Are Small Hydrogen-Containing Gas Molecules Essential for Maintaining Low Deuterium in Mitochondrial Water?

1. Introduction

Deuterium is a heavy isotope of hydrogen, having a neutron as well as a proton in the nucleus, making it essentially twice as heavy. Deuterium is pervasive in nature, found in seawater at a concentration of 156 parts per million (ppm). There is a growing awareness in the research community that deuterium has biological properties that can disrupt metabolism [1]. In particular, the ATPase nanomotors in the mitochondria are very sensitive to deuterium, as it reduces the efficiency of ATP production and increases the release of reactive oxygen species (ROS) [2]. While 156 ppm may seem rare, because hydrogen is so common, deuterium levels in the blood are five or six times as high as calcium levels.

Human metabolic policy has evolved to have a sophisticated strategy for maintaining very low deuterium in the mitochondrial intermembrane space, to minimize exposure of the ATPase nanomotors to deuterium. The process likely depends heavily on the gut microbiome to recycle nutrients by producing abundant molecular hydrogen gas and using the hydrogen gas as a reducing agent to create short chain fatty acids (SCFAs) and methyl groups to fuel methylation pathways [3]. It has been suggested that these microbial products based on recycling hydrogen gas would likely have significantly reduced deuterium content, due to the properties of specialized flavoproteins that exploit proton tunneling, which deuterons are less capable of, and to the fact that deuterium tends to stay behind in the aqueous phase when a gas is formed [4].

A seminal paper published in 1961 showed that molecular hydrogen produced by a marine pseudomonad contained 80% less deuterium than the deuterium fraction in the seawater [5]. The hydrogenase enzyme expressed by this microbe bears significant homology with hydrogenase enzymes expressed by multiple microbes inhabiting the human gut. It is therefore likely that the hydrogen gas produced in the gut is similarly depleted in deuterium.

Interestingly, methane gas formed at high temperature during volcanic eruptions has also been found to be extremely depleted in deuterium [6]. This suggests a pattern that is plausible, given that deuterium is twice as heavy as hydrogen. Like methane and molecular hydrogen, trimethylamine (TMA) is also a gas at room temperature. This may mean that it is also naturally reduced in deuterium content, since we believe this may be a general property of hydrogen-containing gases, due to deuterium’s tendency to resist leaving the aqueous phase. We will have more to say about TMA later in this paper, since gut microbes produce it from dietary sources such as L-carnitine, choline, and betaine. Other hydrogen-containing gases produced by gut microbes include hydrogen sulfide (H₂S), hydrogen peroxide (H₂O₂), methanethiol (CH₃SH), and dimethyl sulfide (CH₃) ₂SH. Might it be the case that all these small molecular gases are enriched in ¹H? Furthermore, does human biology exploit this property? This is the topic that this paper addresses.

Protons bound to carbon, nitrogen, oxygen, and sulfur differ in their ability to freely exchange with deuterons in the medium, and their ability to exchange also depends on the surrounding molecular context. Protons bound to oxygen in carboxyl groups or hydroxyl groups exchange with deuterons from the aqueous medium much more readily than protons bound to nitrogen in amide groups, such as the C(=O)NH₂ amide group in glutamine [7]. Glutamine is the most abundant free amino acid in human plasma [8], and it plays a powerful role in protecting from ammonia toxicity [9,10]. We will present an argument in this paper that glutamine’s amide group may be enriched in ¹H, a feature that may be exploited by human biology.

Generally, carbon atoms are very good at holding onto protons. The protons bound to carbon atoms in organic molecules for the most part remain securely bound and never exchange with deuterons or protons from the aqueous medium, although there are some interesting exceptions such as the protons in bis-allylic carbon atoms and in rings containing carbon and nitrogen, such as the pyrrolidine ring in proline and the imidazole ring in histidine [11].

2. Methanogens, Methanotrophs, Methylotrophs, and Trimethylamine

Gut microbes ferment complex carbohydrates such as dietary fiber into small organic molecules such as glucose and formate that are then metabolized anaerobically to produce hydrogen gas [12]. This hydrogen gas can then be used by methanogens (anaerobic archaea) to generate methane gas directly by reduction of carbon dioxide (CO₂). [13]. For an excellent review on the unique metabolic profile of methanogenic archaea, please see Costa and Whitman (2023) [14]. Methane oxidizing genes are a common feature of the human gut microbiome. Methanotrophs such as Methylocystis intestini help reduce methane content in the gut while producing useful energy in the form of nicotinamide adenine dinucleotide (NADH) [15]. Due to its origins, this NADH is likely a good source of ¹H rather than ²H protons released into metabolic water by the action of NADH dehydrogenase in the mitochondria.

Methylotrophs metabolize methylated amines, most notably, TMA, to produce formaldehyde (derived from the methyl groups) and ammonia as a byproduct. Formaldehyde can then be processed through assimilatory pathways to yield biomass or through dissimilatory pathways, producing carbon dioxide gas and useful energy as NADH [16]. Methylotrophs significantly outcompete methanogens for hydrogen gas. Methylotrophs have a much lower threshold for hydrogen gas, consuming it at partial pressures as low as 0.1 Pascal, whereas methanogens require a range from 2.8 to 10 Pascals [17]. The activity of methylotrophs is thus primarily limited by the availability of methyl groups. The proton in NADH that they produce is unlikely to be a deuteron.

Together with Professor László Boros, I recently published a paper hypothesizing that hydrogen gas produced by gut microbes plays an essential role in reducing the deuterium load in host mitochondria. Furthermore, we argued that trimethylamine oxide (TMAO) a metabolite produced by the liver through oxidation of TMA, may serve as a marker for deuterium overload in the mitochondria, systemically [18]. TMA that remains unmetabolized by gut microbes is an indicator of a deficiency in methylotrophs. Methylotrophs likely play a powerful role in supplying deupleted methyl groups to the methylation pathways. Therefore, elevated TMAO is a potential indicator of deuterium overload in the mitochondria as well as a disrupted microbiome. TMAO has been well recognized as a risk factor for cardiovascular disease as well as several other chronic diseases [19,20].

3. Do Hydrogenases Produce Low-Deuterium Molecular Hydrogen?

Hydrogenase enzymes are found in thousands of genomes from over 30 different phyla [21,22]. They are common in aquatic, terrestrial, and even host-associated ecosystems. They are all metalloenzymes that use iron and nickel as catalysts, with [NiFe]-hydrogenases, [FeFe]-hydrogenases and, less commonly, [Fe]-hydrogenases all having been identified. Their reversible reaction catalyzes the oxidation of molecular hydrogen (H₂) into 2H⁺ and 2e^-. It has been hypothesized that, during prehistoric times on Earth, the atmosphere was rich in hydrogen gas, which has been proposed to have been the first electron donor leading to ATP synthesis. H₂ is ubiquitous throughout the biosphere, and it readily diffuses through microbial cells and has a low activation energy requirement. H₂ metabolizing microbes are keystone species driving carbon cycling along oxygen gradients in hydrogen-rich soil ecosystems [23].

