Development of an LC-MS/MS Method to Measure Sphingolipids in CSF from Patients with Multiple Sclerosis

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary-progressive-multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis(pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed LC-MS/MS method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n=10), PPMS (n=27), and RMS (n=17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared to the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week-52 after-treatment with ocrelizumab in RMS patients, but not in PPMS patients. Specifically, C16 Glc Cer (-26%;P=0.004) and C18 Cer (-13%;P=0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 Glc Cer levels correlated with CSF neurofilament heavy levels at baseline (Rho:0.532;P=0.004) and after treatment (Rho:0.424;P=0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and that treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. Significance Statement This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF of pwMS when compared to healthy controls. Distinct sphingolipid signatures between patients with different clinical disease courses were observed and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other CNS disorders.