This observational study explored the connection between the gut microbiota and the brain in patients with amyotrophic lateral sclerosis (ALS), specifically the modulation of short-chain fatty acids during disease progression and after following a Mediterranean diet for 6 months. Recent research suggests that the gut microbiome-the community of bacteria and other microorganisms living in our intestines-may influence how ALS develops and progresses. The hypothesis was that changes in the gut microbiome and the substances it produces, such as short-chain fatty acids (SCFAs), may play an important role in ALS progression. Additionally, the effect of the Mediterranean diet on circulating short-chain fatty acid concentrations was assessed.

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.

This prospective, single-center study enrolled adults from Romania diagnosed with ALS at Neurology Clinic 1, Mureș County Clinical Emergency Hospital , with an ALS diagnosis within the past 12 months, disease duration under 1 year, El Escorial (1996) criteria-clinically probable or clinically definite ALS. ALS cohort: N=44 at baseline (T0, Aug-Oct 2022); mean age 58.4 ± 12.5 years; 28 men / 16 women. Follow-up: T1 (6 months): N=36 (8 deaths by T1). T2 (12 months): N=30 (6 additional deaths between T1 and T2). Healthy controls: N=40, age- and sex-matched community volunteers without known neurodegenerative or major systemic disease. The patients were recruited through consecutive recruitment at the Neurology Clinic 1, Mureș County Clinical Emergency Hospital (Târgu Mureș, Romania) over a 3-month window (01 Aug-31 Oct 2022).

Given the small, regionally identifiable ALS cohort and the rarity of the condition, the risk of re-identification remains high even after de-identification.