cc 2 lab

• Enzymes are proteins within cells
• Enzymes are macromolecules measured in terms of activity
• General properties of enzymes:

  • Active Site: Water Free
  • Allosteric site: Cavity other than the active site
• Factors affecting enzyme activity:

  1. Enzyme Concentration

  2. Substrate Concentration

  3. Cofactors are non-proteins that must bond to a particular enzyme before a reaction occurs

  4. Inhibitors: Competitive, Non-competitive, Uncompetitive

  5. Isoenzymes

  6. Temperature ranges: 25-37C, 38-50C, 60-65C

  7. Storage temperature: -20C
• Enzyme reaction methods:

  1. Fixed time - reacts at a designated time

  2. Kinetic assay - multiple measurements of absorbance changes are made during the reaction
• Creatine Kinase (CK):

  • Important in the regeneration of adenosine triphosphate
  • Dimeric with 2 different monomers: “M” and “B”
  • Predominantly found in skeletal muscles, heart muscles, brain tissues
  • Transfers Phosphate group between Creatine phosphate and ADP
  • Used in the diagnosis of acute myocardial infarction, muscular dystrophy, and central nervous system disorders
• Isoenzymes of Creatine Kinase:

  1. CK-MM (Muscle Type, CK-3): Abundant in Cardiac and Skeletal muscles, major activity in the heart

  2. CK-BB (Brain Type, CK-1): Normally found in neonatal sera, elevated in brain injury and carcinomas

  3. CK-MB (Hybrid Type, CK-2): Most specific for Myocardial damage (AMI), peaks at 12-24 hours, normalizes in 48-72 hours, falsely elevated in hemolysis

  4. Macro-CK: Common in older women, migrates middle of MM and MB

  5. Mitochondrial CK (CK-Mi): Migrates cathodal to CK-MM, indicates severe illness
• Methods for Creatine Kinase:

  1. Forward - Tanzer and Gilvarg Assay: pH 9.0, Absorbance 340nm

  2. Reverse - Oliver Rosalki: pH 6.8, Absorbance 340nm

  3. Electrophoresis
• Lactate Dehydrogenase (LDH):

  • A transferase that hastens the interconversion of lactic acid and pyruvic acid
  • Composed of 4 peptide chains of two types: “M” (A) and “H” (B)
  • Elevated levels associated with several diseases
  • Highest levels detected in pernicious anemia and hemolytic disorders
  • Other diseases associated with elevations are hepatic disorders, acute myocardial infarction, pulmonary infarct, acute lymphoblastic leukemia
• Isoenzymes of Lactate Dehydrogenase:

  1. LDH1 (HHHH): Heart, RBC, Kidneys

  2. LDH2 (HHHM): Major isoenzyme in healthy people, most abundant and heat stable

  3. LDH3 (HHMM): Lungs, pancreas, spleen, lymphocytes

  4. LDH4 (HMMM): Skeletal muscle, liver, intestine

  5. LDH5 (MMMM): Liver, skeletal muscle, intestine

  6. LDH6: An arteriosclerotic Cardiovascular failure marker
• LDH Clinical Significance:

  1. Myocardial Infarction: Elevates at 12-24 hours after onset, peaks at 48-72 hours, remains elevated for 10 days

  2. Hepatitis

  3. Hemolysis

  4. Lung and muscle disorders
• Methods for Lactate Dehydrogenase:

  1. Wacker method - Forward or direct: pH 8.8, Absorbance 340nm

  2. Wroblewski Ladue - Reverse or indirect: pH 7.2, Absorbance 340nm
• Other Cardiac Markers:

  1. Myoglobin:

  • Transport and stores oxygen to intracellular respiratory enzymes of contractile cells
  • Marker of AMI, elevates 2-3 hours after onset, peaks at 8-12 hours, normalizes 18-30 hours

  2. Troponin I:

  • Found in myocardium with greater cardiac specificity
  • Elevates in AMI, elevates 3-6 hours after onset, peaks at 12-18 hours after onset, normalizes in 6 days

  3. Brain-type natriuretic peptide:

  • A Heart Failure Biomarker, derived from a pro-hormone, “Pro-BNP”
• Pancreatic Enzymes:

  Amylase:

  • For the breakdown of Glycogen and Starch
  • Considered as the smallest enzyme
  • Activators: Calcium and Chloride
  • Major sources: Acinar cells of the pancreas, Salivary glands
  • Clinical significance in Acute Pancreatitis, Intestinal obstruction, Cholecystitis, Acute appendicitis
• Methods for Amylase:

  1. Saccharogenic: measures reducing sugar from starch breakdown

  2. Amyloclastic: also known as Iodometric method, measures amylase activity by following decrease in substrate concentration, uses Iodide as an indicator

  3. Chromogenic: uses dye to check amylase activity

  4. Coupled enzyme
• Lipase:

  • A single-chain glycoprotein with a molecular weight of 48 kDa
  • Needs the presence of Bile salts and colipase
  • Function: Hydrolyze glycerol esters of long-chain fatty acids
  • Most lipase activity found in serum are usually from the pancreas and some are secreted by gastric and intestinal mucosa
• Lipase Clinical Significance:

  1. Acute Pancreatitis

  2. Perforated or duodenal ulcer

  3. Intestinal obstruction

  4. Mesenteric vascular obstruction
• Lipase Method:

  Cherry Crandal - reference method, utilizes Olive oil for hydrolysis, indicator: Phenolphthalein