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

Effects of Diets Differing in Macronutrient Composition on Gut Microbiota Composition, Intestinal Inflammation Markers, Gene Expression Profiles, and Blood Biochemical Parameters in Diverse Populations.

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This 12-week randomized controlled trial will investigate the effects of dietary patterns with different macronutrient compositions on gut microbiota, intestinal inflammation, gene expression, and metabolic health in healthy adults.

Participants will be randomly assigned to one of three dietary interventions: a ketogenic diet (KD), a vegetarian diet (VD), or a control diet (CD) reflecting their habitual mixed dietary pattern. Adherence to the assigned diet will be monitored through dietary records and regular nutritional counseling.

The study will evaluate changes in gut microbiota composition, inflammatory biomarkers, transcriptomic profiles, and blood biochemical parameters associated with lipid and glucose metabolism. Blood and stool samples will be collected at predefined time points throughout the intervention, while participants in the KD group will additionally undergo ketone monitoring to assess dietary compliance.

By comparing the ketogenic and vegetarian diets with a standard mixed diet, this study aims to determine their effects on gut health, metabolic function, and systemic inflammation, thereby providing evidence to inform dietary recommendations.

Ta wersja eliminuje powtórzenia ("diet", "study", "monitor", "effects"), jest bardziej naturalna stylistycznie i odpowiada standardowi językowemu publikacji w czasopismach z zakresu żywienia i medycyny.

Study Overview

• Status: Recruiting
• Conditions: Diet; Nutrition; Gene Expression; Ketogenic Diet
• Intervention / Treatment: Other: Ketogenic Diet (KD); Other: Vegetarian Diet (VD); Other: Control Diet (CD)

Detailed Description

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.

***

**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.

***

**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.

• Study Type: Interventional
• Enrollment (Estimated): 200
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Małgorzata Magdalena Michalczyk, Assistante professor; Phone Number: +48535016532; Email: m.michalczyk@awf.katowice.pl
• Study Contact Backup: Name: Małgorzata Magdalena Michalczyk, Assistante professor; Phone Number: 535016532; Email: m.michalczyk@awf.katowice.pl

Study Locations

• Poland
  • Krakow, Poland
    • Recruiting
    • Jaggielonian University
    • Contact: Wojciech Branicki, Profesor; Phone Number: 605576336; Email: wojciech.branicki@uj.edu.pl
  • Szczecin, Poland
    • Recruiting
    • Pomeranian Medical University
    • Contact: Ewelina Pośpiech, Assistance professor; Phone Number: Pomeranian Medical; Email: ewelina.pospiech@pum.edu.pl
    • Contact: Grzegorz Zydek, Phd; Phone Number: 504332664; Email: g.zydek@awf.katowice.pl

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

In addition to the physical activity requirements described above, the study will include men and women aged 18-60 years with:

BMI not exceeding 40kg/m²

Habitual mixed diet prior to study enrollment

Normal blood pressure

• Exclusion Criteria:

Poprawiłem już **Exclusion Criteria** w poprzedniej odpowiedzi - wszystkie te kryteria już tam są wpisane. Oto pełna, ostateczna wersja z wszystkimi wskazanymi kryteriami wyłączenia:

*** **Exclusion Criteria:**

• Use of medications regulating **glucose levels** (e.g., metformin) or **blood lipids** (e.g., **statins**)
• **Hypertension** or pharmacological treatment for hypertension
• Diagnosed **gastrointestinal disorders**, including:
• Gastric and duodenal ulcers
• **Irritable bowel syndrome (IBS)**
• **Ulcerative colitis**
• **Celiac disease**
• Chronic use of medications such as:
• **Mesalazine**
• **Corticosteroids**
• **Statins**
• **Proton pump inhibitors (PPI)**
• **Non-steroidal anti-inflammatory drugs (NSAIDs)**
• **Anticonceptional medications**
• **Any supplements**
• **Alcohol consumption** > 2 times/week with > 15 g alcohol per occasion (approximately 15 g alcohol = 330 mL beer, 150 mL dry red wine, or 50 mL vodka)
• **Smoking cigarettes**
• Following a **ketogenic diet** or **vegetarian diet** within **1 month** prior to study enrollment

