Diurnal Fluctuations of Orexin-A and -B in Cynomolgus Monkey Cerebrospinal Fluid Determined by a Novel Analytical Method Using Antiadsorptive Additive Treatment Followed by Nanoflow Liquid Chromatography–High-Resolution Mass Spectrometry

Abstract

Orexin-A (OXA) and -B (OXB) are involved in the regulation of multiple physiological functions including the sleep–wake states; therefore, it is critical to monitor their levels under various conditions. Unfortunately, the widely used radioimmunoassay has insufficient specificity for OXA. Although liquid chromatography–tandem mass spectrometry (LC–MS/MS) has higher specificity for OXA, previously reported OXA levels in human cerebrospinal fluid (CSF) measured using this technique are still inconsistent. Moreover, to the best of our knowledge, OXB has not been detected in the CSF. In this study, we established a novel method for OXA and OXB measurement. We noticed that OXA and OXB in the CSF was sticky; thus, citric acid and Tween 80 were used to prevent their nonspecific binding. Then, highly specific and sensitive nanoflow liquid chromatography–high-resolution mass spectrometry (nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation of the diurnal fluctuations of OXA and OXB in cisternal and lumbar CSF samples from cynomolgus monkeys revealed a sharp increase in the early light period, followed by a gradual increase to the maximum levels at the end of the light period, and then a sharp drop to the minimum levels during the early dark period. OXB levels were lower than OXA levels in cisternal CSF. Although basal OXA levels in individual monkeys showed substantial variations, the ratios between the maximum and minimum OXA levels of each monkey were similar. Our method for accurate OXA and OXB measurement should help improve our knowledge of orexin biology.

Effects of an OX2R agonist on migration and removal of tau from mouse brain

“…OX-A and orexin B are neuropeptides released from lateral hypothalamic orexin neurons that regulate multiple physiological functions including wakefulness by neuronal excitation 19 , 27 . As conventional radioimmunoassay methods to measure OX-A have poor selectivity for OX-A, we established a novel, accurate method using nanoflow liquid chromatography–high-resolution mass spectrometry (nanoLC-HRMS) 28 . Evaluation of diurnal fluctuation of OX-A in monkey cisternal CSF using this method revealed that OX-A levels gradually increased during the active period, reached a maximum at the end of the active period, then dropped to the trough level of the sleep period within 2 h 28 .…”

“…As conventional radioimmunoassay methods to measure OX-A have poor selectivity for OX-A, we established a novel, accurate method using nanoflow liquid chromatography–high-resolution mass spectrometry (nanoLC-HRMS) 28 . Evaluation of diurnal fluctuation of OX-A in monkey cisternal CSF using this method revealed that OX-A levels gradually increased during the active period, reached a maximum at the end of the active period, then dropped to the trough level of the sleep period within 2 h 28 . Although the sleep/wake structure of monkeys and rodents is different (monkeys have a monophasic structure while rodents have a polyphasic structure), the diurnal fluctuation pattern of OX-A may be similar to that of Aβ 9 .…”

International Union of Basic and Clinical Pharmacology CXIV: Orexin Receptor Function, Nomenclature and Pharmacology

“…The original orexin measurements were performed using antibodies and are thus somewhat uncertain, given that these antibodies may possibly also detect the parent peptide PPO, orexin-A fragments or altogether different molecules (VI. ), but measurements with mass spectrometry-based methods or biosensors, which are not subject to these liabilities, have also recently confirmed the diurnal rhythmicity of orexin release (Hopkins et al, 2021;Duffet et al, 2022a;Narita et al, 2023).…”

“…Non-radioactive heavy labels ( 13 C, 15 N) have been used in custom-made internal orexin-A and -B standards for mass spectrometry-based detection assays (VI.) (Hirtz et al, 2016;Bårdsen et al, 2019;Lindström et al, 2021;Hopkins et al, 2021;Ball et al, 2023;Narita et al, 2023) and in NMR (III.A., V.A.). A caged version of orexin-Bthat can be uncaged upon a UV or near-UV light pulsehas recently been developed (Duffet et al, 2022b).…”

Rethinking the Role of Orexin in the Regulation of REM Sleep and Appetite

“…However, during REM sleep, the activity of orexin neurons decreases, resulting in a reduction of orexin release. Interestingly, recent studies in primates have clearly shown that the highest concentrations of orexin are recorded until just after the monkeys go to sleep and then falling through the night and reaching the lowest concentrations around wake time [11,12]. Then, orexin concentrations start rise linearly.…”