The effects of sample handling and N-phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma.

REASONS FOR PERFORMING STUDY: Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. OBJECTIVES: This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. STUDY DESIGN: Experimental study. METHODS: Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemiluminescent assay. Plasma samples were then: 1) centrifuged immediately, 2) centrifuged immediately with the addition of maleimide, or 3) allowed to separate by gravity followed by the addition of maleimide, before all samples were stored at 22°C and analysed at 4, 8, 24 and 48 h post collection. A linear mixed effects model and Bland-Altman analyses were performed. Significance was set at P < 0.05. RESULTS: No significant effect of plasma treatment (P = 0.1) on change in ACTH concentration was identified. However, significant effects of horse health status (P < 0.001) and time (P < 0.001) on change in ACTH concentration were identified. No significant interactions were found. Significant decreases in ACTH concentration occurred in horses with PPID between 4 and 8 h after blood collection. In non-PPID horses, the decrease in ACTH concentration over time was not significant. Agreement with baseline values decreased over time and was greater for non-PPID horses than for PPID horses. CONCLUSIONS: Clinically useful results are still obtained if ACTH concentration is measured up to 48 h after sample collection. Allowing samples to separate by gravity rather than centrifugation did not have a significant effect on ACTH concentration, and the addition of maleimide was of no benefit.

Investigation of rhythms of secretion and repeatability of plasma adrenocorticotropic hormone concentrations in healthy horses and horses with pituitary pars intermedia dysfunction.

REASONS FOR PERFORMING STUDY: There is little published information on whether measurement of plasma ACTH concentration at a single timepoint is a repeatable indicator of pituitary pars intermedia dysfunction (PPID). OBJECTIVES: To determine whether ultradian or circadian fluctuations in ACTH production influence plasma ACTH concentration in normal horses and horses with PPID. STUDY DESIGN: Prospective observational study. METHODS: Plasma ACTH concentration in 8 non-PPID horses and 8 horses with PPID was measured at 08.00, 11.00, 14.00 and 17.00 h on 5 nonconsecutive days within a 3 week period. In addition, at 08.30 h on one day, 6 samples were collected from each horse at precisely 5 min intervals over a period of 25 min. Descriptive and graphical analysis was performed and a linear mixed effects model was fitted to assess the effect of time of day on ACTH concentration in non-PPID and PPID horses. RESULTS: Evidence of ultradian fluctuation in ACTH production was not identified in either non-PPID or PPID horses. Evidence for circadian fluctuation was identified in non-PPID horses; plasma ACTH concentrations were highest at 08.00 h and decreased through the day. There was no evidence of circadian fluctuation in PPID horses. In non-PPID horses, the magnitude of circadian changes in ACTH concentration was smaller than variations in concentration that occurred at random. Intrahorse variability of ACTH concentration was greater in PPID horses than in non-PPID horses. CONCLUSIONS: Ultradian and circadian fluctuations in ACTH concentration are unlikely to influence clinical decision making; however, variations of potential clinical relevance do occur in individual horses, for reasons that remain to be determined, and increase in magnitude with progression of PPID. Results of the current study indicate that when an ACTH concentration between 19 and 40 pg/ml is measured, further testing should be considered to increase the accuracy of PPID diagnosis.