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

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.

Prospective Case Series of Clinical Signs and Adrenocorticotrophin (ACTH) Concentrations in Seven Horses Transitioning to Pituitary Pars Intermedia Dysfunction (PPID)

“…Two blood samples immediately before and 30 minutes after intravenous administration of 1 mg of TRH ((Sigma-Aldrich Pty Ltd. [subsidiary of Merck], North Ryde BC, Sydney, Australia) [ 2 , 26 , 37 , 38 , 39 , 40 ] were collected for subsequent measurement of basal and TRH-stimulated ACTH concentrations. Although a 10 min post-TRH sampling time is often considered more convenient for equine practitioners, the 30 min post-TRH sampling time was selected as this sampling time is considered to be less impacted by delays of up to 30 s [ 41 ].…”

“…Blood samples were obtained from a jugular vein and collected in plastic EDTA tubes (BD, Belliver Industrial Estate, Plymouth, UK), kept on ice, centrifuged and plasma separated within 2 h of sample collection. Analysis of plasma ACTH concentrations were measured within 4 hours using a chemiluminescent immunoassay immunoassay (Immulite 1000 Chemiluminescent Assay, Siemens, Bayswater, Victoria, Australia) with an interassay coefficient of variation of 4.8% and an intraassay coefficient of variation of 5.4% as previously described [ 38 , 40 ].…”

Pituitary Pars Intermedia Dysfunction (PPID) in Horses

“…The blood sample should be collected in an EDTA tube, centrifuged, kept at 4 °C and analysed as soon as possible after sampling, ideally within 24 h [ 126 , 127 ]. If the sample cannot be tested immediately, the centrifuged plasma should be frozen as heat results in degradation of ACTH [ 128 ].…”

“…Further research is required to determine the variability of ACTH concentrations 30 min post TRH administration [ 134 ]. Prior to the availability of a compounded product in the USA, most research papers utilise the chemical grade TRH (Sigma-Aldrich Pty Ltd. (subsidiary of Merck), North Ryde BC, Australia or synthetic thyrotropin releasing hormone, Sigma-Aldrich Inc, St. Louis, MO, USA) [ 2 , 121 , 124 , 127 , 129 , 132 ]. Although TRH is now available as a compounded product in the USA, in Europe and Australia TRH is not available as a sterile registered product, and use tends to be limited to referral hospitals [ 132 , 133 , 134 ].…”