The reaction catalyzed by hydrogenases is reversible, and our interest is in the use of this enzyme to produce molecular hydrogen from protons and electrons by gut microbes. As documented in a paper published in 1961, MI Krichevsky et al. studied a hydrogenase produced by a marine pseudomonad species and found that it was capable of fractionating hydrogen isotopes to a remarkable degree when producing hydrogen gas. The hydrogen gas it produced was greatly depleted in deuterium compared to ocean water, having a deuterium content that was reduced by a factor of five. This microbe produced molecular hydrogen when it was grown with glucose, pyruvate, or formate as nutrients [5]. While the species investigated was a marine microbe rather than a colonizer of the human gut, its hydrogenase closely resembles hydrogenases synthesized by gut microbes. Furthermore, other studies have shown that hydrogenase enzymes can have extremely high deuterium kinetic isotope effects (KIEs), as high as 43 in an acidic environment [24]. Part of the reason for this may be the biophysical property that deuterium, being twice as heavy, tends to stay behind in solution in the water and resists the gas phase.

4. Microbial Production and Utilization of Hydrogen Gas

Molecular hydrogen is increasingly being recognized as a therapy that has widespread benefits. Yıldız et al., in a review paper published in 2025, wrote in the abstract: “H₂ demonstrates numerous biologically therapeutic properties, including anti-inflammatory, antioxidant, anti-cancer, anti-stress, anti-apoptotic, anti-allergic effects, signaling molecule functions, regulation of redox balance, modulation of antioxidant enzyme gene expression, improvement of blood vessel function, down-regulation of pro-inflammatory cytokines, stimulation of energy metabolism, and protection of the nervous system” [25].

A metagenomics survey revealed that Firmicutes and Bacteroides are the most prevalent hydrogen-producing bacteria in the human large intestine, accounting for 92% of the bacterial species [26,27]. These species break down dietary fiber into small molecules and then extract hydrogen atoms from those molecules. Interestingly, Bacteroides fragilis, an important symbiont, anchor themselves in the colonic mucosa, mediated by immunoglobulin A, where they constantly produce hydrogen gas and also protect from pathogens [26,28]. Every day, as many as ten liters of hydrogen gas are produced in the human intestines [29]. A study based in Japan showed that patients with dementia had a reduced Bacteroides population compared to controls [30].

Seventy percent of the microbial species in the gut have the capacity to metabolize H₂. The three major pathways for disposal of H₂ in the gut are methanogenesis, acetogenesis and sulfate reduction, with methanogenic archaea being the most abundant in the healthy gut [31]. Hydrogen gas that is not captured by the microbes freely enters the blood stream, and it easily penetrates the blood-brain barrier, where it may serve a role in protecting from oxidative stress [26].

Anaerobic metabolism of non-digestible fiber by human gut microbes converts complex carbohydrates into carbon dioxide and hydrogen gas, that, as we have argued, is likely severely depleted in deuterium. Methanogenic archaea then use the hydrogen gas to reduce carbon dioxide to methane gas. This prevents acidification of the gut and facilitates the breakdown of complex carbohydrates, fibers, and resistant starches, increasing the total energy harvest from food [32]. Methanobrevibacter smithii is the dominant archaeon in the human gut, accounting for over 90% of the archaeal species in the gut [33]. If, as we argue, the hydrogen gas has lost 80% of the deuterium present in the aquatic medium, then the methane gas will also be significantly depleted in deuterium.

Methane is further processed in the gut in order to capture the gas as a significant nutrient. After monooxygenase converts methane to methanol, two subsequent steps involving microbial dehydrogenases transform methanol into formaldehyde and, finally, formate, where NAD⁺ picks up ¹H to become NADH (which is then a source of ¹H to fuel the ATPase nanomotors).

methane → methanol → formaldehyde → formate

Formate is widespread as a respiratory energy source in natural habitats [34]. Campylobacter jejuni, a common cause of diarrhea, has a periplasmic formate dehydrogenase enzyme which allows formate oxidation to be coupled with microaerobic oxygen reduction to water [35]. The water is released in the periplasmic space rather than the cytoplasm, and the outer membrane of the bacterium is highly permeable to water. This essentially means that the reaction delivers deuterium-depleted water to the host’s gut lumen.

In addition to its conversion to formate via a dehydrogenase, formaldehyde can also be a source of methyl groups in the methylation pathways. A study investigating metabolism of a methylotrophic bacterium, Methylobacterium extorquens, when fed methanol as a fuel source, revealed that methanol was reduced to formaldehyde which was then utilized to synthesize methylene tetrahydrofolate from tetrahydrofolate, either through a direct nonenzymatic transfer of the methylene group from formaldehyde to tetrahydrofolate, or through an indirect metabolic pathway involving several steps [36]. Formaldehyde is highly reactive, and, by being directed down these pathways, it can be quickly metabolized prior to causing harm through other spontaneous reactions. However, a further implication is that the methyl groups in methyltetrahydrofolate will be enriched in ¹H. The fact that methylation pathways are so important in biology may be connected to their ability to deliver ¹H to mitochondria [3,18].

Acetogenic bacteria also use the hydrogen gas to produce acetate from carbon dioxide, according to the Wood-Ljungdahl pathway:

2CO₂ +4H₂ → CH₃COO^- +H⁺ + 2H₂O

Both the acetate and the water that are produced in this way are likely to be enriched in ¹H. Acetate is a precursor to the four-carbon fatty acid, butyrate, the primary nutrient consumed by the colonocytes lining the gut barrier [37]. Butyrate stimulates human enteroendocrine L-cells to release glucagon-like peptide-1 (GLP-1), the hormone that suppresses appetite to maintain an ideal body weight [38]. GLP-1 analogues (receptor agonists) are widely prescribed to help people lose weight [39]. Butyrate’s beneficial effects in the gut may be primarily due to its low deuterium content.

Finally, hydrogen gas is also used by sulfate-reducing bacteria such as Desulfovibrio sp. to reduce sulfate to sulfide according to the following equation: [40].

4H₂ + SO₄^-2 + H⁺ → HS^- + 4H₂O

The four water molecules are likely enriched in ¹H compared to ²H. Mitochondria eagerly take up H₂S (a gas) and oxidize it through the enzyme sulfide quinone oxidoreductase (SQR), which donates electrons to the electron transport chain (ETC) to produce ATP, while releasing (preferentially ¹H?) protons into the mitochondrial water [41]. We will return to a discussion on H₂S in a later section.

We predict that these products of molecular hydrogen metabolism - acetate, butyrate, methane and its derivatives, including formaldehyde, formate, and the methyl groups carried by the methylation pathways, as well as the water produced in reactions involving oxygen reduction, will all be sources of ¹H that can be used to fuel mitochondria in the human host cells.

