A Sensitive Isotope Selective Nondispersive Infrared Spectrometer for 13CO2 and 12CO2 Concentration Measurements in Breath Samples

We present a nondispersive infrared spectrometer (NDIRS) for the measurement of the ¹³CO₂/¹²CO₂-ratio in breath samples. A commercial NDIR spectrometer for CO₂ concentration measurements in industrial process control was modified using two separate optical channels for the ¹³CO₂ and ¹²CO₂ detection. Cross interference due to overlapping absorption lines of both isotopic gases was successfully eliminated. The sensitivity of this device is ± 0.4‰ of the ¹³CO₂/¹²CO₂-ratio in a range of 2.5 to 5% of total CO₂. This is sufficient for biomedical applications. Our spectrometer is small in size, cheap and simple to operate and thus a true alternative to isotope ratio mass spectrometers (IRMS). Several biomedical applications with breath samples were demonstrated and were compared in very good agreement with IRMS.     

Carbon Isotope Analysis in Urea at High 13C-Abundances Using the 13/12CO2-Breath Test Device Fanci2

Abstract

The increasing application of ¹³C-labelled urea in medicine requires simple and reasonable methods for measuring highly enriched ¹³C in urea. The combination: ultimate organic analysis—mass spectrometry so far prescribed is complicated and expensive. For medical diagnosis, however, isotope selective nondispersive infrared spectrometers (NDIRS) have been available for many years. One of these tools is FANci2 which is very reasonable and easily to be operated. By means of such devices also urea highly enriched in ¹³C can be analysed, provided that the samples are first diluted with a defined amount of urea of natural isotopic composition and then transformed into carbon dioxide by means of urease. The relative abundance of ¹³C in this carbon dioxide, measured by nondispersive infrared spectrometry, is then a measure of the ¹³C abundance in the initial urea sample. Comparison of results of such measurements with those attained by mass spectrometry proves that this procedure is feasible and yields precise results.     

Determination of 13CO2/12CO2 Ratio by IRMS and NDIRS

Abstract

Breath tests using ¹³C-labelled substrates require the measurement of ¹³CO₂/¹²CO₂ ratio in breath gas samples. Next to isotope ratio mass spectrometry (IRMS), which is very sensitive but also complex and expensive, alternatively isotope selective nondispersive infrared spectrometry (NDIRS) can be used to determine the ¹³CO₂/¹²CO₂ ratio in expired breath. In this study we compared NDIRS- with IRMS-results to investigate whether the less expensive and very simply to operate NDIRS works as reliable as IRMS. For this purpose we applicated 1-¹³C-Phenylalanine to patients with advanced liver cirrhosis and healthy volunteers and took duplicated breath samples for IRMS and NDIRS at selected time points. Our data show a good correlation between these two methods for a small number of samples as required for simple breath tests. Longer series, where repeated measurements are required on the NDIRS instrument lead to a decreasing correlation. This indicates the superiority of IRMS concerning ¹³CO₂-kinetics over longer time periods.     

Significance of Diagnostic Parameters in [13C]Octanoic Acid Gastric Emptying Breath Tests*

Abstract

Two novel characteristic parameters, the latency time (t _lat) and the ascension time (t _asc), are proposed for evaluation of non-invasive [¹³C]octanoic acid breath tests for assessment of the gastric emptying of solids. In breath tests performed in control subjects (n = 30) and diabetic patients (n = 100), the usefulness of these parameters was compared to conventional parameters, i.e., gastric half emptying-time t _1/2,b ) and lag phase (t _lag,b ). The proposed parameters were only loosely correlated (controls, r = 0.199; diabetics, 0.616). A strong correlation was found between the conventional parameters (controls, r = 0.891; diabetics, r = 0.962). Based on the conventional method, 36 patients were suspicious of delayed gastric emptying including 24 patients which exhibited a simultaneous delay in both parameters. Using the new parameters, a total of 46 patients were suspicious of delayed gastric emptying with 15 and 20 having isolated delay in t _lat and t _asc, respectively. We conclude that the novel parameters may be more appropriate for examination of the different phases of gastric emptying and for evaluation of gastric emptying disturbances in diabetic patients than the parameters conventionally used for this purpose.     

