The aims of the current exploratory study were to evaluate the operational performance of the developed telemetric gas‐sensing capsules by benchmarking the internal hydrogen measurements against those external hydrogen measurements through breath testing. To do this, we compared the data obtained through this new device to simultaneous readings of breath hydrogen in healthy volunteers after they ingested varying doses of either well‐absorbed or unabsorbed readily fermented carbohydrates. This work also adds to the data on the safety of the capsules in healthy human subjects. Transit times using the changes in oxygen concentrations as the marker were also further evaluated.

A potential advance in better measurement and localisation of gas production in the intestine is the development of a gas‐sensing capsule for use in humans that can measure hydrogen concentrations in the gut at the point of its production and transmit data telemetrically. 23 The gas‐sensing capsules use very low cost electronic components, equipped with an additional temperature sensor and are housed in a 000 capsule (Figure 1 A and 1 B) that wirelessly transmits data to a small pocket size receiver. Information about the concentration of these gases and temperature records are continuously transmitted so that monitoring can occur in real‐time. Initially, the efficacy of this capsule was demonstrated in a pig model. 5 , 24 , 25 A subsequent first‐in‐human study demonstrated its safety and confirmed efficacy. 26 It also allowed for the diet and timing protocols to be tested and provided evidence that simultaneous measurement of oxygen and hydrogen concentrations allowed localisation of the capsule and thus assessment of the sites at which fermentation of carbohydrate substrates was occurring. Altogether, the initial success of the gas‐sensing capsule provides another path for strengthening the global trend towards ingestible electronic devices. 27 - 31

The principle of breath tests is that, following ingestion of a specific carbohydrate, any unabsorbed carbohydrate will remain in the lumen and, during its passage through the small bowel to the large intestine, it will be fermented by the microbiota, thus generating gases as a by‐product. 15 These by‐products of microbial metabolism may be utilised by other metabolic pathways (acetogenesis, sulphide reduction or methanogenesis) and a proportion is absorbed through the mucosa into the bloodstream then recirculated and released through the lungs. 16 - 19 The breath tests primarily measure hydrogen and methane (both specific to microbial gut production) in the parts per million range or the detection of carbon dioxide gas that is produced in response to an isotopically labelled substrate such as 13 C‐xylose. 2 , 3 While breath tests have been increasingly adopted within the clinical setting, this technology has been impacted by interpretive difficulties arising from the low breath concentration of hydrogen causing low sensitivity and specificity. 3 , 20 There are also inherent assumptions and uncertainty around the site of the hydrogen's production, specifically small versus large intestine and this is particularly important in defining SIBO. 21 , 22

A fast growing research area in gastroenterology is the exploration into the correlation between the gas constituents of the gastrointestinal tract and the individual's health, microbial diversity and dietary profile. 1 - 5 These gases, which include hydrogen, methane, carbon dioxide and oxygen, have been used as biomarkers in prevention, diagnostics and a guide for symptom relief. 6 - 8 Gas production within the gut is derived from three sources: (a) chemical reactions; (b) enzymatic breakdown of food; and (c) microbial metabolism. 7 An understanding of the intestinal gas types, their concentrations and where they are produced are fundamental in correctly linking them to the state of health and many disorders, 9 , 10 in addition to providing a window into local functional activity of the gut microbiota. So far, gas profile assessments have been through indirect investigational methods such as tube insertion, whole body calorimetry, breath testing, flatus measurements, in vitro stool gas analysis and direct sampling of gases produced. 1 , 5 , 8 , 11 , 12 However, breath tests are the most commonly adopted method to measure intestinal gases in the clinical setting, where they are used to investigate, for example, carbohydrate malabsorption or small intestinal bacterial overgrowth (SIBO). 13 , 14

For breath hydrogen testing, a cut‐off of 10 ppm above baseline in hydrogen excretion was used to indicate fermentation of the inulin was occurring, as per usual clinical practice. It was assumed that a significant rise in breath hydrogen after inulin occurred as the inulin entered the colon. This enabled an oro‐caecal transit time of the inulin in solution to be measured. For the gas‐sensing capsule, a rapid reduction of oxygen concentration was considered to represent exit from the stomach, the start of near‐anaerobic conditions to represent passage to the caecum, and fall of temperature reading to represent excretion of the capsule, as recently described. 26 These landmarks enabled calculation of the gastric emptying time, and transit times for small intestinal and large bowel for the capsule to be calculated.

