Plasma Orexin-A Levels Do Not Undergo Circadian Rhythm in Young Healthy Male Subjects

Abstract

Orexin-A (OXA) has been originally isolated from a precursor peptide prepro-orexin from the lateral hypothalamus. The orexin system has been attributed to important functions in sleep, arousal and regulation of energy homeostasis. In addition to its high levels in cerebrospinal fluid, OXA is present in blood. However, reported peptide concentrations in plasma vary significantly depending on the method used. Therefore, a specific and sensitive OXA radioimmunoassay (RIA) with solid phase extraction method was developed to determine whether plasma OXA concentrations is affected by acute feeding and/or wake and sleep in young healthy males. Blood samples were collected for 24 h from nine healthy males (aged 20–24 years; BMI 20.7–26.5) every 2 h starting at 11 a.m. Food was served at 12 p.m, 5:30 p.m, 8 p.m and 8 a.m and the sleep time was between 10 p.m and 7 a.m. Plasma samples were analyzed in addition for cortisol and melatonin levels. Blood pressure was monitored through the experimental period. OXA antibody was raised in rabbits. OXA antiserum had only minor cross-reactivity with prepro-orexin precursor (<0.001%), amino-terminal peptide (<0.001%), carboxy-terminal peptide (0.001%), and orexin-B (0.3%) with high sensitivity (0.15 pg/tube). Plasma OXA levels varied between 0.5 and 16 pg/ml in seven subjects and were undetectable (below 0.5 pg/ml) in two subjects. The OXA concentrations did not correlate to feeding nor wake/sleep, whereas cortisol, melatonin and mean arterial blood pressure presented a clear circadian rhythm in each subject. In conclusion, OXA is present in blood in low amounts and its levels do not follow autonomic nor neuroendocrine circadian rhythms. Thereby, studies examining regulatory mechanisms and influences of OXA from blood samples should interpret results very cautiously.

A critical review of melatonin assays: Past and present

“…A recent publication from the group using this assay reported daytime levels in the range of 2 pg/mL. 44 The antibody has not been widely distributed or commercialised to my knowledge.…”

“…With chloroform extraction from a neutral buffer, the authors measured daytime melatonin levels in the order of 4.6 pg/mL. A recent publication from the group using this assay reported daytime levels in the range of 2 pg/mL . The antibody has not been widely distributed or commercialised to my knowledge.…”

Case Report: Reduced CSF Orexin Levels in a Patient With Sepsis

“…In healthy humans, orexin can be detected (range 250-500 pg/ml) in cerebrospinal fluid (CSF), but it is generally not detected (<40 pg/ml) in blood by a standard orexin radioimmunoassay, which is used for the diagnosis of narcolepsy, because it cannot cross the BBB. By using a novel specific and sensitive orexin radioimmunoassay, blood orexin can be detected at very low levels (range 0.5-16 pg/ml), but it does not correlate with the wake/sleep cycle (Makela et al, 2018). Surprisingly, we previously reported that orexin improved survival in mice with endotoxin shock, a well-studied animal model of sepsis, by acting in the brain (Ogawa et al, 2016), indicating that orexin may be involved in the pathophysiology of SAE and be a new agent in targeting the CNS for the treatment of sepsis.…”

(Neuro) Peptides, Physical Activity, and Cognition

“…Messina and colleagues [ 121 ] reported that PA using a calibrated mechanically braked cycle ergometer (at 75 W for 15 min) increased orexin-A levels in plasma of humans from 2610 ± 187 pg/mL to a maximum level (3589 ± 197 pg/mL) within 30 min after exercise. While relative changes in orexin blood levels may be due to experimental differences, absolute levels are still unclear as commercial kits and extraction protocols differ significantly compared to an in-house generated antibody with plasma levels in the range of 1–16 pg/mL [ 122 ].…”