Sensitivity and precision of the 13C-breath test

Sensitivity and precision of the 13C-breath test were assessed by examining a couple of limiting factors caused by the sensitivity of the instrument used for 13CO2 analysis, endogenous fluctuation of 13CO2 abundance, and the residual CO2 in sample storing tubes, vacutainers, etc. For 13CO2 analysis, a mass spectrometer equipped with a dual inlet, a dual collector, and an automated pressure matching system, was used. 15 ml vacutainers were used for sample storage. Endogenous fluctuation of 13CO2 abundance, however, was measured by putting the breath samples directly into the evacuated CO2 purification system, instead of using vacutainers. Endogenous fluctuation (S.D. = 0.202%) was the most significant limiting factor, compared with instrumental limitation (0.085%), or with influence of the residual CO2 (0.136%). Consequently, the sensitivity of the 13C-breath test was figured out to be 0.52%. This sensitivity is about 1,000 times lower than that of 14C-breath test. The precision was linearly dependent on 13C increase from basal 13C observed after administration of 13C enriched compounds, delta 13C, and was determined to be expressed as 1.94 delta 13C.     

Evaluation of 13C-phenylalanine and 13C-tyrosine breath tests for the measurement of hepatocyte functional capacity in patients with liver cirrhosis.

Liver disease is associated with an abnormal elevation of the plasma concentrations of the aromatic amino acids phenylalanine and tyrosine. The liver is the main site of aromatic amino acid metabolism, particularly the hydroxylation of phenylalanine to tyrosine and further tyrosine degradation. In the present study, we have examined the usefulness of the L-[1-13C]phenylalanine breath test (13C-PheBT) and L-[13C]tyrosine breath test (13C-TyrBT) for the detection of hepatic damage in patients with liver cirrhosis. First, the time courses of 13CO2 excretion after the administration of L-[1-13C]phenylalanine and L-[1-13C]tyrosine were compared. The peak times (the time expressed in minutes at which 13CO2 excretion was maximal) were 20 min in both breath tests, but 13C-TyrBT gave a higher peak than 13C-PheBT. Next, the parameters of 13C-PheBT and 13C-TyrBT were compared with biochemical liver function test values. These parameters were well correlated with several liver blood test values conventionally regarded as measures of hepatocyte functional reserve. Therefore, 13C-PheBT and 13C-TyrBT may be useful to assess the degree and progression of hepatic dysfunction.     

On-line measurements of 13C enrichments in rat breath. Non-invasive method for in vivo study of drug enzymatic induction

A differential kinetic study of 13CO2 enrichment of breath after the intake of specific 13C-labelled substrates and co-administration of a drug allows the drug's ability for enzyme induction to be evaluated in vivo. A method and a gas chromatograph-isotope ratio mass spectrometer device for on-line measurements of 13CO2 enrichment in the breath of small animals are described. This system allows on-line breath sample collection from a metabolic cage, purification by gas chromatography, determination of CO2 by thermal conductivity detection and measurement of 13CO2 enrichment by isotope ratio mass spectrometry. Two protocols for phenobarbital-inducible P450 and 3-methylcholanthrene-inducible P1-450 isoenzymes are described.     

(13)C natural abundance in the British diet: implications for (13)C breath tests

Surprisingly little information is available on the natural abundance of the minor isotope of carbon, (13)C, in common foodstuffs in the British diet. This study therefore aimed to examine the (13)C natural abundance of foodstuffs from a small cross-section of the British diet. The isotopic abundance, delta per mil, was calculated by measurement of the isotope ratio (13)C:(12)C by isotope ratio mass spectrometry. Results from this study were also compared with results from a North American study to highlight the difference in isotopic abundance between Northern European foodstuffs and North American foodstuffs. Such data should prove useful to those planning tracer studies using the stable isotope (13)C where enrichment is measured against a large and variable natural abundance in the body. Minimisation of this basal variation, for example in breath CO(2), can be achieved by controlling dietary intake of foods naturally abundant in (13)C.     