Evaluation of Water Removal and Memory Effect in 13CO2 Breath Tests by Isotope Ratio Mass Spectrometry

The usefulness of different ways of water removal in off-line sample preparation of human breath samples for ¹³CO₂ breath tests was examined and compared. Cryogenic water trapping and water removal with common desiccants like silicagel blue, Mg(ClO₄)₂, and molecular sieves were checked for reliability and reproducibility. With silicagel blue and Mg(ClO₄)₂ memory effects for ¹³C content were observed. The use of molecular sieve 4 Å and 5 Å led to tremendous carbon isotope fractionation. Molecular sieve 3 Å was found to be an excellent alternative to the established use of Mg(ClO₄)₂ and of cryogenic water trapping.     

Macroscopic Isotope Separation of 13C by a CO2 Laser

By photochemical dissociation of the rare carbon isotope component of CHClF₂ by means of a CO₂ laser with an average power of 150 W, Q-switched at 10 kHz, we have demonstrated the separation of more than 1 mol of ¹³C, enriched to 50% (2 mol of total carbon). It is contained in about 1 mol (101 g) of the product C₂F₄. The total throughput of the starting material was 29 kg. The experiment was run day and night for 2 weeks, almost only controlled by a computer. We obtained production rates of 5 mmol/h, corresponding to about 0.5 kg ¹³C per year.     

[13C]Aminopyrine and [13C]Caffeine Breath Test: Influence of Gender,Cigarette Smoking and Oral Contraceptives Intake

Abstract

The [¹³C]aminopyrine breath test ([¹³C]ABT) measures the global activity of cytochrome P450 in vivo and is a sensitive indicator of liver metabolic dysfunction. The present study aims to determine whether gender and cigarette smoking influence the results of [¹³C]ABT as well as to confirm the effect of oral contraceptive steroids (OCS) intake on this metabolic test. Hundred and ten healthy subjects, including men and women, smoker and non-smoker, women taking OCS or not, were phenotyped for CYP1A2 using the [¹³C]caffeine breath test and underwent a [¹³C]ABT. Both tests showed large inter-individual variations in accordance with that of CYP450 liver content. [¹³C]ABT was sensitive enough to point out a significant induction or inhibition related to cigarette smoking habits or OCS. The combined effect of smoking and OCS resulted in an overall unchanged metabolic activity. Consequently, the impact of the studied conditions on the [¹³C]ABT parameters must be considered by clinicians or clinical investigators.     

The effect of physical back-diffusion of 13CO2 tracer on the coupling between photosynthesis and soil CO2 efflux in grassland

Pulse labelling experiments provide a common tool to study short-term processes in the plant–soil system and investigate below-ground carbon allocation as well as the coupling of soil CO₂ efflux to photosynthesis. During the first hours after pulse labelling, the measured isotopic signal of soil CO₂ efflux is a combination of both physical tracer diffusion into and out of the soil as well as biological tracer release via root and microbial respiration. Neglecting physical back-diffusion can lead to misinterpretation regarding time lags between photosynthesis and soil CO₂ efflux in grassland or any ecosystem type where the above-ground plant parts cannot be labelled in gas-tight chambers separated from the soil. We studied the effects of physical ¹³CO₂ tracer back-diffusion in pulse labelling experiments in grassland, focusing on the isotopic signature of soil CO₂ efflux. Having accounted for back-diffusion, the estimated time lag for first tracer appearance in soil CO₂ efflux changed from 0 to 1.81±0.56 h (mean±SD) and the time lag for maximum tracer appearance from 2.67±0.39 to 9.63±3.32 h (mean±SD). Thus, time lags were considerably longer when physical tracer diffusion was considered. Using these time lags after accounting for physical back-diffusion, high nocturnal soil CO₂ efflux rates could be related to daytime rates of gross primary productivity (R²=0.84). Moreover, pronounced diurnal patterns in the δ¹³C of soil CO₂ efflux were found during the decline of the tracer over 3 weeks. Possible mechanisms include diurnal changes in the relative contributions of autotrophic and heterotrophic soil respiration as well as their respective δ¹³C values. Thus, after accounting for physical back-diffusion, we were able to quantify biological time lags in the coupling of photosynthesis and soil CO₂ efflux in grassland at the diurnal time scale.     

