Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Abstract

Background/aim: The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology: Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal findings: The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/significance: Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.

Figure 1. OPLS-DA model results for all ALS versus controls.

(A) OPLS-DA model score vector tP vs. matched sample pairs showing separation between controls and ALS (SALS and FALS) in set I. (B) Showing the separation between controls and ALS samples (SALS and FALS) after subtraction of control score values from matched ALS case score value in set I. (C) OPLS-DA model score vector tP vs. matched sample pairs showing separation between controls and ALS samples (SALS and FALS) in set II. (D) The separation between control and ALS samples (SALS and FALS) after subtraction of control score values from matched ALS score values in set II. Red circles, SALS; blue triangles, FALS; black circles, control.

Figure 2. OPLS-DA model of FALS versus controls.

OPLS-DA model score vector tP vs. matched sample pairs showing separation between control (black circles) and FALS samples (blue triangles) for set I (A) and for set II (B).

Figure 3. OPLS-DA model of SALS versus FALS.

(A) OPLS-DA model score vector tP for the model between SALS samples and FALS samples showing a clear difference between the sample groups. Two patients later found to carry SOD1 mutations (D90Amc and D90Amm) were initially diagnosed as apparently SALS. (B) OPLS-DA model score vector tP for the model between SALS samples and FALS samples when excluding the two SALS patients carrying SOD1 mutations (D90Amc and D90Amm). Predictions of the two excluded samples (marked with stars) placed them into the FALS group. Blue triangles, FALS; red circles, SALS.

Figure 4. OPLS-DA results for patients with SOD1 gene mutations.

(A) OPLS-DA score vector tP showing the difference between ALS samples with (blue triangles) and without (red circles) mutation in the SOD1 gene. (B) Prediction of six FALS cases (some replicated) without SOD1 mutation (open blue triangles marked with stars) into an OPLS-DA model between samples with mutation in the SOD1 gene and SALS samples without mutation in the SOD1 gene places the four FALS cases without mutation in the non-SOD1 group and two on the borderline between the groups (samples predicted to have a positive value along tPS [1]).