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Perspective

An Interplay of Gases: Oxygen and Hydrogen in Biological Systems

by
Grace Russell
,
Jennifer May
and
John T. Hancock
*
School of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK
*
Author to whom correspondence should be addressed.
Oxygen 2024, 4(1), 37-52; https://doi.org/10.3390/oxygen4010003
Submission received: 12 January 2024 / Revised: 1 February 2024 / Accepted: 5 February 2024 / Published: 9 February 2024
(This article belongs to the Special Issue Interaction of Oxygen and Other Gases with Haem Containing Proteins)
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Abstract

:
Produced by photosynthesis, oxygen (O2) is a fundamentally important gas in biological systems, playing roles as a terminal electron receptor in respiration and in host defence through the creation of reactive oxygen species (ROS). Hydrogen (H2) plays a role in metabolism for some organisms, such as at thermal vents and in the gut environment, but has a role in controlling growth and development, and in disease states, both in plants and animals. It has been suggested as a medical therapy and for enhancing agriculture. However, the exact mode of action of H2 in biological systems is not fully established. Furthermore, there is an interrelationship between O2 and H2 in organisms. These gases may influence each other’s presence in solution, and may both interact with the same cellular components, such as haem prosthetic groups. It has also been suggested that H2 may affect the structures of some proteins, such as globins, with possible effects on O2 movement in organisms. Lastly, therapies may be based on supplying O2 and H2 together, such as with oxyhydrogen. Therefore, the relationship regarding how biological systems perceive and respond to both O2 and H2, and the interrelationship seen are worth considering, and will be discussed here.

1. Introduction

Since its discovery in the latter part of the 18th century [1], there has been an interest in how oxygen (O2) has roles in biological systems, including its detection [2] and toxicity [3]. O2 makes up approximately 21% of the atmosphere and is essential for aerobic life. O2 is produced by photosynthesis as a by-product of plant physiology and it is used in respiratory processes, being the terminal electron acceptor for the mitochondrial electron transport chain (Complex IV converting O2 to water). Oxygen has many other uses in normal physiology, too, not least being the starting point of reactive oxygen species (ROS), as outlined in Figure 1.
Superoxide (O2•−) is regarded as the cardinal ROS as its formation is known to give rise to other ROS including the hydroxyl radical (•OH), and also RNS such as peroxynitrite (ONOO−). The dismutation of O2•− by superoxide dismutase (SOD) gives rise to hydrogen peroxide (H2O2), which can generate •OH through downstream reactions noted below in Equations (1) and (2):
Haber–Weiss Reaction: O2•− + H2O2 → O2 + •OH + OH− (catalysed by iron ions)
Fenton Reaction: Fe2+ + H2O2 → Fe3+ + •OH + OH−
The further reduction of •OH yields water, and it is the four-electron reduction of O2 by mitochondrial Complex IV which aims to avoid ROS generation. Such reactive molecules are chemically relatively unstable, as many carry unpaired electrons in the outer orbit which have a strong affinity for hydrogen atoms. This can lead to proton extraction from vital biomolecules such as protective lipid membranes, thereby destabilising their structure and altering functionality. ROS/RNS are also able to affect fundamental processes including genetic transcription and mitochondrial activity through the modification of essential proteins and protective membranes and therefore have a significant role in cellular homeostasis. Several excellent reviews on ROS function are available [4,5,6].
Hydrogen was also discovered in the 18th century, and since then has had a rather on-and-off appearance in biological research [7,8]. However, hydrogen gas has been coming to prominence in the scientific literature more recently, especially since the publication of a Nature Medicine paper in 2007 [9], not only as an alternative source of renewable energy but also as a health and lifestyle supplement for humans and animals with potential uses in surgical [10], recuperative [11] and geriatric medicine [12]. Research also suggests incorporating H2 into the agri-food industry, where it can be used in many ways and can enhance stress tolerance [13,14] and act as a growth stimulant [15] or as a preservative for plants and food products [16,17], for example. Table 1 and Table 2 outline the significant effects molecular hydrogen can have in both animals and plants.
Organisms can be exposed to H2 in a variety of ways, with several species containing hydrogenase enzymes, which will produce H2 [33]. Hydrogenase enzymes, some of which are inhibited by O2 (e.g., group IV NiFe hydrogenases [34]), are responsible for catalysing the reversible oxidation/reduction of H2 (H2 ↔ 2H+ + e−). Such enzymes are found in all single-celled and many multicellular organisms (e.g., fungi, plants [35]) and can be categorised into specific phylogenic groups, namely iron only, iron-iron and nickel-iron (Fe, Fe-Fe and NiFe, accordingly). On the other hand, other organisms host an array of H2-producing microflora in the gastrointestinal tract. Hydrogen can also be naturally occurring, for example in thermal vents [36] and other natural sources [37]. As with O2, organisms can also be treated with molecular hydrogen which can be applied directly as a gas, or as hydrogen-enriched water (hydrogen-rich water: HRW: [38]), perhaps in the form of nanobubbles (hydrogen nanobubble water (HNW)). Furthermore, organisms can be exposed to O2 and H2 at the same time in the form of oxygen/hydrogenated solutions and as a stoichiometric mixture of oxyhydrogen gas (66% H2/33% O2) formed through the electrolysis of water.
Although the role of action of O2 in cells is well established, the way H2 effects cells is still unclear under many circumstances. As listed in Table 3, there are several mechanisms possible, but it is unlikely that a single action would be able to account for all the cellular responses reported. H2 was originally thought to be acting as an antioxidant, with many of its effects from scavenging hydroxyl radicals (•OH) but having little or no effect on several significant small reactive signalling molecules such as superoxide (O2•−), H2O2 or nitric oxide (NO). However, as Table 3 exemplifies, several other mechanisms have been proposed, or may at least be possible, and multiple mechanisms are likely to be invoked simultaneously upon rising H2 concentrations. Several of these mechanisms may involve O2, and it seems timely to discuss what any possible ramifications of this may be.
Water (H2O), composed of hydrogen and oxygen, is a universal biological solvent and is essential for the emergence and continuation of carbon-based life [49]. H2O is formed through the thermal catalysis of O2 and hydrogen (H2) gases, (reaction 2H2 + O2 → 2H2O), but of importance here is that it can also be split to yield O2 and H2 in a ratio of 1:2. An inexpensive and relatively simple way to generate H2 is by electro-hydrolysis, with the H2 being exploited and the O2 being vented to waste. However, both gases can be employed together in a mixture known as oxyhydrogen. Oxyhydrogen can be used in many biological arenas, including as a therapy [50]. Such work exemplifies why O2 and H2 should be considered together, as discussed further below.

2. Solubility of Oxygen and Hydrogen in Water

When using either O2 or H2 in solution, the solubility of the gas has to be considered. The solubility of O2 in pure water is reported to be between 1.18 × 10−3 and 1.25 × 10−3 mol dm−3 (at 25 °C and 1.0 atm of O2 pressure) [51]. For H2, the solubility under similar conditions, ~1 atm and 25 °C, is approximately 0.8 × 10−3 mol dm−3.
If water is equilibrated with air, the O2 concentration would be predicted to be approximately 2.56 × 10−4 mol dm−3, whilst the concentration of H2 would be predicted to be negligible—there is little atmospheric H2 to dissolve at ground level. If the concentration of O2 or H2 is increased in water, one of the questions which should be asked is for how long does that elevated concentration last? As can be seen in Figure 2, an elevated level of O2 in solution starts to decline and if the regression line is extrapolated, compared to the baseline concentration, it gives a half-life of elevated oxygen in solution of approximately 420 min. Therefore, if such a solution is going to be used as a treatment for any organism, be it a plant or animal, the loss of oxygen gas with time needs to be considered—it would be suggested that such solutions are made shortly after being prepared.
Similarly, if hydrogen gas in solution is elevated, it will rapidly re-enter the gaseous phase and be lost. Again, representative data are shown (Figure 3), showing that under the conditions used, the half-life of the H2 in solution is only approximately 32 min (Figure 3). Therefore, long-term storage of such a solution before use is not an option unless kept in a sealed container with little head space. Of critical importance here, such measurements of O2 and H2 in solution should be carried out by researchers using their equipment and solutions before being used in a biological setting.
In a very recent study [52] on the proton exchange membrane (PEM) electrolysis of water (a method that infuses only H2 into aqueous solutions) from different sources, the increased H2 in each solution was shown to have a half-life of between approximately 60 and 140 min, which is longer than found in our studies involving alkaline electrolysis that produces and infuses both H2 and O2 (~32 min half-life). Interestingly, the results are widely different depending on the source of water used. The shortest half-life was with what the authors describe as “Holy Spring” water, with the longest being tap water. The study also found that the highest H2 concentration they could obtain was approximately 1.3 ppm. pH rose by about 1 unit over 70 min of electrolysis, and the oxidation-reduction potential (ORP) dropped significantly, from approximately +200 mV to −500 mV. Such work on different waters highlights the need to be careful about knowing what is dissolved in solution and to measure exact working concentrations when reporting data with dissolved gases.
Lastly, it is likely the dissolving of one gas in an aqueous solution (whether pure water, saline or buffer) may affect the presence of other gases. For example, a representative example is shown in Figure 4. Here, it can be seen that the concentration of O2 in solution drops significantly whilst the magnesium-based tablet produces H2 (by the Mg catalysis of water hydrolysis), but then the O2 levels are slowly restored as the oxygen is absorbed from, and hydrogen released into, the atmosphere. Creating a fresh H2 solution in such a fashion may make a relatively anaerobic solution, and should the creation of such an anaerobic solution not be considered as a mode of action when such solutions are used in agricultural and biomedical research, or as therapies in a medical/sports medicine arena?

