Proton NMR Spectroscopy metabolomics in serum and CSF

Gray E*,Larkin JR*, Claridge TDW, Talbot K, Sibson NR, Turner MR

Talk at 24th International Symposium on ALS / MND, Milan, Italy (2013)


Background: Candidate 'wet' biomarkers are urgently sought in ALS. Results from agnostic proteomic approaches have not yet been replicated independently. Metabolomic studies using the non-selective technique 1H NMR spectroscopy have reported serum glutamate as a marker of disease duration (1) and markers in CSF consistent with altered glucose metabolism (2).

Objectives: To determine whether there is a 'signature' of serum or CSF metabolites common to a large group of ALS patients.

Methods: The Oxford Study for Biomarkers in MND (BioMOx) obtained baseline serum and CSF samples in up to 70 patients across a range of initial disability and progression rates, with six-monthly longitudinal collection where possible, for up to two years. Samples were processed within one hour of extraction and centrifuged prior to storage in polypropylene at -80°C. Aliquots (100μL CSF or 150μL ultracentrifuged serum) were mixed with phosphate buffer in D2O containing 1mM TSP as an internal standard (final volume 0.6mL). 1H NMR spectra were acquired using a 700MHz Bruker spectrometer. Partial least squares discriminant analysis (PLS-DA) was used to determine differences between the spectra from patients and control volunteers, and according to progression rate. q2 values above 0.4 were deemed to be significant.

Results: When comparing CSF spectra from age-matched control volunteers with the most advanced of the longitudinal samples (≥12 months from study enrolment), a significant separation was seen (q2 = 0.49; 20 ALS and 17 control cases). This separation was still evident when less advanced patient samples were included (≥6 months from study enrolment, q2 = 0.45; 26 ALS and 17 control cases), but not when the most advanced samples from each patient were modelled against the control cohort (i.e. including some baseline samples, q2 = 0.22; 47 ALS and 17 control cases). No significant separation was seen with serum.

Discussion and Conclusions: CSF proton NMR spectroscopy has potential for distinguishing ALS patients from healthy controls. The process of defining the key metabolites responsible for this separation is underway, and it is possible they will reflect systemic processes associated with the later stages of disease. Further model-building using multilevel PLS-DA will be used in an attempt to phenotypically stratify patients.

References: 1.Kumar A et al. Metabolomic analysis of serum by 1H NMR spectroscopy in amyotrophic lateral sclerosis. International journal of clinical chemistry 2010:411;563-7.