Longitudinal Evaluation of Short-Chain Fatty Acid Profiles During the Natural Disease Course and a Mediterranean Diet Intervention in Amyotrophic Lateral Sclerosis Patients

This study investigates the role of the microbiome-gut-brain axis in the progression of amyotrophic lateral sclerosis (ALS), with a focus on short-chain fatty acids (SCFAs) and their associated biomarker panel (SCFAGGYC-PROF). The investigators hypothesized that alterations in the gut microbiota composition and SCFA metabolism contribute to the heterogeneity of ALS phenotypes, influencing whether the disease follows a rapidly progressive or more slowly progressive course.

Participants with ALS and healthy controls undergo blood sampling and stool collection at baseline and follow-up. Serum analyses include: measurement of circulating SCFA levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

In a subset of ALS patients, dietary interventions consistent with a Mediterranean dietary pattern are introduced to evaluate the impact of nutrition on gut microbiota composition, SCFA production, and clinical disease progression. This 12-month study (6 months natural course + 6 months dietary intervention) evaluated Mediterranean diet effects on ALS progression rate and circulating levels of SCFA. The diet-known for neuroprotective and anti-inflammatory properties-was associated with significant changes in SCFA plasma levels.

A key process stage involved developing and applying an LC-MS/MS method for plasma quantification of acetic (C2), propionic (C3), butyric (C4), 3-hydroxybutyric (3-OH-C4), hexanoic (C6), and iso-hexanoic acid (Iso-C6; 4-methyl-valeric), using acetic acid-C13, butyric-d8 and hexanoic-d11 as internal standards.

After extraction and derivatization of analytes from plasma, samples were analyzed by reversed-phase LC and detected via MS/MS in MRM mode with negative ESI. The method enabled quantitative determination of free plasma carboxylic acids.

Analytical development and performance verification were carried out for simultaneous analysis of 6 short-chain carboxylic acids plus 3 homologous internal standards via LC-MS/MS with prior derivatization using EDC, 3-NPH, and pyridine. Validation covered LOD/LOQ, linear range and signal-concentration dependence (R>0.9900), accuracy (85-115%), inter-series precision (<15%), and carryover (none), ensuring confidence in the method.

Working solutions were prepared from STOCK/STD solutions fresh each day for method/derivatization checks and for spiking blank plasma at defined levels to build daily calibration curves for routine analysis. Solvent water:methanol 3:7 (v/v) was freshly prepared each analysis day.

Samples were prepared fresh on analysis day and immediately loaded in the autosampler. Samples were grouped into 4 batches: Control, T0, T1, T2. Plasmas were thawed once and equilibrated to room temperature. Sample preparation mirrored that of calibration/control plasma, spiked only with internal standards.

Quantification of SCFAs in Control (n=40), T0 (n=44), T1 (n=36), T2 (n=30). Each batch was run independently with fresh daily calibration curves from pooled healthy plasma spiked at method concentrations. Blanks (unspiked plasma and ISTD-only plasma) were also run to assess endogenous signal. Curves were 6-point linear (R>0.9900), used for quantification with internal-standard peak area correction: acetate C2, propionate C3, and 3-OH-C4 with acetate-C13 ISTD; butyrate C4 with butyrate-d8 ISTD; iso-hexanoic (Iso-C6) and hexanoic C6 with hexanoic-d11 ISTD.

Statistical interpretation using descriptive statistics per analyte and batch: means, SD, %CV (RSD), min, max. Chromatographic peaks processed in MultiQuant (Sciex) linked to Analyst 1.7.1; data/statistics in Microsoft Excel 2019. Method validated for sensitivity, selectivity, linearity, LLOQ, carryover, within-run/between-run precision and accuracy, and recovery.

Inclusion criteria

• Patients diagnosed with amyotrophic lateral sclerosis (ALS) in the past 12 months.
• Disease duration of less than 1 year.
• Fulfillment of the El Escorial diagnostic criteria (1996) for clinically probable or clinically definite ALS.

Exclusion criteria

• Patients with an expected survival <18 months.
• Presence of other neurodegenerative diseases.
• Major comorbidities, such as: heart failure, cancer, autoimmune diseases, systemic diseases.
• Gastrointestinal diseases that could influence gut microbiota.
• Recent or ongoing treatment with antibiotics, probiotics, or prebiotics.
• Low compliance with study procedures.