Determination of 13C Enrichment by Conventional GC-MS and GC-(MS)-C-IRMS

Abstract

Isotopic enrichment of branched-chain L-amino acids (BCAA) and branched-chain 2-oxo acids (BCOA) in standard preparations and in human plasma samples withdrawn after oral loads with 1-¹³C labelled BCAA were measured on a conventional GC-MS system and an on-line GC-C-IRMS employing O-TMS quinoxalinol derivatives. It was concluded that the recently introduced GC-C-IRMS, owing to its high sensitivity, is the adequate analytical tool when tracer doses of stable isotope labelled compounds of low enrichment are to be used in biomedical in vivo studies.     

Estimation of Urea Production Rate With [15N2]Urea and [13C]Urea to Measure Catabolic Rates in Diabetes Mellitus

Abstract

For verifying catabolic states in insulin-dependent patients and dogs the method estimating urea production rates with ¹³C and with doubly ¹⁵N labeled urea, respectively, has been established. For a fast steady state of urea tracer dilution, a prime of 600 times the continuous infusion rate had to be injected. Urea was isolated from plasma samples by protein precipitation and cation exchange chromatography with a consecutive derivatization of the dried urea fraction (trimethylsilyl derivatives). The masses of the fragment ions m/z 189 (¹⁴N¹⁴N), 190 (¹⁴N¹⁵N) and 191 (¹⁵N¹⁵N) urea are monitored to estimate the [¹⁵N₂]urea frequency in the overall body urea pool in mol percent excess (MPE). 1 to 15 ng of derivatized urea were measured efficiently. An excellent correlation between expected standard and measured MPE (r = 0.9977) was achieved from solutions containing 1 to 7% [¹⁵N₂]urea. The interassay coefficient of variation amounted to < 10% for a [¹⁵N₂]urea portion of ≥ 3%.

Normoglycemic diabetic patients who were treated with insulin overnight showed significantly higher urea production compared to healthy controls (9.22 ± 2.07 vs. 5.4 ± 0.32 μmol·kg^?1 · min^?1; p < 0.05). Measurements in chronic diabetic dogs proved an increased rate of amino acid catabolism (+ 20% urea production) in systemic versus portal application of insulin in paired studies. This increased nitrogen load in diabetics may accelerate progression of diabetic nephropathy. - Thus, the established stable isotope technique may serve as a sensitive and useful indicator of amino acid catabolism in clinical and experimental research.     

Can an IR Diode Laser Spectrometer be Used for the Determination of Natural Isotope Ratios in Biological Samples?

Abstract

An IR diode laser spectrometer can detect the enrichment of stable isotopes (¹³C [1] and ¹⁵N[2]) in tracer studies. However our system cannot detect differences of the natural abundances of these isotopes. This problem is not a principal limitation of the IR diode laser spectroscopy, but is inherent to our standard system. A new isotope ratio IR diode laser spectrometer has an accuracy high enough for most biological applications. Some advantages like flexibility in measuring different isotopes, insensitivity against other gases in the sample and price are correlated to this system.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments III

Chemical shielding anisotropy tensors have been determined, within the GIAO-RHF formalism using a smaller [6-31+G(d)] and two medium-size basis sets [6-311++G(d,p) and TZ2P], for all elements of the conformational library (altogether 27 structures) of the hydrophobic model peptide For-L-Phe-NH₂. The individual chemical shifts and their conformational averages have been compared to their experimental counterparts taken from the BioMagnetic Resonance Bank (BMRB). At the highest level of theory applied, for all nuclei but the amide proton, deviations between statistically averaged theoretical and experimental chemical shifts are as low as a few percent. One-dimensional (1D) chemical shift - structure plots do not allow unambiguous identification of backbone conformations. On the other hand, on chemical shift - chemical shift plots of selected nuclei, e.g., ¹H ^N with ¹⁵N or ¹⁵N with ¹³C ^α , regions corresponding to major conformational motifs have been found, providing basis for the identification of peptide conformers solely from NMR shift data. The 2D ¹H ^α -¹³C ^α as well as the 3D ¹H ^α -¹³C ^α -¹³C ^β chemical shift - chemical shift plots appear to be of special importance for direct determination of conformations of protein building units from multidimensional NMR experiments. 48 pairs of ¹H ^α -¹³C ^α data for phenylalanine residues have been extracted from 18 selected proteins and compared to relevant ab initio results, supporting the calculated results. Thus, the appealing idea of establishing backbone folding information of peptides and proteins from chemical shift information alone, obtained from selected multiple-pulse NMR experiments (e.g., 2D-HSQC, 2D-HMQC, and 3D-HNCA), has received further support.     

