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

DI Rendle, E Litchfield, S Gough, A Cowling and KJ Hughes

Adrenocorticotrophic hormone (ACTH) is frequently measured as a diagnostic aid in the detection of pituitary pars intermedia dysfunction (PPID). Delays in analysis of blood samples are frequent with this test as external laboratories are often involved in analysis; the effect of delays of up to 48 hours has not previously been measured. This study measured the effect of time, centrifugation vs. gravity separation and the addition of maleimide (a protease inhibitor) on ACTH concentration. Eight healthy horses and eight horses with PPID were recruited, baseline ACTH was measured immediately after sampling using a chemiluminescent assay. Samples taken from each horse were divided and subjected to the following procedures: centrifugation, centrifugation and addition of maleimide, or gravity separated and addition of maleimide. All samples were stored at 22°C. Each sample was then analysed at 4, 8, 24 and 48 hours post collection. There was no effect on the addition of maleimide, there was a significant reduction in ACTH concentration in horses with PPID between 4 and 8 hours post collection and a wider range of ACTH values were identified with increasing time in horses with PPID. The differences in ACTH concentration over time were not of the magnitude to be of clinical relevance and may be similar to the biological variation observed over a similar time period. There was no significant difference in ACTH concentrations in samples separated by gravity compared to centrifugation.

The bottom line: Although there is some variation in ACTH concentration over time this has not been shown to be clinically relevant and may only be of significance in horses which had borderline ACTH concentrations at the time of sampling. Method of separation and the addition of protease inhibitors had no effect on ACTH concentration.

Photo: Pituitary Pars Intermedia Dysfunction (PPID)


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