From radioimmunoassay to mass spectrometry: a new method to quantify orexin-A (hypocretin-1) in cerebrospinal fluid

Abstract

I(125) radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland-Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant.

Figure 1

Panel (A) MS/MS spectra of the [M+5H]⁵⁺ precursor of light orexin-A standard. Panel (B) Light orexin-A standard chromatograms obtained by micro-HPLC-MS in the “full-scan mode” showing 4+ and 5+ charged molecular ions. Panel (C) Orexin-A sequence with modifications (N-ter Pyroglutamination, C-ter amidation, disulfite bridges).

Figure 2

Panel (A) Workflow used for the micro-LC-MRM quantification of orexin-A in human CSF samples. Panel (B) Calibration curves of orexin-A in the 0–200 pg/mL concentration range (0, 4, 7.5, 10, 14, 19, 25, 50, 100, 200 pg/mL). The equation was linear and r² was equal to 0.9885, based on the following equation: y = 0.0117x + 0.0001.

Figure 3. CSF samples from patients with narcolepsy (NT1, n = 22) and various neurological disorders (n = 22) were analyzed using RIA and MRM.

Panel (A) values plotted show the existence of a significant correlation (Spearman’s rank correlation coefficient (rho) 0.898, significance level P < 0.0001) between the two methods. A clear-cut difference between the absolute values was observed, however, the RIA values were 2.5 times higher than the MRM values. Graph of the RIA Panel (B) and MRM values Panel (C) obtained in the two clinical groups presented as medians and interquartile ranges. Statistical pairwise comparisons performed with the non-parametric Mann-Whitney test confirmed the difference observed between the two groups of patients (P < 0.000001). Bland–Altman analysis with Deming regression Panel (D) confirmed that the two methods were interchangeable since the values of the differences between them were in the +/−1.96 SD range.