Radical-Driven Methane Formation in Humans Evidenced by Exogenous Isotope-Labeled DMSO and Methionine

Methane (CH4), which is produced endogenously in animals and plants, was recently suggested to play a role in cellular physiology, potentially influencing the signaling pathways and regulatory mechanisms involved in nitrosative and oxidative stress responses. In addition, it was proposed that the supplementation of CH4 to organisms may be beneficial for the treatment of several diseases, including ischemia, reperfusion injury, and inflammation. However, it is still unclear whether and how CH4 is produced in mammalian cells without the help of microorganisms, and how CH4 might be involved in physiological processes in humans. In this study, we produced the first evidence of the principle that CH4 is formed non-microbially in the human body by applying isotopically labeled methylated sulfur compounds, such as dimethyl sulfoxide (DMSO) and methionine, as carbon precursors to confirm cellular CH4 formation. A volunteer applied isotopically labeled (2H and 13C) DMSO on the skin, orally, and to blood samples. The monitoring of stable isotope values of CH4 convincingly showed the conversion of the methyl groups, as isotopically labeled CH4 was formed during all experiments. Based on these results, we considered several hypotheses about endogenously formed CH4 in humans, including physiological aspects and stress responses involving reactive oxygen species (ROS). While further and broader validation studies are needed, the results may unambiguously serve as a proof of concept for the endogenous formation of CH4 in humans via a radical-driven process. Furthermore, these results might encourage follow-up studies to decipher the potential physiological role of CH4 and its bioactivity in humans in more detail. Of particular importance is the potential to monitor CH4 as an oxidative stress biomarker if the observed large variability of CH4 in breath air is an indicator of physiological stress responses and immune reactions. Finally, the potential role of DMSO as a radical scavenger to counteract oxidative stress caused by ROS might be considered in the health sciences. DMSO has already been investigated for many years, but its potential positive role in medical use remains highly uncertain.


Introduction
Methane (CH 4 ) is an important and highly abundant carbon molecule in the Earth's atmosphere that affects the Earth's radiative balance. Around 600 to 700 million tons of CH 4 are released into the atmosphere annually by natural and anthropogenic sources, mostly of biological origin [1]. For a long time, biological CH 4 formation was considered to only occur from the metabolism of microorganisms-methanogens that belong to the domain Archaea-living under strictly anaerobic conditions in natural wetlands; landfills; rice fields; or in the alimentary tract of vertebrates, including ruminants and humans. Fe(II) + H 2 O 2 → Fe(III) + OH − + •OH (1) 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 (•CH 3 ), of which a portion reacts to CH 4 through abstraction of a hydrogen atom from hydrocarbons, hydrogen peroxide, or hydrogen carbonate. Alternatively, the methyl radicals form oxidized C1 species, such as methanol, formaldehyde, or formic acid. Thus, it is conceivable that there is in vivo formation of C1 compounds as a result of ROS formation and interaction with methylated compounds. Therefore, we consider the administration of isotopically labeled DMSO and methionine as ideal model compounds to confirm the occurrence of ROS-driven CH 4 formation in humans.

Application of DMSO to Humans
Dimethyl sulfoxide (DMSO) is an organic polar aprotic molecule that was first synthesized in 1866. It was used as an important solvent for many decades before being proposed for use as a pharmaceutical in the 1960s by Stanley Jacob. Because of its ability to rapidly penetrate through human skin and its properties as a free radical (•OH) scavenger, it was widely used as an anti-inflammatory, antipain, and neuroprotective agent. A wide range of biological and pharmacological effects of DMSO were described by Jacob and Herschler [62] for the interested reader. Since 1978, DMSO has been approved by the United States Food and Drug Administration (FDA) for the treatment of interstitial cystitis. Other medical applications, as well as potential physiological and pathological effects of DMSO, are highly controversially discussed. For example, Amemori et al. [63] found that the oral administration of DMSO is an effective treatment for amyloid A amyloidosis. On the other hand, experiments with rats found that DMSO might induce retinal apoptosis [64]. Despite the differing results of the various studies, it is generally assumed that DMSO is nontoxic below 10% (v/v) [65] with an oral medium lethal dose of 28,300 mg/kg (rat) and a dermal medium lethal dose of 40,000 mg/kg (rat).

Aims and Postulates
Recent results showed that CH 4 might be formed in all organisms and that the formation of methyl radicals induced by ROS is a prerequisite for the generation of CH 4 . The experiments described in this paper were undertaken in order to unambiguously demonstrate (as a first proof of principle) that CH 4 is endogenously formed in humans via a radical-driven process without the involvement of the well-known microbial sources (methanogens) living in the gastrointestinal tract. Therefore, a volunteer-the first author of this study-applied isotopically labeled ( 2 H or 13 C) DMSO on the skin (arm), consumed it via the mouth, and applied it to blood samples. In addition, the amino acid methionine (with an isotopically labeled 13 C methyl group) was also applied to the blood samples. The released gases were analyzed for their isotopic composition to unambiguously identify the formation of CH 4 from the precursor compounds DMSO and methionine. Based on the results and the formation patterns observed, we discuss several hypotheses concerning the origin of cell-based CH 4 production and its potential physiological role in mammals. Finally, as DMSO has already been investigated for many years while its potential positive role for medical use is highly uncertain, we briefly discuss the potential application of DMSO to reveal and counteract oxidative stress. All experiments and measurements were conducted by the principal investigator (PI) and first author of this study (F.K.) from June 2018 to October 2020. The subject was a healthy 55-year-old man without known disease, prescribed medications, or drug intake. The average breath CH 4 production value of the subject was 9 ± 6.7 ppmv, measured over 72 weeks [38], and he was thus classified as a medium-to-high emitter (see the explanation above). Air and blood samples were provided by the PI, as shown in Section 2.1.3 below. A surrogate investigator (D.P.) was designated to obtain informed consent from the self-experimenter (F.K.), in agreement with the ethics relevant to solitary self-experimentation [66]. The work described was carried out in accordance with The Code of Ethics of the World Medical Association. The research was reviewed by the Medical Research Council of Hungary (ETT-TUKEB) and it was approved as part of the protocol "Mapping metabolic pathways of endogenous gas formation by isotopic analysis of the gas composition of human samples" (6420-8-2023/EUIG/768).

