The Measurement of Muscle Protein Synthesis in Broilers with a Flooding Dose Technique: Use of 15N-Labelled Phenylalanine,GC-MS and GC-C-IRMS

Abstract

An experiment was carried out to measure fractional muscle protein synthesis rates (k _s ) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [¹⁵N]phenylalanine). K _s was calculated from the [¹⁵N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period.

The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [¹⁵N]APE and 0 to 0.62 [¹⁵N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [¹⁵N]APE and from 0.0004 to 0.0018 [¹⁵N]APE in GC-MS and GC-C-IRMS, respectively.

K _s was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K _s in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation.

It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [¹⁵N]phenylalanine, GC-MS and GC-C-IRMS.     

In vivo NMR spectroscopy: in situ 15N pulse labelling NMR spectroscopy with photoautotrophic microorganisms

For studying the nitrogen metabolism in plants ¹⁵N NMR spectroscopy can be used. For in vivo ¹⁵N NMR (natural abundance of ¹⁵N: 0.37%) enrichment of the sample with the isotope ¹⁵N is compulsory. The detection of time courses of ¹⁵N assimilation from cells, which are enriched in culture is restricted in scope. Here, a method, the ¹⁵N pulse labelling NMR spectroscopy, is demonstrated, which permits labelling of different nitrogen compounds in photoautotrophic microorganisms during the NMR spectroscopic measurement. Using an effective illumination system it is possible to maintain photosynthesis in plant samples of high biomass densities in the magnet necessary for ammonia assimilation. The technique thus enables to directly observe ammonia assimilation pathways by application of a ¹⁵NO₃ ^? or ¹⁵NH₄ ^? pulses.

Für das Studium des Stickstoffstoffwechsels der Pflanzen kann die ¹⁵N-NMR-Spektroskopie herangezogen werden. Hierzu ist bei der in-vivo-¹⁵N-NMR (natürliche Häufigkeit von ¹⁵N: 0.37%) eine Anreicherung der Probe mit dem Isotop ¹⁵N unerläßlich. Eine Verfolgung der ¹⁵N-Assimilationskinetik mit Zellen, die in der Kultur angereichert wurden, ist jedoch nur bedingt möglich. In dieser Arbeit wird die ¹⁵N-Pulsmarkierungs-NMR-Spektroskopie als eine Methode vorgestellt, die es erlaubt, eine Markierung von Stickstoffverbindungen in photoautotrophen einzelligen Mikroorganismen während der NMR-Messung im Magneten vorzunehmen. Es wird ein spezielles Beleuchtungssystem verwendet, das eine für die Stickstoffassimilation ausreichende Photosyntheseleistung der Zellen unter NMR-Bedingungen bei hoher Biomassedichte ermöglicht. Diese Technik erlaubt durch die Applikation eines ¹⁵NO₃ ^?-oder ¹⁵NH₄ ⁺-Pulses eine direkte Verfolgung von Ammonium-Assimilationswegen.     

Experimental plant for simultaneous production of 14N and 15N by 15N/14N exchange in NO,NO2–HNO3 system under pressure

An experimental study on ¹⁴N and ¹⁵N simultaneous separation using the chemical exchange in NO, NO₂–HNO₃ system under pressure is presented. The influence of the pressure and of the interstage 10 M HNO₃ flow rate on the separation of ¹⁴N and ¹⁵N was measured on a packed column with product and waste refluxers. At steady state and 1.8 atm (absolute), the isotopic concentration at the bottom of the separation column was 0.563 at% ¹⁵N, and in the top of the column was 0.159 at% ¹⁵N. The height equivalent to a theoretical plate and interstage 10 M HNO₃ flow rate values, obtained in these experimental conditions, allows the separation of ¹⁴N highly depleted of ¹⁵N and of ¹⁵N at 99 at% ¹⁵N concentration.     

13C and 15N Abundances in the Sediment Core (VER 92/1-St-10-GC2) from Northern Lake Baikal

Abstract

Carbon and nitrogen stable isotope compositions of organic matter, TOC/TN ratio, and manganese concentration in a sediment core that was collected in northern part of Lake Baikal (VER92ST10-GC2, water depth at 922 m, about 3 m long) were investigated to elucidate the origin of the sedimentary organic matter and its associated environmental factors.

