2.3. Adrenal function testing

For ACTH stimulation testing, plasma cortisol concentrations were measured immediately before and 1 hour after administration of a standard IM dose of 125 μg (for dogs < 5 kg) or 250 μg (for dogs > 5 kg) synthetic ACTH (Tetracosactide, Synacthen; Alliance Pharmaceuticals, Chippenham, UK). A positive ACTH stimulation test was defined as a post‐ACTH cortisol concentration > 21.7 μg/dL.

For the LDDST, plasma cortisol concentrations were measured immediately before, 3 or 4 hours and 8 hours after IV administration of 0.015 mg/kg of dexamethasone. To encourage acclimatization and minimize stress, the LDDST routinely was performed after at least 1 day of hospitalization, with no other procedures scheduled during the test. Dogs were not fasted during the test.

A positive LDDST result was defined as either or both a t ₃ or t ₈ cortisol concentration > 1 μg/dL (therefore, including the inverse pattern as positive). For the purpose of our study, each LDDST result was retrospectively classified as complete suppression (both t ₃ and t ₈ cortisol concentrations < 1 μg/dL), lack of suppression (both t ₃ and t ₈ cortisol concentrations > 1 μg/dL and > 50% t ₀ cortisol concentration), partial suppression (t ₃ and t ₈ cortisol concentrations > 1 μg/dL but either or both < 50% t ₀), escape (t ₃ cortisol concentration < 1 μg/dL and t ₈ cortisol concentration > 1 μg/dL), or inverse (t ₃ cortisol concentration > 1 μg/dL and t ₈ cortisol concentration < 1 μg/dL) pattern.