Dietary Macronutrients and Gut Microbiota, Inflammation, and Metabolic Markers (DIETMICRO)

This 12-week randomized controlled trial will evaluate the effects of diets differing in macronutrient composition on gut microbiota, intestinal inflammation markers, gene expression profiles, and blood biochemical parameters. All participants will undergo comprehensive measurements and laboratory assessments at four time points: before the study begins (baseline), after 4 weeks, after 8 weeks, and at 12 weeks (study completion).

At each assessment point (baseline, 4, 8, and 12 weeks), all participants will visit the laboratory in a fasting state and undergo the following measurements:

1. Body mass and body composition assessment
2. Transcriptomic analysis
3. Cellular metabolism studies
4. Biochemical blood parameters
5. Gut microbiome analysis

Additionally, participants in the ketogenic diet (KD) group will measure blood ketone levels once weekly throughout the entire study period to monitor dietary adherence. All participants will complete questionnaires assessing stress levels and appetite before, during, and after the study intervention.

**Body Mass and Body Composition Measurement**

Body composition will be measured on an empty stomach in the supine position using Dual-Energy X-ray Absorptiometry (DEXA). This method utilizes the phenomenon of ionizing radiation beam attenuation as it passes through tissues of different densities. The difference in absorption of two energy levels (43 and 110 keV) by soft tissue and bone tissue enables assessment of both bone mineral density (BMD) and fat tissue content. DEXA is characterized by high accuracy and reproducibility. The examination lasts up to a few minutes, and radiation exposure is minimal. DEXA can be performed during hormonal therapy, hormonal disorders, dietary restrictions, or physical exercise.

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**Transcriptomic Studies, Cellular Metabolism, Gene Polymorphism, and Biochemical Analyses**

For transcriptomic, cellular metabolism, and biochemical studies, 10 mL of venous blood will be collected from the antecubital vein. Simultaneously, at the beginning of the study only, an oral swab will be collected from each participant for gene polymorphism analysis.

Blood and oral swabs will be collected by a nurse at the Functional Examination Laboratory of the Academy of Physical Education in Katowice.

**Transcriptomic Analysis**

Transcriptomic studies will be conducted in mononuclear blood cells (PBMC) using microarrays. PBMCs will be obtained by centrifuging whole blood in a Ficoll gradient. To obtain sufficient cell quantity, 8 mL of blood will be collected into two 4 mL tubes. The plasma obtained after centrifugation will be stored for biochemical assays and isolation of free-circulating RNA molecules (for potential subsequent studies of selected molecules based on transcriptomic results).

PBMCs will be counted using an automatic cell counter (TC20 cell counter, Biorad) and divided into two parts. From 8 mL of blood, 4-10 mL of live mononuclear cells are obtained. One million cells will be cryopreserved in appropriate medium for subsequent cellular metabolism studies (Seahorse, Agilent). This cell quantity enables performance of each of three possible tests (ATP rate assay, glycolytic rate, mito stress), with test selection based on transcriptomic study results.

Remaining cells will immediately undergo total RNA isolation using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA concentration will be assessed using a Qubit4 fluorometer (Thermo), and integrity using a Bioanalyzer 2100 (Agilent) with RNA 6000 Nano Assay kits (Agilent). Microarray studies require at least 4 μL RNA at 67 ng/μL concentration with RIN coefficient ≥7. From the planned blood quantity, we obtain 200-400 ng/μL in 30 μL volume.

Gene expression will be evaluated using SurePrint G3 Human Gene Expression 8x60K v2 Microarray Kits. The procedure will follow the manufacturer's protocol and include: cDNA synthesis, cRNA synthesis and amplification, sample preparation for hybridization, hybridization (17 h, 65°C), washing, scanning (SureScan, Agilent), and image analysis (Feature Extraction software, Agilent). Results will be analyzed using GeneSpring software.

**Cellular Metabolism Analysis**

Cellular metabolism studies (ATP-rate assay, Cell Mito Stress, Glycolytic Rate) will be conducted in previously cryopreserved PBMCs using the Seahorse device (Agilent). After thawing, the number of live cells will be counted, then cells will be seeded onto poly-D-lysine-coated measurement plates. Analyses will be performed according to the manufacturer's protocol.

**Gene Polymorphism Analysis Related to Macronutrient Metabolism**

At study initiation, genetic material will be collected from each participant as an oral swab. This material will be used to analyze polymorphism of genes FTO, GATA3, PPRG2, TCF7L2, ADRB2, APOA2, APOA, and FABP2, associated with protein, fat, and carbohydrate metabolism. The analysis will be performed at Genomix4Life S.r.l. laboratory, a spin-off of the University of Salerno in Italy.

