2024 Volume 47 Issue 6 Pages 1106-1112
Ultrafine bubbles (UFBs), which are bubbles with diameters of less than 1 µm, are widely recognized for their ability to exist stably in liquid as a result of the effects of Brownian motion. In this study, we focused on hydrogen, known for its antioxidant potential, and explored the function of H2-filled UFBs, which encapsulate hydrogen, to determine their potential use as oral carriers for the delivery bioactive gases to living organisms. To this end, rats were orally administered ethanol to induce hepatic oxidative stress, and the effects of drinking H2-filled UFBs (H2 NanoGAS®) water for two weeks were evaluated to assess the reduction of oxidative stress. Continuous alcohol consumption was found to significantly increase the blood lipid peroxidation levels in the control group, confirming the induction of oxidative stress. An increase in blood lipid peroxidation was significantly inhibited by the consumption of concentrated H2 NanoGAS® (C-HN) water. Furthermore, the measurement of mitochondrial activity in the liver revealed that drinking H2 NanoGAS® water helped to maintain at a normal level and/or boosted the functional activity of the electron transport system in mitochondria affected by ethanol intake. To our knowledge, this study is the first to provide evidence for the use of orally ingested UFBs as carriers for the delivery gases to tissues, thereby exerting their physiological activity in the body. Our findings highlight the potential for the application of UFBs to various physiologically active gases and their utilization in the medical field in the future.
Ultrafine bubbles (UFBs) are widely recognized for their stability in liquids due to the effects of Brownian motion, demonstrating an ability to remain unaffected by buoyant force owing to their fineness.1) The gas enclosed in these bubbles can be selected based on its application, making UFBs attractive for use in various fields. Currently, UFBs are being studied in various scientific fields, including medicine,2) agriculture,3,4) hygiene,5) food science,6,7) and nutrition.8) In particular, UFBs have shown significant potential for use in medical applications owing to their stability and safety.2,9) However, research on their use in these fields is still nascent. Although hydrogen,10) oxygen,11) and nitric oxide12) are gases with physiological activities, their use in living organisms is complex because of the need for elaborate equipment and specialized instruments for their production. The complexity and limitations associated with their generation and use make the biomedical application of these gases less convenient. In contrast, the use of UFBs to encapsulate these gases allow for the administration of bioactive gases through their ingestion, thereby facilitating their use in living organisms. However, few studies have examined the bioactive effects of gases in detail using UFBs enclosed within these gases in vivo. Therefore, in this study, we focused on hydrogen, which is known for its antioxidant potential, and investigated the function of H2-filled UFBs encapsulating hydrogen to assess their utility as carriers for use in the delivery bioactive gases to living organisms.
Hydrogen is a gas with a strong reduction capacity and has bioactive effects as a therapeutic and preventive antioxidant due to its nontoxicity to organisms, as well as its ability to exhibit cytoprotective effects against oxidative stress.13,14) Reports have suggested that hydrogen removes ethanol-induced reactive oxygen species (ROS)15) and mitigates mitochondrial dysfunction.16) In rats, the oral administration of hydrogen water has been reported to protect hippocampal neurons and mitochondria from oxidative stress. Therefore, it is considered to be a highly valuable gas in the medical field. Currently, the most common method of ingesting hydrogen by organisms is through saturated hydrogen water, in which hydrogen is dissolved to saturation and stored. Although this is a common approach, maintaining the concentration of dissolved hydrogen in water is difficult. Its half-life is approximately two hours under normal temperature and pressure, making it unsuitable for general distribution because of its low stability. Therefore, we focused on UFBs that could stably retain gas for a long period of time. In this context, we hypothesized that if hydrogen could be ingested orally through water-containing H2-filled UFBs, it would be possible to induce the antioxidant effect of hydrogen in a simple manner in living organisms.
