MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Measurement of Enzymes & Their Clinical Significance Measurement of Enzyme Activity • Often measured by catalytic activity then related to concentration • Enzyme concentration is best measured by its activity or its rate of activity by observing: – Substrate depletion – Product production – Increase/decrease in cofactor/coenzyme • Usually performed in zero-order kinetics Measurement of Enzyme Activity Fixed time Continuous Monitoring/Kinetic • Measurement of the • Multiple measurements of amount of substrate utilized absorbance change are over a fixed amount of time made or by a fixed amount of • Advantages serum – Depletion of substrate is observable • Problems – – – – Long incubation times Possible enzyme denaturation Often a lag phase Possible substrate depletion – Improved accuracy Reporting Enzyme Activity • Originally reported as activity units • IUB standardized these as international units (IU) – IU: the amount of enzyme that will convert one micromole of substrate per minute in an assay system – Expressed as units per liter or U/L – Conditions: pH, temperature, substrate,activators • Katal units(SI): express as moles/second Other Methods to Measure Enzymes • Using Enzyme Mass – Measure protein mass NOT catalytic activity • Electrophoresis – Used to differentiate isoenzymes – Time-consuming ENZYMES OF CLINICAL SIGNIFICANCE Creatine Kinase (CK) • Action – Associated with the regeneration and storage of ATP – Catalyses the following reaction: Creatine Kinase (CK) • Purpose – Allows the body to store phosphate energy as creatine phosphate – Energy can be released/ provided to muscles by reversing the reaction • Source – – – – Skeletal muscle Heart Brain Other Creatine Kinase (CK): Structure – Dimer consisting of two subunits – Two subunits are further divided into 3 molecular forms or isoenzymes • CK-BB: (CK-1)brain type – Migrates fast on electrophoresis – Small amount found in tissue (brian, lung, bladder, bowel) • CK-MB: (CK-2)hybrid type – Heart, Skeletal • CK-MM: (CK-3)Muscle type – Mostly found in healthy people – Striated muscle and normal serum Creatine Kinase (CK) • Diagnostic Use – Appearance of CK (MB) very sensitive indicator of MI – Skeletal muscle disorders such as muscular dystrophy – CNS Disorders • Cerebrovascular accident(CVA) • Seizures • Nerve degeneration CK Isoenzymes What’s in a Number? Creatine Kinase: Specimen Collection • Sources of Error – Hemolysis • Interference of adenylate kinase on CK assays • Results in false elevations – Exposure to light • CK is inactivated by light Creatine Kinase: Reference Range • Affected by: – Age – Physical activity – Race – Bed rest (even overnight can decrease CK) • Total CK – Men: 46-180 U/L – Female: 15-171 U/L Creatine Kinase • Isoenzyme Testing – Fractionation of CK • Immunoinhibition • Mass Assay • Electrophoresis Lactate Dehydrogenase (LDH/LD) • Action – Catalyzes a reversible reaction between pyruvate and lactate with NAD as a coenzyme – Reaction: Lactate Dehydrogenase (LDH/LD) • Source – Skeletal muscle – Cardiac muscle – Kidney – RBCs – Widely distributed in the body Lactate Dehydrogenase (LDH/LD): Structure • Tetramer – Four polypeptide chains, two subunits (heart & muscle) – Five combinations of Isoenzymes Lactate Dehydrogenase (LDH/LD) • Diagnostic Significance – Nonspecific – Increased • Hematologic and neoplastic disorders • Liver disease • Heart problems Lactate Dehydrogenase (LDH/ LD): Specimen Collection • Sources of Error – Hemolysis • RBCs contain 100-150 times that found in serum – Analyte stability • Run assay asap or store at room temperature – Prolonged contact of serum and cells • Reference Range • 140- 280 U/L Liver Enzymes • Transaminases – AST – ALT • Phosphatases – ALP Transaminases • Retain amino groups during the degradation of amino acids • Types – Aspartate transaminase (AST) • Aka: Glutamic Oxalocetic transaminase (SGOT) – Alanine transaminase (ALT) • AKA: Glutamic pyruvic transaminase (SGPT) Aspartate