Enriched Oxygen Mixtures in Athletes
Condition: Oxidative Stress; Stem Cell Research
Study type: Interventional
Phase: Phase 2

Summary: 
      Currently, Hyperbaric Oxigen (HBO) is a widely used treatment for several conditions. There
      are 14 indications for HBO, officially recognized by the Undersea and Hyperbaric Medical
      Society (UHMS), but research is discovering other interesting applications.

      HBO plays an important role in enhancing antioxidant defense mechanisms by increasing radical
      oxygen species (ROS) and nitric oxide species (NOS). This controlled oxidative stress has
      been shown to stop the vicious circle of inflammation - damage - hypoxia already seen in
      several diseases. Increased neoangiogenesis has been demonstrated at pressures of 2
      atmospheres absolute (ATA), while effects helping ischemic tissues need pressures between 2.5
      and 2.8 ATA to develop. Also, stem cell proliferation and mobilization have been demonstrated
      after HBO treatments.

      During sports activities, metabolism generates waste products - mostly CO2, lactic acid, but
      also ROS. HBO could be useful in modulating antioxidant mechanisms and increasing stem cell
      mobilization, thus helping cells in the recovery after training and sportive competitions.

      The authors hypothesize that:

        1. HBO can reduce oxidative stress and induce stem cell mobilization in healthy
           professional athletes;

        2. hyperoxic mixtures can reduce oxidative stress and induce stem cells mobilization in
           healthy professional athletes;

        3. HBO at low pressures (L-HBO at 1.45 ATA) is at least comparable to conventional HBO (at
           2.5 ATA) in reducing oxidative stress and increasing stem cell mobilization.

      The Authors will include healthy athletes. These will be randomly assigned to a control
      group, a L-HBO group, a HBO group, a 30% O2 group, or a 50% O2 group.

      The Authors will assess oxidative stress changes and stem cells proliferation before and
      after 20 L-HBO/HBO/30% O2 mix/50% O2 mix treatments, and after 2 months after the end of
      treatments.
    

Detailed description: 
      Subjects will be recruited through public announcements in local gyms and gathered to explain
      the protocol. Those willing to participate will sign a written informed consent and
      recruited. To be included, all the subjects will undergo a general medical screening to allow
      hyperbaric treatments. This will include weight, height, non-invasive arterial blood
      pressure, and heart rate measurements.

      After inclusion, subjects will be randomly assigned to three arms using an electronic number
      generator by personnel not directly involved in the experiment:

        -  Arm 1(control): no intervention.

        -  Arm 2 (L-HBO): treated with oxygen at 1.45 ATA for 60 min (inclusive of compression and
           decompression times, and an air break of 3 minutes breathing air);

        -  Arm 3 (HBO): treated with oxygen at 2.5 ATA for 60 min (inclusive of compression and
           decompression times, and an air break of 3 minutes breathing air).

        -  Arm 4 (30% O2): breathing an air mixture with 30% of oxygen at atmospheric pressure (1
           ATA).

        -  Arm 5 (50% O2): breathing an air mixture with 50% of oxygen at atmospheric pressure (1
           ATA).

      Subjects included in Arm 2, 3, 4, 5 will undergo a total of 20 treatments. They will follow a
      personalized diet proportional to their energetic expenditure.

      The Authors will identify 3 time-points in the protocol:

      TIME 0 (T0): immediately after inclusion, before any treatment or experiment; TIME 1 (T1): at
      the end of HBO treatments; TIME 2 (T2): 2 months after the end of HBO treatments.

      The following exams will be performed on the included subjects:

        -  a standardized panel including Complete Blood Count (CBC), creatinine, Blood Urea
           Nitrogen (BUN), C reactive protein, and VES will be performed at T0, T1, and T2.

        -  oxidative stress markers will be analyzed on blood, urine, and saliva samples. On blood
           samples (T0; T1; T2), the Authors will measure IL-1 beta, IL-6, TNF-alfa, reactive
           oxygen species and total antioxidant capacity (by paramagnetic resonance), total (tot)
           and reduced (red) aminothiols (by fluorescence spectroscopy), 3-nitrotyrosine (3-NT) (by
           competitive immunoassay).

      On urine samples (T0; T1; T2), the Authors will assess lipid peroxidation by measuring
      8-isoprostane concentration (by competitive immunoassay), nitrite and nitrate (NO2/NO3)
      concentration (by colorimetry based on the Griess reaction), inducible Nitric Oxide Synthase
      (by ELISA commercially available kit), creatinine, neopterine, and uric acid concentrations,
      8-oh-2-deoxyguanosine (by competitive immunoassay).

      On saliva samples (T0; T1; T2) the Authors will measure reactive oxygen species and total
      antioxidant capacity (by paramagnetic resonance), and cortisol (by competitive immunoassay).

        -  stem cells will be analyzed on blood samples (at T0, T1, T2) (by flow cytometry).

      Blood samples (approximately 6-12 ml) will be drawn from the veins of the forearms
      (preferentially on the non-dominant limb); plasma and erythrocytes will be separated by
      centrifuge at 1000×g for 10 min at 4°C. Urine samples will be collected by voluntary voiding
      in sterile containers. 1 mL of saliva will be obtained by Salivette devices (Sarstedt,
      Nümbrecht, Germany). The subjects will be instructed to refrain from drinking, eating,
      smoking, brushing their teeth, and using mouthwash in the 30 min before salivary collection.

      All samples will be stored in multiple aliquots at - 80 °C until assayed and thawed only once
      before analysis.

      With this setting, blinding of patients and investigators will be impossible due to different
      structural characteristics. However, outcome assessors will be blinded to patients'
      allocation.
    

Intervention name(s): L-HBO; HBO; 30% O2; 50% O2
Intervention type(s): Combination Product; Combination Product; Combination Product; Combination Product
Intervention description(s): as previously described.; as previously described.; as previously described; as previously described
Other intervention name(s): Low Pressure Oxygenation; Standard Pressure Oxygenation; 30% O2 mixture; 50% O2 mixture
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Five arms: one control without treatment; one at low pressure (1.45 ATA); one at standard pressure (2.5 ATA); one at atmospheric pressure (1 ATA) breathing air mixture with 30% O2; one at atmospheric pressure (1 ATA) breathing air mixture with 50% O2;
Masking: Single (Outcomes Assessor)
Primary purpose: Treatment
Keywords: Hyperbaric Oxigenation; Sport Medicine; Training; Recovery; Oxidative Stress; Stem Cell Research