Pancreatic Enzymes: Amylase, Lipase, Trypsin, Chymotrypsin, and Elastase-1

Pancreatic Enzymes

Amylase (AMY)

  • Definition: A hydrolase that catalyzes the breakdown of starch and glycogen.
  • Starch Composition: Starch consists of amylose and amylopectin.
    • Amylose: Long, unbranched chain of glucose molecules linked by α, 1-4 glycosidic bonds.
    • Amylopectin: Branched-chain polysaccharide with α, 1-6 linkages at branch points.
  • Glycogen: Similar structure to amylopectin but more highly branched.
  • Action of α-AMY: Attacks only α, 1-4 glycosidic bonds.
    • Produces glucose, maltose, and dextrins (intermediate chains containing α, 1-6 branching linkages).
  • Specificity: Does NOT attack cellulose and other structural polysaccharides with different linkages.
  • Physiological Role: Important enzyme in the digestion of starches.
  • Activation: Requires calcium and chloride ions for activation.

Tissue Source

  • Major Source: Acinar cells of the pancreas and salivary glands.
  • Minor Sources: Skeletal muscle, small intestine, and fallopian tubes.
  • Urine AMY: Due to its small size (MW 50,000-55,000 Da), it is filtered by the renal glomerulus.
  • Salivary AMY (Mouth): Initiates starch digestion but is inactivated by the acidity of gastric contents after swallowing.
  • Pancreatic AMY: Performs the major digestive action of starches in the intestine.

Diagnostic Significance

  • Elevated Serum AMY (Hyperamylasemia):
    • Salivary gland lesions (e.g., mumps & parotitis).
    • Intra-abdominal diseases (serum AMY level <500 Somogyi U/dL):
      • Perforated peptic ulcer
      • Intestinal obstruction
      • Cholecystitis
      • Ruptured ectopic pregnancy
      • Mesenteric infarction
      • Acute appendicitis
    • Renal insufficiency
    • Diabetic ketoacidosis
    • Neoplastic diseases (S. AMY - 50X ULN).
  • Macroamylasemia:
    • AMY molecules combine with Ig, forming a large complex.
    • Reduces renal clearance, leading to increased serum AMY levels and abnormally low urinary excretion.
    • Important to differentiate from other causes of hyperamylasemia.
  • AMY Isoenzymes:
    • Separated by electrophoresis, chromatography, or isoelectric focusing.
    • P isoamylase: From pancreatic tissue.
    • S isoamylase: From salivary gland tissue, fallopian tube, & lung.
    • Salivary isoenzymes (S1, S2, S3) migrate faster.
    • Pancreatic isoenzymes (P1, P2, P3) migrate slower.
    • In acute pancreatitis, P-type (P3) activity increases (but not specific).
    • S-type isoamylase represents approximately ⅔ of AMY activity in normal serum, while P-type predominates in normal urine.

Amylase Methodologies

  • Amyloclastic: Measures the disappearance of starch substrate.
  • Saccharogenic: Measures the appearance of the product (reducing sugars).
  • Chromogenic: Measures increasing color from product production coupled with a chromogenic dye.
  • Continuous Monitoring: Coupling of several enzyme systems to monitor amylase activity.
Amyloclastic Method
  • AMY acts on a starch substrate with attached iodine.
  • Hydrolysis releases iodine, decreasing the dark-blue color intensity.
  • The decrease in color is proportional to AMY concentration.
Saccharogenic Method
  • Classic reference method.
  • Starch substrate is hydrolyzed by AMY into reducing sugars.
  • The amount of reducing sugars is measured; the concentration is proportional to AMY activity.
  • Somogyi units: mg of glucose released in 30 min at 37°C under specific assay conditions.
Chromogenic Methods
  • Starch substrate with a chromogenic dye attached, forming an insoluble dye-substrate complex.
  • AMY hydrolysis produces water-soluble dye-substrate fragments.
  • The increase in color intensity of the soluble dye substrate solution is proportional to AMY activity.
Coupled-Enzyme Systems
  • Continuous monitoring technique.
  • Measures the change in absorbance of NAD^+ at 340 nm.
  • Optimal pH for AMY activity is 6.9.
  • Salivary AMY is preferentially inhibited by wheat germ lectin; salivary and pancreatic AMY can be estimated by measuring total AMY in the presence and absence of lectin.
  • Specific immunoassays are also available for measuring isoenzymes of AMY.