3. Oxyhydrogen

Further to the discussion of O2 and H2 solubility above, as already mentioned, the two gases are often used together, in what is referred to as oxyhydrogen gas. Brown’s gas, H2/O2, HHO and hydroxy are all terms for the same stoichiometric chemical mixture of oxyhydrogen gas (66% H2 and 33% O2). Oxyhydrogen can be inhaled as a gas (using a nasal cannula or face mask), applied ingested in aqueous solutions, or supplied as a gas to sealed containers for plants, for example. Clearly, under these conditions, there is significantly increased O2, but also a very high concentration of H2. It is known that high H2 is well tolerated by humans, as long as the O2 concentration is maintained (something early researchers of gases such as Humphry Davy failed to realise [7]). As a deep-sea diving gas, H2 has been used since the 1940s [53], and there are no known detrimental health effects. Very early experimentation with what was referred to as “medical gases” did have potentially lethal effects, partly because the researchers were never sure of the purity or exact content of their experimental gases. Humphry Davy nearly killed himself on several occasions [7]. For oxyhydrogen, if such an O2/H2 gas mixture is to be used by humans, or in an agricultural or veterinary scenario, it is important to know exactly what is being used, and that there are no erroneous or toxic components present. However, the oxyhydrogen gas mixture, at least in our hands, has a composition as indicated in Table 4. Therefore, there are no significant concentrations of toxic gases or impurities, but both O2 and H2 are elevated compared to breathing air (as atmospheric at low altitudes).
Gas analysis such as in Table 4 identifies a little over 10% of N2 present in oxyhydrogen gas. Although this would not be intuitively expected, N2 is not toxic and only serves to lower the “working” concentrations of O2 and H2, although these are both higher than atmospheric concentrations (29.5% and 60% respectively) in the analysis. Considering that (i) atmospheric air contains 78% nitrogen, (ii) the evaluation of gases is inherently at risk of atmospheric contamination, and (iii) the HydroVitality device is a sealed unit (applying Henry’s Law, the amount of air dissolved in a fluid is proportional to the pressure in the system, so dissolved gases such as N2, O2 and CO2 in the reservoir are to be expected) it is assumed that the nitrogen content is probably a result of carryover from the gases originally dissolved in the water used to supply the HydroVitality™ machine.
It should also be noted that the adaption of oxyhydrogen-generating machines can yield only H2, wherein a membrane is used to restrict the diffusion of the gas in the electrolyte, and O2 is vented as a waste gas. Therefore, this relatively inexpensive and easy-to-use technology can be used to generate oxyhydrogen or H2, and both can then be used for inhalation or to create an enriched solution for treatments.
When considering the direct inhalation of H2 and oxyhydrogen gases and the effect of H2 displacement on O2 to calculate the percentage of H2 inhaled, a simple formula can be applied.
(mL/s): H2/(Breath−H2) × 100
To illustrate, the average person (female: height 1.64 m, weight 72.7 kg and BMI 27.1; male: height 1.78 m, weight 86.7 kg and BMI 27.4; UK data) [54] breathes in 500 mL of air with every breath, with inhalation lasting approximately 2 s. Taking the maximum flow rate of the device tested, HydroVitality™ produces 450 mL/min oxyhydrogen. Thus, females will inhale 2.5% H2 with every breath, whilst males will intake approximately 2%. If the same formula, adjusted for the flow rate and percentage increase (33%) in O2 consumption, is applied, the values of oxygen intake increase by 1.27% (22.27%, O2) for females and 1.01% (22.01%, O2) for males, Table 5.
A reduction in O2 consumption can have unwanted effects on metabolic processes; as mentioned above, O2 also has effects in biological systems, being the terminal electron acceptor in aerobic respiration and ATP production, and the starting point of ROS production. Therefore, a prolonged decrease can lead to tissue damage.
Due to the addition of O2, oxyhydrogen gas could alleviate conditions relating to hypoxia and reduce the incidence of hyperoxia as a result of H2 application. This assumption is supported by clinical data obtained during the COVID-19 pandemic by healthcare professionals in the People’s Republic of China, where oxyhydrogen inhalation was shown to reduce airway resistance and improve the serious burden of disease (NCT04336462—Results: [55]). In contrast, for the treatment of chronic obstructive pulmonary disease (COPD) where hyperoxia from pure O2 inhalation can cause physiological distress, oxyhydrogen was demonstrated to be more effective in reducing breathlessness, cough and sputum. In total, 14.8% fewer adverse events were also reported in the oxyhydrogen-inhalation group when compared with O2 inhalation (NCT04000451—Results: [56]). In clinical investigations (NCT02961387—Results: [57]) of tracheal stenosis, a condition that presents with severe inflammation of the airways, treatment with oxyhydrogen was superior to O2 in reducing inspiration effort. These results suggest that the combination of H2 and O2 has a favourable effect in remediating hypoxia, without instigating hyperoxic effects, delineated by the above studies reporting no adverse events.
It is known that H2 has profound effects on organisms, as exemplified in Table 1 and Table 2. Research also has been carried out utilising both H2- and O2-enriched water in biological systems. As an example, Shin et al. [18] fed broiler chickens either oxygenated or hydrogenated water (i.e., enriched in O2 or H2 respectively) and then measured a variety of characteristics as the chicks developed. A summary of their data is given in Table 6.
The data show that drinking H2 (as a dissolved solution) has a range of effects on the chicks as they develop. This is not a great surprise, considering the data in Table 1 and Table 2. However, drinking O2-enriched solutions also had a similar range of effects. Therefore, on drinking oxyhydrogen-enriched solutions, is it the H2 or O2 that is causing the effects seen? Or is it better to have both O2 and H2 enriched together? Perhaps, as with oxyhydrogen inhalation detailed above, elevating levels of both gases is better than elevating just H2, such as through preparation with Mg tablets. Comparative studies of such solutions, where the exact concentration of gases used is recorded, are required to answer this question. After all, it is not just in chickens that the positive effects of oxygenated water have been reported. Handajani et al. [58] found that drinking such a solution was beneficial for diabetes mellitus, although other reports are sceptical. For example, Gruber et al. [59] said that although there were no long-term detrimental effects on the liver, blood or immune system in humans, the presence of radicals in the blood was increased, albeit transiently. Piantadosi [60] says, “Ergogenic claims for oxygenated water cannot be taken seriously”, and then reviews the evidence. All the claims for oxygenated water need to be taken with caution and more work may need to be undertaken. Interestingly, Shin et al. [18] prepared their enriched solutions using bamboo stems, so that the O2 and H2 entered the water as nanobubbles. This should lead to better stability for the solutions and may perhaps partly account for their effects. The use of nanobubbles has been successful by others, both in vitro and in vivo [39].

