title: The effects of sample handling and N-phenylmaleimide on concentration of adrenocorticotrophic hormone in equine plasma.
abstract: BACKGROUND
  Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
authors: D I
authors: DI
authors: Rendle
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
authors: E
authors: E
authors: Litchfield
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
authors: S
authors: S
authors: Gough
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
authors: A
authors: A
authors: Cowling
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
authors: K J
authors: KJ
authors: Hughes
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000097:blood*; D000818:Animals; D001800:Blood Specimen Collection / Q000662:veterinary*; D016022:Case-Control Studies; D006734:Horse Diseases / Q000097:blood* / Q000175:diagnosis; D006736:Horses; D008301:Maleimides / Q000737:chemistry*; D010900:Pituitary Diseases / Q000097:blood / Q000662:veterinary*; D052716:Pituitary Gland, Intermediate / Q000378:metabolism
chemical_list: D008301:Maleimides; D000324:Adrenocorticotropic Hormone; C028525:N-phenylmaleimide
keywords: adrenocorticotrophic hormone; clinical pathology; endocrinology; horse; pituitary pars intermedia dysfunction