To verify this hypothesis, we used a rat model in which oxidative stress was induced by ethanol to elucidate the utility of UFBs as oral carriers of hydrogen with antioxidant properties. The experimental rats were provided with H2-filled UFBs water for an extended period of time, and the effect of H2-filled UFBs water on reducing oxidative stress was assessed by measuring the blood and liver levels of biomarkers associated with oxidative stress, namely superoxide dismutase (SOD)-like activity and malondialdehyde (MDA) levels. In addition, because the MDA results showed a reduction in oxidative stress in the liver during the course of the study, mitochondrial respiration measurements were performed to evaluate the effects of H2-filled UFBs on liver mitochondrial function. Should a favorable effect in the organism be observed after the ingestion of H2-filled UFBs water, such as a reduction in oxidative stress, the utility of UFBs as new delivery carriers for bioactive gases, which are traditionally inconvenient, will become evident.
H2-filled UFBs (H2 NanoGAS®) water was produced using the rotary shear method.17) H2 NanoGAS® water was obtained from Shinbiosis Corp. (Osaka, Japan). Concentrated H2 NanoGAS® water was prepared by concentrating H2 NanoGAS® water 10-fold using a rotary evaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The water bath temperature was 45 °C and cooling water temperature was set to 3–7 °C. The compression pressure was set to −1 MPa and the rotation speed to 126 rpm. The average size and number of H2 NanoGAS® water were reported to be 124.3 ± 6.8 nm and 0.16 ± 0.05 × 108/mL (mean ± standard error of the mean (S.E.M.)), respectively.9) For concentrated H2 NanoGAS® water, these values were 112.5 ± 8.4 nm and 5.12 ± 0.96 × 108/mL (mean ± S.E.M.), respectively. These data were analyzed by nanoparticle tracking using a NanoSight NS300 (Malvern Panalytical Ltd., Worcestershire, U.K.) (Fig. 1). By considering the size and number of the UFB particles, the concentration of the hydrogen in the H2 NanoGAS® and concentrated H2 NanoGAS® water was calculated as 5.58 × 10−4 and 1.29 × 10−2 mmol/L, respectively.18,19) In contrast, the hydrogen concentration of water is much lower, 4.29 × 10−7 mmol/L based on Henry’s Law at Standard Ambient Temperature and Pressure.
Particle size distribution of HN water (A) and particle size distribution of C-HN water (B) in which is prepared concentrating HN water 10-fold using a rotary evaporator. The peaks represent the concentration of dominant particles with similar sizes, and numbers indicate the particle size. The line and shade indicate the mean ± S.E.M. Data were analyzed by nanoparticle tracking analysis using NanoSight NS300. Data in (A) are based on Morishita et al. (Biocontrol Sci., 27 (2022)).
Natural mineral water was purchased from SUNTORY Holdings, Ltd. (Tokyo, Japan). Concentrated H2 NanoGAS® water was stored at 4 °C and brought to room temperature 30 min before use. H2 NanoGAS® water and natural mineral water were stored at room temperature. Ethanol (80%) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and diluted with tap water to obtain a 30% ethanol solution. Pentobarbital sodium salt was purchased from NACALAI TESQUE, Inc. (Kyoto, Japan).
Animal StudiesThis study was conducted at Kobe Gakuin University in accordance with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals (Approval No. A21-44). Five-week-old male Wistar rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). All rats were housed in rooms maintained at 23 ± 1 °C and 55 ± 5% relative humidity under a 12 h light/dark cycle with free access to water and food during the one-week acclimation and throughout the study period.
Because this study aimed to assess the physiological effects of H2-filled UFBs on daily alcohol consumption, a preliminary experiment was conducted. The rats were orally administered an ethanol solution equivalent to the Ministry of Health, Labour and Welfare index on “moderate and responsible alcohol consumption”20) (20 g/60 kg of body weight = 0.3 g/kg/d) once a day for two weeks using a probe. However, as no changes in SOD-like activity and MDA levels were observed (data not shown), this was followed by the administration of an ethanol dosage of 0.6 g/kg/d, which is twice the recommended dose. This dose corresponds to the amount of alcohol consumed and increases the risk of lifestyle-related diseases.
