Welcome to the all-new Vetlearn

  • Vetlearn is getting a new home. Starting this fall,
    Vetlearn becomes part of the NAVC VetFolio family.

    You'll have access to the entire Compendium and
    Veterinary Technician archives and get to explore
    even more ways to learn and earn CE by becoming
    a VetFolio subscriber. Subscriber benefits:
  • Over 500 hours of interactive CE Videos
  • An engaging new Community for tough cases
    and networking
  • Three years of NAVC Conference Proceedings
  • All-new articles (CE and other topics) for the entire
    healthcare team

To access Vetlearn, you must first sign in or register.


  • Registration for new subscribers will open in August 2014!
  • Watch for additional exciting news coming soon!
Become a Member

Veterinarian Technician June 2007 (Vol 28, No 6) Focus: Endocrine Disorders

Understanding Common Endocrine Tests

by Laura McLain Madsen, DVM

    CETEST This course is approved for 0.5 CE credits

    Start Test

    Key Points

    • The endocrine system works through a complex process of hormone secretion and feedback.
    • Endocrine tests have varying sensitivity and specificity, sometimes requiring the use of more than one test for diagnosis.
    • Proper sample collection and handling are important in obtaining accurate test results.

    Click here for Glossary

    Endocrine diseases are common diagnoses in small animal practice. Veterinary technicians are instrumental in preparing patients for testing, acquiring samples, and processing samples in-house or submitting them to a reference laboratory. Because the results of endocrine tests can be affected by patient and sample preparation, having a basic understanding of the physiology behind these tests can help technicians ensure that the most accurate results are obtained.

    Endocrine diseases typically seen in general practice include diabetes mellitus, hyperadrenocorticism (HAC; Cushing's disease), hypoadrenocorticism (Addison's disease), hypothyroidism (dogs), and hyperthyroidism (cats). Other endocrine diseases occasionally seen include parathyroid disorders, growth hormone disorders, and diabetes insipidus; these are not covered in this article.

    The Endocrine System

    The endocrine system is a tightly regulated complex of hormone-secreting organs (see figure). These organs, which include the hypothalamus (a region of the brain), the pituitary gland, the adrenal glands, the thyroid gland, and the pancreas, produce more or less hormone in response to the concentration of specific substances in the blood. In some cases, they regulate each other through feedback loops, in which the hormone secreted by one organ is the substance that controls hormone production in another organ. The pituitary gland (sometimes called "the master gland") releases hormones that control several other organs in the endocrine system.

    For example, the feedback loop that regulates the adrenal glands begins with the production of corticotropin-releasing hormone (CRH) by the hypothalamus. This hormone stimulates the pituitary gland to produce adrenocorticotropic hormone (ACTH). ACTH stimulates the adrenal glands to produce glucocorticoids and mineralocorticoids, which play important roles in the function of tissues as diverse as the kidney and liver as well as in immune system function. The hypothalamus and pituitary gland respond to the increased blood levels of these hormones by decreasing the production of CRH and ACTH, respectively. This is known as a negative feedback loop. The thyroid gland is also governed by a feedback loop, in which the hypothalamus produces thyrotropin-releasing hormone (TRH), the pituitary gland produces thyrotropin-stimulating hormone (TSH), and the thyroid gland produces thyroxine (T4) and triiodothyronine (T3), which help regulate cell metabolism.

    Not all endocrine functions are controlled by hormones. In the normal glucose metabolic pathway, high levels of blood glucose stimulate b cells in the pancreas to release insulin. Insulin transports glucose across the cell membrane of most cells, thereby lowering blood glucose levels. As blood glucose levels decrease, insulin secretion also decreases.

    Adrenal Gland Disorders


    Hypoadrenocorticism (see figure) is characterized by failure of the adrenal glands to produce sufficient mineralocorticoids (e.g., aldosterone) and glucocorticoids (e.g., cortisol). This is usually due to immune-mediated destruction of the adrenal gland (primary hypoadrenocorticism); rare cases of secondary hypoadrenocorticism are also described, which are caused by damage to the pituitary gland.1 Some cases of hypoadrenocorticism may show only glucocorticoid deficiency ("atypical" hypoadrenocorticism).1 In the absence of feedback from these hormones, the hypothalamus and pituitary gland secrete excessive CRH and ACTH, respectively, but the atrophied adrenal glands are incapable of responding appropriately. Clinical signs of hypoadrenocorticism include anorexia; acute onset or chronic vomiting or diarrhea; generalized weakness and collapse, particularly in stressful situations; and shock.

