Measurement of 24-h continuous human CH4 release in a whole room indirect calorimeter

Abstract

We describe the technology and validation of a new whole room indirect calorimeter (WRIC) methodology to quantify volume of methane (VCH₄) released from the human body over 24 h concurrently with the assessment of energy expenditure and substrate utilization. The new system extends the assessment of energy metabolism by adding CH₄, a downstream product of microbiome fermentation that could contribute to energy balance. Our new system consists of an established WRIC combined with the addition of off-axis integrated-cavity output spectroscopy (OA-ICOS) to measure CH₄ concentration ([CH₄]). Development, validation, and reliability of the system included environmental experiments to measure the stability of the atmospheric [CH₄], infusing CH₄ into the WRIC and human cross-validation studies comparing [CH₄] quantified by OA-ICOS and mid-infrared dual-comb spectroscopy (MIR DCS).Our infusion data indicated that the system measured 24-h [CH₄] and VCH₄ with high sensitivity, reliability, and validity. Cross-validation studies showed good agreement between OA-ICOS and MIR DCS technologies (r = 0.979, P < 0.0001). Human data revealed 24-h VCH₄ was highly variable between subjects and within/between days. Finally, our method to quantify VCH₄ released by breath or colon suggested that over 50% of the CH₄ was eliminated through the breath. The method allows, for the first time, measurement of 24-h VCH₄ (in kcal) and therefore the measurement of the proportion of human energy intake fermented to CH₄ by the gut microbiome and released via breath or from the intestine; also, it allows us to track the effects of dietary, probiotic, bacterial, and fecal microbiota transplantation on VCH₄.NEW & NOTEWORTHY This is the first time that continuous assessment of CH₄ is reported in parallel with measurements of O₂ consumption and CO₂ production inside a whole room indirect calorimeter in humans and over 24 h. We provide a detailed description of the whole system and its parts. We carried out studies of reliability and validity of the whole system and its parts. CH₄ is released in humans during daily activities.

Keywords: gas infusion; reliability; spectroscopy; validation; whole room indirect calorimeter.

Graphical abstract

Figure 1.

Variability of environmental methane concentration ([CH₄]) as function of the air inflow pipeline location. A: [CH₄] assessment over 1 wk, CH₄ was measured with an off-axis integrated-cavity output spectroscopy (OA-ICOS). B: [CH₄] measured simultaneously during 33.5 h on three different environmental locations and from the reference air tank. Each dot represents two-minute [CH₄] [n = 5,040 time points (A); n = 1,000 time points (B)]. Coefficients of variation (CV) between single points of the same category [weekday (A) or location (B)] and areas under the curve (AUC) were utilized to compare the variability the [CH₄] between days (A) and locations (B).

Figure 2.

A–F: methane volume (VCH₄) measured during 1,000-min infusion studies in whole room indirect calorimeter (WRIC). Dashed lines represent expected CH₄ volumes infused with a blender and measured by mass flow controllers (MFCs); solid lines are CH₄ volumes measured by off-axis integrated-cavity output. A shows a short circuit dynamic infusion. B–F are two-step dynamic infusion in two different WRIC (#2 and #1) at the same time point (B and E); in the same WRIC (#1) 1 yr apart (E and D); in the same WRIC (#2) in 2 consecutive days [C (day 1) and F (day 2)].

Figure 3.

Comparison of human-microbiome methane concentration ([CH₄]) production by different measurement methods. Solid black line represents [CH₄] quantified directly from the air outflow of a whole room indirect calorimeter (WRIC) by off-axis integrated-cavity output spectroscopy (OA-ICOS) CH₄ analyzer. The open and closed circles represent measurements done with broadband mid-infrared laser spectroscopy (MIR DCS) at the end of the line prior to entering the multi-inlet unit (MIU) and inside the WRIC, respectively. Data from one participant.

Figure 4.

Concordance correlation coefficient diagram between methane concentration ([CH₄]) measured by broadband mid-infrared laser spectroscopy (MIR DCS) and off-axis integrated-cavity output spectroscopy (OA-ICOS). Solid line is the identity line (45°); dashed line represents regression between [CH₄] measured by OA-ICOS and MIR DCS. ρ_c = concordance coefficient of correlation; Cb = bias correction factor. Green dotted line marks [CH₄] atmospheric (1.8–1.9 ppm). Red line marks the lowest limit of detection for each methodology (AO-ICOS = 0.003 ppm and MIR DCS = 0.0001 ppm). r, precision. Dots represent 47 samples of random time points from 19 participants.

Figure 5.

Bland and Altman plot for the agreement analysis between CH₄ concentration ([CH₄]) measured by broadband mid-infrared laser spectroscopy (MIR DCS) and off-axis integrated-cavity output spectroscopy (OA-ICOS). Solid line represents mean difference between methodologies (−0.4002 ppm); dotted lines are upper (ULA) and lower (LLA) limit of agreement (ULA: 0.7576 and LLA: –1.558, respectively); dashed line is the mean simple regression line between differences and mean values as indicator of proportional bias. Dots represent 47 samples of random time points from 19 participants.

Figure 6.

Profiles of 23-h VCH₄ release in humans living inside a whole room indirect calorimeter (WRIC). A shows simultaneous measurement of VCH₄, V̇o₂, and V̇co₂ from a participant during a single day. Vertical dotted lines split scheduled activities: Blue solid line is V̇o₂ profile; red dashed line is V̇co₂ profile; green solid line is VCH₄ profile. B shows VCH₄ profiles of the same participant during two consecutive days (solid blue and red dashed lines represent days 1 and 2, respectively). BF, breakfast time; EX, exercise.