. Experiments and Sampling of Air
A graphical representation of the set-up of the three individual experimental series (oral intake, arm exposure to sunlight, and blood experiments), including the collection of samples and the applied measurements, is outlined in Figure 2. Table S1 shows the timeline of the experiments.

Experiments and Sampling of Air
A graphical representation of the set-up of the three individual experimental series (oral intake, arm exposure to sunlight, and blood experiments), including the collection of samples and the applied measurements, is outlined in Figure 2. Table S1 shows the timeline of the experiments. The volunteer of the study swallowed 100 µL 13 C-CH4 DMSO (4% 13 C-content, dissolved in 300 mL H2O) or 1 mL of 2 H-CH4 DMSO (10% 2 H-content, dissolved in 300 mL H2O), respectively. Subsequently, the breath CH4 concentration and isotope values of CH4 (δ 13 C or δ 2 H, respectively) were monitored for 130 min. The breath samples were collected using 1 L Tedlar bags. The breath CH4 sampling procedure was performed in a consistent manner. During the breath air collection, the volunteer breathed normally, stopped breathing for around 5 s, and then filled the Tedlar bag with expired air (range of 0.8 to 1  The volunteer of the study swallowed 100 µL 13 C-CH 4 DMSO (4% 13 C-content, dissolved in 300 mL H 2 O) or 1 mL of 2 H-CH 4 DMSO (10% 2 H-content, dissolved in 300 mL H 2 O), respectively. Subsequently, the breath CH 4 concentration and isotope values of CH 4 (δ 13 C or δ 2 H, respectively) were monitored for 130 min. The breath samples were collected using 1 L Tedlar bags. The breath CH 4 sampling procedure was performed in a consistent manner. During the breath air collection, the volunteer breathed normally, stopped breathing for around 5 s, and then filled the Tedlar bag with expired air (range of 0.8 to 1 L). Depending on the study parameter, the gaseous sample was analyzed using cavity ringdown spectroscopy (CRDS), gas chromatography flame ionization detection (GC-FID), or gas chromatography temperature conversion isotope ratio mass spectrometry (GC-TC-IRMS) immediately after sampling (see analytical measurements below).

Arm Incubations and Exposure to Solar Light
For the CH 4 skin emission analysis, the forearm of the subject was placed inside a cylindrical chamber (see photo documentation 1 in the Supplementary Materials) made of polytetrafluorethylene (PTFE) foil (transparent for UV light), with a diameter of 18.5 cm and a length of 42.5 cm (volume = 11.7 L). The round opening at the back was sealed with an elastic PTFE foil tied to the chamber and fixed along the upper arm. A gas inlet and outlet PTFE tube system was attached to the chamber. The pressure of the chamber was constant during the whole monitoring phase. Ventilation at the inside front of the chamber provided a homogeneous air mixture. The outlet tube was directly connected to the CRDS system (see analytical measurements below) for in situ online analysis of CH 4 and CO 2 concentrations and δ 13 C values. First, the empty chamber (filled with laboratory air) was measured as a control. Next, the volunteer thoroughly washed his arm with tap water and dried it with a paper towel before placing it in the chamber for 30 min to obtain a control value. Then, 13 C-CH 4 -labeled DMSO (a mixture of 400 µL DMS0 + 100 µL 13 C-labeled DMSO + 500 µL H 2 O) was thoroughly distributed on the skin of the left upper forearm (penetrated area of around 30 cm 2 ) and the air in the chamber was connected to the CRDS measurement system for a monitoring period of 1 h. Afterward, the forearm was exposed to natural solar light in the field for a period of 1 h (from 10 to 11 am, in July in Heidelberg, Germany). After returning from the field to the laboratory (within 5 min) the left arm was again placed in the chamber and monitored for changes in the δ 13 C-CH 4 values for 1 h. The same procedure was repeated the following two days and the untreated right arm served to record control values.

Blood Samples and Incubation with DMSO and Methionine
Approximately 20 mL of whole-blood samples were collected from the PI through venipuncture by using 4 × 7.5 mL S-Monovettes ® containing Ethylenediaminetetraacetic acid (EDTA) to prevent coagulation. Samples were immediately processed for isotope label experiments. Therefore, 13 C-labeled DMSO and methionine were added to 1 mL of blood in autoclaved 40 mL headspace vials (Supelco 27184) so that the final concentration of the added compound was 1 mM or 10 mM. Vials were sealed using a hole-type screw cap (Supelco) fitted with a PTFE/silicone septum (Supelco). The control samples were prepared in the same way, except that the added DMSO and methionine were isotopically not enriched in 13 C. All samples were prepared in triplicates and incubated at 36 • C for 24 h before the gas phase in the vials was analyzed (first day). Afterward, the vials were opened to equilibrate with the air in a fume cupboard. After 30 min, the samples were again sealed with a PTFE/silicone septum and incubated at 36 • C for 24 h before the gas phase was analyzed (second day).

Analytical Measurements
The analytical laser technique applied in this study to obtain online stable carbon isotope measurements and concentrations of CH 4 was almost identical to that described previously [22]. In addition, stable carbon and hydrogen isotope analyses were conducted by applying GC-IRMS, as described in Einzmann et al. [67]. However, we briefly describe the applied analytical techniques in the sections below. For more analytical details, and the application of stable isotope techniques, please refer to the studies by Keppler et al. [22] and Einzmann et al. [67] and to the Supplementary Materials.
2.2.1. Natural Abundance of 13 C/ 12 C and 2 H/ 1 H, Definition of δ Values, Isotopic Excess, and Keeling Method Throughout this paper, the "delta" (δ) notation-which is the relative difference of the isotope ratio of a material to that of a standard V-PDB (Vienna Pee Dee Belemnite, 13 C/ 12 C ratio of 0.011108) or V-SMOW (Vienna Standard Mean Ocean Water, 2 H/ 1 H ratio of 0.00011576)-is used; values of δ 13 C and δ 2 H relative to those of V-PDB and V-SMOW, respectively, are defined by the following equations: δ 13 C = (( 13 C/ 12 C) sample /( 13 C/ 12 C) standard ) − 1. (2) To comply with the guidelines of the International System of Units (SI), we followed the proposal of Brand and Coplen [68] and used the term urey, after H.C. Urey (symbol Ur), as the isotope delta value unit. In such a manner, an isotope-delta value expressed traditionally as −60‰ can be written as 60 mUr. For natural sources of CH 4 , typical δ 13 C-CH 4 and δ 2 H-CH 4 values are in the range of −20 to −100 mUr [12] and −100 to −400 mUr [44], respectively.
The isotopic difference (∆) between the control and sample is defined as The 13 C % and 2 H % excesses were calculated as follows: 13

Measurements of CH 4 Concentrations and Stable Carbon Isotope Values
Cavity ringdown spectroscopy is a highly sensitive optical spectroscopic technique for the measurements of both the stable carbon isotope value (δ 13 C-CH 4 ) and the concentration of CH 4 . The Tedlar gas sample bag (from breath air) or the arm incubation Teflon chamber (see the Supplementary Materials) was connected to the CRDS, and the flow rate to the analyzer was 23 mL/min. Before entering the analytical system, the gas was passed through two chemical traps filled with AscariteII ® (sodium hydroxide coated silica) and Drierite ® (anhydrous CaSO 4 ) to remove the carbon dioxide (CO 2 ) and water, respectively. This was necessary due to the higher concentrations of CO 2 and water (up to 6%) in the breath sample, which can cause interferences with the spectroscopic CH 4 measurements.
Stable carbon isotope values and concentrations of CH 4 were measured with a G2201-i cw-CRDS-Analyzer (Picarro, Inc., Santa Clara, CA, USA). This instrument enables simultaneous measurements of the CH 4 concentration, δ 13 C-CH 4 value, and water content in a gas sample. The concentration precision (1σ, 2 min average) specified by the manufacturer was 50 ppbv + 0.05% of reading ( 12 C) and 10 ppbv + 0.05% of reading ( 13 C) in the high dynamic range mode, and 5 ppbv + 0.05% of reading ( 12 C) and 1 ppbv + 0.05% of reading ( 13 C) in the high precision mode. The δ 13 C-CH 4 precision provided by the manufacturer was <0.8 mUr. However, typical standard deviations (SD) of measurements of breath samples and standards (using filled Tedlar bags) were in the ranges of ±1.2 ppbv and ±0.3 mUr (1σ, 2 min average measurement interval) for the concentration and stable isotope measurements, respectively (see also [22]).
In order to quality assure the δ 13 C-CH 4 values, some gas samples were measured using both CDRS and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) (for details, see Section 2.2.4. below). Samples measured via IRMS were analyzed three times (n = 3) and the average standard deviations of the analytical measurements were in the range of 0.1 to 0.3 mUr. The measured difference between the two analytical systems was used to normalize the isotope data of the CRDS.

Measurements of CH 4 Concentrations Using Gas Chromatography Flame Ionization Detection
An aliquot (5 mL) of headspace gas was taken from the incubation vials (40 mL) or gas bags (1 L) using a gastight syringe. Before entering the analytical system, the gas sample was passed through a chemical trap filled with Drierite ® to remove water. The sample gas was separated via gas chromatography using a GC-14B (Shimadzu, Japan) equipped with a 2 m column (Ø = 3.175 mm inner diameter) packed with a Molecular Sieve 5A 60/80 mesh from Supelco. Methane was recorded using an FID, and its concentration was quantified by using two reference gases containing 9837 ppbv and 2192 ppbv CH 4 .

Measurement of δ 13 C-CH 4 Values
Gas from the Tedlar gas bags (from breath samples) or 40 mL glass vials (headspace of blood samples) was transferred to an evacuated sample loop (40 mL). Interfering compounds were separated via GC and CH 4 was trapped on Hayesep D. Afterward, CH 4 was separated from the interfering compounds via GC and transferred to a gas chromatography-combustion-isotope ratio mass spectrometer (Deltaplus XL mass spectrometer, ThermoQuest Finnigan, Bremen, Germany) via an open split. The working reference gas was CO 2 of high purity (carbon dioxide 4.5, Messer Griesheim, Frankfurt, Germany) with a known δ 13 C value of −23.64 mUr (calibrated at MPI for Biogeochemistry in Jena, Germany). All δ 13 C-CH 4 values were corrected using two CH 4 working standards (isometric instruments, Victoria, BC, Canada) calibrated against the National Institute of Standards and Technology (NIST) and International Atomic Energy Agency (IAEA) reference substances. The calibrated δ 13 C-CH 4 values of the two working standards in mUr vs. V-PDB were −23.9 ± 0.2 and −54.5 ± 0.2. All samples were normalized via two-scale anchor calibration according to Paul et al. (2007). 4 values were determined via GC-TC-IRMS. The same analytical set-up was applied as for stable carbon isotope measurements (see Section 2.2.2 above) with the following modifications: The flow rate was 0.6 mL min −1 and instead of combustion to CO 2 and H 2 O, CH 4 was thermolytically converted (at 1450 • C) to produce hydrogen (H 2 ) and carbon. After IRMS measurements of the hydrogen, the obtained δ 2 H values were normalized using two reference standards of high-purity CH 4 with δ 2 H values of-190.6 ± 0.2‰ (in-house) and-149.9 ± 0.2‰ (T-iso2, Isometric Instruments).

Statistics
Data analysis was performed using R 4.1.2 software. For data smoothing, the Loess method was used. For data analysis with CRDS (sampling rate = 1 s), mean values were taken for those periods in which the data variation was less than 5% (measurement periods of 20-30 min). The ∆δ 13 C-CH 4 values for both experiments are presented as the arithmetic means of the respective replicates, together with their standard deviations (SD). The arithmetic means and SDs were calculated using Microsoft Excel (Microsoft Excel for Office 365 MSO).  In addition, Figure 3c compares the excess of the isotopic labels in the released breath CH4 from the supplemented 13 C-and 2 H-DMSO. The excess in both 13 C-CH4 and 2 H-CH4 gradually increased, with maximum values observed at 40 min for 2 H-CH4 (~0.68‰) and 50 min for 13 C-CH4 (~0.028‰). The calculated time integral (area under the curve) values were 37 and 2.14 for 2 H-CH4-and 13 C-CH4-excess, respectively. Both isotope tracers evidently indicated partial conversion of the methyl group of DMSO to CH4 by processes within the human body. The calculated time integral found for 2 H-CH4 was by a factor of around 17 higher when compared with 13 C-CH4. In this context, it should be noted that the amount of applied isotope 13 C-labeling of DMSO was much lower for the 13 C experiments (see the Discussion section).

Blood Samples and Addition of Isotopically Labeled DMSO and Methionine
The supplementation of 13 C-labeled DMSO and methionine at equimolar concentrations of 1 mM to the blood samples incubated for 24 h (first day) at 36 °C resulted in mean Δδ 13 C-CH4 values of 95 ± 36 mUr and 2.2 ± 0.5 mUr for DMSO and methionine, respectively ( Figure 4). Repeated measurements of the same samples (after equilibration with laboratory air; see the Materials and Methods section) and another incubation period of 24 h (second day) exhibited lower mean Δδ 13 C-CH4 values, producing 70 ± 10 mUr and 0.24 ± 0.4 mUr for DMSO and methionine, respectively. The application of ten-fold higher concentrations of DMSO and methionine (10 mM) enhanced the formation of isotopically labeled CH4, with the Δδ 13 C-CH4 values producing 748 ± 362 mUr and 4.9 ± 3.5 mUr for DMSO and methionine, respectively. Again, repeated measurements of the same samples In addition, Figure 3c compares the excess of the isotopic labels in the released breath CH 4 from the supplemented 13 C-and 2 H-DMSO. The excess in both 13 C-CH 4 and 2 H-CH 4 gradually increased, with maximum values observed at 40 min for 2 H-CH 4 (~0.68‰) and 50 min for 13 C-CH 4 (~0.028‰). The calculated time integral (area under the curve) values were 37 and 2.14 for 2 H-CH 4 -and 13 C-CH 4 -excess, respectively. Both isotope tracers evidently indicated partial conversion of the methyl group of DMSO to CH 4 by processes within the human body. The calculated time integral found for 2 H-CH 4 was by a factor of around 17 higher when compared with 13 C-CH 4 . In this context, it should be noted that the amount of applied isotope 13 C-labeling of DMSO was much lower for the 13 C experiments (see the Discussion section).

Blood Samples and Addition of Isotopically Labeled DMSO and Methionine
The supplementation of 13 C-labeled DMSO and methionine at equimolar concentrations of 1 mM to the blood samples incubated for 24 h (first day) at 36 • C resulted in mean ∆δ 13 C-CH 4 values of 95 ± 36 mUr and 2.2 ± 0.5 mUr for DMSO and methionine, respectively ( Figure 4). Repeated measurements of the same samples (after equilibration with laboratory air; see the Materials and Methods section) and another incubation period of 24 h (second day) exhibited lower mean ∆δ 13 C-CH 4 values, producing 70 ± 10 mUr and 0.24 ± 0.4 mUr for DMSO and methionine, respectively. The application of ten-fold higher concentrations of DMSO and methionine (10 mM) enhanced the formation of isotopically labeled CH 4 , with the ∆δ 13 C-CH 4 values producing 748 ± 362 mUr and 4.9 ± 3.5 mUr for DMSO and methionine, respectively. Again, repeated measurements of the same samples after another incubation period of 24 h (second day) exhibited lower mean ∆δ 13 C-CH 4 values, producing 588 ± 10 mUr and 1.4 ± 0.1 mUr for DMSO and methionine, respectively. Thus, the change in 10-fold concentrations was closely reflected by the change in ∆δ 13 C-CH 4 values (factor of~8) for both days, whilst for methionine, the change in ∆δ 13 C-CH 4 values was lower (factors of 2.2 and 5.7 for day 1 and day 2, respectively). All control samples including blood without the addition of isotopically labeled compounds did not show any measurable difference in δ 13 C-CH 4 values over the incubation time.
Antioxidants 2023, 12, x FOR PEER REVIEW 12 of 23 after another incubation period of 24 h (second day) exhibited lower mean Δδ 13 C-CH4 values, producing 588 ± 10 mUr and 1.4 ± 0.1 mUr for DMSO and methionine, respectively. Thus, the change in 10-fold concentrations was closely reflected by the change in Δδ 13 C-CH4 values (factor of ~8) for both days, whilst for methionine, the change in Δδ 13 C-CH4 values was lower (factors of 2.2 and 5.7 for day 1 and day 2, respectively). All control samples including blood without the addition of isotopically labeled compounds did not show any measurable difference in δ 13 C-CH4 values over the incubation time.    3.3. Skin Application of Isotopically Labeled DMSO and Incubation of Arm with Exposure to Natural Sunlight Figure 5 shows the isotope difference as δ 13 C-CH 4 values relative to the control values after the application of 13 C-labeled DMSO on the left forearm. Subsequent to the DMSO application, the δ 13 C-CH 4 values increased by 30 mUr within 1 h. After the volunteer exposed his left forearm to natural sunlight in the field, a maximum ∆δ 13 C-CH 4 value of 50 mUr was observed. Please note that direct measurements during exposure to sunlight in the field were not possible. For experimental details, we refer the reader to the Materials and Methods section. After 24 h, δ 13 C-CH 4 values measured for CH 4 release from the skin of the left forearm were still enriched by 4 mUr, whilst the control values (the incubation of the untreated right forearm) did not show any measurable changes. Again, exposure to sunlight in the field and subsequent laboratory measurements of CH 4 release from the skin of the forearm increased the ∆δ 13 C-CH 4 value to 6 mUr. After 48 h, the δ 13 C-CH 4 values monitored from the release of the skin still showed a marginal but measurable 13 C enrichment of 1 mUr. After sunlight irradiation, no measurable increase in δ 13 C-CH 4 values was noted. The associated CH 4 concentrations of the chamber measurement series showed changes in the range of 1.96 to 2.08 ppmv, which were close to the variations observed for the control measurements. of the left forearm were still enriched by 4 mUr, whilst the control values (the incubation of the untreated right forearm) did not show any measurable changes. Again, exposure to sunlight in the field and subsequent laboratory measurements of CH4 release from the skin of the forearm increased the Δδ 13 C-CH4 value to 6 mUr. After 48 h, the δ 13 C-CH4 values monitored from the release of the skin still showed a marginal but measurable 13 C enrichment of 1 mUr. After sunlight irradiation, no measurable increase in δ 13 C-CH4 values was noted. The associated CH4 concentrations of the chamber measurement series showed changes in the range of 1.96 to 2.08 ppmv, which were close to the variations observed for the control measurements.

Conversion of Methylated Sulfur Compounds to Methane
The three sets of experiments-involving the application of two potential CH4 precursor compounds, DMSO and methionine, with isotopic labels-provided independent lines of evidence for partial conversion of the supplemented methyl group to CH4 in the human body. The combination of the three experiments (oral intake, blood incubations, and skin application) was undertaken to confirm that CH4 is endogenously formed in humans via a ROS-driven process without involvement of the well-known microbial sources (methanogens) occurring under anoxic conditions in the gastrointestinal tracts. However, we are aware that it is almost impossible to exclude the contribution of microbes during the screening of humans for CH4 emissions.

Oral Administration of 13 C-Labeled DMSO
The measured isotopic changes for the two labeling experiments (Figure 3) unambiguously demonstrated that the methyl group of DMSO was converted to CH4. The 2 H and 13 C excess values indicated that only a marginal fraction (0.68‰ and 0.028‰) of the CH4 concentration measured in the subject's breath air (~2 to 16 ppmv) was actually derived from the isotopically labeled precursor methyl groups of DMSO. The observed variabilities in concentrations during the individual experiments (Figure 3a,b, top panels) were in the range of the intraday fluctuations. The observed difference in CH4 base levels of approximately 10 ppmv between the experiments with 13 C DMSO and 2 H DMSO reflected usual changes in the individual's breath CH4 state, as the two experiments were performed a few months apart. For details regarding the variabilities of CH4 base levels of the volunteer, see Polag and Keppler [37,38]. The small concentration changes indicated by the supplementation of 13 C-labeled DMSO would be nondetectable when using conventional measurement techniques, and can only be traced using isotopic labeling techniques. To better compare the conversion of the two labeling approaches, it is necessary to consider the 2 H/ 13 C excess values, as shown in Figure 3c. The calculated time integrals of the 2 H-CH4-and 13 C-CH4 excesses were 37 and 2.14, respectively, and thus, the time integral found for the 2 H-CH4 excess was higher by a factor of around 17 when compared with the

Conversion of Methylated Sulfur Compounds to Methane
The three sets of experiments-involving the application of two potential CH 4 precursor compounds, DMSO and methionine, with isotopic labels-provided independent lines of evidence for partial conversion of the supplemented methyl group to CH 4 in the human body. The combination of the three experiments (oral intake, blood incubations, and skin application) was undertaken to confirm that CH 4 is endogenously formed in humans via a ROS-driven process without involvement of the well-known microbial sources (methanogens) occurring under anoxic conditions in the gastrointestinal tracts. However, we are aware that it is almost impossible to exclude the contribution of microbes during the screening of humans for CH 4 emissions.

Oral Administration of 13 C-Labeled DMSO
The measured isotopic changes for the two labeling experiments (Figure 3) unambiguously demonstrated that the methyl group of DMSO was converted to CH 4 . The 2 H and 13 C excess values indicated that only a marginal fraction (0.68‰ and 0.028‰) of the CH 4 concentration measured in the subject's breath air (~2 to 16 ppmv) was actually derived from the isotopically labeled precursor methyl groups of DMSO. The observed variabilities in concentrations during the individual experiments (Figure 3a,b, top panels) were in the range of the intraday fluctuations. The observed difference in CH 4 base levels of approximately 10 ppmv between the experiments with 13 C DMSO and 2 H DMSO reflected usual changes in the individual's breath CH 4 state, as the two experiments were performed a few months apart. For details regarding the variabilities of CH 4 base levels of the volunteer, see Polag and Keppler [37,38]. The small concentration changes indicated by the supplementation of 13 C-labeled DMSO would be nondetectable when using conventional measurement techniques, and can only be traced using isotopic labeling techniques. To better compare the conversion of the two labeling approaches, it is necessary to consider the 2 H/ 13 C excess values, as shown in Figure 3c. The calculated time integrals of the 2 H-CH 4 -and 13 C-CH 4 excesses were 37 and 2.14, respectively, and thus, the time integral found for the 2 H-CH 4 excess was higher by a factor of around 17 when compared with the 13 C-CH 4 excess. Please note that the 2 H-CH 4 excess time integral of 37 included three deuterium atoms from a 2 H-labeled methyl group and a fourth, unlabeled hydrogen atom (see Figure 6). To correct for this effect, the time integral of 2 H increased to 49, and the differences between the excess values of 2 H-CH 4 and 13 C-CH 4 changed to a factor of 23. This value closely reflected the relationship between orally administered 2 H and 13 C isotope tracers (factor of 34). The reason for applying different amounts of 2 H/ 13 C DMSO isotopic labels was due to financial issues, as 2 H-labeled DMSO is considerably cheaper than 13 C-labeled DMSO. Nevertheless, both isotope tracers independently and clearly indicated similar conversion rates of the methyl group of DMSO when normalized to the amount of applied isotopic tracer. We suggest that the observed CH 4 formation is indicative of the formation of methyl radicals from DMSO induced by hydroxyl radicals or oxo-iron(IV) species, as recently proposed by Ernst et al. [17], Benzing et al. [52], and Althoff et al. [51] for biological and abiotic chemical systems. Once methyl radicals are formed, they can react with a hydrogen atom from hydrocarbons, hydrogen peroxide, or hydrogen carbonate to form CH 4 . The formation of 13 C-enriched CH 4 was already measurable a few minutes after the oral intake of the labeled substance for both isotope labeling experiments ( 2 H and 13 C). However, around 2 h after the oral administration, CH 4 formation from DMSO was barely detectable in the breath air, potentially implying that most of the DMSO was converted in the human body within this timespan. A possible decay mechanism is the conversion of DMSO to dimethyl sulfide (DMS) by the molybdoenzyme DMSO reductase, which is widespread in all domains of life [69]. A recently proposed mechanism of DMSO reductase can be found in Le et al. [70].
Antioxidants 2023, 12, x FOR PEER REVIEW 14 of 23 13 C-CH4 excess. Please note that the 2 H-CH4 excess time integral of 37 included three deuterium atoms from a 2 H-labeled methyl group and a fourth, unlabeled hydrogen atom (see Figure 6). To correct for this effect, the time integral of 2 H increased to 49, and the differences between the excess values of 2 H-CH4 and 13 C-CH4 changed to a factor of 23. This value closely reflected the relationship between orally administered 2 H and 13 C isotope tracers (factor of 34). The reason for applying different amounts of 2 H/ 13 C DMSO isotopic labels was due to financial issues, as 2 H-labeled DMSO is considerably cheaper than 13 Clabeled DMSO. Nevertheless, both isotope tracers independently and clearly indicated similar conversion rates of the methyl group of DMSO when normalized to the amount of applied isotopic tracer. We suggest that the observed CH4 formation is indicative of the formation of methyl radicals from DMSO induced by hydroxyl radicals or oxo-iron(IV) species, as recently proposed by Ernst et al. [17], Benzing et al. [52], and Althoff et al. [51] for biological and abiotic chemical systems. Once methyl radicals are formed, they can react with a hydrogen atom from hydrocarbons, hydrogen peroxide, or hydrogen carbonate to form CH4. The formation of 13 C-enriched CH4 was already measurable a few minutes after the oral intake of the labeled substance for both isotope labeling experiments ( 2 H and 13 C). However, around 2 h after the oral administration, CH4 formation from DMSO was barely detectable in the breath air, potentially implying that most of the DMSO was converted in the human body within this timespan. A possible decay mechanism is the conversion of DMSO to dimethyl sulfide (DMS) by the molybdoenzyme DMSO reductase, which is widespread in all domains of life [69]. A recently proposed mechanism of DMSO reductase can be found in Le et al. [70].  2+ or hydroxyl radicals, depending on the reaction conditions. Subsequently, CH4 is formed through the reaction of a methyl radical with a hydrogen atom derived from hydrocarbons, hydrogen peroxide, or hydrogen carbonate. Red and green indicate hydrogen and carbon atoms, respectively, of methylated sulfur compounds labeled with 2 H and 13 C, as applied in this study to subsequently trace the formation of CH4 in humans.

Supplementation of 13 C-Labeled DMSO and Methionine to Blood Samples
The experiments with blood samples were conducted to further demonstrate the nonmicrobial formation of CH4 when different S-methylated compounds were supplemented. When equimolar amounts of DMSO and methionine were added to the blood samples, the conversion of S-methyl-bonded groups to CH4 was much higher for DMSO than for methionine, with factors ranging from 43 to 423. It is well known that DMSO is a potent hydroxyl radical scavenger [71] that forms CH4; ethane; and oxidized C1 compounds, such as formaldehyde and formate, depending on the experimental conditions [72][73][74]. The observed differences between the application of DMSO and methionine are in line with previous experiments conducted by Althoff et al. [51] and Ernst et al. [17], who showed the preferential formation of ROS-induced formation of CH4 from DMSO relative to methionine in chemical systems and living organisms, respectively. However, in our study, the Figure 6. Simplified reaction scheme for endogenous CH 4 formation in humans. Methylated S-/N-compounds produced via metabolism or externally supplemented act as •OH scavengers or react with oxo-iron(IV) ([Fe IV =O] 2+ ) to produce methyl radicals. Activation of hydrogen peroxide by ferrous iron (Fenton systems) leads to several oxidizing agents, such as [Fe IV =O] 2+ or hydroxyl radicals, depending on the reaction conditions. Subsequently, CH 4 is formed through the reaction of a methyl radical with a hydrogen atom derived from hydrocarbons, hydrogen peroxide, or hydrogen carbonate. Red and green indicate hydrogen and carbon atoms, respectively, of methylated sulfur compounds labeled with 2 H and 13 C, as applied in this study to subsequently trace the formation of CH 4 in humans.

Supplementation of 13 C-Labeled DMSO and Methionine to Blood Samples
The experiments with blood samples were conducted to further demonstrate the non-microbial formation of CH 4 when different S-methylated compounds were supplemented. When equimolar amounts of DMSO and methionine were added to the blood samples, the conversion of S-methyl-bonded groups to CH 4 was much higher for DMSO than for methionine, with factors ranging from 43 to 423. It is well known that DMSO is a potent hydroxyl radical scavenger [71] that forms CH 4 ; ethane; and oxidized C1 compounds, such as formaldehyde and formate, depending on the experimental conditions [72][73][74]. The observed differences between the application of DMSO and methionine are in line with previous experiments conducted by Althoff et al. [51] and Ernst et al. [17], who showed the preferential formation of ROS-induced formation of CH 4 from DMSO relative to methionine in chemical systems and living organisms, respectively. However, in our study, the difference between DMSO and methionine was even more pronounced and might be explained by the specific composition of the blood samples, i.e., amounts and availability of iron species and ROS. In addition, methionine needs to be oxidized to methionine sulfoxide before the methyl groups can be cleaved off [51]. Human blood and plasma contain high amounts of iron species, particularly in the form of hemoglobin, and the range of H 2 O 2 might be in a normal concentration range of 1-5 µM but increases to 30-50 µM during chronic inflammation in certain disease states [75]. Thus, the interplay between iron species and ROS in blood might be highly supportive for the formation of CH 4 given that the required methyl precursor compounds are also available. Interestingly, a ten-fold higher DMSO supplementation was well reflected by the amounts of formed labeled CH 4 (factor of~8), whilst a considerably lower increase was observed (mean factor of~4) for the addition of methionine. It was also obvious that CH 4 formation from DMSO was observable for much longer (at least for 48 h) in the blood samples when compared with the oral administration of DMSO (see section above), indicating that different degradation processes in the human body might have contributed to the observed pattern.

Dermal CH 4 Emissions after Treatment of Isotopically Labeled DMSO
The application of 13 C-labeled DMSO on the volunteer's forearm clearly showed the release of isotopically labeled CH 4 immediately after incubation of the penetrated skin section ( Figure 5) under laboratory conditions. Based on our current understandingincluding knowledge of ROS-driven CH 4 formation, and that DMSO rapidly penetrates through human skin-this observation is highly indicative of methyl radical formation induced by ROS that occurs in the epidermis or dermis of the skin. There is frequent formation of ROS in the cells and it is well known that skin exposure to light-including wavelengths of visible, UVA/UVB, and IR light -induces and increases ROS levels [76,77]. After the volunteer exposed his left forearm to natural sunlight in the field for 1 h, a strong isotope change in δ 13 C-CH 4 values (~70% higher relative to laboratory light exposure) was measured, even though these measurements were conducted after exposure to direct solar radiation. This implies that enhanced levels of ROS were caused by the irradiation of solar light, leading to the formation of CH 4 , which could only be made visible by the administration of 13 C-labeled DMSO. After around 24 h, the release of 13 C-labeled CH 4 from the skin under laboratory incubation conditions was still measurable and increased again (by about 50%) after the exposure of the skin to natural sunlight. When repeating the same procedure after 48 h, a small but indicative change in δ 13 C-CH 4 values was still observed for the laboratory exposure incubations of the forearm. No additional increase in δ 13 C-CH 4 values could be measured for the effect of natural sunlight. However, it was remarkable to observe DMSO-related liberation of CH 4 from the skin even 50 h after the application of 13 C-labeled DMSO. There exist only a few studies that dealt in detail with the release of CH 4 from human skin, and in general, these emissions are considered to be much smaller than those measured for breath release [35]. This was recently confirmed by Li et al. [78], who quantified dermal and exhaled CH 4 of 20 volunteers using climate chambers and reported that the average estimated exhaled CH 4 release rate was about 19 (max. range 13-37) times higher than the average dermal CH 4 emission rate. For completeness, it should be noted that Mochalski et al. [79] measured emission rates of selected volatile organic compounds from the skin of healthy volunteers. However, the researchers did not detect CH 4 , as they screened for larger carbon compounds, including C4 to C10 substances, and found relatively large emissions for three volatiles: acetone, acetaldehyde, and 6-methyl-5-hepten-2-one.

ROS-Induced non-Microbial Formation of CH 4 from Methylated S-/N-Compounds in Humans: A Hypothesis
The observed formation of CH 4 from the S-bonded methyl groups of DMSO or methionine provides strong support for a radical-driven process of CH 4 formation. Based on the three applied isotopic labeling experiments and a previous study demonstrating ROS-driven CH 4 formation from in vitro experiments of many organisms [17], we propose a reaction scheme showing the interplay of methyl precursors, ROS, and iron species that eventually leads to the formation of CH 4 in humans ( Figure 6).
The three major players in this reaction scheme are ROS, iron, and methyl groups bonded to sulfur and nitrogen compounds. Below, we briefly summarize their role in humans with respect to non-microbial CH 4 formation.
Initially considered principally toxic, today, ROS are well-known for having beneficial or deleterious effects in aerobic organisms [59,[80][81][82]. The concentration of H 2 O 2 in the normal cytoplasm, mitochondrial matrix, and endoplasmic reticulum (ER) lumen varies by several orders of magnitude (from 80 pM to 700 nM) [83] and is even higher in blood and plasma at normal concentrations of 1-5 µM, but increases to 30-50 µM during chronic inflammation in certain disease states [75]. On the one hand, ROS play various roles in the cellular functioning of aerobic organisms, are involved in many redox-governing activities of the cells for the preservation of cellular homeostasis, and are required for many important signaling reactions. On the other hand, elevated ROS levels can lead to severe damage in cells. In this context, it was suggested that frequently increased oxidative stress leads to an overproduction of ROS, causing many diseases and a variety of age-related disorders, such as Parkinson's disease, Alzheimer's dementia, chronic inflammatory diseases, atherosclerosis, heart attacks, cancer, ischaemia/reperfusion injury, and arteriosclerosis. Thus, it can be easily envisaged that CH 4 might be formed at highly fluctuating levels in different organelles and might potentially serve to monitor enhanced ROS levels in humans. This hypothesis is supported by the results of several recent monitoring studies: (I) The observation that breath CH 4 levels increase with advanced age [24] might be an indication of the human age-related increase in systemic inflammation accompanied by enhanced ROS levels. (II) Long-term monitoring studies of breath CH 4 from several volunteers provided evidence that abrupt deviations in breath CH 4 levels from baseline were linked to inflammatory processes and immune reactions [37]. In this context, infectious diseases were mostly accompanied by temporarily elevated breath CH 4 formation. Next, it was hypothesized that vaccinations as induced perturbations of the immune system might cause substantial fluctuations in the breath CH 4 level of people, indicating individual immune responses and immune states. (III) This was recently shown by Polag and Keppler [38], who investigated the breath CH 4 levels after COVID-19 vaccination. They clearly found large deviations from the average breath CH 4 values after vaccination and concluded that these deviations were likely related to immune reactions and may have also originated from redox homeostasis in cells. A change in the breath CH 4 levels from individual baseline values could be used to monitor changes in levels of ROS and oxidative stress, and could potentially be used to classify immune responses. (IV) Finally, Tuboly et al. [84] investigated the possibility of CH 4 generation in low-CH 4 emitters that consumed high doses of ethanol with the aim to increase oxidative stress. A transient, significant CH 4 production was noted after an excessive ethanol intake. The researchers found similar results when they repeated the ethanol experiments with rats. They further investigated the hypothesis that L-alpha-glycerylphosphorylcholine (GPC) may influence CH 4 formation through the modulation of alcohol-induced mitochondrial dysfunction.
This brings us to the next point: to counteract oxidative stress, aerobic cells possess many antioxidative systems that function to keep the ROS level in a non-toxic range. Methyl precursors-particularly those where the methyl group is bonded to sulfur and nitrogen compounds-can readily be cleaved off to produce CH 4 or oxidized C1 species [17,51,52]. The various available S-/N-methylated compounds in biological systems will cause different efficiencies of CH 4 production and consumption of ROS. DMSO is not produced in humans and is only consumed via the diet in relatively small quantities [85]. However, this effective radical scavenger was ideally suited to test the hypothesis of non-microbial CH 4 formation in humans. It is non-toxic in the applied doses, penetrates rapidly through human skin, and is easily distributed in the body, as it dissolves in both polar and nonpolar compounds. On the other hand, the other applied S-methylated compound, namely, methionine, is an essential amino acid in humans that has an important role in metabolism and health. It is the precursor of other important compounds, such as cysteine, S-adenosyl methionine (SAM), and glutathione. It was also shown to produce CH 4 , albeit at much lower conversion rates when compared with DMSO.
Nitrogen-methylated substances, such as choline (2-Hydroxyethyl-trimethylammonium), are formed in humans but are also essential compounds for maintaining health. Therefore, they must be consumed by diet as choline or as choline phospholipids. Large amounts of choline are stored in the human cell membranes and organelles as phospholipids, and inside cells as phosphatidylcholines and GPC. Choline was shown to form CH 4 in a chemical model system containing iron and hydrogen peroxide [51,86] but this was not confirmed in bacterial culture experiments [17]. Tuboly et al. [84] showed that exogenous GPC protected against ethanol-induced mitochondrial electron transport chain dysfunction in rat liver, which is the primary target of alcohol-induced oxido-reductive stress. Therefore, the exogenous addition of methylated compounds might strongly increase CH 4 production and ROS consumption. In this context, it is of interest to further discuss the potential role of DMSO as an effective scavenger of radicals to counteract enhanced oxidative stress induced by ROS. DMSO has already been investigated for many years, but its beneficial role for medical use remains highly uncertain (see the Introduction section).
Finally, the concentration of free iron (in the form of iron(II)) is of importance for the enhanced production of hydroxyl radicals (Fenton-type reactions) in biological systems [59]. However, inappropriately low or high levels of iron are detrimental and contribute to a wide range of diseases [87]; therefore, understanding the dysregulation of iron metabolism is crucial in the search for therapeutics [88]. Harmful oxidative distress could be observed in states of both iron deficiency (anemia) and overload (ferroptosis) [89]. It is plausible that appropriate supplementation of iron is beneficial to health, which may be related to its role in contributing to the homeostasis of cellular ROS through the production of CH 4 .
A detailed understanding of the interplay between ROS, iron, and methylated substrates in humans is necessary to better understand radical-driven CH 4 and to answer the question of whether the cellular formation of CH 4 has a physiological role in humans. In this context, monitoring CH 4 as an indicator for ROS-driven processes could be a promising approach in biochemical research, where breath CH 4 could be used as a diagnostic tool in the fields of system biology and precision medicine. This could include the application of isotopic labeling experiments of methylated precursor substances (with a 13 C or 2 H label), as this approach could specifically visualize ROS-related CH 4 generation, and thus, overcome the problem of higher breath CH 4 background concentrations derived from microbial sources. These changes may be interesting for diagnostic purposes. Moreover, the possibility exists that such changes may affect the overall cellular response to intracellular hypoxia. Simple asphyxiants, such as CH 4 , act by physically limiting the utilization of oxygen and can modify the symbiosis with other gaseous compounds within the internal milieu of aerobic cells. Although CH 4 is conventionally believed to be physiologically inert, a comprehensive view of its biological effects in various hypoxic and inflammatory scenarios was demonstrated [90]. Notably, it was shown that CH 4 can modulate the pathways involved in key events of inflammation via master switches, such as Nrf2/Keap1 and NF-κB (for a review, see [18]). Several studies also demonstrated that exogenous CH 4 modulates the intrinsic, mitochondrial pathway of pro-apoptotic activation in model experiments [91]. Furthermore, sequential in vitro studies with exogenous normoxic CH 4 in simulated ischemia-reperfusion environments provided evidence that CH 4 preserves the mitochondrial respiratory capacity in cells exposed to anoxia [92]. In a similar protocol, CH 4 treatment restricted the forward electron transfer within complex I in control mitochondria while effectively restricting reverse electron transport (RET) in post-anoxic mitochondria. In parallel studies, CH 4 influenced several components of the endoplasmic reticulum-mitochondria-related pro-apoptotic signaling pathways, the oxidative phosphorylation capacity was more preserved, and the relative mRNA expression for hypoxia-and ER stress-associated genes (including HIF-1α) was significantly reduced [93]. For a detailed discussion regarding the potential applications of monitoring CH 4 in medical research and health sciences, see [18,38,94,95].

Conclusions
We are aware that the investigation of ROS-driven cellular CH 4 formation in only one subject is too low of a sample size to draw broad and general conclusions. However, this study represents the first proof of concept that cellular CH 4 formation occurs in the human body and is most likely a result of the interplay between ROS and methylated substrates. This process can currently only be made clearly visible by applying stable-isotope-tracing techniques to distinguish CH 4 formation in humans from that of microbes living in the gastrointestinal tract. Together with other recently published studies [17,38,94], it is becoming obvious that ROS-driven CH 4 formation might be a necessary phenomenon of aerobic life. Consequently, non-microbial aerobic CH 4 formation should be highly variable in time and source strength, as it may be an integral part of the cellular responses toward changes in oxidative status present in humans. Large changes in human breath levels were observed by several recent monitoring studies [38,78,84], and some suggested that variations in CH 4 breath levels are unlikely to be explained by microbial formation in the human digestive system. However, additional investigations are required to obtain unambiguous evidence of non-microbial CH 4 formation in humans and the underlying processes of its generation. This will be a significant challenge because in the case of high emitters-where CH 4 formation by methanogens is the dominant process-it is difficult to distinguish between the non-microbial and microbial pathways of CH 4 production. Nevertheless, for low and medium CH 4 emitters, who comprise about 70% of the global population, we suggest that substantially changed human breath CH 4 levels from individual baseline values may be used to detect changes in oxidative stress and ROS levels, and could potentially be used to classify immune responses, as recently suggested by Polag and Keppler [38]. Therefore, future investigations should focus on deciphering the potential physiological role of CH 4 formation in humans, as well as on the monitoring of CH 4 as an indicator for individual immune states and a potential biomarker of oxidative stress. In addition, revisiting and studying in greater detail the potential role of DMSO as an effective hydroxyl radical scavenger and its use for human medical research might be worthwhile.
Supplementary Materials: The Supplementary Materials for this article can be downloaded from https://www.mdpi.com/article/10.3390/antiox12071381/s1. Figures S1 and S2: Determination of stable isotope source signatures of CH 4 using keeling plots: Photo S1: Arm incubation chamber for online measurements using CRDS; Table S1: Overview and timeline of isotope labeling experiments.
Author Contributions: F.K. conceived the study, conducted the experiments, and analyzed the samples. F.K. and D.P. discussed and evaluated the data and prepared graphical illustrations of the data. M.B. provided critical discussion on the medical aspects of the experiments and results. The manuscript was written under the lead of F.K., with contributions from M.B. and D.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and it was approved by the Medical Research Council of Hungary (ETT-TUKEB) as part of the protocol "Mapping metabolic pathways of endogenous gas formation by isotopic analysis of the gas composition of human samples" (6420-8-2023/EUIG/768).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data used in this publication are available to the community and can be accessed by request to the corresponding author.