    The primary endocrine test that is used for diagnosing hypoadrenocorticism is the ACTH stimulation test. In this test, a serum sample is obtained and the baseline level of cortisol measured. Synthetic ACTH or ACTH gel is administered, and a second blood sample is obtained 1 hour (with synthetic ACTH) or 2 hours (with ACTH gel) later. In a healthy dog, the administration of ACTH stimulates the adrenal glands to produce cortisol, and the poststimulation sample shows an elevated cortisol level. Laboratory reference ranges vary, but a pre-ACTH cortisol level of 0.5 to 5.5 µg/dl and a post-ACTH cortisol level of 6.0 to 17.0 µg/dl are typically normal.2 Dogs with hypoadrenocorticism have very low cortisol levels (<2.0 µg/dl) before and after ACTH stimulation because their own ACTH levels are already elevated and the adrenal glands are incapable of responding to stimulation.2,3


    HAC is characterized by an excessive production of cortisol by one or both adrenal glands. This may be caused by a pituitary tumor (pituitary-dependent hyperadrenocorticism [PDH]; see figure) or an adrenal tumor (see figure). In PDH, the pituitary gland secretes excessive ACTH, which in turn stimulates excessive cortisol production by the adrenal glands. The pituitary tumor is resistant to the normal negative feedback effects of elevated cortisol levels. Adrenal tumors produce excess cortisol directly. The hypothalamus and pituitary gland respond to the high cortisol level and decrease production of CRH and ACTH, but the adrenal tumor is not regulated by these hormones; therefore, cortisol production continues. Clinical signs of HAC from PDH or adrenal tumor include polyphagia, polydipsia, polyuria, susceptibility to infections, thin skin, "potbellied" appearance, and hair loss.

    Various tests are used to diagnose HAC; however, all have significant risks for false-positive and false-negative results. Tests that are used in general practice include the urine cortisol:creatinine ratio (UC:CR), the ACTH stimulation test, the low-dose dexamethasone suppression test (LDDST), the high-dose dexamethasone suppression test (HDDST), and measurement of endogenous ACTH.

    Screening Tests

    The UC:CR is a screening test in which the levels of cortisol and creatinine in a single urine sample are compared. Creatinine is excreted at relatively steady levels, so an elevated UC:CR indicates excessive excretion of cortisol. The UC:CR is a good screening test because it is easy to conduct and has a high sensitivity: If the UC:CR is normal, a dog is unlikely to have HAC. However, an elevated UC:CR is not necessarily diagnostic for HAC.2,4 Nonadrenal conditions that can cause elevated cortisol levels and therefore increase urinary excretion of cortisol include stress, diabetes mellitus, pyometra, and hypercalcemia. The veterinarian can distinguish these other diseases from HAC on the basis of history, physical examination, and basic laboratory tests (e.g., complete blood count, chemistry panel, urinalysis). To decrease the likelihood of a false-positive result caused by stress, the owner can be asked to bring in a free-catch urine sample.

    The ACTH stimulation test used to diagnose hypoadrenocorticism can also be used as a screening test for HAC. Although the adrenal glands are already producing excessive cortisol, they may still respond to ACTH and show an exaggerated response. An exaggerated response may also be seen with nonadrenal disease, such as diabetes, liver disease, and renal disease (false-positive result).4 A poststimulation cortisol level of 17.0 to 22.0 µg/dl is considered borderline; a level of greater than 22.0 µg/dl is consistent with HAC.2 However, a normal poststimulation cortisol level does not rule out HAC. Of dogs with PDH, 15% to 40% have a poststimulation level in the normal range and another 30% are in the borderline range.2,4 Among dogs with an adrenal tumor, only 50% to 60% show an exaggerated response.2,4 Therefore, if HAC is suspected based on the history and physical examination but the ACTH stimulation test result is normal, additional tests, such as the LDDST, are required.

