Prelim - CC2 LAB Lesson 1 Pancreatic Enzymes - 01 PDF

SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 MLS 418 [LAB]: ENZYMES OF CLINICAL SIGNIFICANCE: AMYLASE & LIPASE   State the clinical significance of amylase and lipase....

SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 MLS 418 [LAB]: ENZYMES OF CLINICAL SIGNIFICANCE: AMYLASE & LIPASE   State the clinical significance of amylase and lipase.  Describe the principles involved in the methods of enzyme LEARNING determination. OBJECTIVES  Perform procedures to determine enzymatic activities. By the end of the lesson, the  Correlate results with various students are expected to: physiologic and pathologic conditions. SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2  SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE TOPIC CLINICAL CHEMISTRY 2 OUTLINE We will learn the following:  Amylase and Lipase  Reactions Catalyzed  Isoenzymes  Methods of Determination  Reference Values  Clinical Significance  Analytic Variables SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 AMYLASE NOMENCLATURE & FUNCTION AMYLASE (AMS)  EC 3.2.1.1  1,4-D-Glucan Glucanohydrolase  Important in the physiologic breakdown of starch and glycogen  Activators: Calcium ions & Chloride  Smallest enzyme: 50,000 to 55,000 Da  Easily filtered by renal glomerulus (appears in urine) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 NOMENCLATURE & FUNCTION AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Sally Kim | Types of Starch—Structures of Amylose vs. Amylopectin. (n.d.). Retrieved July 24, 2024, from https://app.biorender.com/biorender- CLINICAL CHEMISTRY 2 templates/t-6481d82de6947f8270eb870 TISSUE SOURCE AMYLASE (AMS)  Major Tissue Sources:  Acinar cells of the pancreas (Pancreatic AMS)  Salivary glands (Salivary AMS)  Minor Tissue Sources:  Skeletal muscle  Small intestine  Fallopian tubes SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 NOMENCLATURE & FUNCTION AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Dewangan, N. (2023, December 7). Starch Digestion: Structure, Enzymes, Mechanism, Process. https://microbenotes.com/starch-digestion/ CLINICAL CHEMISTRY 2 DIAGNOSTIC SIGNIFICANCE AMYLASE (AMS)  Increased in:  Acute pancreatitis  Salivary gland lesions: Parotitis, Mumps  Intra-abdominal diseases (e.g., perforated peptic ulcer, intestinal obstruction, cholecystitis, ruptured ectopic pregnancy, mesenteric infarction, acute appendicitis)  Renal insufficiency  Diabetic ketoacidosis  Macroamylasemia  Decreased in:  Chronic pancreatitis, congestive heart failure, certain cancers, pregnancy (second and third trimesters). SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2  McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). DIAGNOSTIC SIGNIFICANCE OF AMYLASE MACROAMYLASEMIA  Amylase combined with immunoglobulins  Increased serum AMY due to reduced renal clearance  Low urinary excretion of AMY  Important to differentiate from other hyperamylasemia causes SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 DIAGNOSTIC SIGNIFICANCE OF AMYLASE ACUTE PANCREATITIS SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Pancreatitis: Symptoms, Causes & Treatment. (n.d.). Retrieved July 24, 2024, from https://my.clevelandclinic.org/health/diseases/8103- CLINICAL CHEMISTRY 2 pancreatitis DIAGNOSTIC SIGNIFICANCE OF AMYLASE ACUTE PANCREATITIS Rises Duration of (Onset of Peaks Normalizes Elevation Elevation) Amylase 5-8 hours 24 hours 3-5 days Lipase 4-8 hours 24 hours 7 days 8-14 days SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 ASSAY FOR ENZYME ACTIVITY AMYLASE (AMS) 2.) SACCHAROGENIC 1.) AMYLOCLASTIC METHOD METHOD  Measures the disappearance of  Measures the appearance of the starch substrate product  AMY acts on starch substrate with  Starch substrate hydrolyzed to attached iodine constituent carbohydrates  Hydrolysis releases iodine  Reducing sugars measured  Decrease in dark-blue color  Concentration proportional to intensity of starch–iodine complex AMY activity  Color decrease proportional to  Classic reference method, AMY concentration reported in Somogyi units SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 ASSAY FOR ENZYME ACTIVITY AMYLASE (AMS) 4.) CONTINUOUS-MONITORING 3.) CHROMOGENIC METHOD METHOD  Measures the increasing color  Coupling of several enzyme