Factors affecting 13C-natural abundance measurement of breath carbon dioxide during surgery: absorption of carbon dioxide during endoscopic procedures

The aim of this paper is to review the factors which may affect breath (13)CO(2)/(12)CO(2) natural abundance in patients undergoing surgery or intensive care. Intravenous glucose administration is a major determinant of the (13)CO(2)/(12)CO(2) of breath as intravenous glucose preparations are almost all derived from cornstarch. In addition, the oxidation of endogenous substrates can affect the (13)CO(2)/(12)CO(2) ratio. During many endoscopic procedures, such as laparoscopic surgery, carbon dioxide insufflation is used to provide a working space. As medical CO(2) is relatively depleted in (13)CO(2) compared with endogenous and exogenous metabolic CO(2) sources, breath (13)CO(2)/(12)CO(2) measurements can be used to estimate CO(2) absorption during these procedures. However, all these factors may also be affected by the bicarbonate pool, making a definitive attribution of changes in breath (13)CO(2)/(12)CO(2) to a single factor problematic.     

Carbon sequestration and estimated carbon credit values as measured using <Superscript>13</Superscript>C labelling and analysis by means of an optical breath test analyser

Recent developments in optical systems (isotope-selective non-dispersive infrared spectrometry) for breath testing have provided a robust, low-cost option for undertaking ¹³C analysis. Although these systems were initially developed for breath testing for Helicobacter pylori, they have an enormous potential as a soil science research tool. The relatively low cost of the equipment, US$15,000–25,000, is within the research budgets of most institutes or universities. The simplicity of the mechanisms and optical nature mean that the equipment requires relatively low maintenance and minimal training. Thus methods were developed to prepare soil and plant materials for analysis using the breath test analyser. Results that compare conventional mass spectrometric methods with the breath test analyser will be presented. In combination with simple ¹³C-plant-labeling techniques it is possible to devise methods for estimating carbon sequestration under different agronomic management practices within a short time frame. This enables assessment of the carbon credit value of a particular agronomic practice, which can in turn be used by policy makers for decision-making purposes. For global understanding of the effect of agricultural practices on the carbon cycle, data are required from a range of cropping systems and agro-ecological zones. The method and the approach described will enable collection of hard data within a reasonable time.     

(13)C/(12)C isotope labelling to study leaf carbon respiration and allocation in twigs of field-grown beech trees

In situ (13)C/(12)C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas-exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO(2) with a constant carbon isotope composition (delta(13)C of -51.2 per thousand) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The delta(13)C values of respiratory metabolites and CO(2) that is subsequently respired during the night were measured. It was found that night-respired CO(2) is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO(2) immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the (13)C isotope occurred during the night respiration of beech leaves.     

Selective conversion of plasma glucose into CO2 by Saccharomyces cerevisiae for the measurement of 13C abundance by isotope ratio mass spectrometry: proof of principle

To study carbohydrate digestion and glucose absorption, time-dependent (13)C enrichment in plasma glucose is measured after oral administration of naturally occurring (13)C-enriched carbohydrates. The isotope enrichment of the administered carbohydrate is low (APE <0.1%) and plasma (13)C glucose measurements are routinely determined with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) or liquid chromatography/combustion/isotope ratio mass spectrometry (LC/C/IRMS). In this study, plasma glucose was converted into CO(2) by an in-tube reaction with yeast permitting direct measurement of (13)CO(2) in the headspace. Saccharomyces cerevisiae incubated under anaerobic conditions was able to convert sufficient glucose into CO(2) to produce a consistent CO(2) peak in IRMS with little variation in peak area and precise delta(13)C(PDB) values for corn glucose: -11.40 +/- 0.16 per thousand, potato glucose: -25.17 +/- 0.13 per thousand, and plasma glucose: -26.29 +/- 0.05 per thousand. The measurement showed high linearity (R(2) = 0.999) and selectivity and was not affected by the glucose concentration in the tested range of 5-15 mM. Comparison with GC/C/IRMS showed a good correlation of enrichment data: R(2) > 0.98 for both sources of glucose and plasma samples. Commercially available, instant dried baker's yeast was qualitatively and quantitatively comparable with freshly prepared yeast: R(2) > 0.96, slope 1.03 and 1.08 for glucose solutions and plasma, respectively. Thus, yeast conversion of plasma glucose into CO(2) and (13)C measurement applying a breath (13)CO(2) analyzer is an inexpensive, simple and equally accurate alternative to the more expensive and laborious GC/C/IRMS and LC/C/IRMS measurements.     