The performance of the capsule was assessed in several ways. First, the reliability of delivering information to the external receiver was determined. Second, the subjects’ tolerance of the capsule following its ingestion and its subsequent passage through the gastrointestinal tract was judged according to direct questioning of participants and reports from them of symptoms or other issues. Third, the quantitative measurements of hydrogen concentrations over time by the gas‐sensing capsule were compared with those measured ex vivo through sampling the exhaled breath. Specific indices of interest were (a) peak hydrogen concentrations after the high‐dose inulin challenges to give an indication of the validity and dilution factors of the hydrogen measurements; (b) dose‐dependent hydrogen concentrations after low doses of inulin and high doses of glucose to enable the limitations for measuring hydrogen following fermentation of small amounts of ingested carbohydrates to be assessed; and (c) the patterns of hydrogen concentrations in vivo and in the breath over time.

The volunteers consumed a diet low in fibre and FODMAPs for 24 hours prior to the study to minimise background hydrogen production, as is routine in breath hydrogen tests. After an overnight fast, breath samples were collected prior to and every 30 minutes after the ingestion of the test carbohydrate for 10 hours. The test carbohydrate with some water was the only breakfast the volunteers consumed. Thirty minutes prior to the first breath sample, the capsule was swallowed. The handheld receiver and mobile phone with the application was given to keep near to the subject during the entirety of the test. The subjects fasted for a further 6 hours after capsule ingestion (as per protocol for a pH‐sensing capsule in routine clinical practice 36 ), then resumed a diet low in fibre and FODMAPs for the remainder of the test period. The subjects were kept in a controlled environment where their exercise, vital signs and general health were monitored regularly. Breath hydrogen was measured using gas chromatography within 3 days after collection (Quintron Instrument Co., Milwaukee, WI, USA). Gas measurements were taken by the capsule every 5 minutes from ingestion until excretion.

In a single‐blinded study, individual subjects were randomly allocated via a computer‐generated list to receive only one solution of either rapidly absorbed glucose, or of indigestible and readily fermentable inulin (degree of polymerisation of 16‐30 sugar units; Zinulin Pty. Ltd., Flinders, Australia). One subject was given 20 g and three subjects 40 g of glucose. A range of 1.25, 2.5, 5, 10 and 20 g of inulin were given to individual subjects with three subjects given 40 g of inulin. These sugars were dissolved in 400 mL water and orally ingested. The subjects were blinded to the type of sugar ingested. No subjects were used for repeated measurements to conform to the ethics approval.

Healthy participants were recruited via advertising. They had to be between 18 and 55 years of ages, in excellent physical and mental health with no history of gastrointestinal issues, abdominal surgery or other serious medical conditions. Exclusion criteria included pregnancy, body mass index >27 kg/m 2 , having an implantable device such as heart pacemaker, smoking or excessive alcohol intake, and the inability to give written informed consent. Prospective volunteers who fulfilled entry criteria were then screened with the use of the patency capsule (Agile Patency Capsule; Given Imaging, Yoqneam, Israel) to ensure passage of the capsule occurred within 48 h of ingestion. 35

The data are then analysed against a pre‐trial calibration of the individual capsules. The pre‐trial calibration is run in a controlled environment with gas mixtures created using a mass flow controller (MKS Instruments, Inc, USA). For the current experiments, gas mixtures used for calibration were made up of varying concentrations of zero air (21% oxygen in nitrogen balance), nitrogen and 3% hydrogen in nitrogen balance. Detailed mixture information can be found in the Supplementary Information (Table S1 ). All concentrations given are calculated absolute values based on these calibrations. Calculations establish oxygen concentration first and that fed into the hydrogen calculations. While the capsule contained the capability of measuring carbon dioxide and methane, these gases were not part of the study aims so the pre‐trial calibrations were not run. The concentrations of such gases cannot, therefore, be accurately calculated. Signal‐to‐noise ratio (SNR), an indicator of the performance of the measurement technique and the noise floor of the sensors, was calculated by the ratio between the averages of peak values of the “signal” at a 40 g fermentable carbohydrate (inulin) and the averages of the control or “noise” of the 40 g absorbable carbohydrate (glucose) using the following equation.