    The LDDST is considered to be the most sensitive screening test for HAC4 (see box on sensitivity and specificity). Because a series of blood samples is required, dogs undergoing this test are kept in the hospital for 8 hours. An initial serum sample to determine the baseline cortisol level is obtained, and dexamethasone is administered at 0.01 mg/kg IV. Dexamethasone is a corticosteroid that exerts negative feedback on the hypothalamus and pituitary gland just as cortisol does. In a healthy animal, dexamethasone administration causes a decrease in CRH and ACTH and thus suppresses cortisol production for up to 48 hours.2 In the LDDST, serum samples are obtained to measure cortisol levels before and 4 and 8 hours after dexamethasone administration to determine whether this suppression occurs. Typical cortisol levels for a healthy dog are 0.5 to 5.5 µg/dl at 0 hours and less than 1.4 µg/dl at 4 and 8 hours.2 Most dogs with HAC — approximately 90% to 95% of those with PDH and all of those with adrenal tumor — have a lesser response to dexamethasone so that cortisol levels remain elevated at 4 and 8 hours.2,4 If the history, physical examination, and basic laboratory tests are suggestive of HAC, the LDDST has a sensitivity of 99% and a specificity of 95%.2 False-positive results may be seen with nonadrenal illness and after the administration of glucocorticoids or phenobarbital.4

    Differentiating Tests

    Once a presumptive diagnosis of HAC is made on the basis of ACTH stimulation and/or LDDST in conjunction with history and clinical signs, it is important to differentiate PDH from adrenal tumor. In some cases, the LDDST allows this differentiation. In 65% of patients with PDH, production of cortisol is suppressed in response to dexa­methasone, but the duration is much shorter than in a healthy dog.2 In this pattern, suppression is seen at the 4-hour mark, but cortisol levels are elevated again (escape) by 8 hours.2,4

    The HDDST can also differentiate PDH from adrenal tumor because high doses of dexamethasone can suppress cortisol in patients with PDH but not in those with an adrenal tumor.5 It is conducted in the same manner as the LDDST, but the dose of dexamethasone used is 10 times higher (0.1 mg/kg IV). At this higher dose, approximately 75% of pituitary tumors are suppressed.2,5 However, if suppression is not seen (i.e., the cortisol level is elevated at 0, 4, and 8 hours), either PDH or adrenal tumor may be present.5

    When the results of the HDDST are inconclusive, the endogenous ACTH level is measured to differentiate PDH from adrenal tumor. In PDH, the pituitary tumor is secreting excessive ACTH; therefore, the endogenous level is high.5 In a patient with an adrenal tumor, the cortisol from the adrenal tumor exerts negative feedback on the pituitary gland; therefore, the ACTH level is low.5 Theoretically, this is an easy and accurate test, but in practice, it can be tricky because the plasma samples must be handled carefully, separated promptly, and shipped frozen.

    Imaging studies can also be used to differentiate PDH from adrenal tumor. In PDH, the adrenal glands, as seen by ultrasonography, magnetic resonance imaging, or computed tomography, are usually bilaterally enlarged. Magnetic resonance imaging or computed tomography can also identify a pituitary tumor. When an adrenal tumor is present, one adrenal gland is usually enlarged whereas the other, normal gland is atrophied from decreased ACTH stimulation.

    Thyroid Gland Disorders

    The hormones produced by the thyroid gland, T4 and T3, are critical in regulating the body's metabolic rate (see figure). T3 is the more biologically active of the two, but more T4 is produced.2 Most (99%) T4 binds to blood proteins, such as albumin. Only the free, unbound hormone is biologically active. Free T4 enters cells, where it is transformed into T3.


    Hypothyroidism in dogs may be primary or secondary. In both cases, the result is failure of the thyroid gland to produce T4 and T3(see figure). In primary hypothyroidism, the thyroid gland is destroyed, either by replacement of thyroid tissue with adipose tissue (idiopathic thyroid atrophy), or by what is suspected to be an immune-mediated process targeting the gland (lymphocytic thyroiditis).2 As in hypoadrenocorticism, the resulting lack of hormones interrupts the negative feedback loop, and although the hypothalamus and pituitary gland produce increased amounts of TRH and TSH, respectively, the thyroid gland is incapable of responding. In secondary hypothyroidism, the pituitary gland fails to release adequate amounts of TSH.

