Determination of (13)CO(2)/(12)CO(2) Ratio by IRMS and NDIRS

Abstract Breath tests using (13)C-labelled substrates require the measurement of (13)CO(2)/(12)CO(2) 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 (13)CO(2)/(12)CO(2) 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-(13)C-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 (13)CO(2)-kinetics over longer time periods.     

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.     

Biomedical Application of an Isotope Selective Nondispersive Infrared Spectrometer for 13CO2 and 12CO2 Concentration Measurements in Breath Samples

A newly developed isotope selective nondispersive infrared (NDIR) spectrometer for the measurement of ¹³CO₂ and ¹²CO₂ concentrations in breath samples was applied as a low cost and very simple to operate alternative to isotope ratio mass spectrometry (IRMS). We used this device for several biomedical applications ([¹³C]urea breath test, [¹³C]leucine metabolism, [¹³C]methacetin catabolism of rats) and found that the results agree very well with IRMS.     

Using a laser-based CO2 carbon isotope analyser to investigate gas transfer in geological media

CO₂ stable carbon isotopes are very attractive in environmental research to investigate both natural and anthropogenic carbon sources. Laser-based CO₂ carbon isotope analysis provides continuous measurement at high temporal resolution and is a promising alternative to isotope ratio mass spectrometry (IRMS). We performed a thorough assessment of a commercially available CO₂ Carbon Isotope Analyser (CCIA DLT-100, Los Gatos Research) that allows in situ measurement of δ ¹³C in CO₂. Using a set of reference gases of known CO₂ concentration and carbon isotopic composition, we evaluated the precision, long-term stability, temperature sensitivity and concentration dependence of the analyser. Despite good precision calculated from Allan variance (5.0 ppm for CO₂ concentration, and 0.05 ‰ for δ ¹³C at 60 s averaging), real performances are altered by two main sources of error: temperature sensitivity and dependence of δ ¹³C on CO₂ concentration. Data processing is required to correct for these errors. Following application of these corrections, we achieve an accuracy of 8.7 ppm for CO₂ concentration and 1.3 ‰ for δ ¹³C, which is worse compared to mass spectrometry performance, but still allowing field applications. With this portable analyser we measured CO₂ flux degassed from rock in an underground tunnel. The obtained carbon isotopic composition agrees with IRMS measurement, and can be used to identify the carbon source.     

Breath water-based doubly labelled water method for the noninvasive determination of CO2 production and energy expenditure in mice

ABSTRACT

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO₂ production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (n?=?11, 43.5?±?4.6?g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2?g H₂¹⁸O kg BM^?1 and 0.4?g ²H₂O kg BM^?1, 99 atom% (AP) ¹⁸O or ²H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The ²H/¹H and ¹⁸O/¹⁶O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO₂ production (RCO₂) was calculated from the ²H and ¹⁸O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R²?=?0.89 for ²H and 0.95 for ¹⁸O) linearly. The RCO₂ determined based on breath water and plasma was not different (113.2?±?12.7 vs. 111.4?±?11.0?mmol?d^–1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO₂ avoiding blood sampling.     

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.     

Use of the optogalvanic effect (OGE) for isotope ratio spectrometry of 13CO2 and 14CO2

The use of isotopic carbon dioxide lasers for determination of carbon (and oxygen) isotope ratios was first demonstrated in 1994. Since then a commercial device called LARA^?, has been manufactured and used for Helicobacter pylori breath tests using ¹³C-labelled urea. The major advantages of the optogalvanic effect compared with other infrared absorption isotope ratio measurement techniques are its lack of optical background and its high sensitivity resulting from a signal gain proportional to laser power. Continuous normalisation using two cells, a standard and sample, lead to high accuracy as well as precision. Recent advances in continuous flow measurement of ¹³C/¹²C ratios of CO₂ in air and extensions of the technique to ¹⁴C, which can be analysed as a stable isotope, are described.     

13C Basal Abundance of Expired CO2 -Definition of Pre-Requisites for Kinetic Breath Tests

Abstract

A sufficiently stable rate of ¹³CO₂ exhalation is necessary when the diagnostic ¹³CO₂ breath tests are performed in healthy subjects and patients. The aim of the research was to define prerequisite conditions for kinetic breath tests in order to ensure a stable ¹³CO₂ background. A 3-part protocol was developed. Part I: a study of the one-day variation of ¹³CO₂ abundance in expired CO₂ confirmed that shifts of the basal ¹³C abundance in breath are inherent in nature. Part II: a study of the variations of ¹³C enrichment after the ingestion of different meals and beverages showed that ingestion of food items containing C₄ plant sugars, such as maize, induces a significant increase in isotopic abundance. Part III: a new test breakfast containing rice grain cereal, milk and orange juice was tested. This test meal induces no significant change on the basal ¹³CO₂ abundance in healthy subjects. This new finding allows to avoid the fasting period normally required prior to a breath test which is sometimes difficult for children and pregnant women.     