Photograph of the full package of the capsule, the handheld receiver and the mobile phone that runs the application is shown in Figure 1 . The developed capsules are near the standard dimensions of a 000 size (25.0 ± 1.0 mm in length and 9.8 mm in diameter) (Figure 1 B). The gas sensors included in the device are capable of monitoring concentrations of methane, carbon dioxide, hydrogen and oxygen in varying gradients of aerobic and anaerobic environments. The capsule also consists of a temperature sensor, a microcontroller, a transmission system and button‐sized silver oxide batteries. The sensor that tracks oxygen is nonspecific and responsive to all oxidising gases and vapours. A highly gas‐permeable membrane with embedded nanomaterials allows for the fast diffusion of gas molecules from the intestinal lumen. Enabling the gases and vapours to be analysed, but blocking external solids or liquids from contaminating the internals. 32 - 34 The outputs from the sensors are coded and transmitted every 5 minutes to a hand‐held monitor (Figure 1 A) that is Bluetooth‐connected to a mobile phone application.

Comparison of hydrogen concentrations in intestinal lumen and breath in individual subjects. A and B, Patterns in three subjects each in response to 40 g inulin (n = 3) for the gas capsule and breath tests (C‐D) and in response to 40 g glucose, where breath hydrogen concentrations showed signal fluctuations as large as 8 ppm (colour coded and numbered to individual participants). The capsule measurements are shown with gastric emptying occurring at t = 0. The chosen gastric duration to display on the graph is based on the fastest time from ingestion to gastric emptying. This is to display the absence of hydrogen within the stomach. Black dots indicate timing of capsule reaching the proximal colon

Comparison of hydrogen concentrations in intestinal lumen and breath in individual subjects after 20 g glucose or varying doses of inulin. A, Temporal patterns of fermentation response in the gastrointestinal lumen in reaction to a range of inulin and glucose doses. Fermentation occurs after gastric emptying therefore the timing has been aligned and t = 0 at this point. The chosen gastric duration to display on the graph is based on the fastest time from ingestion to gastric emptying. This is to display the absence of hydrogen within the stomach. B, Patterns of fermentation response in the breath to a range of inulin and glucose. Black dots indicate timing of capsule reaching the proximal colon (C) Peak values for the gas capsule (left axis) and breath tests (right axis) giving an indication of relative concentration comparison (glucose is shown as 0 g inulin)

Typical profiles of oxygen and hydrogen concentrations during the passage of the gas capsule through the gut after ingestion of 20 g glucose or 20 g inulin are shown in Figures 1 C and D. The patterns of oxygen concentrations were similar to those previously described. 26 Since the oxygen sensor reacts with both oxygen and any oxidising agents, it increased to above 21% after ingestion while within the stomach as explained in our previous work. 26 The oxygen concentration rapidly decreased when the capsule left the stomach, progressively decreased until it almost reached zero as it entered the near‐anaerobic conditions of the large bowel, and remained very low until the capsule was excreted. After glucose ingestion, the concentration of hydrogen did not increase until near‐anaerobic conditions were reached. In contrast, after 40 g of inulin, an early rise in hydrogen concentration occurred in the small intestine.

4 DISCUSSION

Breath testing is an indirect measurement; it only demonstrates the small portion of gases that are absorbed through the intestinal epithelium, diluted within the blood stream distributed through the body and then presented to the lungs where they are further diluted within the breath. In contrast, gas measurements are sampled by the capsule at the point of production in the gut, and before the disposal of hydrogen via metabolic and absorptive processes, and by excretion per rectum. Thus, increased gut accessibility and limiting physiological interferences provided by the capsule measurements potentially offer the ability to increase the selectivity and sensitivity in the clinical setting in comparison to breath testing.

