A pilot study for the intrinsic labeling of egg proteins with 15N and 13C

The aim of this study was to produce intrinsically and uniformly doubly (15)N-(13)C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly (15)N-(13)C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The (15)N and (13)C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the (15)N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the (13)C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The (15)N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ(13)C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the (13)C labeling, adequate (13)C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway.     

Spectral analysis of 1H coupled 13C spectra of the amino acids: adaptive spectral library of amino acid 13C isotopomers and positional fractional 13C enrichments

The natural abundance 1H-coupled 13C NMR spectra of all proteogenic amino acids were measured in D2O at pH* 1. The accurate 1H,13C spin-spin coupling constants were analyzed using total-line-shape fitting. The obtained spectral parameters can be used to establish a spectral library of amino acid 13C isotopomers. The adaptive spectral library principle is introduced and discussed in this article. The simulated spectra can be applied to quantification of 13C isotopomer mixtures of amino acids and, thus, for exploring metabolic pathways. Also a protocol for amino acid 13C isotopomer metabolomic profiling in 13C labeled glucose feeding experiments is outlined. The approach is suggested to give invaluable information about positional fractional 13C enrichments, which are not easily available by any other method.     

Formation of 13C-, 15N-, and 18O-labeled guanidinohydantoin from guanosine oxidation with singlet oxygen. Implications for structure and mechanism

Guanosine labeled with 15N at N1, amino, and N7 and 13C at either C2 or C8 was oxidized by Rose Bengal photosensitization (singlet oxygen) in buffered aqueous solution. At pH > 7, spiroiminodihydantoin was the major product, while at pH < 7, guanidinohydantoin (Gh) was the principal product. 15N and 13C NMR studies confirmed that Gh was formed as a mixture of slowly equilibrating diastereomers. Experiments conducted in H218O indicated that Gh and Sp each contained one oxygen atom derived from O2 and one from H2O. Tandem mass spectrometry was used to identify the C4 carbonyl of Gh as the one labeled with 18O, supporting a mechanism involving attack of water at C5 of a dehydro-8-oxoguanosine intermediate.     

Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

Mass isotopomer analysis is an important technique to measure the production and flow of metabolites in living cells, tissues, and organisms. This technique depends on accurate quantifications of different mass isotopomers using mass spectrometry. Constructing calibration curves using standard samples is the most universal approach to convert raw mass spectrometry measurements into quantitative distributions of mass isotopomers. Calibration curve approach has been, however, of very limited use in comprehensive analyses of biological systems, mainly suffering from the lack of extensive range of standard samples with accurately known isotopic enrichment. Here, we present a biological method capable of synthesizing specifically labeled amino acids. These amino acids have well‐determined and estimable mass isotopomer distributions and thus can serve as standard samples. In this method, the bacterium strain Methylobacterium salsuginis sp. nov. was cultivated with partially ¹³C‐labeled methanol as the only carbon source to produce ¹³C‐enriched compounds. We show that the mass isotopomer distributions of the various biosynthesized amino acids are well determined and can be reasonably estimated based on proposed binomial approximation if the labeling state of the biomass reached an isotopic steady state. The interference of intramolecular inhomogeneity of ¹³C isotope abundances caused by biological isotope fractionation was eliminated by estimating average ¹³C isotope abundance. Further, the predictions are tested experimentally by mass spectrometry (MS) spectra of the labeled glycine, alanine, and aspartic acid. Most of the error in mass spectrometry measurements was less than 0.74 mol% in the test case, significantly reduced as compared with uncalibrated results, and this error is expected to be less than 0.4 mol% in real experiment as revealed by theoretical analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbon isotope analysis of bulk keratin and single amino acids from British and North American hair

