Canis ISSN: 2398-2942

Fecal analysis: proteolytic activity - trypsin

Contributor(s): Helen Milner, James Simpson

Overview

  • Trypsin and chymotrypsin are proteolytic enzymes produced by the exocrine pancreas to digest dietary protein.
  • Some proteolytic enzymes may be found in fecal samples of normal dogs.
  • This proteolytic activity can be measured in fecal samples using various methods.
  • Measurement of fecal trypsin has been used as a method of detecting exocrine pancreatic insufficiency EPI Exocrine pancreatic insufficiency.
  • Several methods of detecting the presence of trypsin in feces have been described:
    • X-ray film test an unreliable test and now considered outdated.
    • Gelatin tube test - an unreliable test and now considered outdated.
    • Radial enzyme diffusion test more reliable compared with the above two tests.
  • These tests have now been superceded by the serum Trypsin Like Immunoassay (TLI) test Blood biochemistry: trypsin-like immunoreactivity.

Sampling

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Tests

Methodologies

X-ray film test

  • Add 1 g of fresh feces to 9 ml 1% sodium bicarbonate solution.
  • Thoroughly mix. This gives a 1/10 dilution.
  • Make serial one in two (1:2) dilutions using sodium bicarbonate solution to give 1/20, 1/40, 1/80 and 1/160 dilutions.
  • Add a strip of x-ray film to each tube and incubate at 37°C for 1 hour.
  • Examine the x-ray film for evidence of digestion; areas of the x-ray film exposed to fecal proteolytic enzyme will appear clear.
  • The dilution at which digestion still occurs is the final titer.
  • Normal titers should be > 1/80.

Gelatin tube test

  • Add 1 g of fresh feces to 9 ml of 1% sodium bicarbonate solution.
  • Thoroughly mix. This gives a 1/10 dilution.
  • Make serial one in two (1:2) dilutions using sodium bicarbonate solution to give 1/20 and 1/40 dilutions.
  • To 2 ml of each of the dilutions (1/10, 1/20 & 1/40), add 2 ml of a 7.5% gelatin solution. This will give final test dilutions of 1/20, 1/40 and 1/80.
  • Mix thoroughly and incubate at 37°C fro 1 hour.
  • Place tubes in the refrigerator for 20 minutes.
  • If the tube contents remains as a liquid, digestion has occurred. If the gelatin sets, no digestion has occurred.
  • Normal titers should be > 1/40.

Radial enzyme diffusion test

  • Add 1 g of agar to 50 ml of PBS (phosphate buffered saline).
  • To this, add 1 gm of calcium paracaseinate and 0.1 g sodium azide in 50 ml of PBS.
  • Mix thoroughly.
  • Pour contents into petrie dishes to 2 mm depth.
  • Cut four 6 mm diameter holes in the agar once set. Ensure these holes are at least 15 mm apart from each other.
  • Fill the first well with a 1/10 dilution of feces in PBS.
  • Into subsequent wells, make serial one in two (1:2) dilutions using sodium bicarbonate solution. This results in 1/10, 1/20, 1/40 and 1/80 dilution test wells.
  • Incubate at 22°C for 24 hours.
  • Digestion is revealed by clearing of the agar around the well.
  • Clearning of > 9 mm is normal.
  • Clearning of < 9 mm is suggestive of EPI.

Technique (intrinsic) limitations

  • Poor laboratory technique.
  • Difficult to interpret the x-ray film, gelatin tube and radial enzyme diffusion tests.
  • Intermittent excretion of proteolytic enzymes in feces.
  • Fecal bacterial protease activity may interfere with the tests.
  • Hence, false positives and false negatives are common.

Further Reading

Publications

Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Simpson J W & Doxey D L (1988) Evaluation of faecal analysis as an aid to detection of exocrine pancreatic insufficiency. Brit Vet J 144, 174-178.
  • Canfield P J, Fairburn A J & Weston I M (1982) A re-evaluation of single faecal tests for the diagnosis of canine exocrine pancreatic insufficiency. Aust Vet Practioner 12, 64-66.
  • Westermarck E & Sandholm M (1980) Fecal hydrolase activity as determind by radial enzyme diffusion: A new method for detecting pancreatic dysfunction in the dog. Res Vet Sci 28, 341-346.

Other sources of information

  • Manual of Small Animal Clinical Pathology (1998) Davidson M, Else R & Lumsden J (eds), BSAVA.
  • Clinical Textbook for Veterinary Technicians (1998) 4th edn McCurnin D M (ed), W B Saunders.
  • BSAVA Manual of Canine and Feline Enterology (1997) Thomas D A et al(eds), Iowa State University Press.
  • Laboratory Procedures for Veterinary Technicians (1997) 3rd edn, Pratt P W (ed), Mosby.
  • Williams D A (1985) The diagnosis of canine exocrine pancreatic insufficiency. In: The Veterinary Annual. Grunsell C S G, Hill F W G & Raw M E (eds). Scientechnica, Bristol. pp 330.


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