Chromatography Combustion Isotope Ratio Mass Spectrometry Research Articles

Threonine dehydrogenase is a minor degradative pathway of threonine catabolism in adult humans.

The threonine dehydrogenase (TDG) pathway is a significant route of threonine degradation, yielding glycine in experimental animals, but has not been accurately quantitated in humans. Therefore, the effect of a large excess of dietary threonine, given either as free amino acid (+Thr) or as a constituent of protein (+P-Thr), on threonine catabolism to CO(2) and to glycine was studied in six healthy adult males using a 4-h constant infusion of L-[1-(13)C]threonine and [(15)N]glycine. Gas chromatography-combustion isotope ratio mass spectrometry was used to determine [(13)C]glycine produced from labeled threonine. Threonine intakes were higher on +Thr and +P-Thr diets compared with control (126, 126, and 50 micromol x kg(-1) x h(-1), SD 8, P < 0.0001). Threonine oxidation to CO(2) increased threefold in subjects on +Thr and +P-Thr vs. control (49, 45, and 15 micromol x kg(-1) x h(-1), SD 6, P < 0.0001). Threonine conversion to glycine tended to be higher on +Thr and +P-Thr vs. control (3.5, 3.4, and 1.6 micromol x kg(-1) x h(-1), SD 1.3, P = 0.06). The TDG pathway accounted for only 7-11% of total threonine catabolism and therefore is a minor pathway in the human adult.

Organic geochemical studies of soils from the Rothamsted classical experiments—V. The fate of lipids in different long-term experiments

Lipid extracts from four long-term experiments (Broadbalk Wilderness, Geescroft Wilderness, Hoosfield Spring Barley and Park Grass) were analysed using a combination of gas chromatography, gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry. The lipid content of the primary organic inputs for each soil were also analysed in order to assess the early diagenetic fate of the various compound classes present. Soil pH was observed to, either directly or indirectly, have a significant effect on lipids with a relative increase in abundance of n-alkanes at higher pH (7.31) and a large relative increase in n-alkanoic and ω-hydroxy acids at low pH (3.74). Triacylglycerols exhibited severe losses irrespective of pH. In an arable soil, n-alkanoic acids showed a temporal decrease in concentration whilst levels of n-alkanols remained static, the difference was ascribed to a more rapid turnover and possible leachate migration of the n-alkanoic acids. The phytosterol, sitosterol, was observed to rapidly diminish in soils possibly as a result of assimilation by soil dwelling invertebrates. Analysis of 5β-stigmastanol (a faecal biomarker) showed that it remained at levels indicative of manuring even after 113 years. Furthermore, analysis of 5β-stanyl esters revealed a manuring signal even more persistent than that exhibited by the free stanols. Knowledge of the biogeochemical cycling of lipids in the soil environment will help facilitate understanding of the processes which underpin carbon cycling in soils.

13C Gas Chromatography–Combustion Isotope Ratio Mass Spectrometry Analysis of N-Pivaloyl Amino Acid Esters of Tissue and Plasma Samples

We present the analysis of the stable carbon isotope compositions of 14 individual N-pivaloyl-isopropyl (NPP) amino acid esters by gas chromatography–combustion isotope ratio mass spectrometry (GC-C-IRMS). The mean reproducibility of derivatization procedure and GC-C-IRMS analysis was 0.45‰ (range, 0.12–0.68), whereas the mean analytical error was 0.26‰ δ13C (range, 0.13–0.42). Furthermore, the δ13C values of N-pivaloyl-isopropyl and N-acetyl-n-propyl (NAP) amino acid esters were compared. Due to a reproducible isotopic fractionation introduced by the derivatization process an empirical correction factor for each individual amino acid was derived separately for both derivatives (NPP, −1.13 to −2.52 (lysine, +2.09)‰ δ13C; NAP, −2.36 to −3.97 (lysine, +1.91)‰ δ13C), and the original δ13C value of the underivatized amino acid was calculated. Further, we performed an animal study where rats (n = 5) ingested a mixed meal containing uniformly 13C-labeled casein (indispensable amino acids 1.3 to 1.7 at.%). One hour after the meal δ13C values of protein-bound amino acids from small intestinal mucosa and liver and of free amino acids from mucosa and plasma were determined. Significant 13C enrichments of indispensable amino acids of the free pools of mucosa and plasma (range, 0.0518 to 0.1700 at.% excess) and in mucosa and liver proteins (range, 0.0021 and 0.0161 at.% excess) were observed. The feasibility of various derivatives for the measurement of carbon isotopic composition is discussed.

Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine

Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine

Analytical pyrolysis of humic substances and dissolved organic matter in aquatic systems: structure and origin

Samples of freeze-dried surface-lake brownwater (HO original), derived fulvic (FA) and humic acids (HA) and ultrafiltrate (K) which represent reference materials of a priority program of the German Research Council on “Refractory Organic Substances in Waters” were investigated using pyrolysis–field ionization mass spectrometry (Py–FIMS), Curie-point pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) and, for the first time, Curie-point pyrolysis–gas chromatography–combustion–isotope ratio mass spectrometry (Py–GC–C–IRMS). Py–FIMS of dissolved organic matter (DOM), FA, and HA gave highly reliable fingerprint mass spectra, thermograms describing the thermal properties and qualitative and quantitative data of 10 classes of characteristic molecular building blocks. Carbohydrates, phenols and lignin monomers, lignin dimers, lipids, alkylaromatics, aromatic nitrogen compounds, sterols, peptides, suberins and loosely bound fatty acids were determined. Complementary molecular structure information was obtained by Py–GC/MS which allowed the identification of a wide range of humic building blocks as main pyrolysis products. Our first results using the novel method of combined Py–GC/MS and Py–GC–C–IRMS indicate that the δ13C-values of the pyrolysis products are in agreement with generally accepted data for carbohydrates, lignin and benzenes from biological sources. This implies that the pyrolytical step does not have an isotopic effect. In contrast, some of the thermal products, e.g. 5-methyl-2-furancarboxaldehyde and benzenemethyl, had stronger depletions in 13C in comparison to the mean δ13C-values of furans and benzenes, respectively. This result contradicts the enrichment of 13C due to trophic effects and therefore suggests the incorporation of light carbon sources, such as respiratory CO2, methane, or anthropogenic pollution by fossil fuels, in the humification processes.

Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols

We propose a new screening method for testosterone (T) doping in sport. The current method for detecting T administration is based on finding a T to epitestosterone ratio (T/E) in urine that exceeds six. The difficulties with T/E are that T administration does not always result in a T/E>6 and that a rare individual will have T/E>6 in the absence of T administration. Our previous studies reveal that carbon isotope ratio helps to determine the origin of the urinary T because the values for T and its metabolites decrease after the administration of exogenous T. In this study, we present a rapid and efficient screening sample preparation method based on three successive liquid–solid extractions, deconjugation with E. coli β-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The 13C/ 12C of two T metabolites (5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol) and one pregnanediol as endogenous reference (5β-pregnane-3α,20α-diol) was measured by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) on 10 ml of urine collected from 10 healthy men before and after T administration. Following T administration, the 13C/ 12C of 5β-androstane-3α,17β-diol diacetate and 5α-androstane-3α,17β-diol diacetate declined significantly from −26.2‰ to −30.8‰ and from −25.2‰ to −29.9‰, respectively and the 13C/ 12C of 5β-pregnane-3α,20α-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.