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C]methacetin- and [methyl-13C]methionine-breath tests

The aim of this study was to investigate the hepatic microsomal and mitochondrial functions by using the ¹³CO₂-breath test in healthy subjects either before or after the consumption of red wine. Fourteen adults received [¹³C]methacetin and [methyl-¹³C]methionine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol/kg/day together with dinner over a 10-day period. Thereafter, ¹³C-tracer administration was repeated under identical conditions. The ¹³CO₂-enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage ¹³C-dose recovery (CPDR) after administration of [¹³C]methacetin and [methyl-¹³C]methionine either without or with red wine consumption amounted to 38.2±6.3 vs. 36.3±6.7% (p=0.363) and 9.5±3.3 vs. 8.8±2.5% (p=0.47), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the mitochondrial functions of the human liver in healthy subjects.     

The effect of resistant starches on fat oxidation in healthy adults as measured by a 13CO2-breath test

A resistant starch (RS) mixture (MIX) consisting of fibre of potatoes (FP) and wrinkled pea starch (WPS), and high amylose maize starch (HAMS) were supplemented in adults to evaluate their effects on fat oxidation by means of a ¹³CO₂-breath test. Sixteen subjects received a regular diet either without or with the supplementation of MIX and HAMS in randomised order. After administration of a [U-¹³C]algal lipid mixture, exhaled air was collected over 14?h in 0.5- and 1-h intervals. The ¹³C abundances were measured by nondispersive infrared spectroscopy. In comparison to the dry run (DR), supplementation with MIX and with HAMS increased the cumulative percentage dose recovery: (DR: 16.7?%, MIX: 16.9?%, HAMS: 18.0?%), but without statistical significance. The colonic degradation of MIX and HAMS to short-chain fatty acids tends to lower the formation of carbohydrate-derived acetyl-CoA and contributes to a postprandial lipid oxidation increase by using fat-derived acetyl-CoA as a compensatory fuel source.     

Exact profiles of 13CO2 recovery in breath air after per oral administration of [13C]methacetin in two groups of different ages

The aim of this study is to determine if age is a factor influencing the results of a [¹³C]methacetin breath test (¹³C-MBT). Two groups of healthy volunteers, each comprising six men and six women, but differing in average age (Y=young, 25.1±0.6 years, MA=middle-aged;, 46.0±2.1 years) orally took 75 mg [¹³C]methacetin. Samples of expiratory air for ¹³CO₂ measurement were collected up to 48 h after intake of the substrate. A maximum momentary ¹³CO₂ breath exhalation of 37.0±2.6%dose/h was observed at 18 min (median, range: 9–30 min) in the young subjects and of 38.4±2.5%dose/h at 18 min (median, range: 12–30 min) in the middle-age volunteers. The cumulative ¹³C elimination in expiratory air was statistically significantly higher in the MA compared with the Y group as from 75 min up to 180 min, indicating a greater microsomal metabolic efficiency of the liver in the middle-aged healthy subjects. Gender, use of hormonal contraception, cigarette smoking, or body mass index did not modify the age-related effect on the cumulative ¹³C elimination in breath air. The study results imply a necessity of composing control groups well matched with regard to the age structure for a proper interpretation of clinical ¹³C-MBT results.     

