Enzymes

• AMYLASE


  ALPHA-1-4-GLUCAN-4-GLUCOHYDROLASE (AMS) - Catalyzes the breakdown of starch and glycogen, important in the physiologic digestion of starch
• AMYLASE


  • Smallest enzyme in size (MW of 50,000 to 55,000 daltons), filtered by the renal glomerulus and appears in urine
  • Earliest pancreatic marker
  • P3 is the most predominant pancreatic amylase isoenzyme in acute pancreatitis
  • Isoenzymes: S-type (ptyalin) and P-type (amylopsin) present in normal healthy sera
  • Major Tissue source: acinar cells of the pancreas and the salivary glands
  • Other tissue sources: adipose tissue, fallopian tubes, small intestine, and skeletal muscles
  • Normal serum contains both salivary and pancreatic AMS
• Reference values for AMS

  • 60-180 SU/dL (somogyi units)
  • 95-290 U/L
• Diagnostic Significance of AMS

  1. In acute pancreatitis, AMS levels rise 2-12 hours after onset, peak at 24 hours, and normalize within 3-5 days

  2. Increased AMS blood levels in AP are accompanied by increased urinary excretion

  3. AMS in urine remains elevated for up to 7 days

  4. Salivary gland inflammation (parotitis) due to mumps can release AMS into circulation causing elevated serum AMS

  5. In renal failure without AP, increased plasma AMS is accompanied by decreased urinAMS
• Methods for AMS


  • Heparin may inhibit AMS activity
  • Triglycerides may inhibit serum AMS activity
  • Samples with high AMS activity should be diluted with NaCl
  • Many endogenous inhibitors of AMS present in serum
  • Administration of morphine and other opiates before blood sampling leads to falsely elevated serum AMS levels
• Substrate for AMS methods

  Starch
• AMS methods


  • Saccharogenic
  • Amyloclastic
  • Chromogenic
  • Coupled enzyme
• Three-fold amylase increase with normal 24 hours urine amylase requires repeating serum AMS after polyethylene glycol precipitation
• Salivary AMS is inhibited by wheat germ lectin
• Enzyme molecules are too large to pass through healthy glomerulus of kidneys, except amylase
• Macroamylasemia = AMS + immunoglobulin (too large to be filtered across the glomerulus)
• Normal Amylase/creatinine ratio: 1%-4% (0.01-0.04)
• 1. C ratio (AP) = > 4% (up to 15%)
• Increased Serum Amylase in
• PROFAME ANALYTICAL REAGENT METHOD: Caraway Method, Modified
• Materials/Reagents for AMS test

  • Amylase Substrate
  • Test Sera/N - AB controls
  • Color developer
  • Deionized water
  • Test tubes, cuvettes, parafilm, micro pipette, pipet tips, centrifuge
• Procedure for AMS test

  Mix amylase substrate and test sera/controls, incubate at 37C, add color developer and deionized water
• Enzymatic colorimetric test for the quantitative determination of lipase


  A synthetically produced lipase substrate is added to a microemulsion which is specifically split by lipase in the presence of colipase and bile acids. The combination of lipase and bile acids ensures the specific determination of pancreatic lipase without any reaction due to lipolytic enzymes or esterases. The reagent composition has been thoroughly optimized so there are no serum matrix effects. The generated methylresorufin-ester is spontaneously degraded to methylresorudin. The absorbance by the red dye is directly proportional to the lipase activity
• Cherry Candral (Reference Method)


  Hydrolysis of olive oil after incubation for 24 hours at 37°C and titration of fatty acids using NaOH
• Lipase


  • Enzyme that hydrolyzes the ester linkages of fats to produce alcohol and fatty acid
  • Catalyzes partial hydrolysis of dietary TAG in the intestine to the 2-monoglyceride intermediate, with the production of long chain fatty acids
  • Most specific pancreatic marker - secreted exclusively in the pancreas; not affected by renal disorders
  • Plasma concentrations are normal in conditions of salivary gland involvement
  • Major tissue source: Pancreas
• Diagnostic Significance of Lipase

