Potential Metabolite Biomarkers of Multiple Sclerosis from Multiple Biofluids

Multiple sclerosis (MS) is a chronic and progressive neurological disorder without a cure, but early intervention can slow disease progression and improve the quality of life for MS patients. Obtaining an accurate diagnosis for MS is an arduous and error-prone task that requires a combination of a detailed medical history, a comprehensive neurological exam, clinical tests such as magnetic resonance imaging, and the exclusion of other possible diseases. A simple and definitive biofluid test for MS does not exist, but is highly desirable. To address this need, we employed NMR-based metabolomics to identify potentially unique metabolite biomarkers of MS from a cohort of age and sex-matched samples of cerebrospinal fluid (CSF), serum, and urine from 206 progressive MS (PMS) patients, 46 relapsing-remitting MS (RRMS) patients, and 99 healthy volunteers without a MS diagnosis. We identified 32 metabolites in CSF that varied between the control and PMS patients. Utilizing patient-matched serum samples, we were able to further identify 31 serum metabolites that may serve as biomarkers for PMS patients. Lastly, we identified 14 urine metabolites associated with PMS. All potential biomarkers are associated with metabolic processes linked to the pathology of MS, such as demyelination and neuronal damage. Four metabolites with identical profiles across all three biofluids were discovered, which demonstrate their potential value as cross-biofluid markers of PMS. We further present a case for using metabolic profiles from PMS patients to delineate biomarkers of RRMS. Specifically, three metabolites exhibited a variation from healthy volunteers without MS through RRMS and PMS patients. The consistency of metabolite changes across multiple biofluids, combined with the reliability of a receiver operating characteristic classification, may provide a rapid diagnostic test for MS

Expanding the Coverage of the Metabolome with Nitrogen-Based NMR

Isotopically labeling a metabolite and tracing its metabolic fate has provided invaluable insights about the role of metabolism in human diseases in addition to a variety of other issues. ¹³C-labeled metabolite tracers or unlabeled ¹H-based NMR experiments are currently the most common application of NMR to metabolomics studies. Unfortunately, the coverage of the metabolome has been consequently limited to the most abundant carbon-containing metabolites. To expand the coverage of the metabolome and enhance the impact of metabolomics studies, we present a protocol for ¹⁵N-labeled metabolite tracer experiments that may also be combined with routine ¹³C tracer experiments to simultaneously detect both ¹⁵N- and ¹³C-labeled metabolites in metabolic samples. A database consisting of 2D ¹H–¹⁵N HSQC natural-abundance spectra of 50 nitrogen-containing metabolites are also presented to facilitate the assignment of ¹⁵N-labeled metabolites. The methodology is demonstrated by labeling <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> metabolomes with ¹⁵N₁-ammonium chloride, ¹⁵N₄-arginine, and ¹³C₂-acetate. Efficient ¹⁵N and ¹³C metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols