Amylase

 
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Physiology

Amylase is a calcium dependent enzyme which hydrolyzes complex carbohydrates at alpha 1,4-linkages to form maltose and glucose. Amylase is filtered by renal tubules and resorbed (inactivated) by tubular epithelium. Active enzyme does not appear in urine. Small amounts of amylase are taken up by Kupffer cells in the liver. In healthy dogs, 14% of amylase is bound to globulins. Because of this polymerization, canine amylase has variable (high) molecular weights and is not normally filtered by the kidney. In dogs with renal disease, this polymerized (macroamylase) amylase is found in higher concentration (from 5-62% of total amylase activity) and contributes to the hyperamylasemia seen in these disorders.

There are four different isoenzymes of amylase in the dog: isoenzyme 3 is found in the pancreas (>50%), whereas isoenzyme 4 is found in all tissues.

Organ specificity:

  • Pancreas: Found in zymogen granules. The pancreas has higher concentrations of amylase than other tissues.
  • Intestine: Duodenum, ileum
  • Ovary and testes
  • Salivary gland: Salivary amylase is found in high concentration in pigs, resulting in high reference intervals for amylase in this species. Dogs lack salivary amylase.

Methodology

Amylase assay

There are several different assays for amylase:

  • Enzymatic
    • Saccharogenic – This methodology measures the rate of appearance of reducing sugars (glucose, maltose). This methodology is invalid in the dog as dog serum contains maltase. Maltase is additive to the activity of amylase and will produce increased numbers of reducing sugars.
    • Amyloclastic – This method measures the hydrolysis of starch and the rate of its disappearance. Valid for dogs and other domestic species. Lipemic samples may show an inhibition of enzyme activity which can be overcome by dilution.
  • Turbidometric: Size of the starch substrate decreases with hydrolysis which reduces light scatter.
  • Chromogenic substrates: These use dyes bound to synthetic starch substrates, with the dye being released (and measured) once the substrate is hydrolyzed. Chromogenic substrate techniques are the current clinical amylase assay.

Reaction type

Enzymatic colorimetric assay

Procedure

α-amylases catalyzes the cleavage of certain oligosaccharides. The resulting fragments are completely hydrolyzed to p-nitrophenol and glucose by α-glucosidase. The color intensity of the p-nitrophenol is directly proportional to the α-amylase activity.

Units of measurement (link to conversion calculator)

The concentration of amylase is measured in U/L (conventional units) and μkat/L (SI units). The conversion formulais shown below:

U/L x 0.0167 = μkat/L

Sample considerations

Sample type (serum, plasma, urine, body cavity fluids)

Serum, plasma, urine

Anticoagulant

EDTA or heparin; citrate and fluoride will inhibit the reaction

Stability

The stability of amylase in human serum, plasma, and urine samples are as follows: 30 days at 2 – 8 °C, 7 days at 20 – 25 °C. It is stable in urine for 10 days at 2 – 8 °C, or 2 days at 20 – 25 °C.

Interferences

  • Lipemia: Severe lipemia (>1500 lipemia index)
  • Hemolysis:  (>500 hemolysis index)
  • Icterus:  (>60 icteric index)
  • Glucose: > 2000mg/dL. Approximately 10% lower recovery at glucose concentrations of 4500 mg/dL
  • Ascorbic acid: Decrease levels of amylase with an ascorbic acid concentration > 100 mg/dL

 

Test interpretation

Increased amylase activity (hyperamylasemia)

  • Pathophysiologic
    • Acute pancreatitis: The increase and decrease of serum amylase tends to parallel that of lipase. Values should be at least double reference values to be considered significant, and often may reach 7-10 times normal. Amylase values peak at 12-48 hours and are normal within 8-14 days after a bout of pancreatitis in dogs. It is rare to observe increased amylase in cats with pancreatitis. Note that amylase concentrations are often higher in ascitic fluid than in blood in animals with pancreatitis (or intestinal disease).
    • Chronic renal insufficiency: As discussed above, the increase in amylase is due to macroamylase formation (polymerization with globulins).
    • Decreased GFR: This can cause increased amylase (up to 2-3 x normal) in the absence of significant pancreatic disease. If an azotemic patient has amylase values greater than 2 to 3 times the reference values, pancreatitis should receive consideration as a diagnosis. Amylase is variably increased in cats with disorders associated with renal azotemia. However, this mechanism may not apply to dogs. Dogs do not typically excrete amylase via the kidney. The increased amylase seen in dogs with conditions associated with decreased GFR (pre-renal, renal, post-renal azotemia) may be due to macroamylase formation.
    • Intestinal disease/obstruction: Moderate elevations in amylase are possible.
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