- Study protocol
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Convenient methods for ingestion of molecular hydrogen: drinking, injection, and inhalation
Medical Gas Research volume 5, Article number: 13 (2015)
Molecular hydrogen (H2) is clinically administered; however, in some hospitals, H2 is given to patients without consideration of its safe use. In the present study, we prepared convenient and safe devices for the drinking of super-saturated H2 water, for intravenous drip infusion of H2-rich saline, and for the inhalation of H2 gas. In order to provide useful information for researchers using these devices, the changes in H2 concentration were studied. Our experimental results should contribute to the advance of non-clinical and clinical research in H2 medicine.
Molecular hydrogen (H2) is a medical gas with beneficial effects on oxidative stress , inflammation , apoptosis , lipid metabolism , and signaling pathways . More than 280 articles, including 24 articles on clinical studies, have demonstrated that H2 ameliorates the pathological conditions in numerous human diseases  or disease models in animals , since Ohsawa et al. reported that H2 could be used in antioxidant therapy .
H2 is clinically administered through the oral intake of H2 water [9–12], intravenous drip infusion of H2-rich saline [12–15], or inhalation of air with 2-4 % H2 gas . However, in some hospitals, H2 is given to patients by intravenous drip infusion and/or inhalation without consideration of its safe use. We have developed and provided various devices for the ingestion of H2 to solve this problem. Furthermore, the beneficial effects of H2 using our devices have been reported in 7 human diseases [9–16].
In the present study, we prepared convenient and safe devices for drinking super-saturated H2 water, for intravenous drip infusion of H2-rich saline, and for the inhalation of H2 gas. We examined the changes in H2 concentrations in these devices in order to provide useful information for researchers. Our experimental results reported in this article should contribute to the advance of non-clinical and clinical research in H2 medicine.
A pressure-resistant 500 mL PET bottle (e.g., a Coke bottle) was used. H2-generating agent (0.65 g) was prepared by mixing aluminum powder and calcium hydroxide at a ratio of 76 to 24 by weight. The agent was entirely wrapped with bags, namely, a gas-permeable film or non-woven fabric. The wrapped agent was then reacted with water to generate H2 as follows:
Preparation of super-saturated H2 water for drinking
As shown in Fig. 1a and b, a pressure-resistant PET bottle (500 mL), in which gas-permeable film had been directly inserted, was filled with water and then tightly closed. Water in the bottle reacted with the H2-generating agent (0.65 g), and the H2 gas produced was emitted into the water in the bottle through the gas-permeable film. Thus, during this procedure, the H2-generating agent as well as the water for the reaction did not come into contact with the drinking water. During the reaction, the H2 gas reduced the height of the water level in the standing bottle, which was gradually pressurized to approximately 4.5 atmospheric pressures by the gas after 24 h at room temperature. After the reaction was terminated, the H2 gas was dissolved by shaking the bottle for about 30 s.
Similarly, H2 water was obtained by the use of non-woven fabric. As shown in Fig. 1c and d, the non-woven fabric containing H2-generating agent (0.65 g) was first inserted into an acrylic resin tube, and 0.5 mL of water was added. The tube was tightly closed with a cap attached to a check valve, and inserted into a pressure-resistant PET bottle filled with water. H2 generated in the tube was transferred to the bottle through the valve. In about 5 min at room temperature, the agent started a reaction in the wet fabric. The H2 gas produced was emitted into the water through the check valve attached to the acrylic resin tube. During the reaction, the PET bottle was gradually pressurized to approximately 6 atmospheric pressures due to the generation of H2 gas. After 24 h, the H2 gas was dissolved by shaking the bottle for about 30 s.
Preparation of H2-rich saline for injection
As shown in Fig. 2a and b, a polyethylene bag for drip infusion, dialysis fluid, or organ storage solution was immersed in a H2-containing water tank where the water was continuously electrolyzed and circulated during the operation. The H2 permeated through the polyethylene film and dissolved in the solution without contamination.
As shown in Fig. 2c, non-woven fabric containing the H2-generating agent was moistened with a small amount of water, and then both a drip infusion bag and the non-woven fabric were wrapped with aluminum foil under reduced pressure. The water reacted with the agent in the non-woven fabric to generate H2, and the H2 gas permeating through the polyethylene film in the bag dissolved into the solution.