New chromatographic, mass spectrometric and stable isotope approaches to the classification of degraded animal fats preserved in archaeological pottery

A new method is described for distinguishing between animal fats preserved in ancient pottery. Analysis of lipid fractions from two morphologically distinct vessel types (lamps and “dripping dishes”) using on-line gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) showed that they could be distinguished by plotting the δ 13C value for n-hexadecanoic acid against that for n-octadecanoic acid. The δ 13C values obtained for modern reference fats from domesticated animals likely to have been important in antiquity showed the lamp extracts to correlate with ruminant animal fat, such as sheep or cattle, whereas the “dripping dishes” had δ 13C values similar to those of non-ruminant animal fat, such as pig. These findings were entirely consistent with distributional information obtained by GC and with positional isomer information gained from analysis of dimethyldisulphide derivatives of the monounsaturated fatty acids. The results indicate that GC–C–IRMS has considerable potential for the classification of animal fats absorbed in ancient pottery particularly where fatty acid distributions have been altered by degradation during vessel use or burial. δ 13C values were also shown to be of value in detecting the use of vessels in the processing of animal products from more than one source.

Lipids as carriers of anthropogenic signals from prehistory

Studies performed during the last two decades have shown that lipids are preserved in association with a wide range of artefacts and ecofacts recovered from archaeological sites, e.g. pottery vessels and skeletal remains. The majority of work in this area has focused on the use of molecular structures (‘biomarkers’) and distributions (‘fingerprints’) to assess the nature and origin of commodities associated with past cultural, economic and agricultural practices. However, since lipids, like all other classes of biomolecule, are affected by degradation (both pre– and post–burial), emphasis is now being placed on the complementary use of diagenetically robust, compound–specific stable isotope measurements to enhance the scope and reliability of archaeological interpretations. A feature of theδ13C values of individual lipids, rather than bulk measurements of biochemically more heterogeneous materials, lies in their capacity to reflect differences in both the isotopic composition of the carbon sources used in their biosynthesis and the routing of dietary lipids and their metabolites in consumer organisms. This isotopic information, accessible by gas chromatography–combustion–isotope ratio mass spectrometry, has opened up new avenues of investigation concerning human activity in prehistory.

Free and bound fatty acid oxidation products in archaeological ceramic vessels

While oxidation products of unsaturated fatty acids, for example dicarboxylic acids (hereafter diacids), must form during the use of unglazed ceramic vessels for the processing of animal and plant products, such components have never been observed during studies of absorbed lipids. Their absence from the extractable lipid fraction is presumed to be the result of their loss from potsherds through groundwater leaching. Lipid oxidation products including short-chain dicarboxylic acids, ω-hydroxy acids and longer-chain hydroxy and dihydroxy acids have now been observed as components probably covalently bound into solvent insoluble residues of potsherds recovered from waterlogged deposits. These components were only revealed following alkaline treatment of the insoluble residues. A similar mixture of diacids was observed in high abundance in the free lipid fraction of vessels recovered from an exceptionally arid deposit where groundwater leaching would never have occurred. These results confirm the formation of oxidation and probable polymerization products of unsaturated fatty acids during vessel use and burial.

Simultaneous Measurement of Desaturase Activities Using Stable Isotope Tracers or a Nontracer Method

We report here methods for simultaneous determination ofin vitrodesaturase activities using stable isotope tracers or using a nontracer method in place of radiotracers. Rat microsomal Δ6- and Δ9-desaturase activities were assayed using standard incubationmedium by monitoring the increase in product dueto the reactions [U-13C]18:2n-6 → [U-13C]18:3n-6 or [U-13C]16:0 → [U-13C]16:1n-7, respectively. The stable isotope measurements were made by high-precision gas chromatography–combustion isotope ratio mass spectrometry. Reaction-dependent changes in product or substrate concentrations were also monitored quantitatively by conventional capillary gas chromatography with flame-ionization detection. Desaturase activities calculated from these data are consistent with tracer results. Microsomal Δ5-desaturase activity was monitored by quantifying the decrease in unlabeled substrate mass using the reaction 20:3n-6 → 20:4n-6. All activities showed the expected dependencies on time, temperature, pH, and microsome and substrate concentrations and were well within the range of published activities. Activities in brain, liver, kidney, and heart microsome preparations measured with either labeled or nonlabeled substrate were not significantly different, but were highly significantly different from organ to organ. These methods demonstrate a means to measure desaturase activities conveniently without radiotracers and for reactions involving substrates which are not available in labeled form.