The sediment core was composed of mainly two parts: turbidite sections and other sections. Constant δ¹³C and δ¹⁵N values of the turbidite sections were observed (- 26.8 ±0.2 ‰ for δ¹³C and 3.2 ± 0.1 ‰ for δ¹⁵N) throughout the core. The higher δ¹³C in turbidite sections (about - 27 ‰) than that of the other sections (- 31 to - 29 ‰) was clearly observed, and δ¹⁵N was different between turbidite sections (about 3‰) and other sections (3 to 5 ‰). δ¹³C of other sections was close to that of pelagic phytoplankton, indicating that sediment other than turbidite sections is composed of autochthonous components. The variation of stable isotopes in other sections may be possibly caused by the changes in either phytoplankton growth rate or contribution ratios of terrestrial to aquatic plants for δ¹³C. Either denitrification or fluctuation of δ¹⁵N in pelagic phytoplankton can be the cause of variable δ¹⁵N in other sections.     

A 15N NMR Study of Some Imidazolidine-2,4-Dichalcogen Derivatives

The ¹⁵N NMR chemical shifts and ¹(¹⁵N-¹H) coupling constants of a series of imidazolidine-2,4-dichalcogen (O, S) derivatives are reported.The ¹⁵N NMR chemical shifts show a linear correlatlon wlth the vNH stretchlng vlbratlons. The influence of the substitution of the oxygen at C₂ and/or C₄ with the sulphur, and of the hydrogen at C₅ wlth the methyls and phenyls has been considered. The ¹J(¹⁵N-¹H)'s found In thls serles of molecules agrees well with the expected values.     

The use of stable isotopes in ecosystem research. First results of a field study with 15N

Terrestrial ecosystems, e.g. forest ecosystems, are characterized by a complex and sensitive network of biotic and abiotic factors and their interactions. By using stable isotopes (e.g. labelled nitrogen compounds), very small addition rates of highly enriched compounds can be applied, which do not change or disturb the investigated system, but provide information about single processes, their interactions and especially about their dynamics.

First results of a field study in the Fichtelgebirge, Northeast-Bavaria, Germany, are presented. The distribution of labelled nitrogen (as ¹⁵N-NH₄ ⁺ and ¹⁵N[sbnd]NO₃ ^?) within a spruce ecosystem (Picea abies (L.) Karst. in competition with understory vegetation of Vaccinium myrtillus, Calluna vulgaris and Deschampsia flexuosa) showed maximum ¹⁵N concentrations in tissues of the understory vegetation. During the first six weeks after the ¹⁵N application, the nitrogen uptake of all investigated species was higher after the ¹⁵N[sbnd]NO₃ ^? treatment than after the ¹⁵N[sbnd]NH₄ ⁺ treatment.     

Magnitudes and Orientations of the N Chemical Shift Tensor of [1-N]-2′-Deoxyguanosine Determined on a Polycrystalline Sample by Two-Dimensional Solid-State NMR Spectroscopy

The magnitudes and orientations of the ¹⁵N chemical shift tensor of [1-¹⁵N]-2′-deoxyguanosine were determined from a polycrystalline sample using the two-dimensional PISEMA experiment. The magnitudes of the principal values of the ¹⁵N chemical shift tensor of the N1 nitrogen of [1-¹⁵N]-2′-deoxyguanosine were found to be ς₁₁ = 54 ppm, ς₂₂ = 148 ppm, and ς₃₃ = 201 ppm with respect to (¹⁵NH₄)₂SO₄ in aqueous solution. Comparisons of experimental and simulated two-dimensional powder pattern spectra show that ς_33N is approximately collinear with the N–H bond. The tensor orientation of ς_33N for N1 of [1-¹⁵N]-2′-deoxyguanosine is similar to the values obtained for the side chain residues of ¹⁵N_ε1-tryptophan and ¹⁵N_π-histidine even though the magnitudes differ significantly.     

Aufnahme von 15NO2 und Einbau des 15NO2-Stickstoffs in verschiedene Stickstofffraktionen bei Sonnenblumen (Uptake of 15NO2 and Metabolic Transfer of the 15NO2 Nitrogen to Various Nitrogen Fractions of Sunflowers)

Abstract

Sunflowers were exposed to ¹⁵NO₂ at a range between 4,9 and 42,0 ppb. The ¹⁵NO₂ uptake was quantified and the fate of the ¹⁵N in different parts and pools of nitrogen was investigated. The very high δ¹⁵N-values of the free amino acid pool can't give an answer to the question whether the NO₂-nitrogen is incorporated by a different pathway, compared to the nitrogen which derives from root uptake. Or does a compartmentation of the nitrate pool in the plant cell cause a dilution of the ¹⁵N enrichment in the mineral nitrogen pool during sample preparation?     