from production of product systems to monitor amylase coupled with a chromogenic dye activity  Starch substrate attached to  Change in absorbance of NAD+ at chromogenic dye forming insoluble 340 nm measured dye–substrate complex  Optimal pH for AMY activity: 6.9  Hydrolysis produces water-soluble dye–substrate fragments  Increase in color intensity of soluble dye–substrate solution  Color intensity proportional to AMY activity SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 ASSAY FOR ENZYME ACTIVITY AMYLASE (AMS) 4.) CONTINUOUS-MONITORING METHOD  Coupling of several enzyme systems to monitor amylase activity  Change in absorbance of NAD+ at 340 nm measured  Optimal pH for AMY activity: 6.9 SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 SOURCES OF ERRORS AMYLASE (AMS)  Stability of AMY (in serum and urine)  Stable at room temperature for 1 week  Stable at 4°C for 2 months  Inhibitors (Falsely ↓ AMS):  ↑ Plasma triglycerides  Wheat germ lectin  Chelating agents (EDTA, Citrate, Oxalate)  Medications  Morphine and Opiates (Falsely ↑ AMS):  Pain relief drugs  Cause constriction of the sphincter of Oddi and pancreatic ducts  Leads to regurgitation of AMY into serum due to increased pressure SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 SPECIMEN HANDLING & REFERENCE RANGE AMYLASE (AMS)  Specimen handling  Specimen: serum or heparinized plasma.  Avoid saliva contamination (700 times higher amylase content than serum).  Hemolysis does not affect most methods except coupled-enzyme methods involving peroxidase reactions.  Reference range:  Serum: 28 to 100 U/L (37°C) (0.5 to 1.7 µkat/L)  Urine: 1 to 15 U/h SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2  McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). PACKAGE INSERT AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 PACKAGE INSERT AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 PACKAGE INSERT AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 PACKAGE INSERT AMYLASE (AMS) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY 2 LIPASE NOMENCLATURE & FUNCTION LIPASE (LPS)  EC 3.1.1.3  Triacylglycerol acylhydrolase  Hydrolyzes ester linkages of fats  Produces alcohols and fatty acids  Targets fatty acid residues at positions 1 and 3 of triglyceride molecule  Accelerators: colipase and bile salts SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 TISSUE SOURCE LIPASE (LPS)  Primary Source  Pancreas  Secondary Sources  Stomach  Small intestine SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 DIAGNOSTIC SIGNIFICANCE LIPASE (LPS)  Most specific marker of acute pancreatitis  Comparison with Amylase (AMY)  More specific for pancreatic disorders than AMY  Both LPS and AMY levels rise quickly  LPS elevations persist for ~8 days  AMY elevations persist for only 2 to 3 days SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 DIAGNOSTIC SIGNIFICANCE OF LIPASE ACUTE PANCREATITIS Rises Duration of (Onset of Peaks Normalizes Elevation Elevation) Amylase 5-8 hours 24 hours 3-5 days Lipase 4-8 hours 24 hours 7 days 8-14 days SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 ASSAY FOR ENZYME ACTIVITY LIPASE (LPS)  Early Methods  Classic Cherry-Crandall Method  Used olive oil substrate  Measured liberated fatty acids by titration  24-hour incubation period  Lacked stable and uniform substrates  Modified Cherry Crandall Method: use Triolein as substrate SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 ASSAY FOR ENZYME ACTIVITY LIPASE (LPS)  Modern Methods  Turbidimetric Methods  Simpler and more rapid  Fats in solution create a cloudy emulsion  Hydrolysis by LPS disperses particles  Rate of clearing measures LPS activity  Colorimetric Methods  Based on coupled reactions with enzymes (e.g., peroxidase, glycerol kinase) SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 SOURCES OF ERRORS LIPASE (LPS)  Stability  Stable in serum  Negligible loss of activity:  At room temperature for 1 week  At 4°C for 3 weeks  Hemoglobin inhibits LPS activity  Causes falsely low values SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE REFERENCES:  Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: Principles, procedures, correlations (Enhanced 8th ed). Jones & Bartlett. CLINICAL CHEMISTRY 2 SPECIMEN AND REFERENCE RANGE LIPASE (LPS)  Serum/Plasma  Reference range: 