Intravenous Hydrogen Nanobubbles Effect on Cardiac Physiology and Quality of Life: a Single Blind, Dose-response Study
Phase: NA
Conditions: Cardiovascular Diseases, Hypertension, Blood Pressure
Sponsor: Indonesia Molecule Institute

 
Study Protocol with  
Statistical Analysis Plan 
 
 
 
 
 
With Research Title: 
Intravenous hydrogen nanobubbles effect on 
cardiac physiology and quality of life: a single 
blind, dose-response study 
 
 
 
Malang, Indonesia 
19th September, 2022 

Study Title 
Intravenous hydrogen nanobubbles effect on cardiac physiology and quality of life: a single 
blind, dose-response study 
Study Design 
This study is a randomized, single-blind, dose-response clinical trial conducted to assess the effects of 
intravenous hydrogen nanobubbles (HNBs) on multiple health outcomes, including cardiac physiology, 
quality of life, and blood biomarkers. Participants were randomly assigned to one of six groups and 
received either HNB infusion or normal saline (control) over a 5-week period (10 sessions total, twice 
per week). The intervention doses were 0 mL (control), 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL of HNB 
diluted in 500 mL of normal saline. 
Ethical Considerations 
This study followed the ethical principles outlined in the Declaration of Helsinki. Ethical approval was 
granted by the Research Ethics Committee of State Polytechnic of Health Malang (Ref: 410/KPEK-
POLKESMA/2022). All participants provided written informed consent, and participation was entirely 
voluntary. Blinding was maintained by using indistinguishable infusions for HNB and saline solutions. 
Eligibility Criteria 
Inclusion criteria: adults aged 18–65 years, stable health condition permitting participation, and ability 
to provide informed consent. 
Exclusion criteria: known allergies to hydrogen, concurrent participation in other trials, congestive heart 
failure, stage 4 or higher kidney disease, or liver failure. 
Intervention Details 
Group A (Control): 500 mL normal saline 
Group B: 5 mL HNB in 500 mL NS 
Group C: 10 mL HNB in 500 mL NS 
Group D: 15 mL HNB in 500 mL NS 
Group E: 20 mL HNB in 500 mL NS 
Group F: 25 mL HNB in 500 mL NS 
Each group received 10 intravenous sessions over 5 weeks (2 sessions/week). 
Outcome Measures 
Primary outcomes were assessed at baseline (week 0) and post-intervention (week 5): 
1. Cardiovascular physiology: systolic and diastolic blood pressure (mmHg), heart rate (bpm), LVMI 
(g/m²), RWT (unitless), TAPSE (cm), and FMD (% change). 
2. Quality of Life: measured using the SF-36 questionnaire. 
3. Blood profile: inflammatory markers, lipid profile, liver function tests, and hematologic indices. 

Statistical Analysis Plan 
Statistical analyses were conducted to evaluate time-related changes and dose-dependent effects. 
 
- Repeated Measures ANOVA was used to assess within-subject changes (pre- vs post-treatment) and 
interaction effects between dose and time. 
- The Bayesian paired t-test was applied to complement the ANOVA results, providing Bayes Factors 
(BF₁₀) that quantify the strength of evidence supporting the treatment effect. 
- A p-value < 0.05 was considered statistically significant. BF₁₀ values > 3 indicated moderate evidence, > 
10 strong evidence, and > 100 very strong evidence for the alternative hypothesis. 
- Data analysis was performed using appropriate statistical software, and each outcome was treated 
independently without aggregation into composite indices.