4. Is there an Effect of H2 on Haem Groups and a Disruption of Function?

It has been well established that O2 acts as a terminal electron acceptor in cells. As well as accepting four electrons from Complex IV of the mitochondrial electron transport chain (ETC) [45], O2 is also a terminal acceptor of gp91-phox of some NADPH-dependent oxidase (NOX) complexes [61]. Indeed, Kiyoi et al. [62] have described a reduction in both •OH and ONOO− production in murine models when treated by H2 inhalation. Interestingly, the same study [62] noted that the NADPH oxidase (NOX-1) enzyme, responsible for producing the superoxide anion during cellular stress events, was significantly downregulated in the H2 group. The expression of critical activation components p40-phox and p47-phox, however, were unaffected, suggesting that H2 may influence the expression or action of proteins within the NOX-1 complex. Such enzymes facilitate the conversion of O2 to O2•−, utilizing Fe transition metals as catalysts for electron transference, and as such could provide an effectual target for H2 interactions [63], although empirical data will be required to confirm this. Excellent reviews on the NOX enzymes have been written recently [64,65].
In haemoglobin, the O2 binds to the sixth coordinate position of the haem in a manner in which O2 can associate and dissociate depending on the oxygen tension in the solution. Myoglobin has a similar action, but at different O2 binding constants.
H2 can also interact directly with the Fe atom at the centre of the haem group. This was found in cytochrome c3 of Desulfovibrio desulfuricans [44], where there was an electron transfer, and the Fe was reduced from Fe3+ to Fe2+. Such an electron transfer, if it were to take place, would convert methaemoglobin to the haemoglobin (Fe3+ to the Fe2+) state, and so facilitate O2 binding as indicated by Singh et al. [66]. Having said that, physiologically, methaemoglobin is not prevalent in the blood, accounting for <10% in healthy adults, although it can be much higher in individuals with haemoglobinopathies and enzymopathies where methaemoglobin is a feature [67]. However, such a reduction of the Fe in other haem proteins would have the potential to enhance their function, as previously argued [45].
Recently, using density functional theory (DFT) calculations, it has been reported that there is a potential for H2 to interact directly with Fe/haem [40]; in this case, there would also be electron transfer to the haem. The effect on the haem was not the focus, but rather the formation of H• radicals and the likelihood of their interacting with such radicals as •OH. Should H2 with electrons directly interact with the transition metal components of proteins, as described by Hancock and Hancock [46], Kim et al. [40] and Ohta [42], it is possible that the metal group would catalyse the reduction of radicals via reducing the disassociation energy of free H2 (~4.64 eV–~2.35 eV) and the formation of an acceptor/H• complex. Logically, the H2 radicals would readily react with radical oxidants such as •OH, and non-radical species such as peroxynitrite, although this would be regulated by the spatial and temporal availability of the perceived radicals. Interestingly, Kim et al. [40] state that, “From the accumulated studies of Fe–O2 binding, we can expect that H2 also binds to Fe…”. Does this mean that if H2 binds to the haem in a manner analogous to O2, albeit transiently, the binding of O2 is disrupted? Would the same apply to myoglobin? It has been reported that H2 treatment can increase blood oxygenation [68], which is contrary to H2/haem interactions disrupting O2 ligation.
Jin et al. [41] suggested a similar interaction of H2 with haem. Fe-porphyrin, both free and bound to protein, could react with H2, and further they reported the conversion of CO2 to CO, so implicating CO metabolism as being part of the mechanism mediating H2 effects (which may also implicate haem oxygenase). They conclude that “Fe-porphyrin is a redox-related biosensor of H2”. Ohta [42] suggests that a target molecule for H2, Fe-porphyrin conjugated with the -OH group (PrP-Fe(III)-OH), may mediate the activation of the transcription factor Nrf2, and so alleviates oxidative stress effects [69]. Certainly, the interaction of H2 with haem needs further work.
Of further consideration are the metal catalytic centres of such antioxidant enzymes as CAT (haem: [70]) and SOD (either Cu/Zn or Mn-based: [71]), which are reported to have enhanced activity after exposure to H2. It can therefore be surmised that H2 may also improve the function of such metalloenzymes by preserving the reductive capacity of the catalytic metal elements, which may account for the reports of the effects of H2 by Zeng et al. [72] in plants and Yu et al. [73] in small mammals. And of course, there are a host of other metal-based enzyme reactions, not least those of the cytochromes and chlorophyll. Are the mechanisms proposed by Kim et al. [40] and Jin et al. [41] only limited to the haem prosthetic groups they studied, or is there potential for a wide use of such mechanisms?

5. Direct Interaction of H2 with Proteins

Another prevailing theory of H2 somatic distribution and/or action is one of protein pockets. This hypothesis is based on the identification of discrete hydrophobic channels and surface pockets within protein conformations. Such features are typically lined with amino acids of leucine, isoleucine, alanine or valine, characteristically formed in proteins over 100 amino acids in length (reviewed by Roose et al. [74]). Hydrophobic pockets enable non-covalent/van der Waals interactions with noble gases such as argon (Ar), krypton (Kr), the much larger atom, xenon (Xe) [75], and perhaps molecular hydrogen [48]. Research conducted using X-ray crystallography on one of the largest noble gases, Xe, reports that hydrophobicity and the volume of gas delivered are the primary factors in determining gas–protein binding, occurring via weak London dispersion forces [76]. London dispersion forces, a quantum component of van der Waals interactions, are weak forces that describe a temporary intermolecular attraction between atoms. Such atoms are normally electrically symmetric, resulting in the formation of transitory dipoles in non-polar molecules [77].
Similar to H2, noble gases have been deemed inert in biological systems due to having filled electron orbitals, meaning they cannot partake in electron exchanges. However, numerous studies show that Ar [78,79], Kr [80], Xe [81,82] and helium (He) [83,84] can have distinct physiological effects. However, how these effects are initiated is yet to be fully understood. Below, the possibility of direct protein/gas interactions is explored.
Studies of metmyoglobin, the oxidized form of the haem-containing protein myoglobin, derived from whale spermatic fluid, observed four discrete 129Xe binding sites within the protein [85]. Each 129Xe-binding intra-protein channel was situated antithetically from the O2 binding site adjacent to the prosthetic iron. Two channels by which both O2 and Xe pass through the protein to reach their respective binding sites have been identified in the syncopated haemoglobin of Mycobacterium tuberculosis [86]. Atomistic simulations of dioxygen dynamics recognised Channel 1 as serving as an entry channel for these molecules and nitric oxide (NO), whilst Channel 2 was primarily utilised as an exit passage, although this section was noted to be bidirectional. The authors also demonstrate that O2 utilises Xenon pockets Xe1a and Xe2 as it traverses through the cavity to the haem interface and docking site (DS2/active site). Interestingly, NO was noted to occupy only the Xe2 site, which may have further effects, conceivably through inhibiting O2 release through Channel 2.
It was the consideration of the type of research above that allowed the suggestion that H2, being relatively inert, may act in its interaction with proteins in a manner consistent with the noble and hence inert gases such as Xe [47]. However, another consideration that should be accounted for when assessing the non-chemical effects of protein/molecule interactions includes the size and weight of atoms and molecules, as such physical attributes are likely to have key roles in the effects noted. Examples of these data are summarised in Table 7.
As can be seen, Xe, which is known to have physiological effects including as an anaesthetic [92], magnetoreceptive and neuroprotective agent [82] is considerably larger than H2, which has been mooted to have a similar mode of action, i.e., using Xe pockets in proteins. However, H2 is not dissimilar to He, which was suggested to be a therapeutic gas almost 100 years ago [93] and has since been studied for its biological effects [94]. Therefore, this mode of action of H2 would no doubt be worthy of further investigation, as it would for a range of small—some gaseous—signalling molecules [48]. If such gases as H2 are interacting with hydrophobic pockets in proteins such as haemoglobin and myoglobin—the model proteins for studying such effects—is this affecting O2 transport in animals? Certainly, increased O2 saturation, H2 effects and disease models have all been considered together [62]. Is this one of the ways H2 is having biological effects, and could it be mediated by alterations in O2 metabolism?

6. Future Perspectives and Conclusions

Both O2 and H2 were discovered in the 18th century, and early work by a group of eminent scientists such as Antoine Lavoisier, Joseph Priestley, Thomas Beddoes and Humphry Davy were soon examining the biological effects of these and other gases, such as nitrous oxide (discussed by Hancock and LeBaron [7]). Oxygen has become inexorably studied as part of aerobic life, as instrumental to photosynthesis, as well as being linked to host defence in both plants and animals. Lack of O2 conversion into ROS leads to chronic granulomatous disease in humans, for example [95].
Relatively recently, H2 has been mooted as a medical therapeutic [96] and as a useful treatment for plants in agriculture [97], and since the paper by Ohta’s group in 2007 [9] there has been a resurgence in interest in the biological effects of H2 gas.
However, there are relatively few works in the literature considering O2 and H2 together, and how the actions of each of these gases may interact, or even interfere, with each other. Both gases have been used in solution, and sometimes together as an oxyhydrogen mix, but it is not always considered if the dissolving of one gas alters the measurable concentration of the other gas in solution. H2 appears to reduce O2 in solution, and both gases have relatively short half-lives of elevated concentrations in water. This is rarely considered, and such solutions need to be used in a timeframe well before their predicted half-lives of elevated concentrations.
H2 is thought to have its action, at least in part, by scavenging •OH, and a mechanism to account for this is mediated by H2 interactions with haem. However, it is known that O2 also interacts with many haem proteins, including that of the globin family and the ROS-generating NADPH oxidase. Therefore, what is the interaction of H2 and O2 here? Does one affect the action of the other? If it does, does this account for the effects seen by H2 treatments?
Furthermore, H2 can be considered as an inert gas, and as such does it alter protein function in a manner commensurate with the other inert gases, such as Xe and Ar? Here, the model proteins used for research are the globins, which are so instrumental in oxygen transport and storage. Plants, too, have globins [98]. It has already been suggested that there is an interaction between globins, oxygen and an important signalling gas, that is nitric oxide (NO). Could H2 be involved here too?
The exact action of H2 is not well known. The scavenging action of •OH is often suggested to be the mode of action, but several other mechanisms have been suggested, and some of these would impinge on O2 metabolism. It is suggested here that the interplay between H2 and O2 should be further considered as H2 gas research on biological systems carries on in the future.