Determination of δ13C values of valine in protein hydrolysate by gas chromatography–combustion isotope ratio mass spectrometry

A gas chromatographic–combustion isotope ratio mass spectrometric (GC–C-IRMS) method for the determination of [1- 13C]valine enrichments in protein hydrolysates is described. Using a quick derivatization method, δ 13C values of the N-methoxycarbonyl methyl ester of valine can be determined from baseline separated GC peaks. Evaluation studies with respect to precision, accuracy, linearity, reduction capacity of the CuO combustion furnace and isotope dilution as a result of derivatization, showed that our GC–C-IRMS system allows robust measurement of enrichments of [1- 13C]valine in the range 0 to 1.5 MPE (S.D.±0.01 MPE, n=3). Therefore this method is suited to determine fractional synthetic rates (FSRs) of proteins as low as one-tenth of the FSR of human albumin, in studies using a primed, continuous (6 h) infusion with [1- 13C]valine plasma enrichments of approximately 15 MPE and an hourly sampling schedule.

δ13C analyses of vegetable oil fatty acid components, determined by gas chromatography–combustion–isotope ratio mass spectrometry, after saponification or regiospecific hydrolysis

The δ 13C values of the major fatty acids of several different commercially important vegetable oils were measured by gas chromatography–combustion–isotope ratio mass spectrometry. The δ 13C values obtained were found to fall into two distinct groups, representing the C 3 and C 4 plants classes from which the oils were derived. The δ 13C values of the oils were measured by continuous flow elemental isotope ratio mass spectrometry and were found to be similar to their fatty acids, with slight differences between individual fatty acids. Investigations were then made into the influence on the δ 13C values of fatty acids of the position occupied on the glycerol backbone. Pancreatic lipase was employed to selectively hydrolyse fatty acids from the 1- and 3-positions with the progress of the reaction being followed by high-temperature gas chromatography in order to determine the optimum incubation time. The 2-monoacylglycerols were then isolated by thin-layer chromatography and fatty acid methyl esters prepared. The δ 13C values obtained indicate that fatty acids from any position on the glycerol backbone are isotopically identical. Thus, whilst quantification of fatty acid composition at the 2-position and measurement of δ 13C values of oils and their major fatty acids are useful criteria in edible oil purity assessment, measurement of δ 13C values of fatty acids from the 2-position does not assist with oil purity assignments.

Determination of low 13C-glutamine enrichments using gas chromatography–combustion–isotope ratio mass spectrometry

Glutamine is an essential fuel for tissues with high rates of cell replication, such as enterocytes and lymphocytes. Infusion of 13C-labeled glutamine tracers allows for measurement of the rates of production, utilization and oxidation of glutamine’s carbon skeleton in vivo. The use of this tracer, however, has been limited by its high cost and/or the difficulty in measuring low enrichments in biological fluids using conventional gas chromatography–mass spectrometry (GC/MS) techniques. We have developed a method using gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) that allows for the determination of low 13C enrichments (down to 0.06 mol.% excess) with a precision of 2% or better, and a within-day and between-day variability better than 5%, in plasma free glutamine. The method was applied to measuring the incorporation of 13C in plasma glutamine over the course of infusion of 13C-labeled acetate in a human subject. © 1997 John Wiley & Sons, Ltd.

Detection of exogenous hydrocortisone in horse urine by gas chromatography–combustion–carbon isotope ratio mass spectrometry

A gas chromatography–combustion–isotope ratio mass spectrometry method for confirmation of hydrocortisone abuse in horseracing and equine sports is proposed. Urinary hydrocortisone was converted to a bismethylenedioxy derivative which presents good gas chromatographic properties and brings an extra carbon contribution of only two carbon atoms. Synthetic hydrocortisone has a different 13 C abundance from that of natural urinary horse hydrocortisone and the difference is significant, therefore exogenous and endogenous hydrocortisone can be distinguished.

Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells

Docosahexaenoic acid (22:6) decreases blood platelet function and is highly concentrated in the brain where its depletion leads to functional impairments. Because the platelets and blood brain barrier capillary endothelium cannot hydrolyze the complex lipids for fatty acid (FA) uptake, nonesterified FA (NEFA) bound to albumin are assumed to be the delivery route of FA to these cells. The supply of 13C-labeled 22:6 to blood cells by plasma albumin was studied in humans after a single ingestion of this FA esterified in a triglyceride (TG). The 22:6 13C/12C ratio, measured by gas chromatography combustion-isotope ratio mass spectrometry was measured in lipid classes from albumin, platelets, leukocytes, and erythrocytes (taken as a tentative index of the brain uptake). Nonesterified [13C]22:6 bound to albumin was rapidly produced after ingestion, as a result of the hydrolysis of very low density lipoprotein (VLDL) plus chylomicron TG. We found that albumin carried another source of 22:6, lyso-phosphatidylcholines (lyso-PC), in which [13C]22:6 accumulated while the nonesterified [13C]22:6 reached its minimal plasma concentrations. Computation of the relative contribution of NEFA and lyso-PC for the [13C]22:6 delivery to platelets and erythrocytes showed that the [13C]22:6 supply to platelets occurred uniquely through NEFA, whereas this pool was weakly involved in the delivery to erythrocytes. In contrast, lyso-PC was uniquely concerned with the 22:6 delivery to erythrocytes and represented the major part of this supply. We conclude that plasma albumin carries 22:6 in two lipid forms that are involved differently in the delivery of this FA to target cells.

Measurement of15N/14N Isotopic Composition in Individual Plasma Free Amino Acids of Human Adults at Natural Abundance by Gas Chromatography–Combustion Isotope Ratio Mass Spectrometry

We describe a high-precision methodology to determine the15N/14N isotopic composition of 13 plasma free amino acids out of 500 μl plasma, measured asN-pivaloyl-i-propyl amino acid esters by gas chromatography–isotope ratio mass spectrometry. It is now possible to measure15N isotopic natural abundances in plasma free amino acids, which has not been the case previously because of the minimum requirement of 60 ml plasma to supply, e.g., 1 mg free leucine, for example, when performing a conventional preparative isolation. Data from seven healthy American and German subjects reveal a similar δ15N pattern in plasma free amino acids, although there are small differences in individual amino acids. Phenylalanine and threonine showed the lowest abundance (−12 and −5‰ δ15N, respectively), whereas alanine, leucine, proline, and ornithine ranged between +10 and +15‰ δ15N. Lysine ranged between +1 and +4‰ δ15N and showed the lowest variation of all measured amino acids. The metabolically related aromatic amino acids, phenylalanine and tyrosine, differ by 15‰ in their15N abundance. The isolation of amino acids by cation-exchange chromatography resulted in a small alteration of15N/14N isotopic composition in the amino acids glycine, lysine, and glutamic acid of maximal 1.2, 0.4, and, 0.4‰, respectively, but the remaining amino acids were unchanged. The method described offers new possibilities for the investigation of the amino acid metabolismin vivo.

Influence of gas chromatographic parameters on measurement of 13C/ 12C isotope ratios by gas-liquid chromatography-combustion isotope ratio mass spectrometry. I

A potential influence of gas chromatographic parameters on measured 13C/ 12C-isotope ratios of saturated fatty acid methyl esters in gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has been studied. A dependence of measured isotope ratios on temperature gradients was observed and differences in measured δ 13C-values for individual fatty acid methyl esters varied by 0.5 to 3‰. Possible reasons for this isotopic fractionation and its implications for 13C natural abundance work are discussed.