Results of L-[1-13C]phenylalanine breath test with air isotope ratio mass spectrometry can reflect the activity of phenylalanine hydroxylase in cirrhotic rat liver

The L-[1-13C]phenylalanine breath test (PheBT) could potentially advance the evaluation of hepatocyte function and liver functional reserve. However, because the factors influencing PheBT results have not been clarified, the clinical application of the test has been limited. This study investigated the relationship between the parameters of PheBT, performed with air isotope ratio mass spectrometry, and the activity of phenylalanine hydroxylase (PAH), the phenylalanine metabolism rate-limiting enzyme, in rat liver, and proposes valid parameters for the assessment of liver function. Chronic injury to the liver was induced by the administration of CCl4 to male Sprague-Dawley rats for either 8 or 12 weeks. Livers from rats in the two cirrhotic groups were discolored, enlarged and roughly textured, with cells filled with fat granules of various sizes, pseudolobuli formations, and regenerated tubercles. Of the 12 parameters tested, only the unit liver weight (LW) breath test parameters, including the maximum abundance of 13C in breath (13Cmax/LW), 13C abundance in breaths 2 and 7 min after administration of L-[1-(13)C]phenylalanine (13C-phe) (13C2/LW and 13C7/LW), cumulative 13C excretion 10 and 30 min after 13C-phe administration (AUC10/LW and AUC30/LW), and the 13C excretion rate constant (PheBT-k/LW) were significantly affected in the chronic liver injury groups. There was no significant difference in the total PAH activity in liver among the three groups, but there was significant difference in unit LW PAH activity. Total PAH activity in the liver was significantly correlated with 13Cmax, 13C2, 13C7, AUC10, AUC30 and PheBT-k, while the unit LW PAH activity was significantly correlated with 13Cmax/LW, 13C2/LW, 13C7/LW, AUC10/LW, AUC30/LW and PheBT-k/LW. PheBT-k/LW was also correlated with biochemical indices that are used to assess liver function. The present findings indicate that the PheBT results based on air isotope ratio mass spectrometry can quantitatively reflect the change in total PAH activity in the livers of chronically injured rats. PheBT-k and PheBT-k/LW are the most sensitive among the test parameters, and can be used to assess liver functional reserve and hepatocyte damage at the molecular level.     

Measurement of gastric emptying in man using deuterated octanoic acid.

Gamma scintigraphy is considered the gold standard for the measurement of gastric emptying in humans. Recently, it has been proposed that a [(13)C]octanoate breath test can be used as an alternative technique for measuring gastric emptying of the solid phase, but the results from the two methods are not directly equivalent since in the breath test the label is subject to post-absorptive processing and consequently the emptying functions cannot be observed directly. This work investigates an alternative stable isotope method using deuterated octanoate where the kinetics of redistribution between and elimination from the various body pools are much more easily modelled. Gastric emptying was studied in healthy human volunteers by simultaneous measurement using both [(13)C]octanoate and [(2)H]octanoate as well as gamma scintigraphy. Comparison of the gastric emptying functions from the deuterium method and scintigraphy indicated that the two methods gave equivalent results. The new method can therefore be used in populations considered too vulnerable to ionising radiation to allow gamma scintigraphy to be performed, or as a proxy gold standard in laboratories where scintigraphic methods are unavailable, allowing further comparisons with the breath test method to be made to validate the latter in different population groups.     

Measuring the 13C content of soil-respired CO2 using a novel open chamber system

Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.     