Author Contributions

This article was conceived, authored and edited by both J.T.H. and G.R. J.M. added academic input and editing. All authors have read and agreed to the published version of the manuscript.

Funding

J.M. and J.T.H. research received no external funding, although G.R. is partly funded by Water Fuel Engineering.

Institutional Review Board Statement

No ethical approval was required for this study.

Informed Consent Statement

Not applicable.

Data Availability Statement

Any preliminary data given in this paper will be made publicly available.

Acknowledgments

The authors acknowledge the University of the West of England, Bristol, for their time and the availability of literature for this study, and Water Fuel Engineering for part-funding G.R.’s research.

Conflicts of Interest

G.R. is part-funded by Water Fuel Engineering who manufacture oxyhydrogen generating equipment.

References

  1. Hancock, J.T. A brief history of oxygen: 250 years on. Oxygen 2022, 2, 31–39. [Google Scholar] [CrossRef]
  2. Renger, G.; Hanssum, B. Oxygen detection in biological systems. Photosynth. Res. 2009, 102, 487–498. [Google Scholar] [CrossRef]
  3. Diguiseppi, J.; Fridovich, I.; McCord, J.M. The toxicology of molecular oxygen. CRC Crit. Rev. Toxicol. 1984, 12, 315–342. [Google Scholar] [CrossRef] [PubMed]
  4. Di Meo, S.; Venditti, P. Evolution of the knowledge of free radicals and other oxidants. Oxid. Med. Cell. Longev. 2020, 2020, 9829176. [Google Scholar] [CrossRef]
  5. Murphy, M.P.; Bayir, H.; Belousov, V.; Chang, C.J.; Davies, K.J.; Davies, M.J.; Dick, T.P.; Finkel, T.; Forman, H.J.; Janssen-Heininger, Y.; et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat. Metab. 2022, 4, 651–662. [Google Scholar] [CrossRef]
  6. Sies, H.; Belousov, V.V.; Chandel, N.S.; Davies, M.J.; Jones, D.P.; Mann, G.E.; Murphy, M.P.; Yamamoto, M.; Winterbourn, C. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol. 2022, 23, 499–515. [Google Scholar] [CrossRef] [PubMed]
  7. Hancock, J.T.; LeBaron, T.W. The early history of hydrogen and other gases in respiration and biological systems: Revisiting Beddoes, Cavallo, and Davy. Oxygen 2023, 3, 102–119. [Google Scholar] [CrossRef]
  8. LeBaron, T.W.; Ohno, K.; Hancock, J.T. The on/off history of hydrogen in medicine: Will the interest persist this time around? Oxygen 2023, 3, 143–162. [Google Scholar] [CrossRef]
  9. Ohsawa, I.; Ishikawa, M.; Takahashi, K.; Watanabe, M.; Nishimaki, K.; Yamagata, K.; Katsura, K.; Katayama, Y.; Asoh, S.; Ohta, S. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat. Med. 2007, 13, 688. [Google Scholar] [CrossRef] [PubMed]
  10. Danilova, D.A.; Brichkin, Y.D.; Medvedev, A.P.; Pichugin, V.V.; Fedorov, S.A.; Taranov, E.V.; Nazarov, E.I.; Ryazanov, M.V.; Bolshukhin, G.V.; Deryugina, A.V. Application of molecular hydrogen in heart surgery under cardiopulmonary bypass. Mod. Med. Technol. 2021, 13, 71–75. [Google Scholar] [CrossRef]
  11. Kalocayova, B.; Kura, B.; Vlkovicova, J.; Snurikova, D.; Vrbjar, N.; Frimmel, K.; Hudec, V.; Ondrusek, M.; Gasparovic, I.; Sramaty, R.; et al. Molecular hydrogen: Prospective treatment strategy of kidney damage after cardiac surgery. Can. J. Physiol. Pharmacol. 2023, 101, 502–508. [Google Scholar] [CrossRef] [PubMed]
  12. Fu, Z.; Zhang, J.; Zhang, Y. Role of molecular hydrogen in ageing and ageing-related diseases. Oxid. Med. Cell. Longev. 2022, 2022, 2249749. [Google Scholar] [CrossRef] [PubMed]
  13. Wu, Q.; Su, N.; Cai, J.; Shen, Z.; Cui, J. Hydrogen-rich water enhances cadmium tolerance in Chinese cabbage by reducing cadmium uptake and increasing antioxidant capacities. J. Plant Physiol. 2015, 175, 174–182. [Google Scholar] [CrossRef]
  14. Cui, W.; Yao, P.; Pan, J.; Dai, C.; Cao, H.; Chen, Z.; Zhang, S.; Xu, S.; Shen, W. Transcriptome analysis reveals insight into molecular hydrogen-induced cadmium tolerance in alfalfa: The prominent role of sulfur and (homo) glutathione metabolism. BMC Plant Biol. 2020, 20, 58. [Google Scholar] [CrossRef] [PubMed]
  15. Guan, Q.; Ding, X.W.; Jiang, R.; Ouyang, P.L.; Gui, J.; Feng, L.; Yang, L.; Song, L.H. Effects of hydrogen-rich water on the nutrient composition and antioxidative characteristics of sprouted black barley. Food Chem. 2019, 299, 125095. [Google Scholar] [CrossRef] [PubMed]
  16. Alwazeer, D.; Özkan, N. Incorporation of hydrogen into the packaging atmosphere protects the nutritional, textural and sensorial freshness notes of strawberries and extends shelf life. J. Food Sci. Technol. 2022, 59, 3951–3964. [Google Scholar] [CrossRef] [PubMed]
  17. Yun, Z.; Gao, H.; Chen, X.; Duan, X.; Jiang, Y. The role of hydrogen water in delaying ripening of banana fruit during postharvest storage. Food Chem. 2022, 373, 131590. [Google Scholar] [CrossRef]
  18. Shin, D.; Cho, E.S.R.; Bang, H.T.; Shim, K.S. Effects of oxygenated or hydrogenated water on growth performance, blood parameters, and antioxidant enzyme activity of broiler chickens. Poult. Sci. 2016, 95, 2679–2684. [Google Scholar] [CrossRef]
  19. Qi, D.D.; Ding, M.Y.; Wang, T.; Hayat, M.A.; Liu, T.; Zhang, J.T. The therapeutic effects of oral intake of hydrogen rich water on cutaneous wound healing in dogs. Vet. Sci. 2021, 8, 264. [Google Scholar] [CrossRef]
  20. Zhao, J.; Zhang, M.; Li, Y.; Zhang, Z.; Chen, M.; Liu, T.; Zhang, J.; Shan, A. Therapeutic effect of hydrogen injected subcutaneously on onion poisoned dogs. J. Vet. Res. 2017, 61, 527. [Google Scholar] [CrossRef]
  21. Wang, M.; Wang, R.; Zhang, X.; Ungerfeld, E.M.; Long, D.; Mao, H.; Jiao, J.; Beauchemin, K.A.; Tan, Z. Molecular hydrogen generated by elemental magnesium supplementation alters rumen fermentation and microbiota in goats. Br. J. Nutr. 2017, 118, 401–410. [Google Scholar] [CrossRef] [PubMed]
  22. Lin, Y.; Kashio, A.; Sakamoto, T.; Suzukawa, K.; Kakigi, A.; Yamasoba, T. Hydrogen in drinking water attenuates noise-induced hearing loss in guinea pigs. Neurosci. Lett. 2011, 487, 12–16. [Google Scholar] [CrossRef] [PubMed]
  23. Yu, S.; Zhao, C.; Che, N.; Jing, L.; Ge, R. Hydrogen-rich saline attenuates eosinophil activation in a guinea pig model of allergic rhinitis via reducing oxidative stress. J. Inflamm. 2017, 14, 1–12. [Google Scholar] [CrossRef]
  24. Tsubone, H.; Hanafusa, M.; Endo, M.; Manabe, N.; Hiraga, A.; Ohmura, H.; Aida, H. Effect of treadmill exercise and hydrogen-rich water intake on serum oxidative and anti-oxidative metabolites in serum of thoroughbred horses. J. Equine Sci. 2013, 24, 1–8. [Google Scholar] [CrossRef]
  25. Yamazaki, M.; Kusano, K.; Ishibashi, T.; Kiuchi, M.; Koyama, K. Intravenous infusion of H2-saline suppresses oxidative stress and elevates antioxidant potential in Thoroughbred horses after racing exercise. Sci. Rep. 2015, 5, 15514. [Google Scholar] [CrossRef]
  26. Varga, V.; Németh, J.; Oláh, O.; Tóth-Szűki, V.; Kovács, V.; Remzső, G.; Domoki, F. Molecular hydrogen alleviates asphyxia-induced neuronal cyclooxygenase-2 expression in newborn pigs. Acta Pharmacol. Sin. 2018, 39, 1273–1283. [Google Scholar] [CrossRef]
  27. Ji, X.; Zhang, Q.; Zheng, W.; Yao, W. Morphological and molecular response of small intestine to lactulose and hydrogen-rich water in female piglets fed Fusarium mycotoxins contaminated diet. J. Anim. Sci. Biotechnol. 2019, 10, 9. [Google Scholar] [CrossRef]
