HUS Diagnostic Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands; Sleep-Wake Centre, Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede, The Netherlands.
Clin Biochem. 2021 Apr;90:34-39. doi: 10.1016/j.clinbiochem.2021.01.009. Epub 2021 Feb 1.
Orexin-A and -B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B.
We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months.
Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range < 35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable.
Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.
食欲素-A 和 -B 是参与睡眠-觉醒调节的神经肽。在人类 1 型发作性睡病中，由于下丘脑产生食欲素的神经元丧失，这种循环被打乱。脑脊液（CSF）中食欲素-A 的测量用于 1 型发作性睡病的诊断。目前可用的免疫测定法可能缺乏对准确食欲素定量的特异性。我们开发并验证了一种用于 CSF 食欲素-A 和 -B 的液相色谱-质谱联用分析（LC-MS/MS）。
我们使用了来自 1 型发作性睡病（n=22）和 2 型发作性睡病（n=6）患者以及非发作性睡病对照者（n=44）的 CSF 样本。在固相萃取和 LC-MS/MS 定量之前，向样本中加入稳定同位素标记的食欲素-A 和 -B 内标。还通过商业放射免疫测定（RIA，n=42）和酶免疫测定（EIA，n=72）试剂盒对样本进行了检测。研究了食欲素在 CSF 中的稳定性长达 12 个月。
我们的测定法具有良好的灵敏度（10pmol/L=35pg/mL）和宽线性范围（35-3500pg/mL）。添加的食欲素-A 和 -B 在冷冻时分别在 CSF 中稳定 12 个月和 3 个月。1 型发作性睡病患者 CSF 中的中位食欲素-A 浓度<35pg/mL（范围<35-131pg/mL），低于对照者的 CSF（98pg/mL，范围<35-424pg/mL）。使用我们的 LC-MS/MS 测定法测定的食欲素-A 浓度比使用商业 RIA 测定的浓度低 5 倍。食欲素-B 浓度无法检测到。
与对照组相比，我们的 LC-MS/MS 测定法测定的 1 型发作性睡病患者的食欲素-A 浓度较低。RIA 的平均浓度高于 LC-MS/MS。