The reconstruction of ancient diets using isotopic measurements of bone collagen, and other tissues, which survive in archaeological contexts, relies on known isotopic relationships between diet and body tissues. Examination of these relationships often requires the study of modern human and animal subjects. While hair keratin can act as a useful proxy for bone collagen in isotopic studies on living humans, where it is inappropriate to sample tissues such as collagen, it can, in addition, act as a chronological indicator of dietary change. This study investigates hair keratin delta13C values from current residents of the UK and the USA. Residents in the USA showed a clear bulk hair delta13C enrichment of approximately 3 per thousand over UK individuals, attributed to an elevated C4 dietary input from maize fed to livestock in North America. The keratin delta13C of subjects who moved between the UK and USA showed a pronounced change after relocation, taking approximately 4 months to reach isotopic equilibrium. To investigate these differences further, we measured delta13C values of dispensable and indispensable amino acids as a group, and selected individual amino acids. As a group, enrichment of dispensable amino acids compared with indispensable amino acids occurred in samples from both continents, averaging 7.2 per thousand in the UK and 7.9 per thousand in the USA. Dispensable and indispensable amino acids, as well as all individual amino acids measured, were enriched in samples from the USA compared with those from the UK.     

Advantages of compound‐specific stable isotope measurements over bulk measurements in studies on plant uptake of intact amino acids

Increasing interest in the ability of plants to take up amino acids has given rise to questions on the accuracy of the commonly used bulk method to measure and calculate amino acid uptake. This method uses bulk measurements of ¹³C and ¹⁵N enrichment in plant tissues after application of dual‐labelled amino acids but some authors have recommended the use of compound‐specific stable isotope (CSI) analysis of the plants' amino acids instead. However, there has never been a direct evaluation of both methods. We conducted a field study applying dual‐labelled (¹³C, ¹⁵N) amino acids (glycine, valine, tyrosine and lysine) to soil of a Plantago lanceolata monoculture. Root and shoot samples were collected 24 h after label application and the isotope composition of the plant tissues was investigated using bulk and CSI measurements. Enrichment of ¹³C in the case of CSI measurements was limited to the applied amino acids, showing that no additional ¹³C had been incorporated into the plants' amino acid pool via the uptake of tracer‐derived C‐fragments. Compared with this rather conservative indicator of amino acid uptake, the ¹³C enrichment of bulk measurements was 8, 5, 1.6 and 6 times higher for fine roots, storage roots, shoot and the whole plant, respectively. These findings show that the additional uptake of tracer‐derived C‐fragments will result in a considerable overestimation of amino acid uptake in the case of bulk measurements. We therefore highly recommend the use of CSI measurements for future amino acid uptake studies due to their higher accuracy. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of underivatized amino acid delta(13)C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non-essential amino acid profile on the isotopic signature of individual amino acids in fish

This study provides data for the effect of dietary non-essential amino acid composition on the delta(13)C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of delta(13)C values of individual amino acids from diets and tissues without derivatization. Diet affected the delta(13)C of individual amino acids in fish. For fish on Diets 1-3 amino acid delta(13)C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in (13)C. Improvements are necessary before all amino acid delta(13)C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists.     

Structural scaffold of 18-crown-6 tetracarboxylic acid for optical resolution of chiral amino acid: X-ray crystal analyses and energy calculations of complexes of D- and L-isomers of tyrosine, isoleucine, methionine and phenylglycine

To clarify the structural scaffold of (+)-18-crown-6 tetracarboxylic acid ((+)-18C6H4) for the optical resolution of a chiral amino acid, the crystal structures of its equimolar complexes with L- and D-isomers of tyrosine (Tyr), isoleucine (Ile), methionine (Met) and phenylglycine (PheG) were analysed by X-ray diffraction methods. (+)-18C6H4 took very similar conformations for all complexes. Although the chemical structure of (+)-18C6H4 is C2-symmetric, it took a similar asymmetric ring conformation of radius ca. 6.0 A. In all complexes, the amino group of chiral amino acids was located near the center of the ring and formed three hydrogen bonds and five electrostatic interactions with eight oxygen atoms of the ether ring and carboxyl groups. Also, the Calpha atom of chiral amino acids participated in Calpha-H...O interaction with the oxygen atom of (+)-18C6H4. In contrast, the carboxyl group of chiral amino acids did not directly interact with (+)-18C6H4. These results indicate that the structural scaffold of (+)-18C6H4 for the optical resolution of chiral amino acids is mainly based on the mode of interaction of (+)-18C6H4 with the amino and Calpha-H groups of chiral amino acids. The differences in interaction pattern and binding energy between the L- and D-isomers of each amino acid are discussed in relation to the chiral recognition of (+)-18C6H4.     

