Immune-Mitochondrial Correction in Military Recruits (IMMU-MITO)

Study Rationale In Kazakhstan, infectious morbidity among military personnel is 4 times higher than in the civilian population, with severe cases accounting for 15-30% and mortality of 15-25% (Kasimov et al., 2019). Current prevention strategies (physical exercise, hardening, balanced nutrition, vitamin prophylaxis) have not sufficiently reduced infection rates. Stress-dependent immunosuppression during the adaptation period (4-6 weeks to 6 months) may explain this vulnerability.

Scientific Premise Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, increasing cortisol levels, which suppresses cellular immunity and increases susceptibility to respiratory pathogens. Mitochondrial dysfunction in immune cells (granulocytes) further impairs phagocytosis and bacterial killing. The experimental intervention targets both neuroendocrine stress response (magnesium, vitamin D) and mitochondrial function (sodium nucleinate as an electron donor for complexes III-IV of the respiratory chain).

Study Design Details This is a prospective, randomized, single-blind, parallel-group trial. Randomization will be performed using a computer-generated random number sequence with a 1:1 allocation ratio. Allocation concealment will be achieved using sequentially numbered, opaque, sealed envelopes.

Blinding:

• Participants are blinded to group assignment (both interventions are administered as oral tablets of similar appearance)
• Laboratory personnel performing blood analyses are blinded to group assignment
• The statistician performing final data analysis will receive a de-identified database with groups labeled as "Group 1" and "Group 2"
• The principal investigator is not blinded (responsible for safety monitoring) Intervention Details

Control group (Standard of care):

• Multivitamin containing: Vitamin A 800 mcg, Vitamin D3 5 mcg (200 IU), Vitamin E 10 mg, Vitamin C 60 mg, Vitamin B1 1.4 mg, Vitamin B2 1.6 mg, Vitamin B6 2 mg, Vitamin B12 1 mcg, nicotinamide 18 mg, pantothenic acid 6 mg, folic acid 200 mcg
• Dosage: 1 tablet orally once daily in the morning for 30 days

Experimental group (Immune-mitochondrial correction):

• Sodium nucleinate 12 mg + Magnesium citrate 400 mg + Pyridoxine hydrochloride 6 mg + Vitamin D 5000 IU
• Dosage: 3 tablets orally once daily in the morning for 30 days Laboratory Methods Mitochondrial function assessment of granulocytes will be performed using the method described in Patent for Utility Model No. 6180 (Baltabekov N.T., Panov S.A., Saidvakasov R.A., "Method for Assessing Neutrophil Activity in Oncological Patients"). This method uses fluorescent probes (JC-1, TMRM, MitoSOX) to measure mitochondrial membrane potential (ΔΨm) and reactive oxygen species (mitoROS) in live granulocytes by fluorescence microscopy.

Immunophenotyping will be performed by flow cytometry using a 6-pair panel for immune status (CD3/CD4, CD3/CD8, CD16/CD56, CD19, HLA-DR, CD45).

Stress-adaptation status will be assessed using:

• Bioelectroluminescence (BEL): GRV-camera (Bio-Well device) with analysis of gas-discharge photographs of fingers (Note: This device is used for research purposes only, not for clinical diagnosis)
• Cardiointervalography: Bayevsky stress index calculated from 5-minute ECG recordings
• Neutrophil-to-lymphocyte ratio (NLR) from complete blood count Sample Size Justification Based on preliminary data from oncological patients (where the experimental intervention reduced leukopenia from 39% to 14.5%), a sample size of 100 participants per group provides 80% power to detect a 50% reduction in infection incidence (two-sided alpha=0.05), accounting for 15% loss to follow-up.

Data Monitoring No independent Data Monitoring Committee (DMC) is planned due to the minimal risk nature of the study. The Principal Investigator will conduct an interim analysis at 3 months for futility and safety. The study will be terminated early if infection rates exceed 30% in either group or if serious adverse events occur in >5% of participants.

Ethical Approvals

This protocol will be submitted for approval to:

• Local Ethics Committee of NI MIPO
• Ethics Committee of the Ministry of Defense of the Republic of Kazakhstan
• Ministry of Health of the Republic of Kazakhstan (if required) Dissemination Plan Results will be submitted for publication in peer-reviewed journals indexed in Scopus/Web of Science. Authorship will follow ICMJE guidelines. The complete de-identified dataset will be made available upon reasonable request. Positive, negative, and inconclusive results will all be published to avoid publication bias.

Protocol Amendments Any changes to the protocol will be submitted as amendments to ClinicalTrials.gov and to all relevant ethics committees before implementation.