  28. Su, J.; Yang, X.; Shao, Y.; Chen, Z.; Shen, W. Molecular hydrogen–induced salinity tolerance requires melatonin signalling in Arabidopsis thaliana. Plant Cell Environ. 2021, 44, 476–490. [Google Scholar] [CrossRef]
  29. Xie, Y.; Mao, Y.; Lai, D.; Zhang, W.; Shen, W. H2 enhances Arabidopsis salt tolerance by manipulating ZAT10/12-mediated antioxidant defence and controlling sodium exclusion. PLoS ONE 2012, 7, e49800. [Google Scholar] [CrossRef]
  30. Jiang, K.; Kuang, Y.; Feng, L.; Liu, Y.; Wang, S.; Du, H.; Shen, W. Molecular hydrogen maintains the storage quality of Chinese chive through improving antioxidant capacity. Plants 2021, 10, 1095. [Google Scholar] [CrossRef]
  31. Liu, S.; Zha, Z.; Chen, S.; Tang, R.; Zhao, Y.; Lin, Q.; Duan, Y.; Wang, K. Hydrogen-rich water alleviates chilling injury-induced lignification of kiwifruit by inhibiting peroxidase activity and improving antioxidant system. J. Sci. Food Agric. 2023, 103, 2675–2680. [Google Scholar] [CrossRef]
  32. Cheng, P.; Wang, J.; Zhao, Z.; Kong, L.; Lou, W.; Zhang, T.; Jing, D.; Yu, J.; Shu, Z.; Huang, L.; et al. Molecular hydrogen increases quantitative and qualitative traits of rice grain in field trials. Plants 2021, 10, 2331. [Google Scholar] [CrossRef]
  33. Russell, G.; Zulfiqar, F.; Hancock, J.T. Hydrogenases and the role of molecular hydrogen in plants. Plants 2020, 9, 1136. [Google Scholar] [CrossRef]
  34. Marreiros, B.C.; Batista, A.P.; Duarte, A.M.; Pereira, M.M. A missing link between complex I and group 4 membrane-bound [NiFe] hydrogenases. Biochim. Biophys. Acta (BBA) Bioenerg. 2013, 1827, 198–209. [Google Scholar] [CrossRef] [PubMed]
  35. Greening, C.; Biswas, A.; Carere, C.R.; Jackson, C.J.; Taylor, M.C.; Stott, M.B.; Cook, G.M.; Morales, S.E. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival. ISME J. 2016, 10, 761–777. [Google Scholar] [CrossRef]
  36. Petersen, J.M.; Zielinski, F.U.; Pape, T.; Seifert, R.; Moraru, C.; Amann, R.; Hourdez, S.; Girguis, P.R.; Wankel, S.D.; Barbe, V.; et al. Hydrogen is an energy source for hydrothermal vent symbioses. Nature 2011, 476, 176–180. [Google Scholar] [CrossRef] [PubMed]
  37. Zgonnik, V. The occurrence and geoscience of natural hydrogen: A comprehensive review. Earth-Sci. Rev. 2020, 203, 103140. [Google Scholar] [CrossRef]
  38. Jafta, N.; Magagula, S.; Lebelo, K.; Nkokha, D.; Mochane, M.J. The production and role of hydrogen-rich water in medical applications. In Applied Water Science Volume 1: Fundamentals and Applications; Wiley: Hoboken, NJ, USA, 2021; pp. 273–298. [Google Scholar]
  39. Liu, S.; Oshita, S.; Thuyet, D.Q.; Saito, M.; Yoshimoto, T. Antioxidant activity of hydrogen nanobubbles in water with different reactive oxygen species both in vivo and in vitro. Langmuir 2018, 34, 11878–11885. [Google Scholar] [CrossRef]
  40. Kim, S.A.; Jong, Y.C.; Kang, M.S.; Yu, C.J. Antioxidation activity of molecular hydrogen via protoheme catalysis in vivo: An insight from ab initio calculations. J. Mol. Model. 2022, 28, 287. [Google Scholar] [CrossRef]
  41. Jin, Z.; Zhao, P.; Gong, W.; Ding, W.; He, Q. Fe-porphyrin: A redox-related biosensor of hydrogen molecule. Nano Res. 2023, 16, 2020–2025. [Google Scholar] [CrossRef]
  42. Ohta, S. Molecular hydrogen may activate the transcription factor Nrf2 to alleviate oxidative stress through the hydrogen-targeted porphyrin. Aging Pathobiol. Ther. 2023, 5, 25–32. [Google Scholar] [CrossRef]
  43. Lin, Y.; Zhang, W.; Qi, F.; Cui, W.; Xie, Y.; Shen, W. Hydrogen-rich water regulates cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner. J. Plant Physiol. 2014, 171, 1–8. [Google Scholar] [CrossRef]
  44. Peck, H.D. The ATP-dependent reduction of sulfate with hydrogen in extracts of Desulfovibrio desulfuricans. Proc. Natl. Acad. Sci. USA 1959, 45, 701–708. [Google Scholar] [CrossRef] [PubMed]
  45. Hancock, J.T.; LeBaron, T.W.; Russell, G. Molecular hydrogen: Redox reactions and possible biological interactions. React. Oxyg. Species 2021, 11, m17–m25. [Google Scholar] [CrossRef]
  46. Hancock, J.T.; Hancock, T.H. Hydrogen gas, ROS metabolism, and cell signaling: Are hydrogen spin states important? React. Oxyg. Species 2018, 6, 389–395. [Google Scholar] [CrossRef]
  47. Hancock, J.T.; Russell, G.; Craig, T.J.; May, J.; Morse, H.R.; Stamler, J.S. Understanding Hydrogen: Lessons to be learned from physical interactions between the inert gases and the globin superfamily. Oxygen 2022, 2, 578–590. [Google Scholar] [CrossRef]
  48. Hancock, J.T. Are protein cavities and pockets commonly used by redox active signalling molecules? Plants 2023, 12, 2594. [Google Scholar] [CrossRef]
  49. Westall, F.; Brack, A. The importance of water for life. Space Sci. Rev. 2018, 214, 50. [Google Scholar] [CrossRef]
  50. Russell, G.; Nenov, A.; Hancock, J. Oxy-hydrogen gas: The rationale behind its use as a novel and sustainable treatment for COVID-19 and other respiratory diseases. Eur. Med. J. 2021, 21-00027. [Google Scholar] [CrossRef]
  51. Xing, W.; Yin, M.; Lv, Q.; Hu, Y.; Liu, C.; Zhang, J. Oxygen solubility, diffusion coefficient, and solution viscosity. In Rotating Electrode Methods and Oxygen Reduction Electrocatalysts; Elsevier: Amsterdam, The Netherlands, 2014; pp. 1–31. [Google Scholar]
  52. Dmitry Petrov, D.; Panina, E. Characteristics of hydrogen rich water at different stages of electrolysis. BIO Web Conf. 2024, 82, 01006. [Google Scholar]
  53. Bjurstedt, H.; Severin, G. The prevention of decompression sickness and nitrogen narcosis by the use of hydrogen as a substitute for nitrogen, the Arne Zetterstrom method for deep-sea diving. Mil. Surg. 1948, 103, 107–116. [Google Scholar] [CrossRef]
  54. WorldData.info. Available online: https://www.worlddata.info/average-bodyheight.php (accessed on 11 December 2023).
  55. Guan, W.J.; Wei, C.H.; Chen, A.L.; Sun, X.C.; Guo, G.Y.; Zou, X.; Shi, J.D.; Lai, P.Z.; Zheng, Z.G.; Zhong, N.S. Hydrogen/oxygen mixed gas inhalation improves disease severity and dyspnea in patients with Coronavirus disease 2019 in a recent multicenter, open-label clinical trial. J. Thorac. Dis. 2020, 12, 3448. [Google Scholar] [CrossRef]
  56. Zheng, Z.G.; Sun, W.Z.; Hu, J.Y.; Jie, Z.J.; Xu, J.F.; Cao, J.; Song, Y.L.; Wang, C.H.; Wang, J.; Zhao, H.; et al. Hydrogen/oxygen therapy for the treatment of an acute exacerbation of chronic obstructive pulmonary disease: Results of a multicenter, randomized, double-blind, parallel-group controlled trial. Respir. Res. 2021, 22, 149. [Google Scholar] [CrossRef]
  57. Zhou, Z.Q.; Zhong, C.H.; Su, Z.Q.; Li, X.Y.; Chen, Y.; Chen, X.B.; Tang, C.L.; Zhou, L.Q.; Li, S.Y. Breathing hydrogen-oxygen mixture decreases inspiratory effort in patients with tracheal stenosis. Respiration 2018, 97, 42–51. [Google Scholar] [CrossRef]
  58. Handajani, Y.S.; Tenggara, R.; Suyatna, F.D.; Surjadi, C.; Widjaja, N.T. The effect of oxygenated water in Diabetes Mellitus. Med. J. Indones. 2009, 18, 102–107. [Google Scholar] [CrossRef]