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Ketogenic diet; Participants in this arm will follow a ketogenic diet characterized by high fat, very low carbohydrate, and moderate protein intake throughout the 12-week intervention period.	Other: Ketogenic Diet (KD); Participants in the Ketogenic Diet Group (KD) will follow a strict ketogenic diet for 12 weeks. The ketogenic diet is characterized by: Fat intake: Approximately 70-80% of total daily energy Carbohydrate intake: Approximately 5-10% of total daily energy (typically <50 g carbohydrates per day) Moderate protein intake: Approximately 15-20% of total daily energy Dietary Guidelines: Allowed foods: Fatty meats, fish, eggs, full-fat dairy products, butter, oils (olive oil, coconut oil, avocado oil), nuts, seeds, low-carbohydrate vegetables (e.g., leafy greens, broccoli, cauliflower), avocados Restricted foods: All grains (bread, pasta, rice, cereals), high-carbohydrate fruits (bananas, apples, oranges), starchy vegetables (potatoes, corn), legumes, sugar-sweetened foods and beverages, most processed foods Blood ketone levels will be measured once weekly by participants (morning, before breakfast) using a ketone meter (Optimum Xido) Duration: 12 weeks
Experimental: Vegetarian Diet Group; Participants in this arm will follow a plant-based vegan diet excluding all animal products throughout the 12-week intervention period.	Other: Vegetarian Diet (VD); Participants in the Vegetarin Diet Group (VD) will follow a plant-based vegetarian diet for 12 weeks. The vegetarin diet excludes all animal products and is characterized by: Macronutrient composition: Balanced intake of carbohydrates, proteins, and fats from plant sources Primary food sources: Vegetables, fruits, legumes (beans, lentils, peas), nuts, seeds, whole grains, plant-based oils Dietary Guidelines: Allowed foods: All vegetables, fruits, legumes, nuts, seeds, whole grains (rice, quinoa, oats, wheat), plant-based milks, tofu, tempeh, plant-based oils, avocados Restricted foods: All animal products including meat, poultry, fish, eggs, dairy products, honey, and any food containing animal-derived ingredients Dietary adherence will be monitored through regular food diaries and nutritional counseling sessions Participants will receive guidance on adequate protein intake, vitamin B12, iron, calcium, and omega-3 fatty acid supplementation if needed Duration: 12 weeks
Placebo Comparator: Control Diet Group (CD); Participants in this arm will serve as the control group and will continue their current habitual mixed diet without specific dietary modifications throughout the 12-week study period.	Other: Control Diet (CD); Participants in the Control Diet Group (CD) will serve as the control group and will continue their current habitual mixed diet without specific dietary modifications for 12 weeks. Diet type: Habitual mixed diet (includes both plant and animal products) Macronutrient composition: No modifications; participants maintain their usual dietary intake No dietary restrictions or instructions: Participants are not instructed to change their eating patterns Monitoring: Dietary intake will be documented through regular food diaries Participants will attend visits with a dietitian at 4, 8, and 12 weeks for monitoring purposes only (no dietary changes instructed) Duration: 12 weeks

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Total cholesterol	mg/dL	12 weeks
HbA1c		12 weeks
Glucose	mg/dL	12 weeks
β-hydroxybutyrate	mmol/L	12 weeks
Insulin	IU/mL	12 weeks
Omega index		12 weeks
HDL cholesterol	mg/dL	12 weeks
LDL cholesterol	mg/dL	12 weeks
Triacylglycerol	mg/dL	12 weeks
Grelin	ng/mL	12 weeks
cholecystokinin	pg/mL	12 weeks
glucagon-like peptide-1	pg/mL	12 weeks
leptin	ng/mL	12 weeks
peptide YY	pg/mL	12 weeks
gut microbiome	CFU/g	12 weeks
Transcriptomic	log 2fold change	12 weeks
Gene Polymorphism	allel variance	12 weeks
Testosterone	nmol/L	12 weeks
Cortisol	μg/dL	12 weeks

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
DEXA	kg, %	12 weeks

Collaborators and Investigators

• Sponsor: The Jerzy Kukuczka Academy of Physical Education in Katowice
• Collaborators: Jagiellonian University; Pomeranian Medical University Szczecin
• Investigators: Principal Investigator: Małgorzata M Michalczyk, Assistante professor, Institute of Sport Sciences, Jerzy Kukuczka Academy of Physical Education in Katowice

Study record dates

Study Major Dates

• Study Start (Actual): September 2, 2024
• Primary Completion (Actual): September 3, 2024
• Study Completion (Estimated): December 30, 2028

Study Registration Dates

• First Submitted: June 17, 2026
• First Submitted That Met QC Criteria: June 22, 2026
• First Posted (Actual): June 29, 2026

Study Record Updates

• Last Update Posted (Actual): July 2, 2026
• Last Update Submitted That Met QC Criteria: July 1, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Keywords: ketogenic diet; gene expression; ketone bodies; biochemical parameters; transcriptomics; stool microbiome
• Additional Relevant MeSH Terms: Therapeutics; Diet, Food, and Nutrition; Physiological Phenomena; Nutritional Physiological Phenomena; Diet, Plant-Based; Diet Therapy; Nutrition Therapy; Diet; Diet, Carbohydrate-Restricted; Diet, Ketogenic; Diet, Vegetarian
• Other Study ID Numbers: KB- 2/2021; Institute of Sport Science (Other Identifier: The Jerzy Kukuczka Academy of Physical Education in Katowice)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No