The results of the current exploratory comparative study have supported the hypothetical prediction of performance differences of hydrogen measurements by the gas‐sensing capsule and breath testing in two ways. First, the gas‐sensing capsule demonstrated greatly improved SNR and sensitivity compared with those of breath testing. The noisy baseline seen in breath hydrogen concentrations after glucose in the present study are typical of those observed in routine clinical practice. The noise is likely to be related to hydrogen production in the colon from endogenous or dietary carbohydrates, since breath hydrogen is a summation of all sources of the gas. As such, to read above the noise floor generally necessitates changes of 8‐20 ppm in breath hydrogen concentrations in order to define a positive response after ingestion of a target carbohydrate.20 It is this small increase relative to the noise floor to define a positive response that can be problematic in interpreting breath test outcomes. In contrast, using the capsule to measure the hydrogen concentration at the site of production has very low noise with minimal detectable levels after the intake of glucose, which is rapidly absorbed in the proximal small intestine in healthy subjects. Furthermore, the sensitivity of detection is markedly increased by the capsule technology, measuring gas in parts per hundred in contrast to parts per million in the breath. Thus, in all subjects, the capsule detected hydrogen when the indigestible substrate, even after as small as 1.25 g, was ingested, but not following 20 g glucose, underlining both sensitivity and specificity of the measurements. Moreover, even though tested in different subjects, dose‐dependent effects on luminal hydrogen concentrations following ingestion inulin were apparent, while breath hydrogen over 8 ppm were only detected for 5 and above. However, after a large dose of ingested glucose (40 g) the capsule did detect hydrogen concentrations in the colon similar to those after 1.25 g of inulin. This indicates that, after a large bolus dose of glucose, some is malabsorbed. This finding has implications for the interpretation of results from glucose breath hydrogen testing where, after a 75 g bolus of glucose, increases in breath hydrogen are considered indicative of SIBO.38 Indeed, lack of specificity of glucose breath testing for SIBO has recently been indicated by concomitant breath testing and scintigraphy.39

The second difference in performance was that the capsule enabled quantification of small intestinal transit times, which may be critical to the interpretation of the significance of hydrogen concentrations. The ability of the capsule to sense oxygen concentrations concomitantly with hydrogen gas provides likely anatomical insights, which is not provided by breath testing. In our pilot observations using abdominal ultrasound to define the capsule's location, oxygen concentrations appeared to be a marker of definition between the stomach and small intestine and between the small intestine and caecum,26 as anticipated given current physiological understanding, enabling small intestinal transit time to be estimated. The relatively short small bowel transit times observed were consistent with the low fibre diet before and during the test (as observed by us previously with the capsule),26 and the ingestion of osmotically active substrates. While breath hydrogen tests after, for example, lactulose have also been used as a measure of oro‐caecal transit time,40 the current study showed that large doses of fermentable substrate were needed to identify when the substrate reached the caecum due to the low sensitivity of breath testing. These large doses have the potential to falsely represent the transit to the caecum via hastening transit due to osmotic effects of an unabsorbed small molecule.41 Indeed, transit to the caecum was very rapid. When the same index (hydrogen levels) are used as the marker for transit, which may be very different (see below), circular arguments are used for the interpretation of the site of fermentation. Concomitant scintigraphy, with its high cost and radiation exposure, is needed to show that the increase of hydrogen after lactulose does indeed represent transit into the caecum, a method that assumes lactulose and the radiolabeled substrate have identical transit.42-44

In contrast, the gas‐sensing capsule measurements carry its own marker of anatomical position at no additional cost. The predictive accuracy of measured oxygen concentrations for anatomical location requires further validation. However, the current exploratory studies did show that the transit of the capsule from the stomach was very different from that of a large load of inulin in solution, as per the breath hydrogen data. This longer gastric emptying time was similar to the experience with the pH‐sensing capsule36 and is consistent with current understanding of the differential emptying of solids and liquids from the stomach. While the average gastric emptying time shown in this study is slightly longer than the pH‐sensing capsule, it is important to note that the methodology of this study is different and this is taken without the use of a food bar. While understanding fermentation sites may come as an advantage to the gas‐sensing capsule, there are potential difficulties in the interpretation of individual responses based on transit discrepancies of the capsule and test substrates. Due to the more rapid gastric emptying of the of the test substrate (provided in liquid form), it is unlikely to passage together with the capsule. After bacteria take up the substrate, evidence indicates that its fermentation will commence rapidly.12 However, the duration of hydrogen production is uncertain, since such investigation has been performed in static, not flowing systems. When a diet high in FODMAPs is fed to healthy subjects or those with irritable bowel syndrome, high levels of breath hydrogen continues continuously through the day, despite the intermittent intake of food associated with meals only45 and the likelihood that fermentation is largely restricted to the proximal colon.46 It is possible, therefore, that uptake of the inulin will lead to a sufficiently long fermentation process and subsequent persistence of hydrogen in the luminal environment to enable its detection by the capsule that is trailing behind the leading front of the substrate. Indeed, single doses of inulin in increasing amounts (albeit in different individuals) were associated with higher hydrogen concentrations in the intestine despite the considerable lag time between the arrival of the substrate and the capsule. Such issues require further study.

