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

Sophia L. Hinrichsen,Ka Y. Yuen,François-René Bertin,Allison J. Stewart,Elizabeth L. Dryburgh

doi:10.3390/ani12030324

Abstract

Simple SummaryApproximately 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.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.

Highlights

Pituitary pars intermedia dysfunction (PPID) is a common disease of older horses and ponies, affecting 21% of horses over 15 years of age [1,2]
The stability of equine ACTH has been investigated at room temperature (20 or 22 ◦C) and with archived samples (−20 or −80 ◦C), but as far as the investigators are aware, there have been no studies investigating the stability of ACTH when exposed to high environmental temperatures [15,16]
We found that individual horses with and without PPID had small random and unpredictable rises or declines in the measured immunoreactive ACTH concentration in response to short-term exposure to high temperatures (>20 ◦C)

Summary

Introduction

Pituitary pars intermedia dysfunction (PPID) is a common disease of older horses and ponies, affecting 21% of horses over 15 years of age [1,2]. In order to obtain accurate values of plasma ACTH concentrations in humans, it has been recommended that if samples cannot be transported to a laboratory for analysis within 2 h (at room temperature), they should be kept at 4 ◦C and analyzed within 8 h [13]. The stability of equine ACTH has been investigated at room temperature (20 or 22 ◦C) and with archived samples (−20 or −80 ◦C), but as far as the investigators are aware, there have been no studies investigating the stability of ACTH when exposed to high environmental temperatures [15,16]. Environmental temperatures might occasionally exceed 40 ◦C and temperatures inside vehicles might exceed 70 ◦C [17,18] It is uncertain if ACTH post-TRH stimulation has the same stability as basal ACTH. Previous research could not detect an effect of TRH stimulation on the stability of equine immunoreactive ACTH after up to five freeze/thaw cycles, suggesting that ACTH post-TRH stimulation would have a similar stability to basal ACTH; further investigation is warranted to determine if there is an effect of TRH stimulation on the stability of ACTH after exposure to high temperatures [18]

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