Preparation of H2-containing gas for inhalation
As shown in Fig. 3, inhalation gas was prepared by the mixing of H2 gas and air, where the H2 gas was produced by the electrolysis of water, and the concentration was controlled under the detonation limit of the mixture of H2 gas and air (below 4 %).
Measurement of H2 concentration
The concentration of H2 gas in the water was measured using the methylene blue platinum colloid reagent-based titration method, as described previously , and verified using an electrochemical gas sensor (model DHD1-1, DKK-TOA Corp., Tokyo, Japan). On the other hand, the concentration of H2 in the air was measured using an H2 gas sensor (FIS Inc., Hyogo, Japan).
The concentration of H2 gas in the water or air is presented as ppm (mg/L, weight/volume) or % (volume/volume), respectively. Most of the experimental data are expressed as mean ± standard deviation (mean ± SD) of more than three individual measurements. However, in the examination of H2-rich saline, the H2 concentration is expressed as an individual measurement to examine the differences between each bag and plastic vessel. The statistical significance was assessed by Student’s paired or unpaired t-test for single comparisons or by one-way analysis of variance (ANOVA) followed by Fisher’s LSD test for multiple comparisons. A p value of less than 0.05 was considered to be statistically significant.
H2 concentration of super-saturated H2 water prepared by Method I
H2 concentrations in the super-saturated H2 water prepared by Method I were measured at 10 °C, 15 °C, and 25 °C. As shown in Fig. 4a, at the same temperature, each H2 concentration after 24 h was significantly increased compared with each H2 concentration after 12 h (p < 0.001). After 12 h, H2 concentration at 25 °C was significantly increased compared with the concentration at 15 °C (p < 0.001), and the concentration at 15 °C was significantly increased compared with that at 10 °C (p < 0.001). In addition, after 24 h, H2 concentration at 25 °C showed a significant increase compared with that at 10 °C (p < 0.01). The H2 concentration after the opening of the PET bottle was also measured at room temperature. As shown in Fig. 4b, H 2 concentration of the water was maintained at approximately 7 ppm (7.13 ± 0.22 ppm) after 24 h without opening the bottle; after the cap had been opened, the concentration after 1 h was significantly decreased compared with the concentration after immediately opening (p < 0.01). In addition, H2 concentration after 2 h was significantly decreased compared with that after 1 h (p < 0.05). In our preliminary experiment after opening the bottle, the H2 concentrations in the bottle after 1 and 3 h were 4.53 ± 0.15 ppm and 2.10 ± 0.10 ppm (each n = 3), respectively, when 150 mL of water was removed immediately after the termination of H2 gas production, and the same volume of water additionally removed after 1 h (data not shown). Furthermore, to examine the stability without opening, H2 concentration was measured after 7 days. As shown in Fig. 4c, the H2 concentration of the water was maintained above 8 ppm (8.30 ± 0.98 ppm) after 7 days without the opening of the bottle. These results suggest that the H2 concentration is maintained for at least 7 days without opening, but the H2 water should be drunk within 2 h after opening. In addition, it is important that after opening, the bottle should not contain space for air in order to avoid the reduction of H2 concentration.
H2 concentration of super-saturated H2 water prepared by Method II
H2 concentrations in the super-saturated H2 water prepared by Method II were also measured at 10 °C, 15 °C, and 25 °C. As shown in Fig. 5a, at the same temperature, each H2 concentration after 24 h was significantly increased compared with each H2 concentration after 10 min (p < 0.001). After 10 min, H2 concentration at 15 °C was significantly increased compared with the concentration at 10 °C (p < 0.01), and the concentration at 25 °C was significantly increased compared with that at 10 °C (p < 0.01). As shown in Fig. 5b, H 2 concentration of the water was maintained at approximately 10 ppm (10.08 ± 0.34 ppm) after 24 h without opening of the bottle; after the cap had been opened, the concentration after 1 h showed significant decrease compared with that after immediately opening (p < 0.001), and the concentration after 2 h also showed significant decrease compared with that after 1 h (p < 0.001). As shown in Fig. 5c, the H2 concentration of the water was maintained at approximately 10 ppm (10.10 ± 0.21 ppm) after 7 days without opening of the bottle. These results suggest that the H2 concentration prepared by this method is maintained for at least 7 days without opening, but the water should be drunk within 2 h of the cap being opened.