    Diagnosis of hypothyroidism in dogs can be challenging because dogs with hypothyroidism and those that are healthy can have overlapping test results. Clinical signs of hypothyroidism include lethargy, weight gain, heat-seeking behavior, and hair loss. Thyroid tests that are available through reference laboratories include measurement of total and free T4, total and free T3, TSH, and autoantibodies against thyroglobulin (a precursor of T4 and T3).

    Total T4 tests measure bound and unbound T4, whereas free T4 tests measure only the unbound form. Total T4 is usually measured by radioimmunoassay. Again, normal ranges vary depending on the reference laboratory, but there is an overlap in the range between healthy dogs (e.g., 1.0 to 3.3 µg/dl) and those with hypothyroidism (e.g., 0.0 to 1.5 µg/dl).2 Total T4 is best used to rule out hypothyroidism: If the total T4 is 1.5 to 2.0 µg/dl, hypothyroidism is unlikely; if it is greater than 2.0 µg/dl, hypothyroidism is very unlikely.2 Total T4 may be affected by many factors, including age, breed (it is normally lower in sight hounds), and concurrent medications, including common medications such as clomipramine, propranolol, NSAIDs, furosemide, glucocorticoids, phenobarbital, and sulfa antibiotics.2,6 A significant decrease in total T4 may also be seen in dogs with nonthyroidal illness (e.g., renal disease, hepatic disease, heart failure, immune-mediated hemolytic anemia, infections, diabetic ketoacidosis), trauma, or surgery.2,7 The degree of suppression of T4 correlates with the severity of the nonthyroidal disease.2 This phenomenon is known as "euthyroid sick syndrome."

    The free T4 test may be a more accurate test than the total T4 because it measures only the biologically active component. Free T4 is measured either by radioimmunoassay or by equilibrium dialysis; only equilibrium dialysis has been shown to be sensitive.2 Free T4 measured by equilibrium dialysis has been reported to have a sensitivity of 98% (2% false negatives) and a specificity of 93% (7% false positives).2 Free T4 is not as affected by nonthyroidal illness as is total T4.2,6,7

    Thyroid panels from reference laboratories may also measure total T3, free T3, or reverse T3 (an inactive form). None of these has been shown to be useful in diagnosing canine hypothyroidism.2

    A baseline TSH level is also included on many thyroid panels. In theory, TSH should be elevated in a dog with hypothyroidism because of decreased negative feedback to the pituitary gland. Unfortunately, 20% to 40% of such dogs have normal TSH levels.2,7 Evaluating the TSH along with total or free T4 can increase the diagnostic accuracy of the test.2,7

    In human medicine, TSH or TRH stimulation tests are conducted (analogous to the ACTH stimulation test); these tests are prohibitively expensive in veterinary medicine and have not been shown to be accurate.7

    Autoantibodies are occasionally measured when an immune-mediated process is suspected. If the dog is clinically healthy, the presence of autoantibodies does not necessarily mean that hypothyroidism will develop.7


    Hyperthyroidism is characterized by excessive secretion of T4 and T3 by the thyroid gland, usually due to hyperplasia or tumor.2 The clinical signs include polyphagia, weight loss, hyperactivity, tachycardia, and hypertension. As in animals with adrenal tumors, the excessive T4 and T3 exert negative feedback on the hypothalamus and pituitary glands, causing a decrease in TRH and TSH, but the hyperplastic gland or thyroid tumor functions independently of hormone control.

    Hyperthyroidism is usually diagnosed on the basis of elevated total T4 or free T4 levels. However, the sensitivity of total T4 for diagnosing hyperthyroidism in cats is only 91% (i.e., 9% of cats with hyperthyroidism will have a normal T4 level).2 In some cats with hyperthyroidism, total T4 may be within the normal range because of normal fluctuations, or it may be suppressed by concurrent illness. If the T4 is normal but hyperthyroidism is still suspected, a T3 suppression test can be conducted. Like the LDDST, this test suppresses the pituitary gland and hypothalamus. Levels of T4 are compared before and after administration of T3, which suppresses TSH and therefore suppresses T4 in healthy cats. In cats with hyperthyroidism, the thyroid continues to produce T4 independently; therefore, no suppression is seen.