Reaction of Cl atoms with C6F13CH2OH, C6F13CHO, and C3F7CHO

The Cl atom initiated oxidation of C(6)F(13)CH(2)OH, C(6)F(13)CHO, and C(3)F(7)CHO was investigated at 298 K and 1000 mbar pressure of air in a photoreactor using in situ Fourier transform infrared (FTIR) analysis. The rate coefficient for the reaction Cl + C(6)F(13)CH(2)OH (reaction 2) was measured using a relative method: k(2) = (6.5 +/- 0.8) x 10(-13) cm(3) molecule(-1) s(-1). C(6)F(13)CHO was detected as the major primary product, while CO and CF(2)O were found to be the major secondary products. A fitting procedure applied to the concentration-time profiles of C(6)F(13)CHO provided a production yield of (1.0 +/- 0.2) for this aldehyde in reaction 2, and the rate coefficient for the reaction Cl + C(6)F(13)CHO (reaction 4) was k(4) = (2.8 +/- 0.7) x 10(-12) cm(3) molecule(-1) s(-1). A high CO yield observed in the oxidation of C(6)F(13)CH(2)OH, (52 +/- 1)%, is attributed to the Cl atom initiated oxidation of C(6)F(13)CHO. High CO yields, (61 +/- 2)% and (85 +/- 5)%, were also measured in the Cl atom initiated oxidation of C(3)F(7)CHO in air and nitrogen, respectively. These high CO yields suggest the occurrence of a decomposition reaction of the perfluoroacyl, C(6)F(13)CO, and C(3)F(7)CO radicals to form CO which will compete with the combination reaction of these radicals with oxygen to form perfluoroacyl peroxy radicals in the presence of air. The latter radicals C(n)F(2)(n)(+1)CO(O)(2) (n = 6-12), through their reaction with HO(2) radicals, are currently considered as a possible source of persistent perfluorocarboxylic acids which have been detected in the environment. The consequences of the present results would be a reduction of the strength of this potential source of carboxylic acids in the atmosphere.     

Diagnosis of Helicobacter pylori infection with the (13)C-urea breath test by means of GC-MS analysis

The diagnosis of Helicobacter pylori (H. pylori) infection by GC-MS detection of the (13)CO(2) enrichment in (13)C-urea breath test ((13)C-UBT) samples is reported. This study aimed to optimize the (13)C-UBT with regards to the diagnostic cut-off value, sampling time, and frequency. The H. pylori status of 103 dyspeptic patients was obtained by histological examination, the rapid urease test as well as with the GC-MS (13)C-UBT. Analytical and diagnostic accuracies were determined by comparison of the GC-MS (13)C-UBT results with that of the analytical and diagnostic gold standards, namely GC-isotope ratio MS (IRMS) and histology. The (13)CO(2) enrichment values obtained with GC-MS analysis, correlated favorably (r(2) = 0.993) with those obtained by GC-IRMS analysis. When compared to histology, the GC-MS (13)C-UBT had a diagnostic sensitivity of 92% and a specificity of 93%. The positive predictive value (PPV), negative predictive value (NPV), and accuracy were 95, 89, and 92%, respectively. It was concluded that SIM GC-MS is capable of analyzing nonradioactive (13)C-UBT samples, with a precision and accuracy sufficient to distinguish between H. pylori positive and negative patients.     

Turnover of stable carbon isotopes in the muscle, liver, and breath CO2 of alpacas (Lama pacos)

Stable carbon isotope analysis of animal liver and muscle has become a widespread tool for investigating dietary ecology. Nonetheless, stable carbon isotope turnover of these tissues has not been studied in large mammals except with isotopically labelled tracer methodologies, which do not produce carbon half-lives analogous to those derived from naturalistic diet-switch experiments. To address this gap, we studied turnover of carbon isotopes in the liver, muscle, and breath CO2 of alpacas (Lama pacos) by switching them from a C3 grass diet to an isonitrogenous C4 grass diet. Breath samples as well as liver and muscle biopsies were collected and analyzed for up to 72 days to monitor the incorporation of the C4-derived carbon. The data suggest half-lives of 2.8, 37.3, and 178.7 days for alpaca breath CO2, liver, and muscle, respectively. Alpaca liver and muscle carbon half-lives are about 6 times longer than those of gerbils, which is about what would be expected given their size. In contrast, breath CO2 turnover does not scale readily with body mass. We also note that the breath CO2 and liver data are better described using a multiple-pool exponential decay model than a single-pool model.     