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**Biochemical Studies**

From the 10 mL of blood collected for all studies, 2 mL will be designated for biochemical analyses. The following parameters will be measured:

• Triacylglycerol (TG, mg/dL)
• LDL cholesterol (LDL-C, mg/dL)
• HDL cholesterol (HDL-C, mg/dL)
• Total cholesterol (tCh, mg/dL)
• Omega index
• Glucose (Gl, mg/dL)
• Glycated hemoglobin (HbA1c, %)
• Insulin (I, IU/mL)
• Testosterone (nmol/L)
• Cortisol (μg/dL)
• Inflammatory protein (CRP, mg/L)
• β-hydroxybutyrate (β-HGB, mmol/L)

Additionally, the HOMA-IR index will be calculated using the formula: fasting insulin concentration × fasting glucose concentration / 22.5.

1 mL of blood will be used for serum determination of factors playing essential roles in appetite control: grelin, cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), leptin, and peptide Y-Y (PYY). These determinations will be performed using ELISA method. Diagnostic kits from Randox (UK), Roche Diagnostic, Diagnostic System Laboratories (Webster, TX, USA), and Beckman Coulter, R&D, Biotechne will be used. The Omega index will be measured using gas chromatography GC-FID with flame ionization detection.

**Ketone Body Measurement as Ketosis Indicator**

For ketogenic diet control, participants in the KD group will measure blood ketone levels once weekly, in the morning before breakfast, by collecting 1 drop of blood from the finger using a glucose meter with ketone measurement function (Optimum Xido or Optimum Xido Neo, USA Abbott Laboratories, Chicago, Illinois) or using commercially available urinary ketone test strips (Ketur-Test, Roche Diagnostics, Basel, Switzerland). Results will be discussed with a dietitian during visits at 4, 8, and 12 weeks.

**Stool Sample Analysis**

On the day of laboratory visit for body mass and composition measurements, participants will deliver biological materials (approximately 10 g of stool) for gut microbiota composition analysis.

Stool microflora analysis will enable qualitative and quantitative assessment of selected bacterial types and species in the gastrointestinal tract. For quantitative analysis of bacteria, yeasts, and molds, a specified stool weight is placed in appropriate volume of liquid culture medium and subjected to careful homogenization (using BagMixer® S&SW) to achieve homogeneous suspension. From the suspension, a series of 10-fold dilutions is performed, then plated on selective-differential microbiological media.

Bacterial, yeast, and mold cultures are conducted under appropriate conditions:

• Aerobic bacteria: incubation 24 hours, temperature 37°C, aerobic conditions
• Anaerobic bacteria: incubation 48-72 hours, temperature 37°C, anaerobic conditions
• Campylobacter spp.: incubation 72 hours, temperature 42°C, microaerophilic conditions
• Yeast-like fungi and molds: incubation 48 hours, temperature 37°C, aerobic conditions

After specified incubation time, cultures undergo microbiological diagnostics using culture methods, biochemical methods, and MALDI TOF mass spectrometry. For yeast-like fungi and molds, mycological diagnostics are performed using chromogenic media.

The following microorganisms will be evaluated:

1. **Anaerobic bacteria:** Bacteroides spp. and Bifidobacterium spp., Lactobacillus spp. (including hydrogen peroxide-producing strains), proteolytic microflora bacteria: Clostridium spp. (including Clostridium difficile)
2. **Proteolytic microflora bacteria:** Campylobacter spp. **Aerobic bacteria:** Enterococcus spp., Gram-negative E. coli
3. **Proteolytic microflora bacteria:** potentially pathogenic E. coli biovar form, Providencia spp., Salmonella spp., Shigella spp., Yersinia spp., Hafnia alvei, Enterobacteriaceae family (Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus spp., Morganella spp., Serratia spp.), and Pseudomonas spp.
4. **Total bacterial count:** Bacillus spp.
5. **Yeast-like fungi:** Candida albicans, Candida non-albicans, and mold fungi

Additionally, a sample for qualitative analysis of indicator bacteria using PCR method will be extracted. This assesses presence of enterocyte-feeding bacteria: Akkermansia muciniphila and Fecalibacterium prausnitzii.

In stool samples, inflammatory markers will also be measured: calprotectin, eosinophilic protein X, and alpha-1-antitrypsin.