The major diagnostic criterion for SIBO (beyond culture of small intestinal fluid) has been the measurement of hydrogen breath generation after fermentation in the small intestine of well‐absorbed glucose or nonabsorbable lactulose.40 A significant rise in breath hydrogen production within 90 min or the presence of double peaks after ingestion of lactulose are common indices used.38 However, concomitant scintigraphy has shown that the vast majority of assumed small intestinal fermentation after lactulose and even glucose ingestion is in fact resulting from the osmotically induced rapid transit.39, 43, 44 Additionally, SIBO has been largely considered a categorical phenomenon (ie, present or absent). In contrast, the capsule, by virtue of its in‐built location marker (oxygen concentration), demonstrated increased concentrations of hydrogen as it moved more distally, in line with the knowledge that small intestinal microbiota increases in density in the ileum. It is difficult, however, to interpret the localisation of fermentation sites within the small intestine as there is limited information on the gas flow dynamics and possible proximal diffusion within this region. Speculative interpretation would conclude that diffusion of hydrogen proximally in the small intestine seems limited given the active propulsion of gas distally and the relative rapid, one‐way flow of the contents in the small intestine.47, 48 Further work on small intestinal fermentation sites and gas dynamics within this system needs to be undertaken. It can be assumed that hydrogen concentrations >1% in the small intestine (Figure 2A and 3A) are not due to fermentation within the colon diffusing back through to the small bowel; Hernando‐Harder et al have shown this diffusion to be minimal and would not account for such a large concentration.47 Methods of localising the capsule within the different regions of the small intestine are needed to further refine interpretation of the gas concentrations. Nevertheless, the current findings with the capsule show fermentation does occur in a dose‐dependent manner after inulin in the distal small intestine and this is to be expected where many of the mucosa‐associated microbiota in this region are capable of fermenting the inulin substrate.49 This would seem to indicate that SIBO causes a measurable difference in small intestinal fermentation to that observed in nonaffected individuals.

Additionally, there are some direct associations that can be seen when comparing the breath tests and capsule measurements during the extreme fermentable carbohydrate challenge. While the peak values for both the gas capsule measurements and breath tests are subject‐dependent, the ratios of the peak value are relatively stable across the three different subjects. The measured peak values of 16.5%, 12% and 8.5% obtained using the capsule associate with the breath tests peak values of 67, 46 and 31 ppm, resulting in the ratio of ~0.26 ± 0.2%/ppm. It provides evidence that, when the hydrogen concentration is high, both methods are relatively reliable and that saturation of the measurement does not occur. However, the reliability and reproducibility of such a conclusion requires additional data.

The results of this study suggest that the gas‐sensing capsule can be potentially used for diagnosing those gut disorders for which hydrogen breath testing is currently widely implemented, including SIBO and carbohydrate malabsorption. The trial provides clear validation that, for the use of the capsule in future clinical trials, 24 hours of a low FODMAP, low‐fermentable fibre diet can be used for establishing a baseline as it produces minimal hydrogen concentrations in both small and large intestines. While further validation and optimisation of protocols are needed, the results from the present study are very promising. The >3000 times increase in concentrations of gases measurable by the capsule inside the gut compared to breath testing can potentially eliminate falsely positive and falsely negative results, offering a safe and reliable tool for diagnosing gastrointestinal disorders or physiological states that are amenable to dietary or other manipulation.

In conclusion, the gas‐sensing capsule would appear reliable, well‐tolerated and safe. The benchmarking exercise of the current study has shown a high correlation between the patterns of hydrogen concentrations produced by the capsule and breath testing after acute carbohydrate loading, thus validating the performance of the gas‐sensing capsule. Given the superior performance of the capsule in terms of sensitivity and SNR, and its ability to measure gas concentrations at the source with minimisation of physiological confounders that besets breath testing, further studies with the new technology are warranted in defining its clinical application.