Carbon assimilation and turnover in grassland vegetation using an in situ (13)CO(2) pulse labelling system

A mobile laboratory was developed to administer a controlled flow of (13)C labelled CO(2) at ambient concentrations ( approximately 350 ppm) in the field. The stable isotope delivery (SID) system consists of an isotope-mixing unit with flow control to a series of 12 independent labelling chambers. In-line CPU controlled infrared gas analysers allow automated measurement of chamber CO(2) concentrations and gas flow management. A preliminary experiment was established on an upland pasture located at the NERC Soil Biodiversity experimental site, Sourhope, UK, in August 1999. The objective of this investigation was to determine the magnitude of pulse-derived C incorporation into a typical upland plant community. To achieve this, the SID system was deployed to pulse-label vegetation with CO(2) enriched with (13)C (50 atom %) at ambient concentrations ( approximately 350 ppm) on two consecutive days in August 1999. Samples of headspace CO(2), shoot and root were taken on four occasions over a period of 28 days after (13)C labelling. These materials were then prepared for (13)C/(12)C ratio determination by continuous-flow/combustion/isotope ratio mass spectrometry (CF-C-IRMS). Results showed that pulse derived CO(2)-C was assimilated at a rate of 128 +/- 32 microg g shoot-C hour(-1). Dynamic samplings showed that pulse-derived (13)C concentrations in the labelled plant tissues declined by 77.4 +/- 6% after 48 hours. The rapid decline in (13)C concentrations in plant matter was the result of C loss from the plant in the form of respired CO(2) and root exudates, and dilution by subsequent unlabelled C assimilates. This novel system offers considerable potential for in situ tracer investigations.     

Determination of13C in breath sample by PIGE

This paper discusses a novel application of PIGE for the determination of¹³C in breath. Samples of human breath, urea, glucose, benzamide, barium carbonate were analyzed against cylinder CO₂ and graphite standard. An accuracy check of the¹³C determination (with reference to mass spectrometric True results) gave a relative error of only –0.4% for PIGE. The performance of different standards in this determination was assessed. Relative standard deviation for the determination of¹³C isotopic abundance in breath samples were <20%. Then, if a 25% change is conservatively assumed observable in¹³C abundance, an increase in¹³C percent isotopic abundance from the natural 1.11% (average) to only 1.39% may be detected.     

Mobile, outdoor continuous-flow isotope-ratio mass spectrometer system for automated high-frequency 13C- and 18O-CO2 analysis for Keeling plot applications