One and two-photon detachment cross-sections of Ps-

We present detailed calculations for one- and two-photon above-threshold detachment (ATD) cross-sections of the negative positronium ion Ps ^- ( e ⁺ e ^- e ^- ), below the threshold of Ps(n = 2), using a configuration interaction (CI) method on a B splines basis. Both the one- and two-photon detachment cross-sections have a form similar to the corresponding spectra of the H^- ion, scaled accordingly. The peak value of the one-photon cross-section agrees very well with the calculations by Bathia and Drachman [1], while it differs from those by Igarashi et al. [2], which give a value of 15% lower. Two-photon detachment cross-sections are also reported. Received 24 January 2002 / Received in final form 9 April 2002 Published online 19 July 2002     

Intermolecular Vibrational Coupling A Model Calculation for the Out-of-Plane Vibration of CO3 −- in Compounds with the Aragonite Structure

Abstract

¹³CO₃ ^?- substituted compounds with the aragonite structure show a fine structure of the out-of-plane vibration in the infrared spectra. A simple quantum mechanical model of coupled oscillators is developed to describe quantitatively the observed spectra. The spectra can be interpreted in terms of isolated ^12CO ₃ ^?- and ¹³CO₃ ^?- clusters, whose eigenfunctions interact. The heavy ions have localized vibrations, whereas clusters of the light ion with more than one ion show only localized vibrations at small coupling; stronger coupling results in resonant modes. Intensitytransfer from light to heavy ion bands is explained and the consequences of interaction between different clusters are discussed.     

Compartmental Approach for Evaluation of Plasma Kinetics and 13Co2-Exhalation after Oral Loading with L-[1-13C]Leucine

Abstract

A seven compartment model was applied for evaluation of oral L-[1-¹³C]leucine loading tests (38 μmol/kg body wt.) in healthy volunteers. The model comprises transport and absorption in stomach and gut into a central L-leucine-compartment which is connected to a protein compartment and to the compartment of the corresponding 2-oxo acid. CO₂ release from the latter occurs in a fast and a slow compartment into the central CO₂ compartment for exhalation. Using the fmins routine of MATLAB for parameter estimation, a good agreement was obtained between calculated and actually measured kinetics of ¹³C-labelled metabolites and a mean in vivo L-leucine oxidation of 0.365 ± 0.071 μmol/kg per min (n = 5) was computed. Plausibility of the model was checked by predicting in vivo leucine oxidation rates from primed continuous infusion tests (priming: L-[1-¹³C]leucine, 5 μmol/kg; NaH¹³CO₂, 1.2 μmol/kg; infusion: L-[1-¹³C]leucine, 5 μmol/kg per h). In 5 tested volunteers, the experimental L-leucine oxidation rate amounted to 0.358 ± 0.105 μmol/kg per min versus predicted 0.324±0.099 μmol/kg per min. Possible causes for some observed intraindividual variations are discussed.     

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.     

The Position Dependent 15N Enrichment of Nitrous Oxide in the Stratosphere

Abstract

The position dependent ¹⁵N fractionation of nitrous oxide (N₂O), which cannot be obtained from mass spectrometric analysis on molecular N₂O itself, can be determined with high precision using isotope ratio mass spectrometry on the NO⁺ fragment that is formed on electron impact in the source of an isotope ratio mass spectrometer. Laboratory UV photolysis experiments show that strong position dependent ¹⁵N fractionations occur in the photolysis of N₂O in the stratosphere, its major atmospheric sink. Measurements on the isotopic composition of stratospheric N₂O indeed confirm the presence of strong isotope enrichments, in particular the difference in the fractionation constants for ¹⁵N¹⁴NO and ¹⁴N¹⁵NO. The absolute magnitudes of the fractionation constants found in the stratosphere are much smaller, however, than those found in the lab experiments, demonstrating the importance of dynamical and also additional chemical processes like the reaction of N₂O with O(¹D).     

Elemental Analyzer-Quadrupole MS Coupling—A Low Cost Equipment for the Simultaneous Determination of Carbon/13C and Nitrogen/15N in Isotopically Enriched Soil and Plant Samples

Abstract

Since the end of the 80s elemental analyzer-isotope ratio mass spectrometer connections have been used for the fast, automatic and highly precise determination of carbon and nitrogen content as well as their isotopic composition in one run. But for artificially enriched stable isotopes as tracer in biological processes and since these processes have a high biological variability anyway (e.g. soil processes) the use of these highly precise but also sophisticated and expensive instruments is not required. In this case the use of a quadrupole mass spectrometer connected with an elemental analyzer can offer a low cost alternative. As shown, such coupling is suitable for automatic simultaneous routine analysis of total nitrogen and carbon and their isotopic enrichment (¹⁵N, ¹³C) in plant material and soils. The relative standard deviation for ¹⁵N and ¹³C determination is 2% To meet this precision a careful sample homogenization by grinding is very important. The duration of one measurement is 6–8 min. depending on whether nitrogen alone or both nitrogen and carbon are determined. This enables a high sample throughput.     