High-yield synthesis of 14C labeled alicyclic amino acids with high specific radioactivity using potassium (14C) cyanide

In order to study the tumor specificity of synthetic nonmetabolizing amino acids, 10 different 14C labeled alicyclic amino acids (3a-3j) were synthesized in high yield and with high specific radioactivity. Carbon-14 labeled alicyclic hydantoins (2a-2j) were synthesized from a small amount of radioactive potassium [14C] cyanide and corresponding ketones (1a-1j). Th 14C hydantoins (2a-2j) thus obtained were hydrolyzed without isolation to give 14C labeled alicyclic amino acids (3a-3j). The overall radiochemical yields of the amino acids (3a-3j) from potassium [14C] cyanide were 55.6-93.2% with radiochemical purity more than 99%. Specific activities of these 14C labeled compounds (3a-3j) were 209- 250 MBq/mmol (5.66-6.75 mCi/mmol). When non-radioactive potassium cyanide was not added as a carrier, 1-aminocyclopentane[14C]-carboxylic acid (3b) and 1-aminocyclooctane[14C]carboxylic acid (3e) were synthesized in the yield of 64.9 and 19.0% respectively with the specific activity exceed more than 1.85 GBq/mmol (50mCi/mmol).     

Identification of the D(3h) isomer of carbon trioxide (CO3) and its implications for atmospheric chemistry.

The CO3 molecule is considered an important reaction intermediate in the atmospheres of Earth and Mars for quenching electronically excited oxygen atoms and in contributing to the anomalous 18O isotope enrichment. The geometry of the CO3 intermediate plays an important role in explaining these effects; however, only the cyclic (C(2v)) isomer has been experimentally confirmed so far. Here, we report on the first spectroscopic detection of the acyclic (D(3h)) isomer of carbon trioxide (12C16O3) via its nu1 and nu2 vibrational modes centered around 1165 cm(-1) under matrix isolation conditions; the identification of the 12C18O3, 13C16O3, 13C18O3, 16O12C18O2, and 18O12C16O2 isotopomers of the acyclic isomer confirms the assignments.     

Top-down quantitation and characterization of SILAC-labeled proteins

Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation of their relative expression levels and of the degree of modification between different samples. Here we show through modeling and experiments that SILAC is suitable for intact protein quantitation and top-down characterization. When SILAC-labeling lysine and/or arginine, peaks of light and heavy SILAC-doublets do not interfere with peaks of different charge states at least between 10 and 200 kDa. Unlike chemical methods, SILAC ensures complete incorporation-all amino acids are labeled. The isotopic enrichment of commercially available SILAC amino acids of nominally 95% to 98% shifts the mass difference between light and heavy state but does not lead to appreciably broadened peaks. We expressed labeled and unlabeled Grb2, a 28 kDa signaling protein, and showed that the two forms can be quantified with an average standard deviation of 6%. We performed on-line top-down sequencing of both forms in a hybrid linear ion trap orbitrap instrument. The quantized mass offset between fragments provided information about the number of labeled residues in the fragments, thereby simplifying protein identification and characterization.     

Simplification of protein NOESY spectra using bioorganic precursor synthesis and NMR spectral editing

A novel method is proposed for the analysis of protein NOEs in solution. In this approach, chemically synthesized precursor compounds for the amino acids valine, leucine, and isoleucine are used for amino acid specific labeling of these hydrophobic residues. The methodology is based on a novel synthetic route to 12C,1H,2H Val, Leu, and Ile side chains selectively labeled with 13CH3 only at the terminal methyl group. In an otherwise 12C,1H labeled protein, discrimination between protons bound to 12C and 13C (or 15N) can be achieved using standard isotope-editing NMR pulse schemes. This strategy significantly relieves problems with spectral overlap through selective observation of interresidue methyl NOEs and will thus be a powerful extension of existing biomolecular NMR methodology.     