Sources of Error

  • Stability: AMY in serum and urine is stable.
    • Little loss of activity at room temperature for 1 week or at 4°C for 2 months.
  • Hyperlipidemia: Plasma triglycerides suppress or inhibit serum AMY activity; AMY values may be normal in acute pancreatitis with hyperlipidemia.
  • Opiates: Falsely elevated serum AMY levels can result from the administration of morphine and other opiates before blood sampling.
    • These drugs cause constriction of the sphincter of Oddi and pancreatic ducts, elevating pressure and causing regurgitation of AMY into the serum.

Reference Range

  • Serum: 28 to 100 U/L (37°C) (0.5 to 1.7 µkat/L)
  • Urine: 1 to 15 U/h
  • Activity is expressed according to each procedure.
  • No uniform expression of AMY activity exists, although Somogyi units are frequently used.
  • Approximate conversion factor between Somogyi units and IUs is 1.85.

Lipase (LPS)

  • Definition: Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids.
  • Action: Catalyzes the partial hydrolysis of dietary triglycerides in the intestine to the 2-monoglyceride intermediate, producing long-chain fatty acids.
  • Specificity: Specific for fatty acid residues at positions 1 and 3 of the triglyceride molecule.
  • Requirement: Substrate must be an emulsion for activity to occur.
  • Acceleration: The reaction rate is accelerated by the presence of collapse and a bile salt.

Tissue Source

  • Primary: Pancreas
  • Lesser amounts: Stomach and small intestine.

Diagnostic Significance

  • Acute Pancreatitis: Serum LPS is almost exclusive for diagnosis.
    • Increases 4 to 8 hours after an attack, peaks at 24 hours, and decreases within 8 to 14 days.
    • Elevations persist for approximately 8 days, while AMY elevations persist for 2 to 3 days.
  • Other Conditions:
    • Increased in cases of penetrating duodenal ulcers, perforated peptic ulcers, intestinal obstruction, and acute cholecystitis.
  • Salivary Gland Involvement: Unlike AMY levels, LPS levels are normal in conditions of salivary gland involvement.
  • Isoenzymes: Of the three LPS isoenzymes, L2 is thought to be the most clinically specific and sensitive. Useful in differentiating serum AMY elevation as a result of pancreatic versus salivary involvement.

Lipase Methodologies

Cherry-Crandall Method
  • Used an olive oil substrate and measured the liberated fatty acids by titration after 24 hours of incubation.
Titrimetric Method
  • Estimation of liberated fatty acids
Turbidimetric Methods
  • Simpler and more rapid than titrimetric assays.
  • Fats in solution create a cloudy emulsion.
  • As the fats are hydrolyzed by LPS, the particles disperse, and the rate of clearing is measured as an estimation of LPS activity.
Colorimetric Methods
  • Based on coupled reactions with enzymes peroxidase or glycerol kinase.

Sources of Error

  • Stability: LPS is stable in serum, with negligible loss in activity at room temperature for 1 week or for 3 weeks at 4°C.
  • Hemolysis: Should be avoided because hemoglobin inhibits the activity of serum LPS, causing falsely low values.

Reference Range

  • LPS: <38 U/L (37°C) (<0.6 µkat/L)

Trypsin (TRY)

  • Electrophoretic Mobility:
    • TRY-1: Cationic (MW: 25.8 kDa)
    • TRY-2: Anionic (MW: 22.9 kDa)
  • Inhibitors: Small polypeptides & α2-macroglobulin are present in pancreatic juice, serum, and urine.
    • These inhibitors protect plasma and other proteins against hydrolysis by TRY & other proteases.
  • α1-Antitrypsin Deficiency: Associated with an increased tendency toward panlobular emphysema in early life, illustrating the effects of uninhibited proteases on organ function.

