0624 A Mass Spectrometry Method Evaluating Orexin Metabolites Suggest Abnormalities in Hypersomnias of Central Origin

Abstract Introduction Narcolepsy type 1 (NT1) is caused by orexin (hypocretin) deficiency with 85-95% loss of orexinergic neurons reported in the hypothalamus. Patients with NT1 have cerebrospinal fluid (CSF) orexin-A immunoreactivity deficiency ≤110 pg/ml, as detected using a polyclonal anti-orexin-A radioimmunoassay (RIA). Although some narcolepsy type 2 patients (NT2) have been reported to have a partial loss of orexinergic neurons, the large majority of patients with NT2 have normal levels of CSF orexin-A. Previous work has shown that the RIA measures < 10% of the intact orexin-A peptide, suggesting that the RIA for CSF orexin-A primarily measures unauthentic orexin-A-related metabolites. In this study, we developed a novel mass spectrometry assay to measure intact prepro-orexin, orexin-A, and orexin-B, and their metabolites, in CSF collected from patients with NT1, NT2, idiopathic hypersomnia (IH), and controls. Methods Using a novel sequential immunoprecipitation/mass spectrometry method, all orexin species (intact-long and short-metabolite forms) were comprehensively measured in CSF collected from patients diagnosed with NT1 (N=15), NT2 (N=15), IH (N=15), and controls (N=15). Orexin peptides were analyzed by a nanoAcquity ultra-performance liquid chromatography system coupled to an Orbitrap Tribid Eclipse mass spectrometer. Results NT1 showed pan-orexin deficiency with lower predicted levels of prepro-orexin, orexin-A, and orexin-B species compared to NT2, IH, and controls (all p< 0.05 after Tukey correction for multiple comparisons). Short-form metabolites of orexin-B differentiated both NT2 and IH from NT1 and from controls (all p< 0.05 after Tukey correction for multiple comparisons). Further, the ratio of the shortest form of orexin-B/longer orexin-B species was decreased in NT2 compared to IH and controls (all p< 0.05 after Tukey correction for multiple comparisons). Conclusion A novel mass spectrometry assay comprehensively measuring different forms of CSF orexin differentiated among the hypersomnias of central origin and may have diagnostic utility. As expected, all forms of orexin were lower in CSF collected from NT1 patients compared to other groups. We also found that short metabolites of orexin-B are reduced in participants with NT2 and IH. Abnormal orexin transmission may also be involved in the pathophysiology of NT2 and IH, with differential impact on orexin peptide species. Support (if any) R21 AG074151 and Eisai