Metabolite Profiling Reveals Predictive Biomarkers and the Absence of β-Methyl Amino-l-alanine in Plasma from Individuals Diagnosed with Amyotrophic Lateral Sclerosis

Abstract

By employing chip-based capillary zone electrophoresis coupled to high-resolution mass spectrometry, we profiled the plasma metabolome of 134 patients diagnosed with sporadic amyotrophic lateral sclerosis (ALS) (81 males and 53 females) and 118 individuals deemed healthy (49 males and 69 females). The most significant markers (p < 0.01) were creatine, which was 49% elevated, and creatinine and methylhistidine, which were decreased by 20 and 24%, respectively, in ALS patients. The ratio of creatine versus creatinine increased 370 and 200% for male and female ALS patients, respectively. In addition, male ALS patients on an average had 5–13% lower amounts of seven essential amino acids, whereas females did not significantly differ from healthy controls. We developed two models using the metabolite abundances: (1) a classification model for the separation of ALS and healthy samples and (2) a classification model for the prediction of disease progression based on the ALS functional rating score. Utilizing a Monte Carlo cross-validation approach, a linear discriminant analysis model achieved a mean area under the receiver operating characteristic curve (AUC) of 0.85 (0.06) with a mean sensitivity of 80% (9%) and specificity of 78% (10%) for the separation of ALS and controls, respectively. A support vector machine classifier predicted progression categories with an AUC of 0.90 (0.06) with a mean sensitivity of 73% (10%) and a specificity of 86% (5%). Lastly, using a previously reported assay with a stable isotope-labeled (13C3 15N2) spike-in standard, we were unable to detect the exogenous neurotoxic metabolite, β-methylamino-l-alanine, in the free or protein-bound fraction of any of the 252 plasma samples.

Metabolic Profile and Pathological Alterations in the Muscle of Patients with Early-Stage Amyotrophic Lateral Sclerosis

“…Furthermore, multivariate analysis significantly distinguished ALS patients from controls in muscle and serum, with excellent performances in muscle. Different profiles of metabolites were also described by other groups in the serum [ 29 , 30 ] and in the plasma [ 31 , 32 , 33 ] of ALS patients compared to healthy subjects, which supports the hypothesis of different metabotypes [ 27 ]. A recent study also highlighted different pathways altered in the serum of ALS patients, including alanine, aspartate and glutamate metabolism, confirming our results [ 34 ].…”

Plasma taurine is an axonal excitability-translatable biomarker for amyotrophic lateral sclerosis

“…Creatinine formed from creatine phosphate can pass through the cell membrane and finally into the blood. Recently, a 49% increase of plasma creatine, with associated 20% and 24% decreases in creatinine and methylhistidine, respectively, was reported in the serum of ALS patients 16 , consistent with previous reports 18 , 19 . Additionally, serum creatinine levels were related to clinical progression and a worse time-to-death prognosis 18 , 19 , 26 .…”

“…Recent advances in metabolome analysis have enabled us and others to make important progress in understanding both the pathophysiology of various neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, and how to more accurately diagnose them 9 – 13 . Additional advances also describe various metabolic changes in the blood of ALS patients 14 – 16 . Even with these advances, there are still no clinically established biomarkers that are supported by electrophysiological data such as motor nerve axonal excitability indices.…”

ILB® Attenuates Clinical Symptoms and Serum Biomarkers of Oxidative/Nitrosative Stress and Mitochondrial Dysfunction in Patients with Amyotrophic Lateral Sclerosis

“…In the pre-treatment samples, these parameters were significantly different from those of controls (higher ALA and CITR, and lower ORN/CITR ratio), suggesting higher degradation rate of muscular proteins (ALA) and confirming higher nitric oxide production through higher activity of the iNOS enzymes (CITR and ORN/CITR ratio). Previous data indicate various anomalies in the tissue and serum levels of free amino acids [ 61 , 62 , 63 , 64 ], supporting the notion of a profound metabolic derangement induced by ALS. The benefits of ILB ® treatment on these parameters confirm the data obtained with sum of nitrite + nitrate and suggest that the drug may affect positively muscle metabolism perhaps either through its anti-inflammatory activities [ 5 ] or through its capacity to induce an amelioration of energy-related metabolism and of amino acid metabolism dysregulation under conditions of cell sufferance [ 6 ].…”