A continuous-flow isotope-ratio mass spectrometer (CF-IRMS, custom-made GasBenchII and Delta(plus)Advantage, ThermoFinnigan) was installed on a grassland site and interfaced with a closed-path infrared gas analyser (IRGA). The CF-IRMS and IRGA were housed in an air-conditioned travel van. Air was sampled at 1.5 m above the 0.07-m tall grassland canopy, drawn through a 17-m long PTFE tube at a rate of 0.25 L s(-1), and fed to the IRGA and CF-IRMS in series. The IRMS was interfaced with the IRGA via a stainless steel capillary inserted 0.5 m into the sample air outlet tube of the IRGA (forming an open split), a gas-tight pump, and a sample loop attached to the eight-port Valco valve of the continuous-flow interface. Air was pumped through the 0.25-mL sample loop at 10 mL s(-1) (a flushing frequency of 40 Hz). Air samples were analysed at intervals of approx. 2.8 min. Whole system precision was tested in the field using air mixed from pure CO2 and CO2-free air by means of mass flow controllers. The standard deviation of repeated single measurements was 0.21-0.07 per thousand for delta13C and 0.34-0.14 per thousand for delta18O of CO2 in air with mixing ratios ranging between 200-800 micromol mol(-1). The CO2 peak area measured by the IRMS was proportional to the CO2 mixing ratio (r2 = 1.00), allowing estimation of sample air CO2 mixing ratio from IRMS data. A 1-day long measurement cycle of CO2, delta13C and delta18O of air sampled above the grassland canopy was used to test the system for Keeling plot applications. Delta18O exhibited a clear diurnal cycle (4 per thousand range), but short-term (1-h interval) variability was small (average SD 0.38 per thousand). Yet, the correlation between delta18O and CO2 mixing ratio was relatively weak, and this was true for both the whole data set and 1-h subsets. Conversely, the delta13C of all 541 samples measured during the 25.2-h interval fitted well the Keeling regression (r2 = 0.99), yielding an intercept of -27.40 per thousand (+/-0.07 per thousand SE). Useful Keeling regressions (r2 > 0.9, average r2 = 0.96) also resulted from data collected over 1-h intervals of the 12-h long twilight and dark period. These indicated that 13C content of ecosystem respiration was approx. constant near -27.6 per thousand. The precision of the present system is similar to that of current techniques used in ecosystem studies which employ flask sampling and a laboratory-based CF-IRMS. Sampling (and measurement) frequency is greatly increased relative to systems based on flask sampling, and sampling time (0.025 s per sample) is decreased. These features increase the probability for sampling the entire CO2 range which occurs in a given time window. The system obviates sample storage problems, greatly minimises handling needs, and allows extended campaigns of high frequency sampling and analysis with minimal attendance.     

Use of isotopically labelled octanoic acid to assess the effect of meal size on gastric emptying

It has been proposed that the (13)C-octanoic acid breath test (OBT) provides a safe, non-radioactive means of measuring gastric emptying. However, deuterated octanoic acid provides a better marker when compared with scintigraphy, as the kinetics are less complex than those of the (13)C label. The appearance of (2)H in saliva is modelled as a two-compartment body water system, using an asymmetric triangular gastric emptying function. This study compared the (2)H-octanoic acid saliva test (OST) with the OBT in measuring altered states of gastric emptying in the nutritional context of diet manipulation. Gastric emptying was measured using the OST and OBT in a three-way crossover study involving 12 healthy male and female subjects (mean BMI = 23.4 kg/m(2), aged 24-57 years). Following an overnight fast, subjects were given an egg meal, labelled with 10 microL/kg body weight (2)H-octanoic acid and 100 microL (13)C-octanoic acid. The meal was nutritionally manipulated to provide a 1 MJ, 2 MJ or 3 MJ meal. Breath and saliva samples were collected at regular intervals for 6 h, with further saliva samples being collected over four subsequent days. (2)H isotopic enrichment in saliva and (13)C isotopic enrichment in breath were analysed using isotope ratio mass spectrometry and the data fitted to the respective gastric emptying models. The half excretion time (T(1/2) (D)), time to maximum emptying rate (T(1) (D)) and time when emptying is complete (T(2) (D)) were calculated from the (2)H saliva test data, and the lag time (T(lag) (C)), half excretion time (T(1/2) (C)), latency phase (T(lat) (C)) and ascension time (T(asc) (C)) were calculated from the (13)C breath test data. Overall, the OBT correlated well with the OST, with a significant relationship between T(1/2) (C) and T(1/2) (D), a significant relationship between T(lat) (C) and T(1) (D) and finally a significant relationship between T(asc) (C) and T(2) (D). Gastric emptying measured using the OST was significantly faster with the 1 MJ meal (DeltaT(1/2) (D) = -0.77 h vs. 2 MJ, p = 0.004). Increases were also seen when the meal size was increased from 2 MJ to 3 MJ (DeltaT(1/2) (D) = +0.44 h vs. 2 MJ), but these were not significant. These trends were mirrored in the OBT data, with significant differences between 1 MJ and 2 MJ (DeltaT(1/2) (C) = -0.63 h vs. 2 MJ, p = 0.013) and non-significant increases with the larger 3 MJ meal (DeltaT(1/2) (C) = +0.10 h vs. 2 MJ). Total meal calorie content was shown to have an effect on gastric emptying using both the OBT and the OST. The deuterium method allows the direct calculation of the gastric emptying function and could be used as an alternative to gamma scintigraphy, allowing further validation of the (13)C-octanoic acid breath test.     

