Hydroxycholesterol Levels in the Serum and Cerebrospinal Fluid of Patients with Neuromyelitis Optica Revealed by LC-Ag+CIS/MS/MS and LC-ESI/MS/MS with Picolinic Derivatization: Increased Levels and Association with Disability during Acute Attack

Abstract

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS). Hydroxycholesterols (OHCs), metabolites of CNS cholesterol, are involved in diverse cellular responses to inflammation and demyelination, and may also be involved in the pathogenesis of NMO. We aimed to develop a sensitive and reliable method for the quantitative analysis of three major OHCs (24S-, 25-, and 27-OHCs), and to evaluate their concentration in the cerebrospinal fluid (CSF) and serum of patients with NMO. The levels of the three OHCs in the serum and CSF were measured using liquid chromatography-silver ion coordination ionspray tandem mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry with picolinyl ester derivatization, respectively. The linear range was 5-250 ng/mL for 24S- and 27-OHC, and 0.5-25 ng/mL for 25-OHC in serum, and was 0.1-5 ng/mL for 24S- and 27-OHC, and 0.03-1 ng/mL for 25-OHC in CSF. Precision and accuracy were 0.5%-14.7% and 92.5%-109.7%, respectively, in serum, and were 0.8%-7.7% and 94.5%-119.2%, respectively, in CSF. Extraction recovery was 82.7%-90.7% in serum and 68.4%-105.0% in CSF. When analyzed in 26 NMO patients and 23 control patients, the 25-OHC (0.54 ± 0.96 ng/mL vs. 0.09 ± 0.04 ng/mL, p = 0.032) and 27-OHC (2.68 ± 3.18 ng/mL vs. 0.68 ± 0.25 ng/mL, p = 0.005) were increased in the CSF from NMO patients. When we measured the OHCCSF index that controls the effects of blood-brain barrier disruption on the level of OHC in the CSF, the 27-OHCCSF index was associated with disability (0.723; 95% confidence interval (CI)- 0.181, 0.620; p = 0.002), while the 24-OHCCSF index (0.518; 95% CI- 1.070, 38.121; p = 0.040) and 25-OHCCSF index (0.677; 95% CI- 4.313, 18.532; p = 0.004) were associated with the number of white blood cells in the CSF of NMO patients. Our results imply that OHCs in the CNS could play a role in the pathogenesis of NMO.

Fig 1. Obtained product ion spectra and representative chromatograms of 24S-, 25- and 27-OHC.

For OHC_serum, spectra (A) and chromatograms (B) by silver coordination. For OHC_CSF, spectra (C) and chromatograms (D) by picolinyl ester derivatization.

Fig 2. Levels of 24S-, 25-, and 27-OHC in the CSF and serum of patients.

Among the levels of OHC_CSF (A-C) and OHC_serum (D-F), the levels 25- and 27-OHC_CSF were increased in patients with NMO compared with controls (B and C). The levels of 24S-OHC_CSF (A) and OHC_Serum levels did not differ between groups (D–F). The ratio of 27-OHC_CSF over 24S-OHC_CSF, which could be associated with either the disruption of the BBB or increased synthesis of 27-OHC in the CNS, was also increased in the NMO group (G). *p < 0.001; n.s. = not significant.

Fig 3. Association of the level of OHCs with disability at acute attack in NMO patients.

Of the levels of OHC_CSF (A–C) or OHC_Serum (D–F) of patients with NMO, only the levels of 27-OHC_CSF were significantly associated with their disability at acute attacks (C). n.s. = not significant.

Fig 4. Association of the CNS-derived OHCs with disability and inflammation at acute attack of NMO.

The OHC_CSF index was calculated to control the effects of the disruption in the BBB on the levels of these OHCs in the CSF. The associations of the OHC_CSF index with the disability (A–C) and number of inflammatory cells in the CNS (D–E) were assessed. The 27-OHC_CSF index was significantly associated with disability at acute attacks of NMO (C), moreover the 24-OHC_CSF index (D) and 25-OHC_CSF index (E) were associated with the number of the inflammatory cells in the CNS. EDSS = extended disability scale score; n.s. = not significant; OHC = hydroxycholesterol,; WBC_CSF = number of white blood cells in the CSF.