium and thiol source (cysteine) o Inhibited by zinc and manganese; excess magnesium can also inhibit CK o Tissue source: highest in striated muscle and heart muscle; others – brain, GIT, urinary bladder CK-BB CK-MB CK-MM Other terms CK-1, “the brain CK-2, “the hydrid type” CK-3, “the muscle type” type” Used to be the "gold Standard" Clinical ▪Tumor associated ▪ Acute myocardial ▪ Myocardial infarction significance marker – prostatic infarction ▪ Skeletal muscle carcinoma and other ▪ Rise within 4-8 hours disorders adenocarcinomas ▪ Peak at 12-24 hours ▪ Duchenne’s muscular ▪ Returns to normal dystrophy – HIGHEST within 48-72 hours ELEVATION of CK Notes ▪CK-BB level is >5 ▪ CK-MB in the serum is ▪ The major isoenzyme U/L derived only in fraction (usually within 10-50 myocardium. in serum U/L) in these ▪ CK-MB that is 6% of ▪ Abundantly present in conditions the cardiac ▪ Half-life: 2-3 hours total CK – indicative of and skeletal muscles myocardial damage ▪ Half-life: 15 hours ▪ Half-life: 12 hour JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi) Methods: i. Tanzer-Gilvarg (forward reaction): ATP + creatine → ADP + creatine phosphate ADP formed is reacted with pyruvate kinase and lactate dehydrogenase o Note: NADH absorbs light at 340 nm; NAD does not; pH is maintained at 9.0 ii. Oliver-Rosalki (reverse reaction): ADP + creatine phosphate → ATP + creatine o G6PD: glucose-6-phosphate +NADP+ 6-phocphogluconate + NADPH o Note: NADPH absorbs light at 340 nm; NADP does not; pH is maintained at 6.7 o Reverse reaction is six (6) times faster than forward reaction o Note: Addition of adenosine monophosphate to inhibit adenylate kinase ACUTE PANCREATITIS PROFILE Purpose: Diagnose acute pancreatitis and monitor its severity. Key Enzymes: AMYLASE: Elevated in acute pancreatitis, usually within 12 hours of onset. Less specific than lipase, as it can also be elevated in salivary gland disorders. LIPASE: More specific for acute pancreatitis. Remains elevated for longer duration than amylase (up to 14 days). Clinical Significance: Elevated levels of amylase and lipase, especially lipase, strongly suggest acute pancreatitis. Amylase levels may also be elevated in other conditions like salivary gland disorders, bowel obstruction, or ectopic pregnancy. Lipase levels are more specific to pancreatic injury and less affected by extra- pancreatic factors. Methods for Measurement: Amylase: Saccharogenic methods: Measure the decrease in substrate (starch) concentration or the increase in product (maltose or glucose) concentration. Chromogenic methods: Use a coupled enzymatic reaction to produce a colored compound proportional to amylase activity. Immunoassays: Offer high sensitivity and specificity, but may not be as widely available as enzymatic methods. Lipase: Cherry-Crandall (Reference Method) Principle: Measures liberated fatty acids by titration after a 24-hr incubation Substrate: Olive Oil (before); TRIOLEIN (more pure form of TAG) End Products: Fatty Acids Turbidimetric methods: Measure the decrease in turbidity of an olive oil emulsion as lipase breaks down triglycerides. Colorimetric methods: Use a coupled enzymatic reaction to produce a colored compound proportional to lipase activity. JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)  PROSTATIC CANCER PROFILE PROSTATE SPECIFIC ANTIGEN Clinical Significance: Elevated PSA levels: Can indicate prostate cancer, but further testing is needed for confirmation. Methods for Measurement: Immunoassays: The most common methods for measuring PSA, including: Enzyme-linked immunoassay (ELISA) Chemiluminescence immunoassay (CLIA) Radioimmunoassay (RIA) ACID PHOSPHATASE -A hydrolase that catalyzes the same type of reactions with ALP -Difference between ACP and ALP is the pH of reaction -Optimal pH approximately 5.0 -Found in PROSTATE (highest), BONE, LIVER, SPLEEN, KIDNEY, RBCs, and PLATELETS ISOENZYMES: RBC-ACP and prostate-ACP (PACP) Clinical Significance: - Aid in the detection of PROSTATIC CARCINOMA Methods: Very labile, add 5M acetate buffer or citrate tablet to preserve Substrates: i. Organic Phosphate such as B-glyceroPO4 and p-nitropheny/PO4 ii. Thymolphthalein monophosphate (most specific substrate for prostatic ACP) - for quantitative endpoint reactions (Roy and Hillman)-REFERENCE METHOD REFERENCES: 1. Fialová, M., & Vejrazka, M. (n.d.). *Non-protein nitrogen compounds* [Figure 3]. Semantic Scholar. Retrieved August 27, 2024, from https://www.semanticscholar.org/paper/Non- protein-nitrogen-compounds-1-1-Non-protein-of-Fialová- Vejrazka/c32966ab20b7c242b4102fd9734cee1494abe70d/figure/3 2. ScienceDirect. (n.d.). *Bilirubin metabolism*. In *Encyclopedia of Biochemistry, Genetics and Molecular Biology*. Retrieved August 27, 2024, from https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bilirubin- metabolism 3. Korf, H. W., & Greten, H. (2020). *Bilirubin metabolism and recirculation* [Figure 3]. In *ResearchGate*. Retrieved August 27, 2024, from https://www.researchgate.net/figure/Bilirubin-metabolism-and-recirculation_fig3_353125938 4. Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical Chemistry: Principles, Techniques, and Correlations (7th edition ed.). Philadelphia, Pennsylvania, United States of America: Lippincott Williams & Wilkins. JMC-MTAP 2024-2025 | CLINICAL CHEMISTRY| INTERN’S LEARNING GUIDE 4 | Prepared By: Kezia Crizta M. Alfaras RMT, MLS(ASCPi)