Based on their body weight, the rats were randomly assigned to one of four groups. Rats in the natural mineral (NM) water (n = 9), H2 NanoGAS® (HN) water (n = 9), and concentrated H2 NanoGAS® (C-HN) water (n = 6) groups were provided with water bottles. Ethanol was administered orally once daily at a fixed time (10:00 a.m.) during the experimental period. In the negative control group, (TAP) water (n = 6) was administered orally instead of ethanol. In each case, the rats were given free access to the test water for oral ingestion for two weeks.
Water and food intake were measured periodically, blood samples were collected, and the rats were dissected on the last day of the experiment (day 15). In the dissection experiments, after anesthesia with intraperitoneal pentobarbital sodium (75 mg/kg), the animals were fixed in the dorsal position on a hot plate. After confirming the loss of the pain reflex in the toes, total blood was drawn from the posterior vena cava, leading to exsanguination. After euthanasia, the blood of the animals was drained by perfusion with 100 mL of saline solution, and the liver samples were pretreated according to the protocol for each experiment. Plasma was separated from heparinized or ethylenediaminetetraacetic acid (EDTA)-treated blood by centrifugation at 600 × g and 1000 × g for 10 min at 4 °C, respectively, and stored at −80 °C until further analysis.
Measurement of Biochemical Parameters in BloodPlasma samples for the evaluation of the biochemical parameters were blinded and outsourced to Oriental Yeast Co., Ltd. (Tokyo, Japan). The levels of total protein (TP), glutamic oxaloacetate transaminase (AST), glutamic pyruvate transaminase (ALT), creatine kinase (CK), and triglyceride (TG) were measured on a 7180 Clinical Analyzer (Hitachi High-Tech Corporation, Tokyo, Japan) using Total protein-HRII, L-type Wako AST·J2, L-type Wako ALT·J2, L-type Wako LD·J, L-type Wako CK, and L-type Wako TG·M (FUJIFILM Wako Pure Chemical Corporation).
The SOD-like activity in red blood cells (RBCs) and plasma was assessed using the SOD Assay Kit WST (Dojindo Laboratories, Kumamoto, Japan), while the MDA concentrations in the plasma and liver were measured using a thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical, MI, U.S.A.) as a marker for the antioxidant defense system.
Measurement of Hepatic Mitochondrial ActivityOroboros Oxygraph-O2k, the current leading instrument for assessing mitochondrial oxygen consumption, allows for dynamic high-resolution respirometry assessments across tissues, cells, and mitochondrial isolates in response to titrations of substrates, uncouplers, and inhibitors of mitochondrial respiratory function.21) We assessed the activity of mitochondrial complex I (CI)- and II (CII)-linked oxidative phosphorylation capacity and CI- and CII-linked electron transport chain capacity as functions of mitochondrial oxygen consumption using Oroboros Oxygraph-O2k (Oroboros Instruments, Innsbruck, Austria).
Sections of the liver were weighed and washed twice for 1 min in 2 mL of MiR05 buffer (Oroboros Instruments) containing 50 µg/mL Digitonin in a 6-well plate, followed by washing with 2 mL MiR05 buffer for rinsing. The washed liver samples were transferred to 100 µL of MiR05 buffer, homogenized, and diluted with MiR05 buffer to obtain liver samples with a weight of 2 mg/50 µL. All the manipulations were performed on ice.
The liver samples not measured on the same day were frozen following the procedure outlined by García-Roche et al.22) After weighing, the samples were washed with 1 mL of modified MiR05 buffer and transferred to a cryotube along with modified Belzer UW® fluid (Astellas Pharma Inc., Tokyo, Japan) to obtain liver samples with a weight of 10 mg/1 mL. The samples were covered with ice and allowed to stand for approximately 6 min before treating with liquid nitrogen at −80 °C for 20 min and liquid nitrogen at −196 °C for at least 10 min. Finally, the samples were stored at −80 °C until use.
The oxygen consumption rates (OCRs) in the oxidative phosphorylation (Oxphos) and electron transfer (ET) systems of the homogenized liver were measured, and mitochondrial function indices were obtained using the following protocol with modified doses and reagents, based on a previous report.23) CI Oxphos was measured after the addition of pyruvate (5 mM), malate (2 mM), glutamate (10 mM), and ADP (5 mM); CI + II Oxphos was subsequently measured after the addition of cytochrome C (10 µM) and succinate (10 mM). CI + II ET was measured after the addition of the ATP synthase inhibitor oligomycin (10 nM) and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (0.05 mM steps) until the oxygen consumption reached a plateau. CII ET was measured by adding rotenone (0.5 µM). Finally, antimycin A (2.5 µM) was added and residual oxygen consumption (ROX) was subtracted from the OCRs using the higher ROX. All the reagents were purchased from Sigma-Aldrich Co., Ltd. (MO, U.S.A.). The OCRs were calculated using DatLab 4 analysis software (version 7.4.0.4) (Oroboros Instruments). CII Oxphos was obtained by subtracting CI Oxphos from CI + CII Oxphos and CI ET was obtained by subtracting CII ET from CI + CII ET.
Statistical AnalysisAll experiments were performed in triplicate (n ≥ 3). For multiple comparisons, ANOVA with Tukey’s, Games–Howell, or Kruskal–Wallis tests was used. IBM SPSS Statistics (version 27) (IBM Corp., Armonk, NY, U.S.A.) was used for statistical analysis. Statistical significance was set at p < 0.05.
Figure 2A shows the average body weight, Fig. 2B shows the average total food intake, and Fig. 2C shows the average total water intake of each group, respectively. No weight loss was observed in any of the test groups, and no differences in water or food intake were observed owing to the differences in drinking water.
(A) Body weight, (B) total food intake, and (C) total water intake. Each data point represents the mean ± S.E.M. of n = 6–9.
The results of the blood biochemical tests (TP, AST, ALT, CK, and TG) for each group are shown in Table 1. The fact that no significant differences were detected between the TAP and MW groups for any of the liver function parameters indicates that the 0.6 g/kg/d dose used in this study did not cause acute or severe alcoholic liver injury. While a notable upward trend was observed in the AST and CK values, individual variations were noted. Overall, these results confirmed that the effect of ethanol dosage used in this study was not significant.
| Group | TP (g/dL) | AST (IU/L) | ALT (IU/L) | CK (IU/L) | TG (mg/dL) |
|---|---|---|---|---|---|
| TAP water | 4.6 ±0.1 | 60.7 ± 3.1 | 33.8 ± 2.2 | 335.0 ± 58.1 | 55.7 ± 12.0 |
| NM water | 4.6 ± 0.1 | 76.4 ± 17.7 | 31.8 ± 2.3 | 519.0 ± 252.3 | 42.8 ± 4.0 |
| HN water | 4.6 ± 0.1 | 92.5 ± 24.4 | 30.4 ± 1.1 | 717.4 ± 342.7 | 57.1 ± 9.9 |
| C-HN water | 4.8 ± 0.1 | 100.5 ± 32.3 | 33.5 ± 1.9 | 765.5 ± 386.3 | 54.3 ± 11.9 |
Abbreviations: TP, total protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CK, creatine kinase; TG, triglyceride. Each data point represents the mean ± S.E.M. of n = 6–9.
The SOD-like activities of RBCs and plasma are shown in Figs. 3A and B. A slight decrease in the RBCs and plasma activities was observed in the NM group, while a slight recovery was observed in the H2 NanoGAS® groups in a concentration-dependent manner; however, this difference was not significant. This suggests that ethanol-induced oxidative stress had no effect on SOD-like activity.
SOD-like scavenging activity in RBCs (A) and plasma (B). Each data point represents the mean ± S.E.M. of n = 6–9.
The results of the TBARS assay of the plasma and liver are shown in Figs. 4A and B. MDA is an indicator of lipid peroxidation in tissue samples and a marker of oxidative stress since it is formed as a degradation product of lipid peroxides. The plasma MDA concentrations were significantly higher in the NM group than in the negative control TAP group, suggesting that oxidative stress was induced by the continuous oral administration of ethanol. The MDA concentrations were significantly lower in the C-HN group than in the NM group, and the latter were comparable to those in the TAP group. In the liver, although the overall MDA concentrations were low and the effects of oxidative stress were not pronounced, higher MDA concentrations were observed in the NM group than in the negative control TAP group, and lower MDA concentrations were observed in the C-HN group (p = 0.058). Based on the above results, H2-filled UFBs maintain lipid peroxidation levels, which are elevated by oral ethanol intake, at normal levels; this effect depends on the UFB count (the amount of hydrogen enclosed).
Concentration of MDA in plasma (A) and liver (B) in rats after oral ingestion of ethanol for 2-week. Each data point represents the mean ± S.E.M. of n = 6–9. * p < 0.05.
The respective OCR of ET and Oxphos in liver mitochondria are shown in Figs. 5A and B. The OCR in the ET (Fig. 5A) and NM groups were the lowest among the four groups for all measurements, suggesting that oxidative stress decreased mitochondrial oxygen consumption, whereas the HN and C-HN groups showed a significantly higher level of oxygen consumption than in the NM group. Especially for CI + CII ET, the HN group was also significantly higher than for the TAP group, the negative control group. In terms of Oxphos (Fig. 5B), overall, no significant differences nor trends between the test groups were observed; however, a lower OCR of CI was observed in the NM group and a lower OCR of CII was observed in the TAP and HN groups, while the NM group showed activity comparable to that of CI. These results suggest that drinking H2-filled UFB water may have delivered hydrogen to the liver, protecting the liver mitochondria from oxidative stress induced by alcohol administration, thereby maintaining or enhancing their function. These results also indicate that the UFB concentration did not influence this effect.
OCRs of ET (A) and Oxphos (B) measured in frozen liver tissue slices on the Oroboros Oxygraph-O2k. Each data point represents the mean ± S.E.M. of n = 4–5. * p < 0.05, ** p < 0.01. Abbreviations: CI, complex I; CII, complex II; OXPHOS, oxidative phosphorylation; ET, electron transfer; OCR, oxygen consumption rate.
Hydrogen is recognized for its diverse physiological effects, with numerous studies exploring its biological applications in water, where hydrogen is dissolved. However, a disadvantage of conventional hydrogen water, in which hydrogen is dissolved in water until saturation, is ensuring the stability of hydrogen. However, it remains difficult to effectively utilize hydrogen gas in the body using methods other than hydrogen water. In the case that gases can be effectively utilized in the body through the consumption of water-containing UFBs, UFBs could be used as delivery carriers to facilitate the transport of gases to living organisms. To substantiate this concept, we focused on examining the mitigating effect of H2-filled UFBs on alcohol-induced oxidative stress in the liver. Alcohol generates ROS, along with the toxicity of its metabolite, acetaldehyde.24) Mitochondria are intracellular organelles that contain aldehyde dehydrogenase 2, the most important enzyme involved in acetaldehyde metabolism. However, mitochondria are susceptible to damage due to elevated ROS levels resulting from alcohol metabolism, acetaldehyde toxicity, and hypoxia induced by such metabolism.24,25) Therefore, in this study, we assessed the effects of H2-filled UFBs on liver mitochondrial activity directly.