Aminotransferase( AST) • Sources – Major • Heart • Liver • Muscle – Minor • • • • RBCs Kidney Pancreas Lung Aspartate Aminotransferase( AST) • Reaction: AST AST: Specimen Collection • Sources of Error – Hemolysis – Analyte stability • Stable at room temp for 48 hours or 3-4 days refrigerated • Reference Range – 5-30 U/L Alanine Transaminase (ALT) • Transfers an amino group from alanine to alpha-ketoglutarate to form glutamate and ALT pyruvate Alanine Transaminase (ALT) • Sources – Liver (Majority) – Kidney – Heart – Skeletal muscle ALT: Specimen Collection • Sources of Error – Hemolysis – Analyte stability • 3-4 days refrigerated • Reference Range – 6-37 U/L Diagnostic Significance: AST & ALT • Many diseases can cause increases since widely distributed in tissues • Liver – Hepatitis – Cirrhosis – Liver cancer • Myocardial Infarction – AST increases most – ALT normal to slightly increased, unless liver damage accompanies • Other – Pulmonary emboli – Muscle injuries – Gangrene – Hemolytic diseases – Progressive Muscular dystrophy Phosphatases • Removes phosphates from organic compounds • Functions to facilitate transfer of metabolites across cell membranes • Alkaline Phosphatase (ALP) • Acid Phosphatase (ACP) Phosphatases: Sources Alkaline Phosphatase (ALP) Acid Phosphatase (ACP) • • • • • • • • • • • Bone Liver Kidney Placenta Intestines Prostate gland Seminal fluid Liver Spleen RBCs Platelets Alkaline Phosphatase (ALP) • ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an alkaline pH • Maximum activity at pH of 9.0- 10.0 Alkaline Phosphatase (ALP) • Diagnostic Significance – Elevations seen in • During bone activity – Paget’s disease • Liver disease, especially in obstructive disorders • Pregnancy between 16-20 weeks gestation – Decreased levels occur, but not diagnostic Alkaline Phosphatase (ALP): Specimen Collection • Sources of Error – Hemolysis – Delay in processing, false increases can occur • Reference Range (Adult) – 30-90 U/L – NOTE: Normal increases seen in pregnancy, childhood, adolescence Acid Phosphatase (ACP) • Diagnostic Significance – Aids in detection of prostatic carcinoma – Other conditions associated with prostate – Forensic chemistry: Rape cases – Elevated in bone disease Acid Phosphatase (ACP): Specimen Collection • Sources of Error – Separate serum from cells asap – Decrease in activity seen at room temp – Hemolysis – Reference Range (prostatic) • 0-4.5 ng/mL Gamma-Glutamyltransferase (GGT) • Possibly involved in peptide and protein synthesis, transport of amino acids and regulation of tissue glutathione levels • Sources – Kidney – Brain – Prostate – Pancreas – Liver Gamma-Glutamyltransferase (GGT) • Diagnostic Significance – Sensitive indicator of liver damage – Increased in patients taking enzyme-inducing drugs such as warfarin, phenobarbital and phenytoin – Indicator of alcoholism – Elevated in acute pancreatitis, diabetes mellitus and MI GGT: Specimen Collection • Sources of Error – Very stable – Hemolysis not an issue • Reference Range – Male: 10-34 U/L – Female: 9-22 U/L Digestive & Pancreatic Enzymes • Amylase • Lipase Amylase (AMS) • Digestive enzyme that hydrolzes/catalyzes the breakdown of starch and glycogen to carbohydrates • Smallest enzyme • Sources – Acinar cells of pancreas and salivary glands Amylase (AMS) • Diagnostic Significance – Acute pancreatitis • AMS levels rise 2-12 hours after onset of attack, peak at 24 hrs and return to normal within 3-5 days – Salivary gland lesions • Mumps Amylase • Sources of Error – Presence of opiates increases levels – Stabile • Reference Range – Serum: 30-100 U/L – Urine: 1-17 U/h Lipase (LPS) • Hydrolyzes triglycerides to produce alcohols and fatty acids • Source – Pancreas Lipase (LPS) • Diagnostic Significance – Acute pancreatitis • More specific than amylase • LPS persists longer than AMS Lipase: Specimen Collection • Sources of Error – Stabile – Hemolysis results in false decreases • Reference Range – 13-60 U/L References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .