Breath Methane Research Articles

Association of bacterial overgrowth in the small intestine with cortical thickness and functional connectivity in Parkinson's disease involving mild cognitive impairment.

This study explored potential associations of bacterial overgrowth in the small intestine, as detected based on levels of hydrogen and methane in breath after lactulose consumption, with cortical thickness and resting-state functional connectivity in different brain regions. Prospective comparison of 35 patients with Parkinson's disease (PD) involving mild cognitive impairment, 35 patients with PD with normal cognitive function and 17 healthy controls showed the largest level of hydrogen alone and the largest combined level of hydrogen and methane in patients with mild cognitive impairment. The comparison also revealed a significant negative correlation between those levels and thickness of the right insular cortex. Mild cognitive patients showed different functional connectivity between the right insula and cognition-related brain networks from normal cognitive patients. Our results suggest that bacterial overgrowth in the small intestine may contribute to cortical thinning and alterations in resting-state functional connectivity in PD involving mild cognitive impairment. These insights support and deepen previous observations implicating the gut-brain axis in the neurological disorder.

S777 Relationships Between Bowel Movements and Basal Levels of Breath Hydrogen and Methane as Measured by a Portable Handheld App-Connected Device

S777 Relationships Between Bowel Movements and Basal Levels of Breath Hydrogen and Methane as Measured by a Portable Handheld App-Connected Device

Machine learning-based phenogroups and prediction model in patients with functional gastrointestinal disorders to reveal distinct disease subsets associated with gas production.

Symptom-based subtyping for functional gastrointestinal disorders (FGIDs) has limited value in identifying underlying mechanisms and guiding therapeutic strategies. Small intestinal dysbiosis is implicated in the development of FGIDs. We tested if machine learning (ML) algorithms utilizing both gastrointestinal (GI) symptom characteristics and lactulose breath tests could provide distinct clusters. This was a prospective cohort study. We performed lactulose hydrogen methane breath tests and hydrogen sulfide breath tests in 508 patients with GI symptoms. An unsupervised ML algorithm was used to categorize subjects by integrating GI symptoms and breath gas characteristics. Generalized Estimating Equation (GEE) models were used to examine the longitudinal associations between cluster patterns and breath gas time profiles. An ML-based prediction model for identifying excessive gas production in FGIDs patients was developed and internal validation was performed. FGIDs were confirmed in 300 patients. K-means clustering identified 4 distinct clusters. Cluster 2, 3, and 4 showed enrichments for abdominal distention and diarrhea with a high proportion of excessive gas production, whereas Cluster 1 was characterized by moderate lower abdominal discomforts with the most psychological complaints and the lowest proportion of excessive gas production. GEE models showed that breath gas concentrations varied among different clusters over time. We further sought to develop an ML-based prediction model to determine excessive gas production. The model exhibited good predictive capabilities. ML-based phenogroups and prediction model approaches could provide distinct FGIDs subsets and efficiently determine FGIDs subsets with greater gas production, thereby facilitating clinical decision-making and guiding treatment.

Targeted isolation of Methanobrevibacter strains from fecal samples expands the cultivated human archaeome

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes’ adaptability in critical microbiome contexts.

Methane gas in breath test is associated with non-alcoholic fatty liver disease

Although the associations between a patient’s body mass index (BMI) and metabolic diseases, as well as their breath test results, have been studied, the relationship between breath hydrogen/methane levels and metabolic diseases needs to be further clarified. We aimed to investigate how the composition of exhaled breath gases relates to metabolic disorders, such as diabetes mellitus, dyslipidemia, hypertension, and nonalcoholic fatty liver disease (NAFLD), and their key risk factors. An analysis was performed using the medical records, including the lactulose breath test (LBT) data of patients who visited the Ajou University Medical Center, Suwon, Republic of Korea, between January 2016 and December 2021. The patients were grouped according to four different criteria for LBT hydrogen and methane levels. Of 441 patients, 325 (72.1%) had positive results for methane only (hydrogen < 20 parts per million [ppm] and methane ⩾ 3 ppm). BMIs and NAFLD prevalence were higher in patients with only methane positivity than in patients with hydrogen and methane positivity (hydrogen ⩾ 20 ppm and methane ⩾ 3 ppm). According to a multivariate analysis, the odds ratio of only methane positivity was 2.002 (95% confidence interval [CI]: 1.244–3.221, P = 0.004) for NAFLD. Our results demonstrate that breath methane positivity is related to NAFLD and suggest that increased methane gas on the breath tests has the potential to be an easily measurable biomarker for NAFLD diagnosis.

Real-time breath gas analysis of methane using a multipass cell-based near-infrared gas sensor.

We demonstrated a near-infrared exhaled breath sensor for real-time methane measurements by using tunable diode laser absorption spectroscopy (TDLAS), which can enable the noninvasive diagnosis of intestinal tract problems. The core component of the near-infrared TDLAS sensor is a two-mirror-based multipass cell with nine-circle patterns. An optical path length of 23.4 m was achieved in a volume of 233.3 cm3, which effectively improved the detection sensitivity and shortened the gas exchange time. The minimum detection limit was 0.37 ppm by applying wavelength modulation spectroscopy, which was 12.4 times greater than that of direct absorption spectroscopy. In addition, combined with wavelength modulation spectroscopy, the two-mirror-based multipass cell enabled sub-second gas exchange time of 0.6 s. Methane breath experiments were conducted with six volunteers, and the real-time measurement results and concentrations at the end of exhalation were analyzed. This study demonstrates that the developed sensor has high sensitivity, high selectivity, and fast response for breath methane measurements and has promising potential for noninvasive, real-time, and point-of-care disease diagnosis in clinical applications.

Fecal Coprococcus, hidden behind abdominal symptoms in patients with small intestinal bacterial overgrowth

BackgroundSmall intestinal bacterial overgrowth (SIBO) is the presence of an abnormally excessive amount of bacterial colonization in the small bowel. Hydrogen and methane breath test has been widely applied as a non-invasive method for SIBO. However, the positive breath test representative of bacterial overgrowth could also be detected in asymptomatic individuals.MethodsTo explore the relationship between clinical symptoms and gut dysbiosis, and find potential fecal biomarkers for SIBO, we compared the microbial profiles between SIBO subjects with positive breath test but without abdominal symptoms (PBT) and healthy controls (HC) using 16S rRNA amplicon sequencing.ResultsFecal samples were collected from 63 SIBO who complained of diarrhea, distension, constipation, or abdominal pain, 36 PBT, and 55 HC. For alpha diversity, the Shannon index of community diversity on the genus level showed a tendency for a slight increase in SIBO, while the Shannon index on the predicted function was significantly decreased in SIBO. On the genus level, significantly decreased Bacteroides, increased Coprococcus_2, and unique Butyrivibrio were observed in SIBO. There was a significant positive correlation between saccharolytic Coprococcus_2 and the severity of abdominal symptoms. Differently, the unique Veillonella in the PBT group was related to amino acid fermentation. Interestingly, the co-occurrence network density of PBT was larger than SIBO, which indicates a complicated interaction of genera. Coprococcus_2 showed one of the largest betweenness centrality in both SIBO and PBT microbiota networks. Pathway analysis based on the Kyoto Encyclopedia of Genes and Genome (KEGG) database reflected that one carbon pool by folate and multiple amino acid metabolism were significantly down in SIBO.ConclusionsThis study provides valuable insights into the fecal microbiota composition and predicted metabolic functional changes in patients with SIBO. Butyrivibrio and Coprococcus_2, both renowned for their role in carbohydrate fermenters and gas production, contributed significantly to the symptoms of the patients. Coprococcus’s abundance hints at its use as a SIBO marker. Asymptomatic PBT individuals show a different microbiome, rich in Veillonella. PBT’s complex microbial interactions might stabilize the intestinal ecosystem, but further study is needed due to the core microbiota similarities with SIBO. Predicted folate and amino acid metabolism reductions in SIBO merit additional validation.

Chemiresistors Based on Hybrid Nanostructures Obtained from Graphene and Conducting Polymers with Potential Use in Breath Methane Detection Associated with Irritable Bowel Syndrome.

This paper describes the process of producing chemiresistors based on hybrid nanostructures obtained from graphene and conducting polymers. The technology of graphene presumed the following: dispersion and support stabilization based on the chemical vapor deposition technique; transfer of the graphene to the substrate by spin-coating of polymethyl methacrylate; and thermal treatment and electrochemical delamination. For the process at T = 950 °C, a better settlement of the grains was noticed, with the formation of layers predominantly characterized by peaks and not by depressions. The technology for obtaining hybrid nanostructures from graphene and conducting polymers was drop-casting, with solutions of Poly(3-hexylthiophene (P3HT) and Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-bithiophene] (F8T2). In the case of F8T2, compared to P3HT, a 10 times larger dimension of grain size and about 7 times larger distances between the peak clusters were noticed. To generate chemiresistors from graphene-polymer structures, an ink-jet printer was used, and the metallization was made with commercial copper ink for printed electronics, leading to a structure of a resistor with an active surface of about 1 cm2. Experimental calibration curves were plotted for both sensing structures, for a domain of CH4 of up to 1000 ppm concentration in air. A linearity of the curve for the low concentration of CH4 was noticed for the graphene structure with F8T2, presenting a sensitivity of about 6 times higher compared with the graphene structure with P3HT, which makes the sensing structure of graphene with F8T2 more feasible and reliable for the medical application of irritable bowel syndrome evaluation.

Effect of Trimebutine and Rifaximin on Breath Hydrogen and Methane by Glucose Breath Test in Patients With Functional Bloating: A Randomized Double-blind Clinical Trial.

Drugs that stabilize intestinal motility may improve the efficacy of nonabsorbable antibiotics, such as rifaximin, against small intestinal bacterial overgrowth (SIBO). We compared the efficacy of rifaximin alone with that of its combination with trimebutine maleate against SIBO. We performed a randomized double-blind placebo-controlled trial (https://cris.nih.go.kr, no. KCT0004836) that included patients with functional bloating, no constipation, and SIBO using the hydrogen (H2)-methane (CH4) glucose breath test (GBT). Patients were randomized into 2 groups in a 1:1 ratio, namely rifaximin (1200 mg/day) + trimebutine maleate (600 mg/day) group and rifaximin + placebo group, for 2 weeks. Patients completed a symptom questionnaire and underwent a GBT at baseline and at 1 month after treatment withdrawal. The primary outcome was SIBO eradication. The secondary outcomes included changes in the concentrations of exhaled gases, symptoms, and presence of adverse events. The complete eradication rate of SIBO was 35.9% (14/39) in the rifaximin group, and 34.1% (14/41) in the combined group with no significant differences. In both groups, no significant differences were observed in GBT profiles before and after the treatment, respectively. However total breath H2 and CH4 concentration were conspicuously decreased in the combined group after treatment. The combined group exhibited substantial relief of bloating. The adverse events were similar in the 2 groups. While the combination therapy was not superior over rifaximin alone for SIBO eradication, it improves the symptom of bloating with numerically reducing the concentration of breath H2/CH4.

Glucose breath test for the detection of small intestine bacterial overgrowth: Impact of diet prior to the test.

Glucose breath test (GBT) is used for the diagnosis of small intestine bacterial overgrowth. A restrictive diet without fibers and/or fermentable food is recommended on the day before the test. The aim of our retrospective study was to evaluate the impact of two different restrictive diets on the results of GBT. A change of the pretest restrictive diet was applied in our lab on September 1, 2020. The recommended diet was a fiber-free diet before this date, and a fiber-free diet plus restriction of all fermentable food afterward. We thus compared the results of GBT performed before (group A) and after (group B) this pretest diet modification. Demographics, reasons to perform GBT, digestive symptoms, and hydrogen and methane baseline values and variations after glucose ingestion were compared between the two groups. 269 patients underwent GBT in group A, and 316 patients in group B. The two groups were comparable in terms of demographics. Methane and hydrogen baseline values were significantly higher in group A (respectively 14 [18] vs. 8 [14] ppm, p < 0.01 and 11 [14] vs. 6 [8] ppm, p < 0.01). The percentage of positive tests was higher in group A for methane (43% vs. 28%, p < 0.05), and for hydrogen (18% vs. 12%, p = 0.03). This retrospective study suggests the importance of the restrictive diet prior to GBT. A strict limitation of fibers and fermentable food decreased hydrogen and methane baseline values, and the prevalence of positive GBT. Thus a strict restrictive diet should be recommended on the day before the test, in order to limit the impact of food on hydrogen and methane breath levels, and possibly improve the diagnosis quality of GBT.

Influence of Helicobacter pylori Infection and Eradication on Small Intestinal Bacterial Overgrowth and Abdominal Symptoms.

The relationship between Helicobacter pylori (H. pylori) infection and small intestinal bacterial overgrowth (SIBO) has attracted attention recently. To analyze the influence of H. pylori infection and eradication on SIBO, IMO, and abdominal symptoms. Patients with gastrointestinal symptoms were tested for 13C urea breath test and if positive, treated with bismuth-based quadruple therapy. Lactulose hydrogen methane breath test (HMBT) was performed and symptoms were assessed using gastrointestinal symptom rating scale (GSRS) before and 6weeks after eradication. Of the 102 subjects, 53 were H. pylori positive. The prevalence of SIBO and IMO were higher in patients with H. pylori infection than in those without infection (49.1% vs 24.5%, P = 0.019 for SIBO; 24.5% vs 8.2%, P = 0.027 for IMO). GSRS scores were similar between H. pylori-infected and uninfected patients (2 (IQR: 1;3) vs 2 (IQR: 1;2), P = 0.211). Patients with SIBO or IMO presented higher GSRS scores than patients with both SIBO and IMO negative (2 (IQR: 2;3), 2 (IQR: 2;3) vs 2 (IQR: 1;2), P = 0.011, 0.001, respectively). For the 50 patients who successfully eradicated H. pylori, the response rates for SIBO and IMO were 66.7% and 76.9%, respectively. GSRS scores also significantly decreased (2 (IQR: 1;3) to 0 (IQR: 0;1), P < 0.001) after eradication. Helicobacter pylori infection was associated with higher prevalence of SIBO and IMO, both of which led to more pronounced abdominal symptoms. H. pylori eradication also achieved therapeutic effects on SIBO and IMO, accompanied by relief of abdominal symptoms.

Real-Time Measurement of CH4 in Human Breath Using a Compact CH4/CO2 Sensor.

The presence of an elevated amount of methane (CH4) in exhaled breath can be used as a non-invasive tool to monitor certain health conditions. A compact, inexpensive and transportable CH4 sensor is thus very interesting for this purpose. In addition, if the sensor is also able to simultaneously measure carbon dioxide (CO2), one can extract the end-tidal concentration of exhaled CH4. Here, we report on such a sensor based on a commercial detection module using tunable diode laser absorption spectroscopy. It was found that the measured CH4/CO2 values exhibit a strong interference with water vapor. Therefore, correction functions were experimentally identified and validated for both CO2 and CH4. A custom-built breath sampler was developed and tested with the sensor for real-time measurements of CH4 and CO2 in exhaled breath. As a result, the breath sensor demonstrated the capability of accurately measuring the exhaled CH4 and CO2 profiles in real-time. We obtained minimum detection limits of ~80 ppbv for CH4 and ~700 ppmv for CO2 in 1.5 s measurement time.

Measurements of methane and nitrous oxide in human breath and the development of UK scale emissions

Exhaled human breath can contain small, elevated concentrations of methane (CH4) and nitrous oxide (N2O), both of which contribute to global warming. These emissions from humans are not well understood and are rarely quantified in global greenhouse gas inventories. This study investigated emissions of CH4 and N2O in human breath from 104 volunteers in the UK population, to better understand what drives these emissions and to quantify national-scale estimates. A total of 328 breath samples were collected, and age, sex, dietary preference, and smoking habits were recorded for every participant. The percentage of methane producers (MPs) identified in this study was 31%. The percentage of MPs was higher in older age groups with 25% of people under the age of 30 classified as MPs compared to 40% in the 30+ age group. Females (38%) were more likely to be MPs than males (25%), though overall concentrations emitted from both MP groups were similar. All participants were found to emit N2O in breath, though none of the factors investigated explained the differences in emissions. Dietary preference was not found to affect CH4 or N2O emissions from breath in this study. We estimate a total emission of 1.04 (0.86–1.40) Gg of CH4 and 0.069 (0.066–0.072) Gg of N2O in human breath annually in the UK, the equivalent of 53.9 (47.8–60.0) Gg of CO2. In terms of magnitude, these values are approximately 0.05% and 0.1% of the total emissions of CH4 and N2O reported in the UK national greenhouse gas inventories.

Hydrogen and methane breath testing for abdominal bloating of small intestinal origin

Abdominal bloating with or without excessive gas production and with or without abdominal distention is common, very bothersome for patients, and is poorly understood. Bloating can be associated with gastrointestinal disorders, nutrient malabsorption, and systemic illnesses. The origin of abdominal bloating is often unclear, though it is usually associated with food intake and bowel motility dysfunction. Often, food allergies, food intolerance, or food poisoning are considered by the patient or physician as potential causes for bloating. A common concern in the patient’s mind is that healthcare professionals will dismiss or misdiagnose their complaint. The limited understanding of the pathophysiology of bloating leads to current empirical management, and bloating is often characterized as a “functional” disorder. Bloating carries a heavy clinical, psychological, and economic burden. Proper diagnosis will provide the patient with peace of mind and lead to effective treatment. This review focuses on the mechanisms and management of abdominal bloating associated with different small intestinal disorders identified by non-invasive hydrogen and methane breath tests.

Photoacoustic Spectroscopy-Based Breath Analysis for SIBO.

Breath hydrogen (H2) and methane (CH4) monitoring play an important role in the diagnosis of gastrointestinal disorders, such as lactose intolerance and small intestinal bacterial overgrowth (SIBO). In this paper, the photoacoustic spectroscopy method is used for H2 gas and CH4 gas detection. We present a novel approach for H2 gas concentration measurement, which is the linear relationship between the resonant frequency of breath carbon dioxide (CO2) and the H2 concentration in a resonant photoacoustic cell. Experimental results show that the minimum detectable limits of H2, CH4, and CO2 are calculated to be 8.86, 0.56, and 145.14 ppm, respectively, which can meet the requirements of breath diagnosis.

Small intestinal bacterial overgrowth is associated with laryngopharyngeal reflux symptom severity and impaired esophageal mucosal integrity

Objectives: To explore the relationship between small intestinal bacterial overgrowth (SIBO) and laryngopharyngeal reflux (LPR). Methods: Data from patients undergoing high resolution manometry, pH-impedance monitoring, and hydrogen methane breath testing at a single tertiary center were analyzed retrospectively. SIBO was determined by a ≥ 20 ppm rise in breath hydrogen from baseline within 90 minutes after ingestion of lactulose. Patients were grouped by SIBO result and compared for subjective LPR symptoms on the Reflux Symptom Index (RSI) in addition to objective manometric and reflux parameters, including mean nocturnal baseline impedance (MNBI) in the proximal and distal esophagus. Results: Forty-one patients were analyzed, of which 46.3% were positive for SIBO. Patients with SIBO had a significantly greater LPR symptom burden on RSI (26.5 ± 9.3 vs. 16.9 ± 8.9, P = 0.002). Independently, SIBO was associated with throat clearing (P = 0.016), cough (P &lt; 0.001) and globus (P = 0.003). Objectively, there was no difference in manometric or reflux parameters except patients with SIBO had significantly lower MNBI in the proximal esophagus (1970.2 ± 511.6 Ω vs. 2504.2 ± 816.1 Ω, P = 0.026). Conclusion: Patients with SIBO have greater LPR symptom severity and impaired mucosal integrity in the proximal esophagus. Future study should look to determine if treating SIBO improves symptoms of LPR.

S647 Variation in Breath Hydrogen, Breath Methane, and Symptom Severity by Time-of-Day in Response to the Ingestion of FODMAP Containing Foods

S647 Variation in Breath Hydrogen, Breath Methane, and Symptom Severity by Time-of-Day in Response to the Ingestion of FODMAP Containing Foods

An insight into the functional alterations in the gut microbiome of healthy adults in response to a multi-strain probiotic intake: a single arm open label trial.