  59. Gruber, R.; Axmann, S.; Schoenberg, M.H. The influence of oxygenated water on the immune status, liver enzymes, and the generation of oxygen radicals: A prospective, randomised, blinded clinical study. Clin. Nutr. 2005, 24, 407–414. [Google Scholar] [CrossRef] [PubMed]
  60. Kiyoi, T.; Liu, S.; Takemasa, E.; Nakaoka, H.; Hato, N.; Mogi, M. Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress. PLoS ONE 2020, 15, 0227582. [Google Scholar] [CrossRef] [PubMed]
  61. Lambeth, J.D.; Neish, A.S. Nox enzymes and new thinking on reactive oxygen: A double-edged sword revisited. Annu. Rev. Pathol. Mech. Dis. 2014, 9, 119–145. [Google Scholar] [CrossRef] [PubMed]
  62. Vermot, A.; Petit-Härtlein, I.; Smith, S.M.; Fieschi, F. NADPH oxidases (NOX): An overview from discovery, molecular mechanisms to physiology and pathology. Antioxidants 2021, 10, 890. [Google Scholar] [CrossRef] [PubMed]
  63. Piantadosi, C.A. “Oxygenated” water and athletic performance. Br. J. Sports Med. 2006, 40, 740–741. [Google Scholar] [CrossRef] [PubMed]
  64. Nolfi-Donegan, D.; Braganza, A.; Shiva, S. Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement. Redox Biol. 2020, 37, 101674. [Google Scholar] [CrossRef]
  65. Yu, L.; Quinn, M.T.; Cross, A.R.; Dinauer, M.C. Gp91 phox is the heme binding subunit of the superoxide-generating NADPH oxidase. Proc. Natl. Acad. Sci. USA 1998, 95, 7993–7998. [Google Scholar] [CrossRef]
  66. Singh, R.B.; Halabi, G.; Fatima, G.; Rai, R.H.; Tarnava, A.T.; LeBaron, T.W. Molecular hydrogen as an adjuvant therapy may be associated with increased oxygen saturation and improved exercise tolerance in a COVID-19 patient. Clin. Case Rep. 2021, 9, e05039. [Google Scholar] [CrossRef]
  67. Iolascon, A.; Bianchi, P.; Andolfo, I.; Russo, R.; Barcellini, W.; Fermo, E.; Toldi, G.; Ghirardello, S.; Rees, D.; Van Wijk, R.; et al. Recommendations for diagnosis and treatment of methemoglobinemia. Am. J. Hematol. 2021, 96, 1666–1678. [Google Scholar] [CrossRef]
  68. Kawamura, T.; Wakabayashi, N.; Shigemura, N.; Huang, C.S.; Masutani, K.; Tanaka, Y.; Noda, K.; Peng, X.; Takahashi, T.; Billiar, T.R.; et al. Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 2013, 304, L646–L656. [Google Scholar] [CrossRef] [PubMed]
  69. Yu, C.; Xiao, J.H. The Keap1-Nrf2 system: A mediator between oxidative stress and aging. Oxid. Med. Cell. Longev. 2021, 2021, 6635460. [Google Scholar] [CrossRef] [PubMed]
  70. Tehrani, H.S.; Moosavi-Movahedi, A.A. Catalase and its mysteries. Prog. Biophys. Mol. Biol. 2018, 140, 5–12. [Google Scholar] [CrossRef] [PubMed]
  71. Zelko, I.N.; Mariani, T.J.; Folz, R.J. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 2002, 33, 337–349. [Google Scholar] [CrossRef] [PubMed]
  72. Zeng, J.; Zhang, M.; Sun, X. Molecular hydrogen is involved in phytohormone signaling and stress responses in plants. PLoS ONE 2013, 8, e71038. [Google Scholar] [CrossRef] [PubMed]
  73. Yu, Y.; Yang, Y.; Yang, M.; Wang, C.; Xie, K.; Yu, Y. Hydrogen gas reduces HMGB1 release in lung tissues of septic mice in an Nrf2/HO-1-dependent pathway. Int. Immunopharmacol. 2019, 69, 11–18. [Google Scholar] [CrossRef] [PubMed]
  74. Roose, B.W.; Zemerov, S.D.; Dmochowski, I.J. Xenon–protein interactions: Characterization by X-ray crystallography and Hyper-CEST NMR. Methods Enzymol. 2018, 602, 249–272. [Google Scholar]
  75. Prangé, T.; Schiltz, M.; Pernot, L.; Colloc’h, N.; Longhi, S.; Bourguet, W.; Fourme, R. Exploring hydrophobic sites in proteins with xenon or krypton. Proteins Struct. Funct. Bioinform. 1998, 30, 61–73. [Google Scholar] [CrossRef]
  76. Abraini, J.H.; Marassio, G.; David, H.N.; Vallone, B.; Prangé, T.; Colloc’h, N. Crystallographic studies with xenon and nitrous oxide provide evidence for protein-dependent processes in the mechanisms of general anesthesia. Anesthesiology 2014, 121, 1018–1027. [Google Scholar] [CrossRef]
  77. Geyer, M.; Gutierrez, R.; Mujica, V.; Silva, J.F.; Dianat, A.; Cuniberti, G. The contribution of intermolecular spin interactions to the London dispersion forces between chiral molecules. J. Chem. Phys. 2022, 156, 234106. [Google Scholar] [CrossRef]
  78. Ye, Z.; Zhang, R.; Sun, X. Bustling argon: Biological effect. Med. Gas Res. 2013, 3, 22. [Google Scholar] [CrossRef] [PubMed]
  79. Nespoli, F.; Redaelli, S.; Ruggeri, L.; Fumagalli, F.; Olivari, D.; Ristagno, G. A complete review of preclinical and clinical uses of the noble gas argon: Evidence of safety and protection. Ann. Card. Anaesth. 2019, 22, 122. [Google Scholar] [PubMed]
  80. Perov, A.Y.; Ovchinnikov, B.M.; Parusov, V.V.; Bobrovnikov, A.V.; Safronova, V.G.; Holodilin, Y.D. The method of inhalation therapy with micro-doses of mixtures noble gases with oxygen. arXiv 2021, arXiv:2111.10231. [Google Scholar]
  81. Lawrence, J.H.; Loomis, W.F.; Tobias, C.A.; Turpin, F.H. Preliminary observations on the narcotic effect of xenon with a review of values for solubilities of gases in water and oils. J. Physiol. 1946, 105, 197–204. [Google Scholar] [CrossRef]
  82. Maze, M.; Laitio, T. Neuroprotective properties of xenon. Mol. Neurobiol. 2020, 57, 118–124. [Google Scholar] [CrossRef] [PubMed]
  83. Sykes, W.S.; Lawrence, R.C. Helium in anaesthesia. Br. Med. J. 1938, 2, 448. [Google Scholar] [CrossRef]
  84. Manuilov, V.M.; Suvorov, A.V.; Kurkin, S.V.; Olenev, Y.O.; Pavlov, N.B.; Logunov, A.T.; Anikeev, D.A.; Orlov, O.I. Evaluation of the efficiency of oxygen–helium therapy for patients with Covid-19-associated pneumonia. Hum. Physiol. 2022, 48, 863–870. [Google Scholar] [CrossRef]
  85. Tilton, R.F., Jr.; Kuntz, I.D., Jr.; Petsko, G.A. Cavities in proteins: Structure of a metmyoglobin xenon complex solved to 1.9. ANG. Biochemistry 1984, 23, 2849–2857. [Google Scholar] [CrossRef]
  86. Cazade, P.A.; Meuwly, M. Oxygen migration pathways in NO-bound truncated hemoglobin. ChemPhysChem 2012, 13, 4276–4286. [Google Scholar] [CrossRef]
  87. Breck, D.W. Zeolite Molecular Sieves Structure, Chemistry and Use; John Wiley and Sons: New York, NY, USA, 1974; pp. 492–493. [Google Scholar]
  88. Freude, D. Size, mass and kinetics of molecules. In Molecular Physics; Leipzig University (Universität Leipzig): Leipzig, Germany, 2004; Available online: https://home.uni-leipzig.de/energy/freume.html (accessed on 31 January 2024).
  89. Matteucci, S.; Yampolskii, Y.; Freeman, B.D.; Pinnau, I. Transport of gases and vapors in glassy and rubbery polymers. In Materials Science of Membranes for Gas and Vapor Separation; Yampolskii, Y., Pinnau, I., Freeman, B., Eds.; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2006; pp. 1–47. [Google Scholar]
  90. Fauzi, A.; Kailash, I.; Khulbe, C.; Matsuura, T. Gas Separation Membranes: Polymeric and Inorganic; Springer: Cham, Switzerland, 2015. [Google Scholar]
  91. Almasalmeh, A.; Krenc, D.; Wu, B.; Beitz, E. Structural determinants of the hydrogen peroxide permeability of aquaporins. FEBS J. 2014, 281, 647–656. [Google Scholar] [CrossRef] [PubMed]
  92. Smith, J.; Zadeh Haghighi, H.; Salahub, D.; Simon, C. Radical pairs may play a role in xenon-induced general anesthesia. Sci. Rep. 2021, 11, 6287. [Google Scholar] [CrossRef] [PubMed]
  93. Barach, A.L. Use of helium as a new therapeutic gas. Proc. Soc. Exp. Biol. Med. 1934, 32, 462–464. [Google Scholar] [CrossRef]
