Serum Metabolomic Profiling in Possible Early-Stage Multiple Sclerosis: A Targeted 1H-NMR Comparison of OCB Type 1 and Type 2 Patterns

Abstract

Oligoclonal band (OCB) analysis in cerebrospinal fluid (CSF) remains a cornerstone for the early diagnosis of multiple sclerosis (MS), with recent criteria highlighting the κ-free light chain (κ-FLC) index as a sensitive marker of intrathecal immunoglobulin synthesis. However, both approaches rely on lumbar puncture. To address the need for less invasive tools, this study employed proton nuclear magnetic resonance (¹H-NMR)-based serum metabolomics to explore whether peripheral metabolic signatures can distinguish immunophenotypic subgroups, namely OCB Type 1 and Type 2, in patients with suspected early-stage MS. To investigate whether targeted proton nuclear magnetic resonance (¹H-NMR) serum metabolomics can differentiate between individuals with OCB Type 1 (no intrathecal synthesis) and OCB Type 2 (definite intrathecal synthesis), as a step toward non-invasive metabolic stratification in possible early-stage MS. Serum samples (n = 49) were classified by OCB profile and analysed using targeted ¹H-NMR spectroscopy. All spectra were acquired at 298 K on a Bruker Avance Neo 600 MHz spectrometer. Metabolites were identified using Bruker BioRefCode libraries, and absolute quantification was performed using the ERETIC (Electronic Reference To access In vivo Concentrations) signal. Statistical analysis included age-adjusted univariate tests (t-test or Wilcoxon rank-sum, depending on normality) with false discovery rate (FDR) correction, followed by multivariate analyses including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), variable importance in projection (VIP) scores, and ROC-AUC permutations using the pROC package in R. Of the quantified metabolites, Leucine emerged as a robust differentiator, showing significant elevation in OCB Type 2 after both age adjustment and multiple testing correction (p = 0.025, FDR-adjusted; VIP = 2.38; AUC = 0.74), and correlating moderately with the IgG index (r = 0.32, p = 0.026). Histidine, Glycine, Sarcosine, and Succinic acid met VIP > 1.5 thresholds but did not survive FDR correction and are therefore reported as exploratory candidates. This pilot study identifies Leucine as a promising serum biomarker candidate for early intrathecal immune activity. Although additional metabolites showed potential, their findings remain exploratory due to statistical limitations. These results highlight the potential of targeted serum metabolomics as a non-invasive adjunct in early MS risk stratification and support further validation studies incorporating κ-FLC data and longitudinal follow-up.

Keywords: 1H-NMR spectroscopy; Age-adjusted analysis; Biomarker discovery; Early diagnosis; Kappa index; Multiple sclerosis; Oligoclonal bands; Serum metabolomics.