High sensitivity cw-cavity ringdown and Fourier transform absorption spectroscopies of CO2

The absorption spectrum of ¹³CO₂ has been recorded by cw-cavity ringdown spectroscopy with a new set up based on fibered DFB lasers. By using a series of 31 DFB lasers, the spectrum of carbon dioxide could be recorded in the 6130-6750 cm⁻¹ region with a typical sensitivity of 5 × 10⁻¹⁰ cm⁻¹. The spectrum has also been recorded between 4400 and 8500 cm⁻¹ with a Fourier transform spectrometer associated with a multi-pass cell (maximum path length of 105 m). The new observations obtained both by FTS and CRDS represent a significant extension of the available data. For instance, more than 4000 line positions were measured and assigned in the CRDS spectrum while only 232 line positions are listed in the HITRAN database. Altogether, the band by band analysis has led to the determination of the rovibrational parameters of 65, 7, and 24 bands for the ¹³C¹⁶O₂, ¹⁶O¹³C¹⁷O, and ¹⁶O¹³C¹⁸O isotopomers, respectively. As some observed line positions show significant deviations from the predictions of the effective Hamiltonian model, the new observed line positions were gathered with the data available in the literature to refine the set of effective Hamiltonian parameters of the ¹³C¹⁶O₂ isotopic species. The refined set of 96 effective Hamiltonian parameters reproduces more than 14 650 line positions of ¹³C¹⁶O₂ with an RMS=0.002 cm⁻¹. A detailed comparison with the line positions retrieved from Venus spectra and the line list provided by HITRAN is also presented and discussed.     

The rotational spectrum of 13C2HD in the ground and excited bending states

The pure rotational spectrum of ¹³C₂HD was recorded in the range 100–700 GHz. Lines belonging to the ground vibrational state were observed from J^″ = 1 to J^″ = 11. Several absorption lines were also detected in the bending states v₄ = 1 (Π), v₅ = 1 (Π), v₄ = 2 (Σ⁺ and Δ), v₅ = 2 (Σ⁺ and Δ), v₄ = v₅ = 1 (Σ^?, Σ⁺ and Δ), v₄ = 3 (Π and Φ) and v₅ = 3 (Π and Φ). The transition frequencies measured in this work were fitted together with all the infrared lines available in the literature. The global fit allowed a very accurate determination of the vibrational, rotational and ?-type interaction parameters for the bending states of this molecule.     

The 13C bicarbonate method: an inverse end product method for measuring CO2 production and energy expenditure

We reconsider the principle of the ¹³C bicarbonate (NaH¹³CO₃) method (¹³C-BM) for the determination of the CO₂ production to obtain an estimate of energy expenditure (EE). Its mathematical concept based on a three-compartmental model is related to the [¹⁵N]glycine end product method. The CO₂ production calculated by the ¹³C-BM, R_aCO₂(¹³C) is compared to the result from the indirect calorimetry, RCO₂(IC). In an interspecies comparison (dog, goat, horse, cattle, children, adult human; body mass ranging from 15 to 350?kg, resting and fasting conditions) we found an excellent correlation between the results of ¹³C-BM and IC with RCO₂(IC)?=?0.703?×?R_aCO₂(¹³C), (R²?=?0.99). The slope of this correlation corresponds to the fractional ¹³C recovery (RF(¹³C)) of ¹³C in breath CO₂ after administration of NaH¹³CO₃. Significant increase in RF(¹³C) was found in physically active dogs (0.95?±?0.14; n?=?5) vs. resting dogs (0.71?±?0.10, n?=?17; p?=?.015). The ¹³C recovery in young bulls was greater in blood CO₂ (0.81?±?0.05) vs. breath CO₂ (0.73?±?0.05, n?=?12, p?<?.001) and in ponies with oral (0.76?±?0.03, n?=?8) vs. intravenous administration of NaH¹³CO₃ (0.69?±?0.07; n?=?8; p?=?.026). We suggest considering the ¹³C-BM as a ‘stand-alone’ method to provide information on the total CO₂ production as an index of EE.     

Rotational dependences of line half-widths for CO and CO2 confined in SiO2/Al2O3 xerogel

The absorption spectra of CO and CO₂ confined in nanopores of SiO₂/Al₂O₃ xerogel have been measured using a Bruker IFS-125 HR Fourier spectrometer. Dependences of the half-width values on rotational quantum numbers and the line shift mean values are studied and compared with the data available in literature. Possible causes which can affect the rotational dependences are discussed.     