In vivo measurement of normal rat intracellular pyruvate and lactate levels after injection of hyperpolarized [1-(13)C]alanine

Hyperpolarized technology utilizing dynamic nuclear polarization has enabled rapid and high-sensitivity measurements of ¹³C metabolism in vivo. The most commonly used in vivo agent for hyperpolarized ¹³C metabolic imaging thus far has been [1-¹³C]pyruvate. In preclinical studies, not only is its uptake detected, but also its intracellular enzymatic conversion to metabolic products including [1-¹³C]lactate and [1-¹³C]alanine. However, the ratio of ¹³C-lactate/¹³C-pyruvate measured in this data does not accurately reflect cellular values since much of the [1-¹³C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and monocarboxylate transporter activity. In order to measure the relative levels of intracellular pyruvate and lactate, in this project we hyperpolarized [1-¹³C]alanine and monitored the in vivo conversion to [1-¹³C]pyruvate and then the subsequent conversion to [1-¹³C]lactate. The intracellular lactate-to-pyruvate ratio of normal rat tissue measured with hyperpolarized [1-¹³C]alanine was 4.89±0.61 (mean±S.E.) as opposed to a ratio of 0.41±0.03 when hyperpolarized [1-¹³C]pyruvate was injected.     

On the fitting of displaced maxwellians to laser-produced plasma ion velocity distributions

The empirical fitting of displaced maxwellians to ion velocity distributions measured at some distance from the target in laser-produced plasma studies is shown to lack a theoretical basis. A similar ion velocity spectrum to a displaced maxwellian is obtained if a simple self-similar plasma expansion is assumed. The discussion is illustrated by experimental ion velocity distributions measured at 0.8 m from a slab carbon target irradiated by a neodymium laser pulse of duration 4 ns focussed to an intensity of ≈10¹² W cm^-2.     

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).     

A detailed study on the decomposition pathways of the amino acid valine upon dissociative electron attachment

In the present study we investigate free electron attachment to the amino acid valine. Mass spectra and anion efficiency curves are measured in the electron energy range from about zero eV to about 15 eV and the anionic fragments are analyzed with a double focusing mass spectrometer. The high sensitivity of the present setup allows the detection of 10 fragment anions that have not been reported before and the high mass resolution of our sector field mass spectrometer allows us the separation and identification of isobaric anions. Thus the isobaric ion pairs, CN^-/C₂H₂^-_2^-, and O^-/NH₂^-_2^-, can be identified and assigned to individual resonances. For some of the heavier fragment anions formed we have studied collision induced dissociation to collect more information on the structures of these anions.     

Measuring the link between cardiac mechanical function and metabolism during hyperpolarized 13C-pyruvate magnetic resonance experiments

PurposeThe goal of this study was to develop a methodology to investigate the relationship between contractile function and hyperpolarized (HP) [1-¹³C]pyruvate metabolism in a small animal model. To achieve sufficient signal from HP ¹³C compounds, HP ¹³C MRS/MRSI has required relatively large infusion volumes relative to the total blood volume in small animal models, which may affect cardiac function.MethodsEight female Sprague Dawley rats were imaged on a 4.7T scanner with a dual tuned ¹H/¹³C volume coil. ECG and respiratory gated k-t spiral MRSI and an IDEAL based reconstruction to determine [1-¹³C]pyruvate metabolism in the myocardium. This was coupled with ¹H cine MRI to determine ventricular volumes and mechanical function pre- and post-infusion of [1-¹³C]pyruvate. For comparison to the [1-¹³C]pyruvate experiments, three female Sprague Dawley rats were imaged with ¹H cine MRI to determine myocardial function pre- and post-saline infusion.ResultsWe demonstrated significant changes in cardiac contractile function between pre- and post-infusion of [1-¹³C]pyruvate. Specifically, there was an increase in end-diastolic volume (EDV), stroke volume (SV), and ejection fraction (EF). Additionally, the ventricular vascular coupling ratio (VVCR) showed an improvement after [1-¹³C]pyruvate infusion, indicating increased systolic performance due to an increased arterial load. There was a moderate to strong relationship between the downstream metabolic conversion of pyruvate to bicarbonate and a strong relationship between the conversion of pyruvate to lactate and the cardiac mechanical function response.ConclusionThe infusion of [1-¹³C]pyruvate resulted in demonstrable increases in contractile function which was related to pyruvate conversion to bicarbonate and lactate. The combined effects of the infusion volume and inotropic effects of pyruvate metabolism likely explains the augmentation in myocardial mechanical function seen in these experiments. Given the relationship between pyruvate metabolism and contractile function observed in this study, this methodological approach may be utilized to better understand cardiac metabolic and functional remodeling in heart disease.     