H2 concentration of H2-rich saline prepared by Method III
The H2 concentrations of H2-rich saline prepared by Method III in the infusion bags were measured after immersion for 1, 3, 5, and 10 h (Table 1). When the 3 types of bag (No. 1–3) were immersed for 10 h, approximately 1.0 ppm H2-rich saline was obtained (Fig. 6a). There were no differences in the H2 concentration between the types of drip infusion bag. These results demonstrated that it is necessary to immerse the drip infusion bag for at least 10 h in order to obtain 1.0 ppm H2-rich saline. To examine the permeability of H2 for the different polyethylene vessel materials, 5 types of vessels (No. 1–5) were immersed in the water bath for 5 h, and the change in H2 concentration of each vessel was examined (Table 1). The H2 concentration of various infusion bags and polyethylene vessels depends on their thickness and the content of the solution. The H2 easily penetrated into the physiological saline (No. 1), but barely penetrated into the sodium hydrogen carbonate solution (No. 4). In addition, in the physiological saline, the H2 more easily penetrated into the 500 mL drip infusion bag (No. 1) than the 20 mL plastic injection ampoule (No. 5) (Fig. 6b). After the infusion bags had been immersed in the bath for 3, 5, and 10 h, they were removed and the changes in H2 concentration were measured until 5 h later. The H2 concentration of the drip infusion bag decreased from 1.0 ppm to 0.6 ppm after 1 h of removal from the water bath after immersion for 10 h (Fig. 6c). These results suggest that intravenous drip injection with these bags should be completed within 1 h.
H2 concentration of H2-rich saline prepared by Method IV
The H2 concentrations of 4 types of bag (No. 1–4) prepared by Method IV were also measured after 1, 3, 6, and 12 months in order to examine long-term preservation (Table 1). The H2 concentrations in the drip infusion bags (No. 1, 3, and 4) or dialysis fluid bag (No. 2) were maintained for 12 months, suggesting that the H2-rich saline prepared by this method could be used for 12 months (Fig. 7a).
H2 concentration of gas introduced by inhaler
We examined the H2 gas concentration for up to 3 h after starting use of the inhaler, because stability of the gas concentration is required in order to examine the performance of the gas inhaler. The H2 gas concentration in the inhaler was 2.91 ± 0.08 % after 0.5 min, and a H2 gas concentration of approximately 3 % was maintained for 3 h. There was no significant difference among of the time points after starting (Fig. 7b). These results demonstrate that the H2 gas could be supplied stably for 3 h using this inhaler.
In summary, we prepared two types of super-saturated H2 water (7 or 10 ppm) for drinking. The concentrations in these waters were maintained for 7 days without opening, but the waters should be drunk within 2 h of the cap being opened. We also prepared two types of H2-rich saline for injection. Although intravenous drip injection with the H2-rich saline should be completed within 1 h, H2 concentrations in the saline prepared by aluminum foil (Method IV) were maintained for 12 months without opening. Moreover, we prepared H2-containing gas for inhalation. The gas was controlled under the detonation limit of the mixture of H2 gas and air, and the gas could be supplied stably for 3 h. In a recent study, we examined the H2 concentration in rat tissue following administration of H2 via various routes . We demonstrated that H2 concentrations in the tissues depend on the H2 concentration of the administered water or gas, and that the specific uptake of H2 in the tissues is due to the difference in administration route . The present results suggest the importance in choosing the more efficient route of H2 treatment for each disease or tissue . Therefore, we believe that the super-saturated H2 water (10 ppm) prepared by Method II, the H2-rich saline prepared by Method IV, and the H2 gas prepared by our method are convenient and safe preparatory methods. The present results should contribute to the advance of non-clinical and clinical research in H2 medicine.
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The authors are grateful to Dr. Yoshio Okamoto for his excellent advice in the writing of this manuscript.
The authors declare that they have no competing interests.
RK and SH designed the study and analyzed the data. SH wrote the manuscript. RK, BS, and FS developed and prepared the various apparatuses for the ingestion of H2. TS, BS, and FS supported this study by collecting data and giving advice. All authors read and approved the final manuscript.