基于傅里叶变换红外光谱法CO2气体碳同位素比检测研究

介绍了基于傅里叶变换红外技术检测CO₂气体碳同位素比的新方法, 详细介绍了如何从HITRAN红外数据库中提取气体标准吸收截面; 介绍了基于非线性最小二乘法反演CO₂气体碳同位素比和整套实验装置的组成及实验步骤. 从理论和实验分析两方面讨论了温度和气压变化对δ¹³CO₂值的影响规律. 对于同一CO₂标准气体, 采用FTIR和同位素质谱法两种技术进行了δ¹³CO₂值对比检测, 两种测量技术的平均值差异仅为0.25%. 从实验结果可以看出, FTIR技术可以实现对CO₂气体碳同位素比的检测.     

Diode laser absorption spectrometry for 13CO2/12CO2 isotope ratio analysis: Investigation on precision and accuracy levels

Near-infrared laser spectroscopy is used to measure the ¹³C/¹²C isotope abundance ratio in gas phase carbon dioxide. The spectrometer, developed expressly for field applications, is based on a 2 μm distributed feedback diode laser in combination with sensitive wavelength modulation detection. It is characterized by a simplified optical layout, in which a single detector and associated electronics are used to probe absorptions of a pair of ¹³CO₂ and ¹²CO₂ lines, simultaneously in a sample, as well as a reference gas. For a careful investigation of the achievable precision and accuracy levels, we carried out a variety of laboratory tests on CO₂ samples with different isotopic compositions, calibrated with respect to the international standard material by means of isotope ratio mass spectrometry. The 1-σ accuracy of the ¹³CO₂/¹²CO₂ determinations, reported in the so-called δ notation, is about 0.5‰ (including both statistical and systematic errors), for δ-values in the range from -30‰ to +20‰. We show that the major source of systematic errors is a consequence of the non-linearity of the Lambert–Beer absorption law, and can be corrected for to a very high degree of accuracy. PACS 42.62.Fi; 42.55.Px; 33.20.Ea     

Analysis of 13C-mixed Triacylglycerol in Stool by Bulk (Ea-IRMS) and Compound Specific (GC/MS) Methods

Abstract

This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna. Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91).

The ¹³C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion. 20–40% of the ingested ¹³C label is recovered in breath CO₂. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of ¹³C-MTG by children with impaired exocrine pancreatic function and healthy controls. ¹³C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total ¹³C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and ¹³C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the ¹³C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.     

Microcombustion of ng Amounts of Carbon in Non-Volatile Materials for isotope Ratio Evaluation

Abstract

A novel microcombustion technique for carbon isotopic analysis of nanogram amounts of carbon in non-volatile materials based on isotope ratio monitoring (irm) mass spectrometry is described. Liquid or solid samples placed in a quartz sleeve are combusted at 1000°C in a continuous stream of helium and oxygen. CO₂ removed from the carrier gas stream by cryogenic trapping is transferred onto a GC column. Following GC separation, the CO₂ is transferred via an open split to the ion source of a gas isotope ratio mass spectrometer. Reproducibility for samples >25 nmol carbon is <1‰. Problems associated with blanks from various sources and with reproducible deposition of small sample amounts led to variable accuracy, which was dependent on the compound class being analysed. Minimum sample size was in the range from 5 to 10 nmol carbon. Measurements of dissolved organic carbon (DOC) of groundwater from Germany yielded consistent values of δ¹³C = -28.8‰.     

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.     

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ2H and δ18O in human urine

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ²H and δ¹⁸O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R ²=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.     

Evaluation of the performance of high temperature conversion reactors for compound-specific oxygen stable isotope analysis

In this study conversion conditions for oxygen gas chromatography high temperature conversion (HTC) isotope ratio mass spectrometry (IRMS) are characterised using qualitative mass spectrometry (IonTrap). It is shown that physical and chemical properties of a given reactor design impact HTC and thus the ability to accurately measure oxygen isotope ratios. Commercially available and custom-built tube-in-tube reactors were used to elucidate (i) by-product formation (carbon dioxide, water, small organic molecules), (ii) 2nd sources of oxygen (leakage, metal oxides, ceramic material), and (iii) required reactor conditions (conditioning, reduction, stability). The suitability of the available HTC approach for compound-specific isotope analysis of oxygen in volatile organic molecules like methyl tert-butyl ether is assessed. Main problems impeding accurate analysis are non-quantitative HTC and significant carbon dioxide by-product formation. An evaluation strategy combining mass spectrometric analysis of HTC products and IRMS ¹⁸O/¹⁶O monitoring for future method development is proposed.     