Zum Stand der Stoffwechselforschung mit 15N- und 13C-markierten Tracersubstanzen in der Kinderheilkunde (Protein Turnover Research Using 15N and 13C Labelled Substrates in Pediatrics)

Abstract

Measurements in protein turnover and in metabolism of amino acids and their degradation products by means of stable isotope labelled substrates have been increasingly applied in clinical research over the last years. In spite of numerous studies dealing with this topic, quite a few important insufficiently clarified methodical aspects remain. This refers, for instance, to the choice of suitable tracer substances, the difficulties in the determination of the excretion plateau and the validation of the oxidation rates as measured with individual-labelled amino acids with regard to the whole body protein synthesis. Such problems may become of decisive importance in special subjects, such as preterm infants and critically-ill patients.

Investigations into these issues conducted by our group have revealed that the protein turnover in the very small preterm infant is by no means as intensive as previously claimed. The utilisation of urea nitrogen for the whole body protein synthesis of the infant may assume substantial proportions under the conditions of marginal protein intake and of catchup-growth. Studies conducted by means of ¹⁵N-labelled bifidobacteria have pointed at the intensive substrate exchange existing between microflora and host.

Pediatric research has to be non-invasive. Consequently, methods based on arterio-venous differences in tracer concentrations and on muscle biopsies do not have very high priority in pediatric research. A search for references published in the last five years has shown, that ¹⁵N-glycine is still the most frequently used tracer substance. There is a tendency towards a further increase of cell culture experiments run with stable isotope labelled amino acids.

Clinical research groups increasingly turn their attention to stable isotopes and mass spectrometry. This impressively demonstrates the continuing importance of tracerkinetic methods in all branches of medicine.     

Determination of the Structure and Configuration of 16α-Fluoro-5-androsten-17-one by NMR Spectroscopy and X-ray Diffraction

The stereochemistry of 16α-fluoro-5-androsten-17-one (1) was determined by nmr spectroscopy and X-ray diffraction. The γ-effects of the fluorine atom in ¹³C nmr were used as a first stereochemical probe for the α-fluorine configuration. The magnitude of ³J (HH) measured on the H-16 multiplet of the 300 MHz ¹H nmr spectrum of compound 1, was used with Karplus-type plots for the CH₂CHF fragment of the two possible epimers. The plots show that only the α-fluorine configuration fits with the observed ³J (HH), and X-ray diffraction confirms such result. The dihedral angles estimated from the Karplus-type plot were consistent with the angles found by X-ray. Crystallographic data of compound 1 show the following ring conformations: A is a chair, B is an 8β, 9α-half-chair, C is a chair and D is a C(13)β-envelope.     

Opacity broadening as a density diagnostic for spot spectroscopy

A significant advantage of the recently developed spot spectroscopy techniques for plasma diagnostics is that the blowoff resulting from the tracer dots originally imbedded in the target has a well-characterized size. The helium-like resonance line 1s²?1s 2p¹P may have appreciable optical depth in these blowoff regions, especially near the original target surface. The observed width of this line is largely determined by opacity broadening, which depends directly on the density. Calculations are presented for aluminum, which allow density determination as a function of measured line width. Scaling of the calculations to tracer elements other than aluminum is also discussed.     

Incorporation of 15NO2 Nitrogen into Individual Amino Acids by Sunflowers Using Gc-C-Irms

Abstract

The nitrogen isotopic composition of individual amino acids in sunflower leaves after exposures to ¹⁵NO₂ in the range of ambient NO₂ concentrations (5–37 ppb) was analysed by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). Amino acids as well as the amides glutamine and asparagine were converted with MTBSTFA (N-methyl-N-(tert.-butyldimethylsilyl)-tri-fluoroacetamid) in pyridine to their corresponding TBDMS derivatives (N, O-tert.-butyldimethylsilyl) in a simple one-step silylation reaction. The derivatized amino acids were separated by gas chromatography, combusted on-line, and the products were sent continuously to an isotope ratio mass spectrometer. Accurate measurements were obtained, when more than 7 nmol N₂ were introduced into the ion source of the mass spectrometer per gas chromatographically separated and combusted compound. No interferences of the silicate and fluor containing derivatization agents on the performance of the system were observed.

In the range of ambient NO₂ concentrations sunflower leaves predominately incorporate the nitrogen derived from atmospheric NO₂ into soluble amino acids. The highest δ¹⁵N values were measured for alanine. The ¹⁵N enrichments of the detectable amino acids increased with increasing ¹⁵NO₂ concentration.     

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive ¹³C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective ¹³C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-¹³C]glucose preferentially labels the ends of the side chains, while [2-¹³C]glycerol labels the C^α of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively ¹³C labeled protein were performed using ¹⁵N–¹³C 2D correlation spectroscopy. From the time dependence of the ¹⁵N–¹³C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective ¹³C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.