5. Methane Gas: A Broader View

Many toxic metal and chemical exposures can alter the composition of the gut mi- crobiome, including pesticides, toxic metals, persistent organic polutants, and artificial sweeteners [42]. Gut dysbiosis results in a decrease in beneficial microbiota, such as Faecalibacterium and Roseburia, along with an increase in pathogenic species, such as adherent-invasive Escherichia coli (AIEC). This results in a reduction in SCFAs along with an increase in detrimental microbial metabolites [43]. Pathogen overgrowth triggers the immune system to release ROS to neutralize the threat, but this can also be harmful to host cells [44].

Excessive methane in the breath, linked to constipation issues, is considered to be a marker for gut dysbiosis, whereby the metabolism of methane gas cannot keep up with its production by methanogenic archaea [45]. However, since 2012, there has been growing interest in the idea of the potential for methane gas administration to have therapeutic benefits. Beneficial effects have been shown in treating ischemia and reperfusion injury, as well as inflammatory diseases in general. It appears to have anti-inflammatory effects and to protect cells from apoptosis [46,47]. This could be simply because it is a low-deuterium nutrient.

While it had long been believed that methane is primarily synthesized by methanogenic archaea, which reduce carbon dioxide using hydrogen gas as a reducing agent, it has recently become clear that methane can also be generated nonenzymatically from certain common compounds containing methyl groups bound to sulfur or nitrogen atoms. This abiotic mechanism, catalyzed by light and heat, occurs in all living organisms, including cyanobacteria, algae, fungi, plants, and mammals, and it can even take place outside of living cells [48].

Most notably, dimethyl sulfoxide (DMSO) and methionine can be spontaneously oxidized to produce methane under conditions of oxidative stress. Keppler et al. wrote in 2023: “In summary, the reaction of methylated sulfur compounds, such as DMSO and methionine with Fenton-type chemistry involving ROS, carbonate radicals, or oxo-iron(IV) results in the formation of methyl radicals (•CH₃), of which a portion reacts to CH₄ through abstraction of a hydrogen atom from hydrocarbons, hydrogen peroxide, or hydrogen carbonate” [49]. In an earlier paper by the same authors, it was stated that “An in-depth understanding of the various factors that control cellular methane formation and consumption and a thorough understanding of its dual function and bioactivity may bring significant benefits to the domain of human health and disease” [50].

It is intriguing that, under conditions of oxidative stress, which develops in response to pathogen overgrowth, methane can be regenerated nonenzymatically from methylated nitrogen or sulfur sourced from common organic molecules, via the Fenton reaction. This may be especially important because oxidative stress interferes with the enzymatic production of methane by strict anaerobes [51].

6. Does Hydrogen Peroxide Play a Central Role in Deuterium Homeostasis?

Hydrogen peroxide (H₂O₂) production, signaling, and detoxification are central in metabolism, and H₂O₂ acts as a vital second messenger in cellular processes, including growth, inflammation, and wound healing, but it can also cause damage at high levels. Key enzymes like NADPH oxidases (NOX) generate superoxide (O₂•^-) from oxygen [52], and superoxide dismutase (SOD) isoforms convert O₂•^- to H₂O₂ [53]. Catalase and glutathione peroxidase break down H₂O₂, maintaining a balance that dictates whether H₂O₂ signals or damages, impacting gene expression, cell fate, and mitochondrial function [54].

Metabolically generated H₂O₂ has been identified as a central hub in redox signaling and oxidative stress. Major sources include the NOX proteins and complex III of the mitochondrial respiratory chain. H₂O₂ is also generated in the peroxisome during fatty acid oxidation. H₂O₂ serves as a second messenger in insulin signaling and several signaling cascades induced by growth factors [55]. H₂O₂, as a small gas molecule, can easily cross lipid membranes, and its transport across membranes is also facilitated by aquaporins, especially peroxiporins [56]. Peroxiporins are dysregulated in cancer and other diseases associated with inflammatory conditions [57]. O₂•^-, produced in Complex I and Complex III of the respiratory chain, is rapidly dismutated to H₂O₂ and oxygen by mitochondrial superoxide dismutase (SOD2) [58]. H₂O₂, in turn, is metabolized to two molecules of water (DDW) by glutathione peroxidase and peroxiredoxins, particularly PRDX3 [59,60,61,62].

The point we want to emphasize here is that H₂O₂ is a gas, and therefore it is likely to be deupleted. We hypothesize that it essentially selectively removes hydrogen rather than deuterium from the aqueous medium where it is formed, and then it can migrate to another locale where it deposits the deupleted protons that it extracted, as metabolic water. In this way, it fractionates deuterium out and delivers DDW, whenever it is reduced by catalase, glutathione peroxidase, or peroxiredoxins.

Various signaling events due to stressors trigger ROS generation by NADPH oxidases (NOX), whose primary and deliberate product is O₂•^-. Adequate antioxidant systems are essential for protecting cells from tissue damage due to overabundant ROS [63]. Under physiological conditions, a baseline, normal level of ROS formation supports a wide range of processes. NOX is no longer viewed as solely involved with phagocyte antimicrobial activity, but rather as being vital to broad cellular functions and homeostasis [64]. Tissue damage associated with many diseases is attributed to an imbalance between ROS formation and its clearance via antioxidant defenses.

6.1. NOX2 and SOD3

NOX2 is a key enzyme that promotes atherosclerosis, due to the fact that it releases O₂•^- into the extracellular space. O₂•^- spontaneously dismutates to produce H₂O₂ and O₂. NOX2 was originally identified in phagocytes, where it generates the respiratory burst to kill pathogens [65]. Subsequent studies have demonstrated that NOX2 is also expressed in endothelial cells, cardiomyocytes, hematopoietic stem cells, and platelets [66]. Interestingly, macrophages release exosomes containing NOX2 that can then deliver NOX2 to distant cells [67].

Via NOX2, macrophages and endothelial cells release O₂•^- into the vascular wall, where it can react with nitric oxide (NO) to form peroxynitrite (ONOO^-), a highly reactive but short-lived molecule that causes widespread tissue damage by initiating lipid peroxidation chain reactions. The rate constant of the reaction of O₂•^- with NO to form peroxynitrite is 10,000 times faster than spontaneous dismutation [67].

SOD enzymes play an essential role in accelerating the rate of conversion of O₂•^- to H₂O₂, to reduce exposure to ONOO^-. SOD-enhanced catalysis of the dismutation reaction increases the rate of dismutation by 10,000-fold or more [68]. SOD3 (also known as EC-SOD) is the only SOD isoform found predominantly in the extracellular space, anchored to the extracellular matrix through a heparin-binding domain. It is secreted by vascular smooth muscle cells and fibroblasts. It catalyzes the dismutation of extracellular O₂•^- into H₂O₂ and O₂.

