Abstract/Summary

Plasma adrenocorticotrophin (ACTH) is highly labile and far from an ideal analyte for use in the diagnosis of ACTH-dependent endocrine conditions. ACTH is derived, along with several other biologically active peptides, from a larger precursor molecule known as pro-opiomelanocortin (POMC). As the POMC peptides are simultaneously released with ACTH, each concomitant peptide could be used as a marker of ACTH levels. Here we explore the use of the co-secreted POMC-derived peptides, Pro-ƴ-MSH and POMC joining peptide (JP), as more robust biomarkers of ACTH-dependent conditions. In this study, two conditions were investigated; Cushing’s syndrome and Exertional Heat Illness. For detection of Pro-ƴ-MSH, a two-site immunoassay was constructed using antibodies against N-POMC (1-28) and ƴ1-MSH, respectively. For detection of the POMC JP, antiserum was raised in sheep against the full-length peptide, and N- and C-terminal-specific antibodies were affinity purified and subsequently used to develop a two-site immunoassay. Both ELISAs were optimised for the direct measurement of endogenous Pro-ƴ-MSH and POMC JP in unextracted human plasma. The sensitivity of the Pro-ƴ-MSH assay was 1.96 ± 1.6 ng/L (n=29) and the JP assay was 2.36 ± 1.1 μg/L (n=29). The main hypothesis of this research proposed that the POMC peptides, JP and Pro-γ-MSH, are raised alongside ACTH in the ACTH-dependent conditions; Cushing’s syndrome and Exertional Heat Illness. Neither POMC peptide was able to distinguish between normal subjects and patients with Cushing’s syndrome. Nevertheless, interesting findings emerged, particularly the elevated levels and broad distribution of JP found in control subjects and ACTH-independent Cushing’s patients. In contrast, Pro-γ-MSH levels of >3.054 ng/L were revealed as an excellent indicator of Exertional Heat Illness (EHI) in marathon runners by ROC curve analysis (AUC = 0.8830, **p = 0.0006), while JP levels remained unchanged. The identification of Pro-γ-MSH as a novel biomarker of EHI could contribute to improvements in the early diagnosis, prevention and management of EHI outcomes. To conclude, two-site ELISAs were successfully developed in this project for detection of Pro-ƴ-MSH and POMC JP in unextracted plasma, which offer potential use as reliable assays for ACTH-related clinical purposes.