Developing an index of suspicion for an endocrinopathy (Proceedings)

Collection, storage and storage of samples for endocrine testing

In order for a hormone to be measured properly, with valid, repeatable results, the sample must be collected, stored and transported in a manner that is applicable for the hormone being tested. Obviously because various hormones have diverse chemical structures and physical properties, it is imperative that that the laboratory being utilized issue specific instructions and that these instructions be adhered to in order to achieve optimal results. Certain hormones must be run on serum only (gastrin, FT4,TT4, FT3 and TSH; while others yield more accurate results from EDTA or heparinized plasma (ACTH, aldosterone, cortisol, insulin, rennin, parathormone, parathormone related protein, vasopressin 1, 25 dihidroxy Vit. D3,. ACTH is a small, labile protein that must be collected and handled with special considerations; utilizing an EDTA plastic or siliconized glass tubes, centrifuged immediately, quick and prolongedly froze and shipped via an overnight express service. Certain special considerations of the sample must be taken into account.1) LIPEMIA-Usually excessive fat in the serum. Is not a problem for most RIA assays, but may be a problem for colorimetric indicator procedures. Certain FT3 and ACTH assys may yield spurious results in the face of heavy lipemia. If this is the case the clinician may request an 18 hour fast prior to blood draw. 2) HEMOLYSIS-Most RIA assays are unaffected by slight hemolysis. However severe hemolysis as a result of freezing or vigorously shaking a sample to lyse RBC membranes, results innaccurate results With the increased use of ELISA assys and techniques that employ chemoluminescent and colorimetric endpoints hemolysis become a more significant factor. 3) PROTEOLYSIS- Generally, cooling slows the activity of prteolytic enzymes. In Europe the use of aprotinin which inhibits proteolysis, yielding more accurate results. Some of the hormone assays that are affected by proteolysis include:ACTH, PTH, insulin and vasopressin. Unfortunately aprotinin is not readily available in the USA. It is interesting to consider the effects of time and temperature upon various hormonal assays If one considers incubation periods of 4-7 days at temperatures of 4 degreesC, 20-22 degrees C and 37 degreesC; the hormones which degrade the most include:aldosterone, cortisol, TT3, FT3, vassopressin and PTH. Interestingly TT4 concentrations will actually increase because the binding proteins degrade and the free fraction increases. There are various commercially available containers available for the transportation of hormone samples, usually consisting of a polystyrene box and 2 450 gram gel packs which can be frozen to -18 to-24degrees C. Samples for the measurement of vasopressin and plasma rennin should be shipped in dry ice containers.

As stated previously, there are a variety of assays to accurately measure the minute quantities of hormones in the circulation of canines and felines; including RIA, IRMA (immunoradiometric assay, which is similar to RIA but employs the use of a radiolabeled ant-hormone antibody that is present in excessive quantities-the so-called "sandwich" method, useful in the measurement of TSH and ACTH), ELISA and EIA (enzyme immunoassay). The latter two assays offer the advantage of requiring the use of radioisotopes, thus making them feasible to be run in-house, however there have been studies showing a discrepancy of the results between these in-house ELISA kits and validated RIA assays; especially in the measurement of TT4. Measurement of circulating FT4 would certainly appear to give the clinician a more accurate assessment if a dog was hypothyroid and the use of the equilibrium dialysis technique appears to gaining favor amongst the large reference laboratories in the USA.

Clinical assessment of a dog or cat with a suspected endocrinopathy

In order for a clinician to accurately diagnose an endocrinopathy it is important that he or she possess "an index of suspicion". This "6th sense" improves obviously over the years and the number of cases managed by the clinician. If a patient presents with a chief complaint of polyuria and polydypsia many endocrine disorders can be considered (hypothalamic diabetes insipidus-HDI, hyperadrenocorticism, diabetes mellitus, pyometra-unspayed females, hypercalcemia (primary, renal or pseudohyperthyroidism,), hyperthyroidism, hypokalemia and acromegaly. Alopecia is another important physical sign in a number of endocrinopathies. In the majority of cases the alopecia is symmetrical and devoid of pruritis and cutaneous inflammation. Some of the more important endocrinopathies include: 1) hypothyroidism,2) naturally-occurring hyperadrenocorticism, 3) hyperestrogenism, 4) functional testicular tumor, 5) alopecia X, 6) estrogen-responsive alopecia, 7) testosterone-responsive alopecia and 8) growth hormone-responsive alopecia. In certain instances the presence of fasting hyperlipidemia may signal a hormonal disorder; including diabetes mellitus (often as a result of pancreatitis), hyperadrenocorticism and hypothyroidism. Anemia and lethargy are often associated with hypothyroidism; while polyphagia, nervousness, vocalization, weight-loss, hyperdefecation, and vomition are common symptoms of feline hyperthyroidism. In addition, feline hyperthyroidism and associated thyrotoxicosis can lead to hypertrophic cardiomyopathy and signs of congestive heart failure, ( dyspnea, lethargy, cyanosis, etc.,). Weakness, anorexia and tremors/convulsions are often in hypocalcemic patients as a result of hypoparathyroidism.

References

Drazner, FH: Small Animal Endocrinology Churchill Livingstone. 1984.

Drazner, FH: A Case Report of a Dog with Gastrinoma, Resembling the Zollinger-Ellison Syndrome. California Veterinarian. 1978.

Mooney, CT. and Peterson ME (eds) BSAVA Manual of Canine and Feline Endocrinology. BSAVA. 2004.

Monroe, WE: Disease of the Endocrine Pancreas and Pituitary Gland in: Leib MS.and Monroe WE.:(eds.) Practical Small Animal Internal Medicine, WB Saunders. 1997.