Probiotic supplements, by definition, provide a benefit to the host, but few studies have investigated the effect of probiotic supplements in healthy adult populations. The present, single arm, open label clinical trial, evaluated compositional and functional changes in the fecal microbiome of healthy adults after supplementation with a 14-strain probiotic. We analysed the effect of a 14-strain probiotic blend (Bacillus subtilis NCIMB 30223, Bifidobacterium bifidum NCIMB 30179, B. breve NCIMB 30180, B. infantis NCIMB 30181, B. longum NCIMB 30182, Lactobacillus helveticus NCIMB 30184, L. delbrueckii subsp. bulgaricus NCIMB 30186, Lacticaseibacillus paracasei NCIMB 30185, Lactiplantibacillus plantarum NCIMB 30187, Lacticaseibacillus rhamnosus NCIMB 30188, L. helveticus NCIMB 30224, Lactobacillus salivarius NCIMB 30225, Lactococcus lactis subsp. lactis NCIMB 30222, and Streptococcus thermophilus NCIMB 30189), on the faecal microbiota of healthy young adults (n=41) in a single arm study. The adults consumed 4 capsules daily of the 14 strain blend(8 billion colony forming units/day) for 8 weeks. Compositional and functional changes in faecal microbiota before and after supplementation were assessed using shotgun metagenomic sequencing. Fasting breath analysis, faecal biochemistry and bowel habits were also assessed. In healthy adult participants, no significant changes to the overall alpha- or beta-diversity was observed after 8 weeks of multi-strain probiotic supplementation. However, in a simplified model that considered only time and individual differences, significant decreases (p < 0.05) in family Odoribacteraceae and Bacteroidaceae abundance and a significant increase (p < 0.05) in genus Megamonas abundance were observed. At a functional level, there were significant changes in functional gene abundance related to several functional pathways, including phenylalanine metabolism, O-antigen nucleotide sugar biosynthesis, bacterial chemotaxis, and flagellar assembly. No significant changes in stool form or frequency, fecal biochemistry, or methane and hydrogen breath tests were observed. In healthy young adults, overall alpha- and beta-diversity did not change in response to probiotic intake even though modest compositional changes at the family and genus level were observed. However, at functional level, results identified changes in gene abundance for several functional pathways.

Comparison of jejunal aspirate culture and methane and hydrogen breath test in the diagnosis of small intestinal bacterial overgrowth.

Small intestinal bacterial overgrowth (SIBO) is still difficult to diagnose. Quantitative culture of small intestine aspirate is recommended to be the gold standard. The methane and hydrogen breath tests are easily repeatable, sufficiently sensitive and highly specific for SIBO diagnosis. Our goal is to contrast the diagnostic value of the breath tests with jejunal aspiration cultures. 40 adult outpatients (age < 60) were enrolled in our study. Randomly, within 2days, both the methane and the hydrogen breath test and jejunal aspiration culture were performed on each patient and the results of both tests were evaluated and contrasted. The jejunal culture was positive (105CFU / mL) in 14/40(35%) subjects, the lactulose breath test (LBT) was positive in 18/40 (45%) subjects, and the glucose breath test (GBT) was positive in 12/40 (30%). The GBT showed good agreement (κ = 0.659) and LBT showed poor agreement (κ = 0.588) with the jejunal aspirate culture. The sensitivity, specificity, positive and negative predictive values of LBT/GBT were 85.7/71.4%,76.9/92.3%, 66.6/83.3% and 90.9/85.7%, respectively. 35% of patients with suspected SIBO are identified using jejunal aspirate cultures. For the identification of SIBO, GBT is more specific than LBT, but has a lower sensitivity. In individuals with suspected SIBO, the breath test should be initially due to its good agreement with the jejunal aspirate culture.

Effect of immune responses on breath methane dynamics

Methane (CH4) which can be detected in human breath has long been exclusively associated with anaerobic microbial activity (methanogenesis) in the gastrointestinal tract. However, recent studies challenge this understanding by revealing that CH4 might also be produced endogenously in cells through oxidative–reductive stress reactions. Consequently, variations in breath CH4 levels compared to an individual’s baseline level might indicate enhanced oxidative stress levels, and, therefore, monitoring breath CH4 levels might offer great potential for ‘in vivo’ diagnostics such as disease diagnosis, monitoring the efficacy of treatments, or during the application of personalized medicine. To evaluate the effects from immune responses triggered by infections, inflammations, and induced perturbation by vaccination on CH4 dynamics in breath, two subjects were monitored over a period of almost 2 years. Breath CH4 levels were measured by gas chromatography equipped with a flame-ionization detector. Both subjects exhibited significant deviations (positive and negative, respectively) from their normal CH4 breath levels during periods of potential enhanced immune activity. Deviations from the ‘healthy state’ were indicated by the exceeding of individual CH4 ranges. Moreover, for the first time we could clearly prove CH4 degradation induced through vaccination by measuring stable carbon isotopes of CH4 using gas chromatograph–combustion–isotope ratio mass spectrometry. Hence, breath CH4 concentration and isotopic analyses may be used as a biomarker to evaluate specific immune responses and individual immune states.