Separation of Anisotropy and Exchange Broadening Using N CSA–N–H Dipole–Dipole Relaxation Cross-Correlation Experiments

Based on the measurement of cross-correlation rates between ¹⁵N CSA and ¹⁵N–¹H dipole–dipole relaxation we propose a procedure for separating exchange contributions to transverse relaxation rates (R₂ = 1/T₂) from effects caused by anisotropic rotational diffusion of the protein molecule. This approach determines the influence of anisotropy and chemical exchange processes independently and therefore circumvents difficulties associated with the currently standard use of T₁/T₂ ratios to determine the rotational diffusion tensor. We find from computer simulations that, in the presence of even small amounts of internal flexibility, fitting T₁/T₂ ratios tends to underestimate the anisotropy of overall tumbling. An additional problem exists when the N–H bond vector directions are not distributed homogeneously over the surface of a unit sphere, such as in helix bundles or β-sheets. Such a case was found in segment 4 of the gelation factor (ABP 120), an F-actin cross-linking protein, in which the diffusion tensor cannot be calculated from T₁/T₂ ratios. The ¹⁵N CSA tensor of the residues for this β-sheet protein was found to vary even within secondary structure elements. The use of a common value for the whole protein molecule therefore might be an oversimplification. Using our approach it is immediately apparent that no exchange broadening exists for segment 4 although strongly reduced T₂ relaxation times for several residues could be mistaken as indications for exchange processes.     

A 15N NMR Study of Protonation and Deprotonation of 4,5-Dicyanoimidazole and Its 2-Amino Analogue

Protonation and deprotonation of the title compounds, was studied by means of ¹⁵N NMR. The shieldings of the ring nitrogen atoms are found to be very sensitive to changes in the amount of protonation. In contrast the ¹⁵N shieldings of the cyano and amino groups are found to be relatively insensitive to protonation effects and are unsuitable for estimating the degree of protonation occurring.     

Dynamic modelling and simulation of 15N separation by chemical exchange in NO,NO2–HNO3 system

The dynamic behaviour of a ¹⁵N separation process by chemical exchange in a NO, NO₂–HNO₃ system has been analysed based on an accurate mathematical model. A nonlinear system of first-order partial differential equations was determined by considering the multiple exchange reactions between the components of the gaseous mixture and the liquid phase constituents. The mathematical model of the process describes the space–time variation of the ¹⁵N mole fraction in gas and liquid phases and provides a better understanding of operating limits and decision support in process design and optimisation.     

3D NMR Experiments for MeasuringN Relaxation Data of Large Proteins: Application to the 44 kDa Ectodomain of SIV gp41

A suite of 3D NMR experiments for measuring¹⁵N–{¹H} NOE,¹⁵NT₁, and¹⁵NT_1ρvalues in large proteins, uniformly labeled with¹⁵N and¹³C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D¹H–¹⁵N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated¹³C/¹⁵N-labeled sample of the 44 kDa ectodomain of SIV gp41.     

Solution Structures of Pyrophthalones,III: Complementary Application of 14N/16N-NMR Spectroscopy to Study Solution Structures of Pyrophthalones

The solution structures of nine pyrophthalone-type substances are determined by ¹⁴N / ¹⁵N-NMR-spectroscopy. Mostly depending on the conditions (solvent, solubility, chemical nature of the compound), both isotopes can be used complementary to obtain reliable data. Additionally, for some compounds ¹⁵N solid state NMR data are available and demonstrate the structural identity in solution and the solid state.     

Solid-state NMR investigations of Si-29 and N-15 enriched silicon nitride

We compare ²⁹Si magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra from the two modifications of silicon nitride, α-Si₃N₄ and β-Si₃N₄, with that of a fully (²⁹Si, ¹⁵N)-enriched sample ²⁹Si₃¹⁵N₄, as well as ¹⁵N NMR spectra of Si₃¹⁵N₄ (having ²⁹Si at natural abundance) and ²⁹Si₃¹⁵N₄. We show that the ¹⁵N NMR peak-widths from the latter are dominated by J(²⁹Si–¹⁵N) through-bond interactions, leading to significantly broader NMR signals compared to those of Si₃¹⁵N₄. By fitting calculated ²⁹Si NMR spectra to experimental ones, we obtained an estimated coupling constant J(²⁹Si–¹⁵N) of 20 Hz. We provide ²⁹Si spin-lattice (T₁) relaxation data for the ²⁹Si₃¹⁵N₄ sample and chemical shift anisotropy results for the ²⁹Si site of β-Si₃N₄. Various factors potentially contributing to the ²⁹Si and ¹⁵N NMR peak-widths of the various silicon nitride specimens are discussed. We also provide powder X-ray diffraction (XRD) and mass spectrometry data of the samples.     