Direct analysis of carbon isotope variability in albumins by liquid flow-injection isotope ratio mass spectrometry

We demonstrate the high precision C isotopic analysis of a series of purified albumins by liquid chromatography-combustion isotope ratio mass spectrometry (IRMS) by using direct aqueous liquid injection. Albumins from 18 species and albumens from chicken and turkey egg were obtained from a commercial source and shown to be of > 98% purity by capillary zone electrophoresis and high-performance liquid chromatography. One microliter of an aqueous protein solution with a total of < 40-pmol protein (2. 5 µg), which contained about 150-nmol C, was injected directly into a flowing stream of high-performance liquid chromatography grade water. The solution passed through a pneumatic nebulizer, was sprayed onto a moving wire, passed through a drying oven, and was combusted in a furnace. After the water of combustion was removed, the resulting CO2 gas was directed to a high precision IRMS instrument operated in continuous flow mode. The average precision across the 20 samples analyzed was SD(δ (13)C)=0.45%., and the average accuracy was δ(13)C < 0.4%. compared to aliquots analyzed by conventional preparation by using combustion tubes and dual inlet analysis. The observed isotope ratio range was about -22.5%. < δ (13)CPDB < -16%. as expected for modern materials from a natural source. These results demonstrate rapid, high precision, and accurate C isotopic analysis of untreated macromolecules in an aqueous stream by liquid source IRMS.

Conversion of alpha-linolenate to docosahexaenoate is not depressed by high dietary levels of linoleate in young rats: tracer evidence using high precision mass spectrometry.

The conversion of alpha-linolenate (18:3n-3) to docosahexaenoate (22:6n-3) in the presence of low and high dietary levels of linoleate (18:2n-6) is reported in young rats using [U-13C]-alpha-linolenic acid (18:2n-3*) and high precision gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS). After consuming an 18:3n-3-deficient diet for 4 weeks, dams were bred and assigned to one of three diet groups: a) 2 g 18:3n-3/kg diet and 17 g 18:2n-6/kg diet (Lo-18:2), b) 2 g 18:3n-3/kg diet and 140 g 18:2n-6/kg diet (Hi-18:2), or c) essential fatty acid-deficient diet (EFAD). Pups were weaned to the maternal diets. At 42 days of age, pups were gavaged with 1 mg 18:3n-3*, and killed 48 h later. Fatty acid composition of liver reflected the diets to a greater extent than did the brain, and 22:5n-6 replaced 22:6n-3 in the brain. About 80% of the label in liver, brain, and plasma was found as 22:6n-3* for the replete groups. The enrichment pattern was similar in liver and plasma except for 18:3n-3, which was higher in liver. Total label detected was 4-fold higher in the EFAD livers and 2-fold higher in the EFAD brains than in the other groups, which were indistinguishable. Conversion of 18:3n-3* to 22:6n-3* was greater in livers from the Hi-18:2 group than from the Lo-18:2 group (P < 0.05). Estimates of overall label recovery in liver and brain were consistent with literature values. These data indicate that high dietary levels of 18:2n-6 do not inhibit conversion of a single dose of 18:3n-3 to 22:6n-3 in young rats, and demonstrate the applicability of high precision GCC-IRMS to fatty acid tracer studies.

High-precision gas chromatography-combustion isotope ratio mass spectrometry at low signal levels

Precision and accuracy of gas chromatography-combustion isotope ratio mass spectrometry are investigated for sample levels down to about 5 pmol C in fatty acid methyl ester mixtures spanning 1000-fold in concentration. Precision and accuracy of isotope ratios diverge rapidly for conventional summation methods, and become unusable below 30 pmol material on column. At lower levels, mean isotope ratios were statistically different from reference values indicating bias as well as poor precision. In contrast, curve fitting, using the exponentially modified Gaussian line shape, gives improved precision for most peaks and useful results down to 3 pmol. The curve-fitting algorithm was also less sensitive to signal integration time than the summation method. These data indicate that curve fitting may be the method of choice for integration of noisy data when high-precision isotope ratios are desired.