O₂•^- cannot cross plasma membranes due to its negative charge, but H₂O₂ can freely enter the cell via diffusion. Furthermore, aquaporins, especially AQP8, actively support the uptake of H₂O₂ by the cells [67]. Internally, H₂O₂ also crosses membranes to reach the peroxisome and the mitochondria. In the peroxisome, it is rapidly converted to oxygen and water through catalase:

2H₂O₂ → 2H₂O + O₂

In the mitochondria, glutathione peroxidase converts H₂O₂ to two molecules of DDW:

2GSH + H₂O₂ → GSSG + 2H₂O

Thioredoxin catalyzes a similar reaction which results in a disulfide bond between two of its cysteine residues. Since H₂O₂ is a gas, it is highly likely that its protons are predominantly ¹H rather than ²H. When H₂O₂ is formed from O₂•^- in the extracellular space, we propose that deuterons are much less likely to leave the aqueous medium, just as is the case for H₂. Thus, the import of H₂O₂ into the cell is a clever way to decrease the deuterium content in the interior while increasing it in the extracellular space. We hypothesize that both the peroxisomes and the mitochondria capitalize on this feature to synthesize DDW. However, we are unaware of any studies that have measured the amount of deuterium in metabolically produced H₂O₂.

SOD3 critically depends on copper as a catalyst, and the copper transporter ATP7A plays an essential role in providing copper to SOD3. Interestingly, in experiments on diabetic mice, it was shown that ATP7A depends on insulin signaling. The studies on these mice showed that ATP7A activity is severely reduced, and this is associated with increased oxidative stress and increased levels of O₂•^- in the vasculature, along with endothelial dysfunction. External administration of insulin ameliorated the effect. In humans, copper deficiency and/or impaired copper transport are associated with cardiac hypertrophy, heart failure, ischemic heart disease, and diabetes mellitus cardiomyopathy [69].

Ceruloplasmin is a protein that is synthesized in the liver and released into the circulation. It is a multi-copper oxidase enzyme, and copper incorporation is crucial for its ability to oxidize Fe⁺² (which triggers the Fenton reaction when H₂O₂ is present) to the nontoxic form, Fe⁺³, which can then be stored in transferrin for safe transport. Copper deficiency would thus also impair ceruloplasmin function, greatly increasing the risk of damage due to the Fenton reaction when H₂O₂ reacts with Fe⁺² [70].

6.2. NOX4

NOX4 is the only NOX isoform that is expressed at the mitochondrial membrane, and it is primarily localized to internal membranes, including in the mitochondria, the ER, and the nuclear envelope. NOX4 is unique among NADPH oxidases because it primarily directly produces H₂O₂ rather than O₂•^-, due to a longer E-loop (the third extracytosolic loop) with a histidine residue that acts as a proton source to facilitate the dismutation of O₂•^- [71]. NOX4 is upregulated in cardiomyocytes under stress conditions such as pressure overload or hypoxia. Nox4-null mice developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload [72]. NOX4 expression is correlated with aging and increased in human atherosclerotic arteries [73].

NOX4 has significant anti-atherosclerotic functions. Deletion or inhibition of NOX4 often leads to increased inflammation and atherosclerosis. Endothelial NOX4 promotes angiogenesis and recovery from hypoxia [74]. In a mouse model of ischemia/reperfusion injury, NOX4 knockout surprisingly revealed that NOX4, despite producing ROS, protects the heart from damage during ischemia [75]. Expression of vascular NOX4 is increased in association with hypertension, atherosclerosis, diabetes, restenosis, ischemia reperfusion injury, fibrosis, vascular aging, and pulmonary hypertension [74]. It is conceivable that all these conditions are associated with excess deuterium in the mitochondria, and that NOX4 helps to ameliorate that problem.

Figure 1 illustrates schematically the processes by which the cell is able to import H₂O₂ into the mitochondria and the peroxisome as a way to deliver ¹H to these organelles.

6.3. NOX4 in Cancer Cells

NOX4 acts as a key cellular energy sensor, linking mitochondrial ATP levels to metabolic states, especially during stress adaptation and in drug resistance in tumor cells. NOX4 is upregulated in many types of cancer, and this leads to chemotherapy drug-resistance in ovarian cancer cells [76]. A well-designed study investigating the role of NOX4 upregulation in renal carcinoma cells revealed that NOX4 in these cells is localized to the mitochondria, and it is inactivated by ATP, through ATP binding to a Walker A binding motif (AXXXXGKT). None of the other NOX isoforms have this motif. When cancer cells switch to aerobic glycolysis as a source of ATP, ATP levels drop in the mitochondria, and this leads to increased production of ROS via NOX4 activation. Pyruvate kinase catalyzes the last step within glycolysis. Pyruvate kinase M2 (PKM2) is the main isoform expressed in cancer cells. Lysine acetylation mediates its clearance through lysosomal degradation. The ROS generated through NOX4 expression suppress acetylation, thereby protecting it from degradation [77]. PKM2 promotes cancer cell migration and invasion, by redirecting metabolism towards the Warburg effect [78]. It is conceivable that deuterium overload impairs mitochondrial ATP synthesis in cancer cells, leading to upregulation of NOX4, both as a source of ROS and as a potential source of DDW.

7. Hydrogen Sulfide Gas

Hydrogen sulfide (H₂S) has long been recognized as a toxic and dangerous gas, but it was much more recently that it also became recognized as an endogenous regulatory transmitter. H₂S, produced by several human enzymes, affects a variety of biological processes, including transcriptional and posttranslational gene modification, activation of signaling pathways, and alterations in metabolic processes. New efforts are being considered around the use of H₂S as a gas transmitter with therapeutic drug potential [79].

7.1. Sulfate Reducing Bacteria in the Gut

Desulfovibrio (DSV) are a resident commensal bacterium present in the human gastrointestinal (GI) tract, and the most common sulfur-reducing bacterium. They are known to be opportunistic pathogens that frequently overgrow under dysbiotic conditions, e.g., in association with inflammatory bowel disease (IBD) [80] and neurodegenerative diseases such as Parkinson’s disease (PD) [81]. They are a gram-negative bacterium that produces H₂S via the dissimilatory sulfate reduction pathway, using molecular hydrogen as a reducing agent [82]. The nematode Caenorhabditis elegans exhibits dietary preferences, often favoring bacterial strains that support its growth and health [83]. In an elegant study involving a transgenic strain of C. elegans which expresses human α-synuclein, researchers found that, when presented with DSV strains from various sources as food, C. elegans greatly preferred the environmental strain D. vulgaris DSM 644 over two human isolates, one of which was obtained from a PD patient. Furthermore, colonization with the environmental strain led to reduced aggregation of α-synuclein, reduced oxidative stress, and increased lifespan (by a median of 36 days). The authors proposed that sublethal stressors may activate protective molecular pathways such as antioxidant defenses that increase the capacity of the host to cope with toxic exposures that could cause cellular damage [84].

Desulfovibrio strains are generally considered to be strict anaerobes. however, interestingly, it has been found that a strain of Desulfovibrio (D. vulgaris Hildenborough) obtained from anoxic ditch sludge could tolerate up to 0.04% oxygen in the medium, which could even support their growth. When exposed to an oxygen gradient, these microbes are drawn to an optimal location where the oxygen levels are favorable to growth, i.e., a hypoxic but not an anoxic environment [85]. This suggests that they colonize the gut wall where the environment is hypoxic, and they may serve a useful role in clearing the oxygen present in the gut wall, where there is a strong oxygen gradient, benefiting other strict anaerobes colonizing the gut. Singhal et al. noted in a paper published in 2020 that the richly vascularized subepithelial mucosa harbor significantly more oxygen than the anaerobic gut lumen, especially in the more distal, colonic regions of the GI tract [86]. These authors wrote: “The pO₂ drops precipitously along the radial axis from the intestinal submucosa to the lumen, which is home to trillions of anaerobic microbes” [86].

D. vulgaris Hildenborough possess membrane-bound terminal oxygen reductases (members of the respiratory cytochrome bd family) that serve as an oxygen scavenger [87,88]. They also express soluble rubredoxin oxidases, which reduce oxygen to water, coupling NADH oxidation to oxygen consumption. Oxygen exposure increases their resistance to killing by H₂O₂ [89]. Bacterioferritin plays a crucial role in protecting D. vulgaris Hildenborough from oxygen toxicity, by sequestering iron. Its gene expression increases dramatically upon oxygen exposure. It also protects both the host cells and other microbes from oxidative stress due to the Fenton reaction [90].

7.2. Mitochondria Metabolize H₂S

Sulfate-reducing bacteria, common in shallow marine sediments and hydrothermal vents, utilize sulfate as the terminal electron acceptor rather than oxygen, and generate H₂S as the main respiratory by-product. The endosymbiotic hypothesis of mitochondrial origin proposes that mitochondria originate from the integration of an endosymbiotic bacterium into the host cell, a primordial Asgard archaeon [91]. This may be why eukaryotic mitochondria are able to oxidize H₂S efficiently to generate ATP. A mitochondrial sulfide oxidation pathway rapidly eliminates H₂S by oxidizing it to sulfite and thiosulfate. Sulfide quinone oxidoreductase (SQR) and persulfide dioxidase, operating in tandem, yield sulfite from H₂S. The overall equation for the reaction is:

Quinone + H₂S + O₂ + H₂O → Quinol + Sulfite + H⁺

where one proton is picked up by the quinone in the inner membrane and the other is released into the aqueous medium. Both should be deupleted, given that H₂S is a gas, derived from molecular hydrogen produced by anaerobic hydrogenogens. Glutathione is involved as an intermediary to temporarily form S-sulfanylglutathione (GSSH). Sulfite is then oxidized to sulfate by sulfite oxidase or to thiosulfate by rhodenase. The electrons that are released by SQR are transferred to the ETC at complex III, promoting ATP synthesis [92].

7.3. H₂S Is a Powerful Signaling Gas

Occupational exposure to high levels of H₂S is toxic, because H₂S blocks the respiratory chain by inhibiting cytochrome c oxidase. However, H₂S is an endogenous signaling gas, acting as a cytoprotectant and promoting cardiovascular homeostasis and health [93]. It is produced in human cells primarily by three key enzymes: cystathionine β-synthase (CBS), Cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), using the sulfur-containing amino acids, L-cysteine and homocysteine as substrates [94].

The administration of physiological or pharmacological levels of H₂S attenuates myocardial injury, protects blood vessels, limits inflammation, and regulates blood pressure [93]. Like NO, H₂S is a potent vasodilator. In mice, genetic deletion of CSE markedly reduces H₂S levels in the serum, heart, aorta, and other tissues [95]. H₂S plays a role in wound healing, and endogenously produced H₂S mediates angiogenesis induced by vascular endothelial growth factor (VEGF) [96]. In in vitro studies, exogenously administered H₂S at physiologically relevant concentrations induced angiogenesis and stimulated endothelial cell proliferation and migration [96,97].

8. Methanethiol, SELENBP1 and Cancer

Hydrogen sulfide, methanethiol, and dimethyl sulfide are all volatile sulfur compounds (VSCs) characterized by their low odor thresholds, significant role in halitosis (bad breath), and toxicity at high concentrations. They act as crucial biological mediators and signaling molecules (gasotransmitters) in mammals, influencing blood pressure, inflammation, and neuronal signaling [98]. Methanethiol (CH₃SH) is a toxic foul-smelling gas that plays interesting but poorly characterized roles in human physiology. It can be derived from methionine or through methylation of H₂S. It is overproduced by cancer cells and often found in excess amounts in tumor microenvironments, associated with accelerated disease progression [99].

Selenium-binding protein 1 (SELENBP1) is a methanethiol oxidase, and it is a critical enzyme for metabolizing methanethiol [100]. It catalyzes the conversion of methanethiol to H₂S, H₂O₂, and formaldehyde.

H₂O + CH₃SH + O₂ → H₂CO + H₂O₂ + H⁺ + H₂S

It depends on both selenium and copper as catalysts [101]. It plays a protective role in ulcerative colitis [102]. Both mouse and human SELENBP1 are abundantly expressed in the liver, lungs, prostate, colon, and pancreas [103,104].

Deficiencies in SELENBP1 cause high levels of methanethiol, leading to a “cabbage-like” breath odor known as extraoral halitosis [105]. SELENBP1 is widely recognized as a tumor suppressor, and it is often downregulated in various epithelial cancers, including lung, colorectal, ovarian, liver, and prostate cancers [103].

Human methyltransferase-like protein 7B (METTL7B; also known as TMT1B) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to H₂S, yielding methanethiol, and to other exogenous thiol-containing small molecules [106]. METTL7B is consistently upregulated in various cancers, acting as a promoter of tumor progression and a marker of poor prognosis [107]. In a study on lung adenocarcinoma cells, high expression levels were linked to enhanced cancer cell proliferation, migration, and invasion, and increased resistance to therapies [108].

Both glucose and methionine are enriched in cancer cells, and the nonenzymatic Maillard reaction between these two metabolites yields methanethiol. Gut microbes also break down methionine via γ-methionine-lyase, to produce methanethiol. Methanethiol-producing strains such as Fusobacterium nucleatum are highly abundant in the gut microbiome of colorectal carcinoma (CRC) patients [99]. Colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) cells produce and release high levels of methanethiol (CH₃SH) into the tumor microenvironment (TME) due to their dysregulated sulfur metabolism. Elevated levels of methionine, along with reduced expression of SELENBP1 and increased expression of METTL7B, lead to high endogenous production of this volatile compound, which serves as a scent-based marker for cancer [99].