  94. Oei, G.T.; Weber, N.C.; Hollmann, M.W.; Preckel, B. Cellular effects of helium in different organs. J. Am. Soc. Anesthesiol. 2010, 112, 1503–1510. [Google Scholar] [CrossRef] [PubMed]
  95. Roos, D. Chronic granulomatous disease. Br. Med. Bull. 2016, 118, 50. [Google Scholar] [CrossRef] [PubMed]
  96. Zhang, J.Y.; Liu, C.; Zhou, L.; Qu, K.; Wang, R.; Tai, M.H.; Lei, J.C.W.L.; Wu, Q.F.; Wang, Z.X. A review of hydrogen as a new medical therapy. Hepato Gastroenterol. Curr. Med. Surg. Trends 2012, 59, 1026. [Google Scholar] [CrossRef] [PubMed]
  97. Li, L.; Lou, W.; Kong, L.; Shen, W. Hydrogen commonly applicable from medicine to agriculture: From molecular mechanisms to the field. Curr. Pharm. Des. 2021, 27, 747–759. [Google Scholar] [CrossRef] [PubMed]
  98. Gupta, K.J.; Hebelstrup, K.H.; Mur, L.A.; Igamberdiev, A.U. Plant hemoglobins: Important players at the crossroads between oxygen and nitric oxide. FEBS Lett. 2011, 585, 3843–3849. [Google Scholar] [CrossRef] [PubMed]
Oxygen 04 00003 g001
Figure 1. Oxygen can be reduced to reactive oxygen species (ROS), such as superoxide anions (O2•−), hydrogen peroxide (H2O2) and the hydroxyl radical (•OH). In mitochondrial respiration, O2 is converted to water by a 4-electron reaction.
Figure 1. Oxygen can be reduced to reactive oxygen species (ROS), such as superoxide anions (O2•−), hydrogen peroxide (H2O2) and the hydroxyl radical (•OH). In mitochondrial respiration, O2 is converted to water by a 4-electron reaction.
Oxygen 04 00003 g001
Oxygen 04 00003 g002
Figure 2. Estimation of the O2 concentration in solution. On extrapolation, this gives the half-life of concentration elevation. The solution was bubbled with oxyhydrogen solution 450 mL/min for 30 min (HydroVitality™, Wakefield, UK). O2 was measured with a Clark-type electrode (Hanna Instruments, Bedfordshire, UK. Cat. #H198198). The red dotted line is an extrapolation, whilst the horizonal line represents the half-way point of increase. The arrow indicates an approximate half-life. Data representative of 3 repeats +/− SEM.
Figure 2. Estimation of the O2 concentration in solution. On extrapolation, this gives the half-life of concentration elevation. The solution was bubbled with oxyhydrogen solution 450 mL/min for 30 min (HydroVitality™, Wakefield, UK). O2 was measured with a Clark-type electrode (Hanna Instruments, Bedfordshire, UK. Cat. #H198198). The red dotted line is an extrapolation, whilst the horizonal line represents the half-way point of increase. The arrow indicates an approximate half-life. Data representative of 3 repeats +/− SEM.
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Oxygen 04 00003 g003
Figure 3. Estimation of the H2 concentration in solution after bubbling with oxyhydrogen gas 450 mL/min for 30 min (HydroVitality™, Wakefield, UK). H2 was measured with a methylene blue-based assay system (H2Blue, H2 Sciences Inc., Henderson, NV, USA). The horizontal dotted line is halfway between the maximal concentration measured and the minimal concentration expected when the solution is equilibrated with atmospheric air. The red dotted line is an extrapolation, whilst the horizonal line represents the half-way point of increase. The arrow indicates an approximate half-life Data representative of 3 repeats +/− SEM.
Figure 3. Estimation of the H2 concentration in solution after bubbling with oxyhydrogen gas 450 mL/min for 30 min (HydroVitality™, Wakefield, UK). H2 was measured with a methylene blue-based assay system (H2Blue, H2 Sciences Inc., Henderson, NV, USA). The horizontal dotted line is halfway between the maximal concentration measured and the minimal concentration expected when the solution is equilibrated with atmospheric air. The red dotted line is an extrapolation, whilst the horizonal line represents the half-way point of increase. The arrow indicates an approximate half-life Data representative of 3 repeats +/− SEM.
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Figure 4. Concentration of O2 and H2 in solution. H2 was produced using a magnesium-based tablet (Drink HRW, Oxnard, USA). The tablet was dropped into deionised water (15 MΩ∙cm2) in a non-sealed conical flask (500 mL). H2 was measured with a methylene blue-based assay system (H2Blue, H2 Sciences Inc., Henderson, NV, USA). O2 was measured using a Clark-type electrode (Hanna Instruments, Bedfordshire, UK. Cat. #H198198). Data are mean 3 repeats +/− SEM.
Figure 4. Concentration of O2 and H2 in solution. H2 was produced using a magnesium-based tablet (Drink HRW, Oxnard, USA). The tablet was dropped into deionised water (15 MΩ∙cm2) in a non-sealed conical flask (500 mL). H2 was measured with a methylene blue-based assay system (H2Blue, H2 Sciences Inc., Henderson, NV, USA). O2 was measured using a Clark-type electrode (Hanna Instruments, Bedfordshire, UK. Cat. #H198198). Data are mean 3 repeats +/− SEM.
Oxygen 04 00003 g004
Table 1. Examples of the effects seen in animals after molecular hydrogen treatment. HRS: Hydrogen-rich saline; HRW: Hydrogen-rich water MDA: Malondialdehyde; SOD: superoxide dismutase; Nrf-2: nuclear factor erythroid 2-related factor 2; HO-1: haem oxidase-1; NQO-1: NAD (P)H quinone oxidoreductase 1; VEGF: vascular endothelial growth factor; PDGF: platelet-derived growth factor; RBC: red blood cell; WBC: white blood cell; COX-2: cyclooxygenase-2; 8-oxo-dG: 8-Oxo-2′-deoxyguanosine; CLDN3: Claudin 3.
Table 1. Examples of the effects seen in animals after molecular hydrogen treatment. HRS: Hydrogen-rich saline; HRW: Hydrogen-rich water MDA: Malondialdehyde; SOD: superoxide dismutase; Nrf-2: nuclear factor erythroid 2-related factor 2; HO-1: haem oxidase-1; NQO-1: NAD (P)H quinone oxidoreductase 1; VEGF: vascular endothelial growth factor; PDGF: platelet-derived growth factor; RBC: red blood cell; WBC: white blood cell; COX-2: cyclooxygenase-2; 8-oxo-dG: 8-Oxo-2′-deoxyguanosine; CLDN3: Claudin 3.
AnimalMethod of TreatmentEffect ReportedReference
ChickensOral HRW:
H2 (1–1.5 mg/L)
(ad libitum)		Increased IgG and IgM antibodies[18]
DogsOral HRW:
H2 (1.6 mg/L)
(800 mL/day)		Improved angiogenesis and dermal thickness
Increased SOD, Nrf-2, HO-1, NQO-1, VEGF, and PDGF
Reduced MDA		[19]
Dogs Injected H2 gas
(0.2 mL/kg)		Increased RBC count, Haemoglobin and Haematocrit
Reduced haemolysis and WBC infiltration		[20]
GoatsMg supplement: either Mg(OH)2 or elemental Mg
(Mg(s) + 2H2O(l) → Mg(OH)2(s) + H2(g))		Altered rumen microflora and fermentation process (increased propionate metabolism); increased methanogenesis[21]
Guinea PigsOral HRW:
H2 (>1.6 mg/L)
(ad libitum)		Attenuated noise-induced hearing loss[22]
Guinea PigsHRS:
H2 (~1.2 mg/L)
10 mL/kg intraperitoneal + 20 µg intranasal		Increased SOD
Decreased eosinophils, IgE antibodies mucous production and MDA		[23]
HorsesNasogastric HRW:
H2 (>1 mg/L)
(2L)		Reduced accumulation of reactive oxygen metabolites[24]
HorsesIntravenous HRS:
H2 (0.6 mg/L)
(2 L)		Elevated antioxidant potential
Suppressed oxidative stress		[25]
PigsH2 gas
(2.1% in air/4 h)		Reduced COX-2 expression and 8-oxo-dG[26]
PigsHRW gavage:
H2 (1.6 mg/L)
(10 mL/kg)		Reduced intestinal leakage and toxin-induced apoptosis
Restored CLDN3 expression		[27]
