Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays

Lindström, Mikael; Schinkelshoek, Mink S.; Tienari, Pentti J.; Kukkonen, Jyrki P.; Renkonen, Risto; Fronczek, Rolf; Lammers, Gert Jan; Itkonen, Outi

doi:10.1016/j.clinbiochem.2021.01.009

Cited by 12 publications

(17 citation statements)

References 23 publications

(44 reference statements)

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“…Prospective inter-laboratory research adhering to the recommendations of the FDA and EMA (or other relevant) guidelines would be preferred. We are aware that recently more sophisticated mass spectrometry-based methods for the determination of hypocretin-1 were published, which is a very promising evolution [ 10 , 11 , 41 ]. At the moment, the field of quantitative clinical chemistry proteomics is developing fast [ 42 ], and the measurement of CSF hypocretin-1 by this technique can overcome some of the limitations of the RIAs.…”

Section: Discussionmentioning

confidence: 99%

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Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Hoeven

Waaij

Bijlenga

et al. 2022

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Study objectives The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cut-off of 110 pg/mL and the inter-and intra-assay variability. Methods Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established, defined as the lowest converted concentration with a CV<25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. Results The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n=41, range 1.6-17.0%), which was significantly higher than in RIAs with higher specific activity (n=36; median 3.2%, range 0.4-11.6%, p=.013). The CV around the 110 pg/mL cut-off was <7%. Conclusions Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cutt-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as “undetectable” when measured with the Phoenix Pharmaceuticals RIA.

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Section: Discussionmentioning

confidence: 99%

“…Still, the RIA remains the only test that is sufficiently validated to assess hypocretin deficiency to diagnose narcolepsy. The enzyme-linked immunosorbent assay (ELISA) cannot be used as a reliable alternative method to measure hypocretin-1 in CSF [ 41 , 43 ].…”

Section: Discussionmentioning

confidence: 99%

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Hoeven

Waaij

Bijlenga

et al. 2022

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“…However, a recent report indicated that conventional RIA for orexin-A largely measures unauthentic orexin-A-related metabolites [44]. Better methods, including recently reported liquid chromatography mass spectrometry assays [45][46][47], should be used for accurately measuring orexin-A levels.…”

Section: Discussionmentioning

confidence: 99%

Orexin 2 Receptor (OX2R) Protein Distribution Measured by Autoradiography Using Radiolabeled OX2R-Selective Antagonist EMPA in Rodent Brain and Peripheral Tissues

Mitsukawa

Kimura

2021

Preprint

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Orexin, a neuropeptide, performs various physiological functions, including the regulation of emotion, feeding, metabolism, respiration, and sleep/wakefulness, by activating the orexin 1 receptor and orexin 2 receptor (OX2R). Owing to the pivotal role of OX2R in wakefulness and other biological functions, OX2R agonists are being developed. A detailed understanding of OX2R protein distribution is essential for determining the mechanisms of action of OX2R agonists; however, this has been hindered by the lack of selective antibodies. In this study, we first confirmed the OX2R-selective binding of [3H]-EMPA in in vitro autoradiography studies, using brain slices from OX2R knockout mice and their wild-type littermates. Subsequently, OX2R protein distribution in rats was comprehensively assessed in 51 brain regions and 10 peripheral tissues using in vitro autoradiography with [3H]-EMPA. The widespread distribution of OX2R protein, including that in previously unrecognized regions of the retrosplenial cortex and suprachiasmatic nucleus of the hypothalamus, was identified. In contrast, negligible/very low OX2R protein expression was observed in peripheral tissues, suggesting that orexin exerts OX2R-dependent physiological functions primarily through activation of the central nervous system. These data would be useful for understanding the wide range of biological functions of OX2R and the application of OX2R agonists in various disorders.

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“…Furthermore, no LC−MS/MS methods have detected OXB in the CSF, although OXA and OXB are produced in the same amount by the cleavage of preproorexin. 17 Thus, little is known about the distribution and physiological functions of OXB. 1 A combination of nanoflow LC and high-resolution mass spectrometry (HRMS) is a promising approach to address this problem because of their higher specificity, sensitivity, and separation efficiency compared to conventional LC−MS/MS.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Theoretically, LC–MS/MS could distinguish OXA from its metabolites like truncated OXA unless the metabolites give remarkably similar m / z to OXA; for example, deamidation at the C-terminal could show quite similar m / z to that of OXA (Δ m / z = −0.984). Thus far, three LC–MS/MS methods have been reported for measuring OXA in human CSF. − However, the median OXA concentrations from those studies differed from each other by factors as high as 10 for healthy human subjects. It is well known that some peptides adsorb to the surface of materials, such as syringes, needles, and sample tubes .…”

Section: Introductionmentioning

confidence: 99%

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

Narita

Yamada

Kakehi

et al. 2023

ACS Chem. Neurosci.

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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.

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Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays

Cited by 12 publications

References 23 publications

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Orexin 2 Receptor (OX2R) Protein Distribution Measured by Autoradiography Using Radiolabeled OX2R-Selective Antagonist EMPA in Rodent Brain and Peripheral Tissues

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

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Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays

Cited by 12 publications

References 23 publications

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Orexin 2 Receptor (OX2R) Protein Distribution Measured by Autoradiography Using Radiolabeled OX2R-Selective Antagonist EMPA in Rodent Brain and Peripheral Tissues

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

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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