Short-Term Effects of Temperature and Thyrotropin-Releasing Hormone Stimulation on Adrenocorticotropin Stability in Horses

SIMPLE SUMMARY: Approximately 20% of older horses develop pituitary dysfunction (PPID), which is associated with haircoat changes, muscle loss, and a higher risk of developing an infection or laminitis. Elevated plasma adrenocorticotropic hormone (ACTH) is used to diagnose PPID; however, ACTH is not stable in blood samples. Therefore, samples should be kept at 4 °C until analysis. In ambulatory veterinary practice, blood samples can be left at room temperature (20 or 30 °C) or inadvertently left in a vehicle without refrigeration where they might be exposed to temperatures of up to 70 °C in hot climates. To evaluate the effects of temperature on ACTH concentrations, we experimentally subjected blood samples from horses with and without pituitary dysfunction to temperatures of 4 (reference), 20, 30, and 70 °C for 1 h prior to laboratory measurement. The stability of ACTH was affected by short-term exposure to high temperatures in horses with and without pituitary dysfunction with both higher and lower ACTH concentrations measured unpredictably. Our results suggest that samples should be kept at 4 °C to reflect the true ACTH concentration. Exposure to temperatures of up to 40 °C for 1 h can still provide an appropriate assessment of pituitary function in most cases, but the ACTH concentration changed by 12% in healthy horses and 5% in horses with PPID. ABSTRACT: Pituitary pars intermedia dysfunction (PPID) is diagnosed by increased basal or post thyrotropin-releasing hormone (TRH) stimulation ACTH concentrations. ACTH is known to be unstable; however, the effect of different temperatures and TRH stimulation on equine ACTH stability is poorly described. In total, 15 horses, including 8 PPID positive (ACTH > 35 pg/mL at baseline or >65 pg/mL 30 min after TRH stimulation), were divided into 2 groups: 9, including 5 PPID positive, with basal ACTH concentrations and 6, including 3 PPID positive, with post TRH stimulation ACTH concentrations. Whole blood was stored for 1 h at 4, 20, 30, 40, or 70 °C. After centrifugation, immunoreactive ACTH concentrations were determined using a chemiluminescent assay. Linear mixed effect models were used to detect the effects of temperature, PPID status, and TRH stimulation on the immunoreactive ACTH concentration. Temperature had a significant effect (p = 0.003) on immunoreactive ACTH concentrations, and this effect was greater in PPID-negative horses (p = 0.01), with the changes in immunoreactive ACTH concentrations being slightly unpredictably higher or lower than samples stored at 4 °C. Even at 20 °C, mean immunoreactive ACTH concentrations minimally changed by 5% in PPID horses and 12% in non-PPID horses after 1 h. No significant effect of TRH stimulation was identified. Although ACTH concentrations should ideally be determined from samples kept at 4 °C, samples inadvertently left at temperatures of up to 40 °C can provide valid results if analyzed within 1 h; however, this increases the risks of altered ACTH concentrations, occasionally influencing the diagnosis of PPID.