Table 2. Examples of the effects seen in plants after molecular hydrogen treatment. APX: ascorbate peroxidase; CAT: catalase; POD: peroxide; SOD: superoxide dismutase.
Table 2. Examples of the effects seen in plants after molecular hydrogen treatment. APX: ascorbate peroxidase; CAT: catalase; POD: peroxide; SOD: superoxide dismutase.
PlantsMethod of TreatmentEffect ReportedReference
Alfalfa
(M. sativa L. victoria)		Seedlings incubated HRW/12 h:
H2 (0.16 mg/L)		Regulated expression of genes relevant to sulphur and glutathione metabolism; enhanced glutathione metabolism[14]
Arabidopsis thalianaHRW and H2-infused growth medium:
H2 (1.6 mg/L)		Enhanced salinity tolerance via modulation of redox homeostasis and upregulation of serotonin N-acetyltransferase and melatonin expression[28]
Arabidopsis thalianaHRW and H2-infused growth medium:
H2 (0.16, 0.4, 0.8, 1.2 and 1.6 mg/L)		Enhanced salinity tolerance via modulation of redox homeostasis and reduced ion influx through modulation of H+ pump and antiporter activity[29]
Bananas
(Musa spp. AAA cv. Baxijiao)		Soaked in HRW/10 min:
H2 (0.8 mg/L)		Delayed ripening as a result of repressed ethylene signalling and respiration[17]
Barley
(Hordeum distichum L.)		Soaked in HRW/6 h:
H2 (2 mg/L)		Increased concentration of vanillic acid, coumaric acid, sinapic acid, Ca and Fe, significantly increasing the germination and growth rates[15]
Cabbage
(Brassica campestris spp. Chinensis L.)		Seeds soaked in HRW/3 h before germination:
H2 (0.8 mg/L)		Improved Cd tolerance, associated with reduced Cd uptake and increased antioxidant defences[13]
Chives
(Allium tuberosum Rottler ex Spreng.)		H2 gas in packaging:
H2 (1%, 2%, 3%)		Here, 3% H2 significantly delayed post-harvest ripening via enhancing antioxidant capacity (APX, CAT, POD, SOD)[30]
Kiwi fruit
(Actinidia deliciosa, cv. Hayward)		Soaked in HRW/5 min:
H2 (1.2 mg/L)		Alleviated chilling injury through enhanced sugar production and repressed peroxidation[31]
Rice
(Oryza sativa L.)		HNW in irrigation system:
H2 (1 mg/L)		Increased grain size, weight and yield; decreased amylose content; significantly reduced Cd accumulation[32]
Strawberries
(variety unknown)		H2 gas in the packaging atmosphere
(10% CO2, 4% H2, 86% N2)		Extended the shelf-life (300–500%) by moderating antioxidant responses[16]
Table 3. Some of the possible mechanisms of the action of H2 in cells and tissues.
Table 3. Some of the possible mechanisms of the action of H2 in cells and tissues.
Proposed MechanismCommentReference(s)
Direct scavenging of •OH•OH will be derived from O2. Little evidence of other ROS scavenged[9]
Scavenging of other ROS, e.g., H2O2Evidence suggests that this is not a possible mechanism[9]
Action of nanobubblesCould remove •OH, hypochlorite (ClO–), ONOO– and O2•−[39]
Scavenging of reactive nitrogen speciesEvidence suggests that this is not a possible mechanism, e.g., no reaction with nitric oxide (NO) for example.[9]
Scavenging •OH in a mechanism that involves haemWould explain some of the effects reported[40,41,42]
Effects mediated by haem oxidase, e.g., HO-1May involve carbon monoxide (CO)-mediated signalling[43]
Effects mediated by the redox poise of the H2 coupleSuggested as a widely used mechanism, but no evidence[44,45]
H2 spin stateSpin states mediate interactions with other biomolecules. No direct evidence for this[46]
Acting through protein hydrophobic polypeptide pocketsGlobin proteins are a model system, where several inert gases are known to interact[47,48]
H2 and O2 interactingMay account for some of the effects seen?Discussed here
Table 4. The gaseous and inferred output of alkaline water electrolysis. Gas was prepared from a HydroVitality™ (Wakefield, UK) oxyhydrogen generator, collected in a Tedlar®® bag and sent for analysis to SGS Gas Analysis Services (Bristol, UK). N/D means not detected.
Table 4. The gaseous and inferred output of alkaline water electrolysis. Gas was prepared from a HydroVitality™ (Wakefield, UK) oxyhydrogen generator, collected in a Tedlar®® bag and sent for analysis to SGS Gas Analysis Services (Bristol, UK). N/D means not detected.
CompoundMeasured Results (%)Inferred Output Percentage (%)
H2 60.16%66%
O2 29.50%33%
N2 10.32%Not expected
Methane (CH4)0.01%0.01%
Carbon dioxide (CO2)0.01%0.01%
Carbon monoxide (CO)N/DN/D
Table 5. Calculated percentages of gases inhaled by the average female and male (www.worlddata.info: accessed 31 January 2024) when breathing regularly. Normal air is compared to oxyhydrogen and hydrogen-only breathing. To match the flow rate of the HydroVitality™ oxyhydrogen generator, a flow rate of pure H2, 300 mL/min was used.
Table 5. Calculated percentages of gases inhaled by the average female and male (www.worlddata.info: accessed 31 January 2024) when breathing regularly. Normal air is compared to oxyhydrogen and hydrogen-only breathing. To match the flow rate of the HydroVitality™ oxyhydrogen generator, a flow rate of pure H2, 300 mL/min was used.
Air
(250 mL/s)		Oxyhydrogen
(450 mL/min)		H2-Only
(300 mL/min)
(% inhaled)O2H2O2H2O2H2
Female21%<0.0001%22.27%2.5%~19%2.5%
Male21%<0.0001%22.01%2%~19.5%2%
Table 6. Examples of some of the effects of oxygenated or hydrogenated water treatments on broiler chickens as they developed (data from Shin et al. [18]).
Table 6. Examples of some of the effects of oxygenated or hydrogenated water treatments on broiler chickens as they developed (data from Shin et al. [18]).
Treatment GivenCharacteristic MeasuredEffect Seen
Oxygenated waterWeightSignificantly increased
Body mass indexSignificantly reduced abdominal fat accumulation
TriacylglycerideReduced
Total cholesterolReduced
LDL cholesterolReduced
IgG antibodiesSignificantly increased
IgM antibodiesSignificantly increased
Hydrogenated waterWeightIncreased
Body mass indexNon-significant reduction
TriacylglycerideReduced
Total cholesterolReduced
LDL cholesterolReduced
IgG antibodiesIncreased
IgM antibodiesIncreased
Table 7. The mass and diameter of gaseous substances, noting physical properties, atomic mass and kinetic diameters of biomolecules.
Table 7. The mass and diameter of gaseous substances, noting physical properties, atomic mass and kinetic diameters of biomolecules.
SubstanceChemical SymbolMolecular Mass (g/mol)Kinetic Diameter (nm)Reference
ArgonAr400.34[87]
Carbon dioxideCO2440.33[88]
Carbon monoxideCO280.37[89]
HeliumHe40.26[89]
HydrogenH220.28[88]
KryptonKr840.36[87]
NeonNe200.28[87]
NitrogenN2280.36[90]
Nitric oxideNO300.32[89]
OxygenO2320.35[88]
XenonXe1310.40[87]
WaterH2O180.20[91]
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Russell, G.; May, J.; Hancock, J.T. An Interplay of Gases: Oxygen and Hydrogen in Biological Systems. Oxygen 2024, 4, 37-52. https://doi.org/10.3390/oxygen4010003

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Russell G, May J, Hancock JT. An Interplay of Gases: Oxygen and Hydrogen in Biological Systems. Oxygen. 2024; 4(1):37-52. https://doi.org/10.3390/oxygen4010003

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Russell, Grace, Jennifer May, and John T. Hancock. 2024. "An Interplay of Gases: Oxygen and Hydrogen in Biological Systems" Oxygen 4, no. 1: 37-52. https://doi.org/10.3390/oxygen4010003

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