1 research outputs found
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
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