We hypothesize that the methanethiol that is produced in abundance in association with colon cancer can be exploited by normal cells expressing SELENBP1 in the gut to produce H₂S, H₂O₂, and formaldehyde, all of which are likely readily available sources of deupleted water to support mitochondrial health. This hypothesis aligns with the general theory we have developed that cancer cells hoard deuterium and supply the resident immune cells with deupleted resources [4,109]. Indeed, it has been confirmed experimentally that SELENBP1 expression levels are significantly lower in colorectal cancer cells than in adjacent healthy tissue cells [110]. Given that it is a gas, it is likely that, like the other small organic gas molecules, methanethiol has reduced levels of deuterium.

Figure 2 illustrates the pathways by which methanethiol is produced, through both microbial and human enzymes, and the pathway by which it is broken down into potentially valuable deupleted molecules by SELENBP1.

9. A Role for Ammonia in Deupletion?

Ammonia management in the body is a critical detoxification process centered on converting ammonia, which can be toxic in elevated amounts, into non-toxic urea in the liver (the urea cycle) to be recycled via the gut microbiome or finally for renal excretion. The kidneys play a dual role: managing acid-base balance by excreting ammonia as ammonium (NH₄⁺) in the urine and producing ammonia from glutamine in the proximal tubules to regulate body pH.

In the remainder of this paper, we will refer to “total” ammonia as T-ammonia, representing the mixture of NH₃ and NH₄⁺. NH₃, being a gas, is likely enriched in ¹H relative to ²H. A question we address here is whether the organism exploits this feature to fractionate deuterium, preferentially retaining NH₃ and exporting NH₄⁺.

T-ammonia is no longer viewed merely as metabolic waste. It is a critical driver of tumor progression. T-ammonia accumulates in the tumor microenvironment, where it is a source of nitrogen to support amino acid and nucleic acid synthesis. It stimulates growth pathways and fuels an immunosuppressive state [111]. Cancer cells actively export ammonia as a gas (NH₃) which is likely low in deuterium simply because it is a gas. Resident cancer-associated fibroblasts take up NH₃ and utilize it to synthesize glutamine, which is then delivered back to the tumor cells [112]. This exchange may serve as a way to preferentially export ¹H over ²H to the external milieu, by taking advantage of the high pH in the tumor cytoplasm to promote the production and export of NH₃ derived from T-ammonia [113].

9.1. Do Gut Microbes Convert Urea to Deupleted Ammonia?

Urea, produced primarily in the liver and excreted as a major component of urine, can be viewed as a storage form of T-ammonia. A reaction of urea with one molecule of water yields ammonia and carbon dioxide:

(NH₂) ₂CO + H₂O → 2NH₃ + CO₂

The metabolism of urea by microbial urease [114], as well as the microbial metabolism of other nitrogen-containing molecules such as the amino acids, purines, and pyrimidines, are all sources of T-ammonia in the gut lumen. Interestingly, ammonia, the gas, (NH₃) readily diffuses across plasma membranes, whereas ammonium, the ion (NH₄⁺) does not. Furthermore, epithelial cells in the gut express transport proteins that actively take up NH₃ but not NH₄⁺ [115]. Villous epithelial cells throughout the gut express two proteins that facilitate the active transport of NH₃ across the gut barrier into the circulation, namely the NH₃ transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) [115].

T-ammonia in the lumen is mainly found in solution as the cation NH₄⁺. At equilibrium, only a small percentage is present as the gas, NH₃, particularly in an acidic environment, because of the high acid dissociation constant (pKa). But only NH₃ can passively cross the gut barrier. Various transport proteins exist to actively carry either NH₃ or NH₄⁺ across membranes, and in different circumstances a preference for transport of one or the other is probably strategically important.

It is likely that an ammonium ion containing deuterium (NH₃D⁺) would leave behind in the lumen the deuteron rather than one of the protons when it gets converted to the gas, ammonia, via acid dissociation: NH₃D⁺ → NH₃ + D⁺. This means that ammonia as a gas is likely a source of low-deuterium protons. As the ammonia is syphoned off by the transporters, more ammonia is produced, as the two species restore chemical equilibrium.

9.2. What Is the Role of RhAG in Red Blood Cells?

RhAG, another member of the Rh glycoprotein ammonia transporter family, is only expressed in red blood cells (RBCs). It has not been easy for researchers to figure out what role this protein plays in RBCs, but it is becoming clear that it is involved in T-ammonia homeostasis in the vasculature [116]. RhAG acts as a scaffold for the Rh complex. It appears to transport T-ammonia exclusively as NH₃ across the RBC membrane [117]. It has been suggested that RBCs act as a storage depot for T-ammonia to reduce the levels of free T-ammonia in the circulation [116]. Thus, RhAG’s role in RBCs may be to support T-ammonia buffering within the RBCs, which can help sequester T-ammonia and protect the plasma from hyperammonemia [118].

Notably, RhAG transports methylamine (CH₃NH₂) as well as ammonia (NH₃). Like NH₃, CH₃NH₂ is a gas, and this suggests that RhAG specifically acts as a gas channel. Ripoche et al. wrote: “The results show that RhAG facilitates CH₃NH₂/NH₃ movement across the RBC membrane and represents a potential example of a gas channel in mammalian cells” [117]. In fact, sequestration of methylamine may be a beneficial role of RhAG. The concentration of methylamine in RBCs is five times that found in plasma. Interestingly, methylamine is not efficiently cleared during dialysis treatment, due to the fact that RBCs sequester it [119]. An analysis of the crystallographic structure of the related bacterial NH₃ transport channel AmtB showed, through reconstitution into vesicles, that AmtB conducts uncharged NH₃ [120], consistent with the analysis of Ripoche et al. for RhAG.

The concentration of T-ammonia in RBCs is significantly higher than that in the plasma. RhAG expressed in RBCs forms a structural unit with anion exchanger 1 (eAE1; a sodium- independent anion exchanger) within the ankyrin core complex. RBCs swell and lyse in isotonic NH₄⁺ buffer. This effect is tightly regulated by chloride concentration. When chloride was replaced by glutamate, NH₄⁺-induced swelling was inhibited, preventing hemolysis. Inhibition of eAE1 also completely inhibited hemolysis [121]. It has been shown that NH₃ transport parallels RhAG expression in RBCs [117].

Westhoff et al. wrote: “Erythrocytes are ideally positioned to then transport ammonium to be exchanged in the liver and kidney, where other Rh-related proteins (RhBG and RhCG) are expressed” [122]. We take this idea one step further: we suggest that RBCs actively import NH₃ as a gas from the medullary interstitium in the kidneys, syphoning it off before NH₄⁺ is exported via the urine, as explained in Section 9.3.