Two-dimensional infrared spectra of the 13C=18O isotopomers of alanine residues in an alpha-helix

The parameters needed to describe the two-dimensional infrared (2D IR) spectra of the isotopically labeled alpha-helix are presented. The 2D IR spectra in the amide-I' spectral region of a series of singly 13C=18O-labeled 25-residue alpha-helices were measured by three-pulse heterodyned spectral interferometry. The dependence of the spectra on the population time was measured. Individual isotopomer levels (residues 11-14) were clearly identified in 2D IR, downshifted by approximately 61 cm(-1) from the main helical band. By analyzing the line shapes of the 13C=18O diagonal peaks that appeared at approximately 1571.3 +/- 0.8 cm(-1) for all four labeled samples, we observed wider structural distributions for residues 14 and 11 than those for 12 and 13. A small fast component in the correlation function was used to estimate the dynamics of these distributions. In all cases, the v = 1 --> 2 transition showed a more Lorentzian-like line shape and also decayed faster than the v = 0 --> 1 transition, indicating that the population relaxation time of the v = 2 state was significantly faster than the v = 1 state. The amide transitions with naturally abundant 13C=16O appeared at approximately 1594 cm(-1), forming very weak and blurred cross-peaks with 13C=18O isotopomer modes. The effects of spectral interferences on the coherence time dependence of the detection frequency spectrum were also investigated. The methods of first moments and Wigner analysis were developed to circumvent the interference effects on the weak isotopomer transitions. The structural origin of the distributions for individual isotopomers was proposed to be an effect of nearby lysine residues on the intrahelical hydrogen-bond network.     

Anomalous oxygen isotope enrichment in CO2 produced from O+CO: estimates based on experimental results and model predictions

The oxygen isotope fractionation associated with O+CO-->CO(2) reaction was investigated experimentally where the oxygen atom was derived from ozone or oxygen photolysis. The isotopic composition of the product CO(2) was analyzed by mass spectrometry. A kinetic model was used to calculate the expected CO(2) composition based on available reaction rates and their modifications for isotopic variants of the participating molecules. A comparison of the two (experimental data and model predictions) shows that the product CO(2) is endowed with an anomalous enrichment of heavy oxygen isotopes. The enrichment is similar to that observed earlier in case of O(3) produced by O+O(2) reaction and varies from 70 0/00 to 136 0/00 for (18)O and 41 0/00 to 83 0/00 for (17)O. Cross plot of delta (17)O and delta (18)O of CO(2) shows a linear relation with slope of approximately 0.90 for different experimental configurations. The enrichment observed in CO(2) does not depend on the isotopic composition of the O atom or the sources from which it is produced. A plot of Delta(delta (17)O) versus Delta(delta (18)O) (two enrichments) shows linear correlation with the best fit line having a slope of approximately 0.8. As in case of ozone, this anomalous enrichment can be explained by invoking the concept of differential randomization/stabilization time scale for two types of intermediate transition complex which forms symmetric ((16)O(12)C(16)O) molecule in one case and asymmetric ((16)O(12)C(18)O and (16)O(12)C(17)O) molecules in the other. The delta (13)C value of CO(2) is also found to be different from that of the initial CO due to the mass dependent fractionation processes that occur in the O+CO-->CO(2) reaction. Negative values of Delta(delta (13)C) ( approximately 12.1 0/00) occur due to the preference of (12)C in CO(2)* formation and stabilization. By contrast, at lower pressures (approximately 100 torr) surface induced deactivation makes Delta(delta (13)C) zero or slightly positive.     

Synthesis and reactions of fluorous carbobenzyloxy (FCbz) derivatives of alpha-amino acids

Fluorous carbobenzyloxy ((F)Cbz) reagents RfCH(2)CH(2)C(6)H(4)CH(2)OC(O)OSu (where Su is succinimidoyl and Rf is C(6)F(13) and C(8)F(17)) have been used to make (F)Cbz derivatives of 18 of the 20 natural amino acids. The potential utility of this new family of reagents in both standard fluorous synthesis with spe separation and fluorous quasiracemic synthesis is illustrated with representative reactions of the (F)Cbz-Phe derivatives.     

Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C-valine isotopic ratios in complex biological samples

On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.     

An isotopic-independent highly accurate potential energy surface for CO2 isotopologues and an initial (12)C(16)O2 infrared line list

An isotopic-independent, highly accurate potential energy surface (PES) has been determined for CO(2) by refining a purely ab initio PES with selected, purely experimentally determined rovibrational energy levels. The purely ab initio PES is denoted Ames-0, while the refined PES is denoted Ames-1. Detailed tests are performed to demonstrate the spectroscopic accuracy of the Ames-1 PES. It is shown that Ames-1 yields σ(rms) (root-mean-squares error) = 0.0156 cm(-1) for 6873 J = 0-117 (12)C(16)O(2) experimental energy levels, even though less than 500 (12)C(16)O(2) energy levels were included in the refinement procedure. It is also demonstrated that, without any additional refinement, Ames-1 yields very good agreement for isotopologues. Specifically, for the (12)C(16)O(2) and (13)C(16)O(2) isotopologues, spectroscopic constants G(v) computed from Ames-1 are within ±0.01 and 0.02 cm(-1) of reliable experimentally derived values, while for the (16)O(12)C(18)O, (16)O(12)C(17)O, (16)O(13)C(18)O, (16)O(13)C(17)O, (12)C(18)O(2), (17)O(12)C(18)O, (12)C(17)O(2), (13)C(18)O(2), (13)C(17)O(2), (17)O(13)C(18)O, and (14)C(16)O(2) isotopologues, the differences are between ±0.10 and 0.15 cm(-1). To our knowledge, this is the first time a polyatomic PES has been refined using such high J values, and this has led to new challenges in the refinement procedure. An initial high quality, purely ab initio dipole moment surface (DMS) is constructed and used to generate a 296 K line list. For most bands, experimental IR intensities are well reproduced for (12)C(16)O(2) using Ames-1 and the DMS. For more than 80% of the bands, the experimental intensities are reproduced with σ(rms)(ΔI) < 20% or σ(rms)(ΔI∕δ(obs)) < 5. A few exceptions are analyzed and discussed. Directions for future improvements are discussed, though it is concluded that the current Ames-1 and the DMS should be useful in analyzing and assigning high-resolution laboratory or astronomical spectra.     

Affinity monolith preconcentrators for polymer microchip capillary electrophoresis

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25 000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.     

A field and laboratory method for monitoring the concentration and isotopic composition of soil CO2

The stable isotope composition of nmol size gas samples can be determined accurately and precisely using continuous flow isotope ratio mass spectrometry (IRMS). We have developed a technique that exploits this capability in order to measure delta13C and delta18O values and, simultaneously, the concentration of CO2 in sub-mL volume soil air samples. A sampling strategy designed for monitoring CO2 profiles at particular locations of interest is also described. This combined field and laboratory technique provides several advantages over those previously reported: (1) the small sample size required allows soil air to be sampled at a high spatial resolution, (2) the field setup minimizes sampling times and does not require powered equipment, (3) the analytical method avoids the introduction of air (including O2) into the mass spectrometer thereby extending filament life, and (4) pCO2, delta13C and delta18O are determined simultaneously. The reproducibility of measurements of CO2 in synthetic tank air using this technique is: +/-0.08 per thousand (delta13C), +/-0.10 per thousand (delta18O), and +/-0.7% (pCO2) at 5550 ppm. The reproducibility for CO2 in soil air is estimated as: +/-0.06 per thousand (delta13C), +/-0.06 per thousand (delta18O), and +/-1.6% (pCO2). Monitoring soil CO2 using this technique is applicable to studies concerning soil respiration and ecosystem gas exchange, the effect of elevated atmospheric CO2 (e.g. free air carbon dioxide enrichment) on soil processes, soil water budgets including partitioning evaporation from transpiration, pedogenesis and weathering, diffuse solid-earth degassing, and the calibration of speleothem and pedogenic carbonate delta13C values as paleoenvironmental proxies.