    Diabetes Mellitus

    Diabetes mellitus is most often characterized by deficient insulin secretion by the pancreatic islet or b cells. All cells, except erythrocytes and neurons, require insulin for glucose uptake; without insulin, they starve for energy while glucose builds to high levels in the bloodstream. Clinical signs of uncomplicated diabetes mellitus include polyuria, polydipsia, polyphagia, and weight loss.

    The initial diagnosis of diabetes mellitus is fairly straightforward, but endocrine tests are frequently used in long-term monitoring. Diabetes is diagnosed by finding persistently elevated fasting blood glucose levels and glucose in the urine. Monitoring tests include serum fructosamine and the glucose curve.

    Fructosaminerefers to serum proteins, primarily albumin, that are irreversibly bound to glucose.8 This binding occurs in all animals, and the level of fructosamine is directly related to the average blood glucose level over the preceding 2 to 3 weeks.2,8 Thus, an elevated fructosamine level indicates that the average blood sugar level has been elevated and that the diabetes is not under control. Because the fructosamine is related to the average glucose level, it is not affected by acute increases in glucose, such as those caused by the stress of venipuncture.2 Normal fructosamine levels are 225 to 365 µmol/L. In diabetic patients, the goal is to maintain a fructosamine level between 350 and 450 µmol/L; a level greater than 500 µmol/L indicates poor regulation.2 A fructosamine level less than 350 µmol/L in a diabetic animal raises concern that the animal may be experiencing hypoglycemic episodes.2

    The glucose curve is used for assessing appropriate insulin dose. To conduct this test, the pet is hospitalized for the day. The owner feeds the pet its normal breakfast and administers its morning insulin. The initial blood glucose level is measured at the time of insulin injection or within 1 hour. Blood glucose levels are then measured every 1 to 2 hours throughout the day, and the results are plotted on a graph. When analyzing the glucose curve, the highest value, the lowest value (nadir), the difference between high and low values (the glucose differential), and the duration (the time between the injection and the increase in the glucose level to >200 to 250 mg/dl) are considered.2,8 Ideally, the nadir is 100 to 125 mg/dl, and the glucose level remains at 100 to 250 mg/dl throughout the day; however, this goal may not be possible in some animals.2 Based on the curve, the insulin dosage or type may be altered.


    Many tests may be required to diagnose an endocrine disorder, and further tests are required to monitor treatment. Technicians should be familiar with the rationale for conducting various tests.

    1. Lathan P, Tyler J: Canine hypoadrenocorticism: Pathogenesis and clinical features. Compend Contin Educ Pract Vet 27(2):110-119, 2005.

    2. Feldman EC, Nelson RW: Canine and Feline Endocrinology and Reproduction, ed 3. St. Louis, Saunders, 2004.

    3. Lathan P, Tyler J: Canine hypoadrenocorticism: Diagnosis and treatment. Compend Contin Educ Pract Vet 27(2):121-131, 2005.

    4. Zerbe CA: Screening tests to diagnose hyperadrenocorticism in cats and dogs. Compend Contin Educ Pract Vet 22(1):17-30, 2000.

    5. Zerbe CA: Differentiating tests to evaluate hyperadrenocorticism in dogs and cats. Compend Contin Educ Pract Vet 22(2):149-156, 2000.

    6. Kull P, Kimmel S, Chaitman J: Canine hypothyroidism. Stand Care 4(5):1-7, 2002.

    7. Diaz Espineira MM, Mol JA, Peeters ME, et al: Assessment of thyroid function in dogs with low plasma thyroxine concentration. J Vet Intern Med 21(1):25-32, 2007.

    8. Behrend EN, Greco DS: Feline diabetes mellitus: Evaluation and treatment. Compend Contin Educ Pract Vet 22(5):440-450, 2000.

    References »

    NEXT: A Question of Conscience: "Once Bitten ... Too Shy?"

    CETEST This course is approved for 0.5 CE credits

    Start Test


    Did you know... Vomiting caused by motion sickness involves M1-cholinergic and H1-histaminergic receptors, and treatment should antagonize both receptors.Read More

    These Care Guides are written to help your clients understand common conditions. They are formatted to print and give to your clients for their information.

    Stay on top of all our latest content — sign up for the Vetlearn newsletters.
    • More