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.

Metabolomic Profiling in Neuromyelitis Optica Spectrum Disorder Biomarker Discovery

“…Although not explicitly self-identified as metabolomic investigations, at least two additional studies have incorporated targeted metabolomic platforms to evaluate NMOSD [ 88 , 89 ]. In the first of these two studies, Tortorella et al 2011 used high performance liquid chromatography-mass spectrometry/more selective (HPLC-MS/MS) to quantify NAA from CSF and sera from 32 NMO patients (13 AQP4-Ab + and 9 AQP4-Ab − ), and compared these results with 48 RRMS and 76 healthy cohorts [ 88 ].…”

“…In the first of these two studies, Tortorella et al 2011 used high performance liquid chromatography-mass spectrometry/more selective (HPLC-MS/MS) to quantify NAA from CSF and sera from 32 NMO patients (13 AQP4-Ab + and 9 AQP4-Ab − ), and compared these results with 48 RRMS and 76 healthy cohorts [ 88 ]. More recently, Cha et al 2016 employed liquid chromatography-electrospray ionization tandem mass spectrometry with picolinyl ester derivatization (LC-ESI/MS/MS + PE) to quantify CSF 24S-, 25-, and 27-hydroxycholesterols (OHCs) in 26 AQP4-Ab + NMO patients and 23 control patients with other non-inflammatory, non-degenerative neurological disorders (ONNDs) [ 89 ]. This publication further reported elevated serum OHC levels in NMOSD when compared to ONNDs, as detected by liquid chromatography-silver ion coordination ionspray tandem mass spectrometry (LC-Ag + CIS/MS/MS) [ 89 ].…”

“…More recently, Cha et al 2016 employed liquid chromatography-electrospray ionization tandem mass spectrometry with picolinyl ester derivatization (LC-ESI/MS/MS + PE) to quantify CSF 24S-, 25-, and 27-hydroxycholesterols (OHCs) in 26 AQP4-Ab + NMO patients and 23 control patients with other non-inflammatory, non-degenerative neurological disorders (ONNDs) [ 89 ]. This publication further reported elevated serum OHC levels in NMOSD when compared to ONNDs, as detected by liquid chromatography-silver ion coordination ionspray tandem mass spectrometry (LC-Ag + CIS/MS/MS) [ 89 ]. Neither of these studies included healthy controls.…”

“…Collectively, these metabolomic investigaations identify significant perturbations of 36 metabolites in plasma, serum, blood, CSF, and urine biopsies collected from NMOSD patients as compared to RRMS and healthy controls ( Table 1 ). Of these 36 metabolites, acetate, lysine, formate/formic acid, glucose, lactate/lactic acid, N -actyl aspartate (NAA), and scyllo-inositol discriminated NMOSD in two or more biopsy sources from two or more independent studies [ 74 , 82 , 85 , 86 , 87 , 88 , 89 ]. Of these eight metabolites, only one inter-study discordance was noted, and this was increased serum lysine [ 85 ] compared to decreased plasma lysine [ 82 ], in NMOSD compared to RRMS.…”

Chromatography of oxysterols

“…Regarding chromatographic performance, there are small differences in the behavior of the derivatized or native oxysterols. Cha et al [48] has analyzed both native oxysterols from serum samples as silver adducts and picolinyl ester derivatized (PED) oxysterols from CFS.…”