9.3. Renal Tubules Preferentially Export NH₄⁺

The ammonium ion (NH₄⁺) and potassium ion (K⁺) show almost identical biophysical characteristics. Since NH₄⁺ is very similar in size and charge to K⁺, cells can exploit K⁺ transport channels to actively transport NH₄⁺ across membranes. The kidneys utilize a complex process to selectively export NH₄⁺ into the urine and retain NH₃ in the interstitium. Cells in the proximal tubules of the kidneys synthesize abundant NH₄⁺ from glutamine, which is secreted into the luminal fluid through active transport by both an apical Na⁺/H⁺ exchanger NHE3 and by various K⁺ transport proteins [123]. However, a substantial amount of ammonia (NH₃) is reabsorbed later in the thick ascending limb of the loop of Henle. Approximately 40% of the NH₄⁺ that enters the loop of Henle is passed to the interstitium as NH₃.

Tubule cells in the thick ascending limb of Henle have a remarkable apical impermeability to NH₃, due to the specific lipid composition of the bilayer [124,125]. However, they actively import NH₄⁺ from the lumen against a concentration gradient, utilizing the Na⁺/H⁺ ion exchanger NHE4, substituting NH₄⁺ for the proton. Defective versions of NHE4 lead to metabolic acidosis [126]. On the basolateral side, these cells release NH₃ but not NH₄⁺ into the medullary interstitial space, to facilitate acid-base balance and to create a concentration gradient used for final waste excretion [127]. The Rhesus glcyoproteins, RhBG and RhCG act as NH₃ transporters in the distal tubule and collecting duct [128]. We hypothesize that RBCs may actively import NH₃ in the medullary interstitium, syphoning it off to capture ¹H for return to the circulation. As we’ve discussed, red blood cells actively take up NH₃ but not NH₄⁺ via their RhAG proteins [117].

The Inner Medullary Collecting Duct (IMCD), located in the deep, inner medulla, is the final, terminal segment of the renal nephron responsible for maximal urine concentration and final electrolyte adjustment before urine enters the renal pelvis. In the IMCD, basolateral Na⁺-K⁺ ATPase enables active basolateral uptake of NH₄⁺ but not NH₃ [128]. The renal excretion of NH₄⁺ acidifies the urine, playing a crucial role in acid/base balance.

However, this process of RBC uptake of NH₃ and export of NH₄⁺ into the urine presumably also selectively retains protium that is returned to the circulation, while deuterium is then selectively excreted in the urine via NH₄⁺. Essentially, through this complex process, deupleted protons reenter the circulation and deuterium has become enriched in the aqueous medium in the loop of Henle. These deuterium-enriched fluids are finally excreted as urine.

10. Does Glutamine Supply Deupleted Protons to the Neuronal Mitochondria?

Glutamate is not only the most abundant excitatory neurotransmitter, but it is also the precursor to γ-aminobutyric acid (GABA), the predominant inhibitory neurotransmitter. Abnormal levels of extracellular glutamate impair neurotransmission and cause neurodegeneration and neuronal apoptosis. One of the most important roles of astrocytes is to assure that glutamate does not reach toxic levels in the synapse [129].

Astrocytes also play an essential role in the brain to clear excess T-ammonia, protecting the neurons from T-ammonia toxicity [130]. In achieving this goal, they also protect from glutamate neuroexcitotoxicity, because they take up glutamate that is released into the synapse and use the T-ammonia to convert the glutamate to glutamine. The so-called astrocyte-neuron glutamate-glutamine cycle prevents overstimulation of neuronal N-Methyl-D-aspartate (NMDA)/ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which can lead to cell death [131]. The process of glutamate uptake by astrocytes, conversion to glutamine, and transport back to neurons is fast, happening in the order of seconds [132]. A question is whether it is fast enough to predominantly avoid picking up a deuteron via exchange with protons in the side chain amide group while in transit. If so, then this whole process might be a mechanism to deliver ¹H to the mitochondria. Note that the amide group in glutamine exchanges protons with deuterons in the aqueous medium at a much lower rate than do protons bound to oxygen in hydroxyl and carboxyl groups [7].

10.1. The Glutamate/GABA-Glutamine Cycle

Glutamate and GABA are the principal excitatory and inhibitory neurotransmitters in the brain. They are both extensively recycled between neurons and astrocytes. Astrocytes take up these neurotransmitters from the synapse following their release by neurons. In the cytoplasm, astrocytes convert glutamate to glutamine via glutamine synthetase, and then return the glutamine to the neurons, which, in turn, resynthesize glutamate from glutamine, in their mitochondria. GABAergic neurons further metabolize glutamate via glutamic acid decarboxylase to produce GABA [133]. Infants with a genetically defective version of glutamine synthetase suffer from severe brain malformations, multi-organ failure and neonatal death, attesting to the importance of this enzyme to metabolism [134].

10.2. Aquaporin 4 and Potassium Channels in Astrocytes

Aquaporin 4 (AQP4) is the primary water channel in the central nervous system, and it is concentrated in the end feet of astrocytes. Curiously, AQP4 channels are localized in close proximity to inward rectifier potassium channels (Kir 4). Potassium is released from neurons into the extracellular space during repolarization of the action potential and is then rapidly taken up by astrocytes via Kir 4. Glutamate is released into the synapse during synaptic activity and is taken up by astrocytes via glutamate transporters on astrocyte processes [135].

AQP4 channels import not only water but also NH₃, because it is an uncharged molecule of similar size. Similarly, Kir 4 takes up NH₄⁺ in addition to K+, again, because of similar size and charge properties. So, both the AQP4 channels and the Kir 4 channels can import T-ammonia.

Experiments on cultured astrocytes have shown that AQP4 is upregulated in the presence of high levels of T-ammonia [136]. This explains the feature of brain swelling due to the uptake of excess water by astrocytes in association with hepatic encephalopathy. But it also implies that astrocytes actively take up more NH₃ via upregulated aquaporin channels. An experiment on cultured astrocytes exposed to ammonium chloride found that AQP4 was sharply upregulated in response to ammonium exposure, and this was mediated through phosphorylation of p38 [137]. On the other hand, both Kir4.1 and Kir5.1 were downregulated in hyperammonemic mice, suppressing the uptake of NH₄⁺ but also disturbing potassium homeostasis [138,139]. We hypothesize that the upregulation of AQP4 and the simultaneous downregulation of Kir4.1 and Kir5.1 in response to excess T-ammonia in the vasculature is a strategy to selectively import deupleted NH₃ rather than deuterium-rich NH₄⁺ when T-ammonia levels are high.

10.3. Astrocytes Clear Glutamate from the Synapse

Astrocytes convert glutamate to glutamine via glutamine synthetase (GS). The reaction catalyzed by GS is carried out through a two-step mechanism. In the first step, γ-glutamylphosphate is formed as an intermediate from glutamate, where phosphate is sourced from ATP. In the presence of this intermediate, NH₄⁺ binds to the active site and, in the second step, it is deprotonated, releasing NH₃ near the γ-glutamyl phosphate intermediate. The ammonia molecule then displaces phosphate, yielding free phosphate and glutamine [140]. We hypothesize that any ammonium ion containing deuterium would preferentially lose the deuteron over a proton in this step, thus rarely delivering an NH₃ molecule containing ²H.