Laser spectroscopic monitoring of gas emission and measurements of the C/C isotope ratio in CO2 from a wood-based combustion

We report on the application of a compact and field-deployable instrument, based on a continuous-wave fiber-coupled Telecom external cavity diode laser, to measure the ¹³C/¹²C isotope ratio in CO₂ from a wood-based combustion. Carbon dioxide, the most important greenhouse gas, is a major product of combustion. The measurements of the ¹³C/¹²C isotopic ratio in CO₂ from combustion emission permit one to identify the CO₂ source and to study the temporal and spatial variations of pollution in the atmosphere. The average value of the ¹³CO₂/¹²CO₂ ratio is found to be (1.1011±0.0024)%. The corresponding δ-value relative to PDB standard is (−20.17±2.14)‰, which is in good agreement with the typical value of (−25±2)‰ for wood. Simultaneous monitoring of multiple species from gas emission has been performed using direct-absorption spectroscopy. The concentrations of C₂H₂, CO, CO₂ and H₂O were determined on the basis of integrated absorbance measured by least-squares fitting a Voigt lineshape to experimental absorption spectra.     

Short-term carbon dynamics in a temperate grassland and heathland ecosystem exposed to 104 days of drought followed by irrigation

Temperate ecosystems are susceptible to drought events. The effect of a severe drought (104 days) followed by irrigation on the plant C uptake, its assimilation and input of C in soil were examined using a triple ¹³CO₂ pulse-chase labelling experiment in model grassland and heathland ecosystems. First ¹³CO₂ pulse at day 0 of the experiment revealed much higher ¹³C tracer uptake for shoots, roots and soil compared to the second pulse (day 44), where all plants showed significantly lower ¹³C tracer uptake. After the third ¹³CO₂ pulse (day 70), very low ¹³C uptake in shoots led to a negligible allocation of ¹³C into roots and soil. During irrigation after the severe drought, the ¹³C tracer that was allocated in plant tissues during the second and third pulse labelling was re-allocated in roots and soil, as soon as the irrigation started. This re-allocation was higher and longer lasting in heathland compared to grassland ecosystems.     

A New Two-Dimensional Pulse Sequence for T2* Measurements of Protons in C Isotopomers

A new two-dimensional pulse sequence for T₂* measurement of protons directly coupled to ¹³C spins is proposed. The sequence measures the tranverse relaxation time of heteronuclear proton single-quantum coherence under conditions of free precession and is therefore well suited to evaluate relaxation losses of proton magnetization during preparation delays of heteronuclear pulse experiments in analytical NMR. The relevant part of the pulse sequence can be inserted as a “building block” into any direct or inverse detecting H,C correlation pulse sequence if proton spin–spin relaxation is to be investigated. In this contribution, the building block is inserted into a HETCOR as well as into a HMQC pulse sequence. Experimental results for the HETCOR-based sequence are given.     

Continuous measurements of stable isotopes of carbon dioxide and water vapour in an urban atmosphere: isotopic variations associated with meteorological conditions

Isotope ratios of carbon dioxide and water vapour in the near-surface air were continuously measured for one month in an urban area of the city of Nagoya in central Japan in September 2010 using laser spectroscopic techniques. During the passages of a typhoon and a stationary front in the observation period, remarkable changes in the isotope ratios of CO₂ and water vapour were observed. The isotope ratios of both CO₂ and water vapour decreased during the typhoon passage. The decreases can be attributed to the air coming from an industrial area and the rainout effects of the typhoon, respectively. During the passage of the stationary front, δ¹³C–CO₂ and δ¹⁸O–CO₂ increased, while δ²H–H₂Ov and δ¹⁸O–H₂Ov decreased. These changes can be attributed to the air coming from rural areas and the air surrounding the observational site changing from a subtropical air mass to a subpolar air mass during the passage of the stationary front. A clear relationship was observed between the isotopic CO₂ and water vapour and the meteorological phenomena. Therefore, isotopic information of CO₂ and H₂Ov could be used as a tracer of meteorological information.