Methods of Analysis

  1. Catalytic Assays: Other proteolytic enzymes present in serum could hydrolyze the same substrates.
  2. Immunoassays: Quantify blood TRY.
    • TRY-1: Immunoassays detect trypsinogen-I, TRY-1, and TRY-α-antitrypsin complex.

Reference Ranges

  • Reference limits are method-dependent because of no assay standardization.

Clinical Significance

  • Acute Pancreatitis:
    • Serum TRY-1 rises in parallel with serum AMY activity to peak values ranging from 2 to 400 times the upper limit.
  • Chronic Renal Failure:
    • Serum TRY-1 is elevated, as is serum AMY and LPS; chronic renal failure must be ruled out.
  • Chronic Pancreatitis:
    • With steatorrhea, fasting concentrations are extremely low.
    • In the relapsing phase, plasma TRY concentration is high in neonates.
    • Without steatorrhea, plasma concentrations of TRY-1 do not differ.

Chymotrypsin (CHY)

  • Definition: Serine proteinase that hydrolyzes peptide bonds involving carboxyl groups of Trp, Leu, Tyr, or Phe, with preference for aromatic residues.

Biochemistry and Tissue Location

  • Synthesis: Acinar cells of the human pancreas synthesize Chymotrypsin 1 & 2 (major) in the form of the inactive proenzymes (or zymogens) - Chymotrypsinogen 1 & 2.
  • Storage: These zymogens are stored in granules and secreted like trypsinogen into the pancreatic duct.
  • Activation: In the intestinal tract, the Chymotrypsinogens are converted to CHY by the action of TRY.
  • Stability: CHY is more resistant than TRY to degradation in the intestine; it is therefore the enzyme of choice for assay in feces.
  • Stool CHY Activity: Remains constant at room temperature for up to 7 days.
  • Molecular Weight: The MW of both CHY 1 & 2 is approximately 25 kDa.
  • Binding: CHY, like TRY, is bound in plasma by α1-antitrypsin and α2-macroglobulin.

Clinical Significance

  • Stool CHY: Used for the investigation of chronic pancreatic insufficiency.
    • Often reduced below the lower reference limit (12 U/g stool) in patients with steatorrhea but not useful to identify early pancreatic insufficiency.
  • Monitoring Enzyme Supplementation: CHY measurement in patients with chronic pancreatic insufficiency, treated with oral pancreatic enzyme supplements, may indicate whether the therapy is adequate or whether increased supplementation is necessary.

Elastase-1 (E1)

*Human pancreatic elastase-1 (EC 3.4.21.36; no systematic name; E1)

  • Definition: a serine protease, a carboxy endopeptidase that catalyzes hydrolysis of native elastin, which is the major structural fibrous protein in connective tissue, with a special affinity for the carboxyl group of Ala, Val, and Leu.
  • Synthesis: Human EI (MW 26 kDa) is synthesized by the acinar cells of the pancreas along with the other digestion enzymes.
  • Activation: The enzyme is synthesized as a pre-proelastase. After processing to proelastase, it is stored in the zymogen granules and later activated to elastase by TRY in the duodenum, undergoing minimal degradation during intestinal transit.
    *Measurement in stool is the most reliable and sensitive noninvasive procedure for the diagnosis of chronic pancreatic insufficiency. However, such a test does not consistently separate mild to moderate insufficiency cases from healthy controls.
    *Unlike fecal CHY, E1 provides no information helpful to the therapeutic management of the patient.
    *The enzyme has been found to be stable in stool samples for up to week at room temperature
    *Fecal E1 concentration lower reference limit is 200 ug/g stool.