The findings presented in this study indicate that persistent alcohol consumption markedly elevates blood lipid peroxidation levels, confirming the induction of oxidative stress (Fig. 4). However, this effect was not sufficiently potent to cause significant alterations in blood parameters (Table 1). A marked increase in the blood lipid peroxidation levels observed in the control group (NM) was significantly attenuated by C-HN water consumption, and a similar trend was observed in the liver. Furthermore, in this study, the mitochondrial CI- and CII-linked oxidative phosphorylation capacity and CI- and CII-linked electron transport chain capacity were evaluated in detail based on the oxygen consumption of liver mitochondria. The OCR on ET in NM group was significantly lower than in the H2 NanoGAS® groups (HN, C-HN) (Fig. 5A). Furthermore, in the CI + II ET group, there was no significant difference between the NM and TAP groups; however, a significant difference was observed between the NM and NanoGAS® groups, with the HN group showing a significantly higher oxygen consumption than the TAP group. This suggests that although the function of the mitochondrial electron transport system was affected by ethanol intake, this was maintained at normal levels and/or boosted by H2 NanoGAS® water intake. In contrast, no significant difference was observed in the CI + II Oxphos group, reflecting ATP synthesis (Fig. 5B). Similarly, there were no differences between the groups in terms of the quantitative measurement of the liver ATP levels (data not shown). In many cases, the treatment of stress-induced groups with hydrogen enhances ATP production26); however, this phenomenon was not observed in the present study. This indicates that the induction of oxidative stress in this study (via the ingestion of ethanol 0.6 mg/kg/d for two weeks) did not cause effects strong enough to impair mitochondrial ATP production, the oxidative phosphorylation process. This suggests that ingesting H2 NanoGAS® water had no effect.
Molecular hydrogen mitigates oxidative stress-induced cell death by reducing lipid peroxidation and alleviating mitochondrial dysfunction.27) Given the ability of hydrogen to diffuse through the cell membranes into the nucleus and mitochondria,14) once it reaches the target tissue, it can easily diffuse into the tissue and elicit its effects. In this context, our results suggest that by ingesting H2 NanoGAS®, hydrogen reaches the liver via the oral route, likely through the portal vein, thereby contributing to the reduction of oxidative stress observed in the liver.
To date, it is unclear how molecular hydrogen internalized in UFBs is transferred to the liver. UFBs can exist stably in water for long periods of time, and it has been reported that nanosized bubbles do not collapse easily, even when a surfactant is added.28) However, under acidic conditions, the bubble surface was found to lose its negative charge,28) rendering it unstable. Consequently, it is presumed that orally ingested H2 NanoGAS® is largely dissolved by gastric acid. The gastrointestinal tract fluid that dissolves the encapsulated hydrogen is expected to migrate to the small intestine, where it is absorbed through the digestive tract mucosa. Subsequently, the hydrogen reaches the liver via the portal venous system.29) Regarding the possibility that UFBs are absorbed as bubbles is unlikely because the tight junction size in the small intestine is approximately 0.4 Å, and UFBs typically measure approximately 100 nm. To further clarify the utility of UFBs as oral carriers for delivering hydrogen, the morphological changes in the bubbles in the gastrointestinal tract and the in vivo kinetics of internalized hydrogen will need to be studied in greater detail.
Although the therapeutic effects of gases with physiological activities have been widely studied, their practical use in the prevention and treatment of diseases remains uncommon. In this study, we hypothesized that by using UFBs, which have excellent gas retention properties in liquid, it would be possible to “drink” gas. To this end, we sought to verify the utility of using H2-filled UFBs (H2 NanoGAS®) as oral delivery carriers for physiologically active gases. Hydrogen is a gas with a strong reduction capacity and has bioactive effects as a therapeutic and preventive antioxidant owing to its cytoprotective effects against oxidative stress. In our experiments, rats were orally administered ethanol to induce hepatic oxidative stress, and the effects of drinking H2 NanoGAS® water for two weeks were evaluated to assess the reduction of oxidative stress. Continuous alcohol consumption was found to significantly increase the blood lipid peroxidation levels in the control group, confirming the induction of oxidative stress. The increase in blood lipid peroxidation was significantly inhibited by the consumption of C-HN. Furthermore, the measurement of mitochondrial activity in the liver revealed that drinking HN water maintained at a normal level and/or boosted the functional activity of the electron transport system in mitochondria affected by ethanol intake. These results indicate that UFBs can serve as carriers for delivering gases to tissues via oral administration, thereby exerting physiological effects in the body. These results highlight the potential of the application of UFBs to encapsulate various physiologically active gases and their utilization in the medical field in the future.
This study was partially supported by the Hyogo COE Research Program and Kobe Gakuin University Research Grant B.
The authors declare no conflict of interest.