  • In acute pancreatitis (AP), LPS levels rise 6 hours after onset of attack, peak at 24 hours, remains elevated for 7 days, and normalize in 8-14 days
  • In chronic pancreatitis, acinar cell degradation occurs resulting in loss of amylase and lipase production
• Methods for Lipase


  1. Uses olive oil as the substrate because other esterases can hydrolyze TAG and synthetic diglycerides

  2. Addition of colipase and bile salts will make assay more sensitive and specific for AP detection

  3. Hemoglobin inhibits the activity of LPS leading to falsely low values

  4. Triolein is used as a substrate for LPS assay
• Reference value for Lipase is 0-1.0 U/mL
• Reagents are stable for 7 days at 20-25C, 3 weeks at 4-8C, and 1 year at -20C
• Specimen


  • Serum or heparinized plasma
  • Discard contaminated samples
• Stability


  • 7 days at 20-25C
  • 3 weeks at 4-8C
  • 1 year at -20C
• Reagents are ready for use and stable until expiration date stated on the label when stored at 4-80C. Avoid freezing and contamination. Mix at room temperature before use.
• Procedure


  1. Pipetting scheme

  2. Pipette into cuvettes

  3. Mix carefully (do not shake), incubate for 5 min

  4. Start reaction by adding substrate

  5. Mix, incubate 2 min. at 37°C, read absorbance again and start the stopwatch

  6. After exactly 1 and 2 min. Read absorbance again and then calculate A/min
• Reference Value: ≤ 60 U/l
• Important Procedural Notes:
• As many other clinical chemistry reagents, specially reagents for determination of triglycerides, HDL - and LDL- cholesterol contain lipase or high concentrations of detergents, carry over should be avoided by cleaning the glassware thoroughly.
• SUB is a turbid orange colored micro-emulsion, if it becomes red discard. Precipitation may also appear on storage, specially if stored lower than the recommended temperature, it does not affect the analysis. It is just necessary to slightly rotate the vial to resuspend before the analysis.
• Sodium azide is a preservative so extra careful not to swallow. Avoid contact with skin and the mucous membranes.
• Lactate Dehydrogenase (LD)


  • An enzyme that catalyzes the interconversion of lactic and pyruvic acids
  • A zinc-containing enzyme that is part of the glycolytic pathway and is found in virtually all cells in the body
  • A hydrogen-transfer enzyme that uses the coenzyme nicotinamide dinucleotide (NAD+)
  • A tetrameric molecule containing four subunits of two possible forms (H and M)
• Tissue sources of LD

  • Heart, RBCs, kidneys (LD-1 & LD-2)
  • Lungs, pancreas, spleen (LD-3)
  • Skeletal muscles, liver, intestine (LD-4 & LD-5)
• Diagnostic Significance of LD:
• Diagnostic Significance of LD

  • Highest serum levels are seen in pernicious anemia and hemolytic disorders
  • In AMI, LD levels begin to rise within 12-24 hours, peak levels within 48-72 hours, and remain elevated for 10-14 days
  • Hepatic carcinoma and toxic hepatitis will have 10-fold increased
  • Viral hepatitis and cirrhosis would give LD slightly increased values (2-3x URL)
  • LD-1 > LD-2 also known as the "flipped pattern" is seen in myocardial infarction and hemolytic anemia
  • LD2: LD1 ratio rises to >0.75 and often exceeds 1.0, which occurs only about 36 hours after the onset of symptoms
  • LD-2, LD-3, LD-4 = LD cancer markers (predominantly LD-3); acute leukemia, germ cell tumors, breast and lung cancers
  • LD-5 is moderately increased in acute viral hepatitis and cirrhosis and markedly increased in hepatic carcinoma and toxic hepatitis
  • It will be clinically significant if separated into isoenzyme fractions
  • An elevated total LD is a nonspecific result because of its presence from several tissues
• LD Isoenzymes as a Percentage of Total LD

  • LD-1 17-27%
  • LD-2 27-37%
  • LD-3 18-25%
• 1. 5 is moderately increased in acute viral hepatitis and cirrhosis and markedly increased in hepatic carcinoma and toxic hepatitis
• It will be clinically significant if separated into isoenzyme fractions