Collection and storage of CO2 for 13C analysis: An application to separate soil CO2 efflux into root- and soil-derived components

Soil surface CO2 efflux is comprised of CO2 from (i) root respiration and rhizosphere microbes and (ii) heterotrophic respiration from the breakdown of soil organic matter (SOM). This efflux may be partitioned between these sources using delta13C measurements. To achieve this, continuous flow isotope ratio mass spectrometry can be used and, in conjunction with 10 mL septum-capped vials, large numbers of samples may be analysed using a Finnigan MAT Delta(plus)XP interfaced to a Gas Bench II. Here we describe a number of advances to facilitate such work, including: (i) a technique for monitoring mass spectrometer performance, (ii) improvements to sample storage, and (iii) a gas-handling system for incubating and sampling the CO2 derived from roots and soils. Mass spectrometer performance was monitored using an automated refillable vial. Compressed air analysed with this system had mean delta13C of -9.61 +/- 0.16 per thousand (+/- 1sigma, n = 28) collected over four runs. Heating the butyl rubber septa used to seal the vials at 105 degrees C for 12 h improved the sample storage. After air transportation over 12 days, the isotope composition of the CO2 at ambient concentrations was unchanged (before: -35.2 +/- 0.10 per thousand, n = 4; after: -35.3 +/- 0.10 per thousand, n = 15); without heat treatment of the septa the CO2 became slightly enriched (-35.0 +/- 0.14 per thousand, n = 15). The linearity of the Gas Bench II was found to decline above 8000 micromol CO2 mol(-1). To stay within a linear range and to allow the incubation of soil and root material we describe a gas-handling system based around a peristaltic pump. Finally, we demonstrate these methods by growing a C-4 grass (Guinea grass, Panicum maximum Jacq.) in a C-3 soil. Root respiration was found to contribute between 5 and 22% to the soil surface CO2 efflux. These methodologies will facilitate experiments aimed at measuring the isotopic composition of soil-derived CO2 across a range of ecological applications.     

Advantages of deuterium-labelled mixed triacylglycerol in studies of intraluminal fat digestion

The (13)C-mixed triacylglcerol (MTG, 1,3-distearyl, 2-[1-(13)C]octanoyl glycerol) breath test is a non-invasive measure of intraluminal fat digestion. Recovery of (13)C in breath CO(2) is incomplete (<50%) owing to sequestration of (13)C into organic molecules via the tricarboxylic acid (TCA) cycle. In addition lack of knowledge of CO(2) production rate (VCO(2)) during the test leads to errors in the calculated percentage dose recovered (PDR). (2)H sequestration into organic molecules is low ( approximately 4%) and is not influenced by factors that affect VCO(2) such as food intake or physical activity. After oxidation of (2)H-labelled macromolecules, the label appears in body water, which can be sampled non-invasively in urine or saliva. After an overnight fast, two healthy adults consumed [(2)H]MTG (1,3-distearyl, 2-[(2)H(15)]octanoyl glycerol) and [(13)C]MTG (1,3 distearyl, 2-[1-(13)C]octanoyl glycerol) simultaneously. Total body water (TBW) was measured by (18)O dilution and also estimated from height and weight. Urine and saliva were sampled at baseline and for 10 h after consumption of the test meal. The abundance of (2)HOH and H(2) (18)O in urine and saliva was measured by continuous-flow isotope-ratio mass spectrometry. Cumulative PDR of (2)H and (18)O was calculated from the plateau enrichment, which was reached by 6 h in both saliva and urine. Recovery of (2)H calculated using measured TBW was compared with that using an estimated value of TBW. Mean recovery of (2)H in saliva was 99.3% and in urine was 96.4%. Errors introduced by estimating TBW were <5%. [(2)H]MTG could provide a simpler, more robust, indirect test of intraluminal fat digestion compared with the (13)C-breath test. Further studies are required in pancreatic insufficient patients.