Interestingly, astrocytic density is reduced in major depressive disorder (MDD). In particular, blood vessels in gray matter from the orbitofrontal area of brains of patients suffering from MDD were specifically significantly impoverished in AQP4 expression compared to normal controls [141]. Low levels of AQP4 could impair glutamate/glutamine cycling, and this could be a factor in symptoms of depression, by reducing the ability of neurons to maintain low deuterium in their mitochondria. Mitochondrial dysfunction in neurons is a prevalent feature of depression [142].

Figure 3 illustrates the glutamate-glutamine exchange between neurons and astrocytes that may serve as a mechanism for glutamine to safely deliver a deupleted ammonia molecule to the mitochondria in the neuron.

10.4. T-Ammonia Handling in the Mitochondrial Matrix: Supplying ¹H to the Intermembrane Space?

Aquaporin-8 (AQP8) is the primary aquaporin expressed in the inner mitochondrial membrane (IMM). It plays a key role in mediating water transport into and out of the mitochondrial matrix, contributing to the organelle’s volume regulation. Because AQP8 is highly expressed in the inner membrane, water freely moves from the matrix into the intermembrane space and vice versa [143]. AQP8 is known as a “peroxiporin” because it can transport not only water but also ammonia and hydrogen peroxide across the membrane. It exclusively transports NH₃ but not NH₄⁺ [144]. The mitochondrial matrix has a very high pH of around 7.9, which means that∼6% of the T-ammonia is present in the gas form. The porous membrane then allows the ammonia to be siphoned off into the intermembranes space, delivering an overabundance of ¹H to it (we hypothesize). As the ammonia is removed, more ammonia appears while dissociation equilibrium is continuously reestablished. Because the intermembrane space is much more acidic, very little of the T-ammonia remains as a gas on that side of the membrane, so ammonia naturally crosses from the matrix into the intermembrane space.

10.5. Liver Failure and Hepatic Encephalopathy

Liver failure results in elevated levels of T-ammonia in the blood, because the damaged liver is unable to detoxify T-ammonia via the production of urea [145]. This T-ammonia readily crosses the blood-brain barrier and causes toxic effects in the brain. Increased T-ammonia concentrations in the brain significantly alter brain glutamate levels, glutamate uptake, and glutamate receptor function. In rat models, acute liver failure causes an upregulation of extracellular glutamate levels. T-ammonia is likely a major contributing factor to the brain edema and seizures associated with hepatic encephalopathy [146].

Glutamate plays a critical role in inducing brain swelling. Activation of the metabolomic glutamate receptors (mGluR) increases the rate of tissue swelling in rat hippocampal slices. During cerebral edema, excessive amounts of glutamate are released, and it causes increased expression of AQP4 [147], promoting the uptake of not only water but also ammonia into astrocytes. The glutamate can then be combined with the ammonia to produce glutamine, which, in our view, is a carrier of deupleted protons. The removal of both the stressor ammonia and the neurotoxin glutamate from the circulation is achieved by the astrocytes through this process.

During hepatic encephalopathy, astrocytes upregulate AQP4 [137] and downregulate Kir4.1 channel expression [138]. This causes both edema (which is primarily due to astrocyte swelling) and excess potassium in the external space, due to impaired potassium uptake by astrocytes [148]. But it also means that T-ammonia is taken up by the astrocytes primarily as the gas rather than the dissolved cation. The fact that glutamate induces these changes in expression makes sense if one acknowledges that glutamate can be converted to glutamine both as a way to detoxify ammonia and to create a carrier molecule for ¹H, which the astrocytes immediately deliver to adjacent neurons. In other words, might it be the case that astrocytes are able to capitalize on excess T-ammonia in the circulation to facilitate the delivery of deupleted protons to neurons?

11. Discussion

In this paper, we have carefully examined metabolism that involves small hydrogen-containing gas molecules, from the perspective of deuterium homeostasis. We suggest that much of the complexity found in the metabolism of these gas molecules revolves around strategies to reduce deuterium content in mitochondrial water. We hypothesize that, when the methanogenic archaea that derive small organic molecules by using molecular hydrogen to reduce carbon dioxide are scarce in the gut, various processes increase the abundance of H₂O₂, as a compensatory mechanism to supply ¹H protons to mitochondria. This results in inflammatory bowel disease that can lead to colon cancer in extreme conditions [149]. Methanogenic archaea are depleted in association with IBD [150] . Hydrogen gas administration is being widely recognized as a treatment option for many diseases and conditions [25].

Chronic inflammation is a leading driver in the progression of many long-term health conditions [151], and it may be that its positive role is to heal deuterium-overloaded mitochondria through the delivery of deupleted H₂O₂ to them to compensate for the insufficiency of deupleted nutrients such as SCFAs and methyl groups supplied by the gut microbes. Mitochondria express both glutathione peroxidase and especially peroxiredoxins [62] that can readily convert H₂O₂ to two molecules of H₂O. High H₂ concentration in the gut stimulates the production of butyrate, an essential nutrient for the colonocytes that is probably deupleted [12].

H₂S , and other sulfur-containing gases, including DMSO and CH₃SH, also appear to play an important role in supplying ¹H protons to the mitochondria. Gut microbes are also heavily involved in this process. H₂S-producing bacteria are upregulated in the gut in association with Crohn’s disease [152]. Cancer cells actively produce CH₃SH and deliver it to tumor-resident cells, potentially as a resource to help restore their mitochondrial health [99].

Ammonia is another gas that can be exploited for its low-deuterium content, but this is a complicated process that involves selective transport proteins to optimize the export of deuterons via NH4+ and the retention of protons in NH₃. Gut microbes play an essential role through their expression of urease that converts urea produced in the liver to T-ammonia. Gut epithelial cells actively take up NH₃, and deliver it back to the liver [115]. Bacterial urease activity is strongly upregulated in association with gut dysbiosis and Crohn’s disease [153]. The kidneys appear to preferentially retain NH₃, the gas, and export NH₄⁺, the cation, into the urine.

Finally, the complex interaction between neurons and astrocytes in glutamate/glutamine exchange may also be a mechanism to deliver ¹H to neuronal mitochondria. The glutamate/glutamine cycle is dysregulated in association with various tauopathies present in neurodegenerative disease [154].

We hope that this paper will inspire others to conduct research to clarify the role of deuterium in metabolic policy and the processes by which organisms protect mitochondria from deuterium overload. If our ideas around the role of small molecular gases, many of which are produced by the gut microbes, in deuterium homeostasis can be proven, it would revolutionize our understanding of the mechanisms of disease processes.