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.     

Towards an Inhalative 13C Breath Test Method

Abstract

Customary ¹³CO₂ breath tests—and also ¹⁵N urine tests—always start with an oral administration of a test substrate. The test person swallows a stable isotope labelled diagnostic agent. This technique has been used to study several pathophysiological changes in gastrointestinal organs. However, to study pathophysiological changes of the bronchial and lung epithelium, the inhalative administration of a stable isotope labelled agent appeared more suitable to us. [1-¹³C]Hexadecanol and [1-¹³C]glucose were chosen. Inhaled [1-¹³C]hexadecanol did not yield ¹³CO₂ in the exhaled air, but [1-¹³C]glucose did. To study the practicability of the [1-¹³C]glucose method and the reproducibility of the results, 18 inhalation tests were performed with healthy subjects. In 6 self-tests, the optimum inhalative dose of [¹³C]glucose was determined to be 205 mg. Using the APS aerosol provocation system with the nebulizer ‘Medic Aid’ (Erich Jaeger Würzburg), a 25% aqueous solution was inhaled. Then, breath samples were collected at 15 min. intervals and analysed for ¹³CO₂. 75–120min after the end of inhalation a well-reproducible maximum δ¹³C value of 6‰ over baseline (DOB) was detected for 12 healthy probands.

Speculating that the pulmonary resorption of the [¹³C]glucose is the rate-limiting step of elimination, decompensations in the epithelium ought to be reflected in changed [1-¹³C]glucose resorption rates and changed ¹³CO₂ output.

Therefore, we speculate that the inhalation of suitable ¹³C-labelled substrates will pave the way for a new group of ¹³CO₂ breath tests aiding investigations of specific pathophysiological changes in the pulmonary tract, such as inflammations of certain sections and decompensations of cell functions.     

Isotope-Ratio-Monitoring Liquid Chromatography Mass Spectrometry (IRM-LCMS): First Results from a Moving Wire Interface System

Abstract

A Liquid Chromatography-Combustion (LC-C) Interface, based on a moving wire technique, has been built and tested. The LC effluent is deposited onto a transport wire, which carries the sample through solvent evaporation and combustion ovens. CO₂ from the combustion step is analysed in an isotope ratio mass spectrometer. Performance of the interface was tested by loop injections of sucrose and glucose into a liquid flow of methanol/water (80/20). Accuracy and precision of δ¹³C_PDB < 1‰ was achieved for sample concentrations > 500 ng/ul (5μl loop), sufficient for studies at natural isotope ratios. In case of ¹³C tracer applications the detection limit was determined to be about 20 pg carbon tracer (on wire).     

Determination of 13C/12C-Ratios in Rumen Produced Methane and CO2 of Cows,Sheep and Camels

Abstract

Naturally produced methane shows different δ¹³C-values with respect to its origin, e.g., geological or biological. Methane-production of ruminants is considered to be the dominant source from the animal kingdom. Isotopic values of rumen methane—given in literature—range between ?80‰ and -50‰ and are related to feed composition and also sampling techniques. Keeping cows, camels and sheep under identical feed conditions and sampling rumen gases via implanted fistulae we compared δ_PDB ¹³C-values of methane and CO₂ between the species. Referring to mean values obtained from 4 or 5 samples at different times of 11 animals (n = 47) we calculated δ_PDB ¹³C-medians resulting in small but not significant differences within and significant differences between the species for CO₂ and methane. The δ_PDB ¹³C-differences between methane and CO₂ were statistically equal within and also between the species. Therefore a linear regression of methane values on CO₂ is appropriate and leads to: δ_PDB ¹³C(methane)‰ = 1,57 * δ_PDB ¹³C(CO₂)‰-47‰ with a correlation coefficient of r = 0,87.     

Continuous measurements of stable carbon isotopes in CO2 with a near-IR laser absorption spectrometer

A near-IR laser absorption spectrometer using a technique of wavelength modulation spectroscopy is used to measure stable carbon isotope ratios of ambient CO₂ (δ¹³C) via the absorption lines ¹²CO₂ R(17) (2ν₁ + ν¹₂ − ν¹₂ + ν₃) at 4978.205 cm⁻¹ and ¹³CO₂ P(16) (ν₁ + 2ν₂ + ν₃) at 4978.023 cm⁻¹. The isotope ratios are measured with a reproducibility of 0.02‰ (1σ) in a 130-s integration time over a 12-h period. The humidity effect on δ¹³C values has been evaluated in laboratory experiments. The δ¹³C values of CO₂ in ambient air were measured continuously over 8 days and agreed well with those from isotope ratio mass spectrometry of canister samples. The spectrometer is thus capable of real-time, in situ measurements of stable carbon isotope ratios of CO₂ under ambient conditions.