Distribution and Utilization of 15N in Cowpeas Injected into the Stem Under Influence of Water Deficit

Abstract

Investigations were carried out on Vigna unguiculata L. Walp. to estimate the distribution and utilization of ¹⁵N in different organs after stem injection during vegetative, flowering and pod filling stage. During flowering effects of water deficit were also examined. In well watered plants, within 4 days after injection, 65% of ¹⁵N accumulated in leaves. This was drastically reduced to 42% by water deficit. ¹⁵N accumulation in stems increased under water deficit. The translocation of ¹⁵N from the stem base to roots were not altered by water deficit. During pod filling 62% of recovered ¹⁵N in the plants had accumulated in seeds, 24% in leaves and 11% in stems within 4 days, whereas the uptake of nitrogen in pod walls and roots remained low (2%). These results demonstrate that the method of injecting very small quantities (1 mg/plant) of ¹⁵N into the stem base allows an exact and detailed quantitative assessment of N translocation/distribution with regard to different organs, different growth stages and different treatments.     

In Vivo Detection of N-Coupled Protons in Rat Brain by ISIS Localization and Multiple-Quantum Editing

Three-dimensional image-selected in vivo spectroscopy (ISIS) was combined with phase-cycled ¹H–¹⁵N heteronuclear multiple-quantum coherence (HMQC) transfer NMR for localized selective observation of protons J-coupled to ¹⁵N in phantoms and in vivo. The ISIS–HMQC sequence, supplemented by jump–return water suppression, permitted localized selective observation of 2–5 μmol of [¹⁵N_indole]tryptophan, a precursor of the neurotransmitter serotonin, through the ¹⁵N-coupled proton in 20–40 min of acquisition in vitro at 4.7 T. In vivo, the amide proton of [5-¹⁵N]glutamine was selectively observed in the brain of spontaneously breathing ¹⁵NH₄⁺-infused rats, using a volume probe with homogeneous ¹H and ¹⁵N fields. Signal recovery after three-dimensional localization was 72–82% in phantoms and 59 ± 4% in vivo. The result demonstrates that localized selective observation of ¹⁵N-coupled protons, with complete cancellation of all other protons except water, can be achieved in spontaneously breathing animals by the ISIS–HMQC sequence. This sequence performs both volume selection and heteronuclear editing through an addition/subtraction scheme and predicts the highest intrinsic sensitivity for detection of ¹⁵N-coupled protons in the selected volume. The advantages and limitations of this method for in vivo application are compared to those of other localized editing techniques currently in use for non-exchanging protons.     

The First in Vivo Observation of C–N Coupling in Mammalian Brain

[5-¹³C,¹⁵N]Glutamine, with ¹J(¹³C–¹⁵N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by ¹³C MRS at 4.7 T. The brain [5-¹³C]glutamine peak consisted of the doublet from [5-¹³C,¹⁵N]glutamine and the center [5-¹³C,¹⁴N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-¹³C,¹⁵N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-¹³C,¹⁵N]glutamine was formed by glial uptake of released neurotransmitter [5-¹³C]glutamate and its reaction with ¹⁵NH₃ catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively¹³C-enriched by intravenous [2,5-¹³C]glucose infusion to ¹³C-label whole-brain glutamate C5, followed by [¹²C]glucose infusion to chase ¹³C from the small and rapidly turning-over glial glutamate pool, leaving ¹³C mainly in the neurotransmitter [5-¹³C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-¹³C,¹⁵N]glutamine arises from a coupling between ¹³C of neuronal origin and ¹⁵N of glial origin. Measurement of the rate of brain [5-¹³C,¹⁵N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

天然丰度的N-磷酰化氨基酸的15N NMR 研究

采用¹⁵N-¹H的2D HSQC、HMBC实验方法,测定了天然丰度的N-磷酰化氨基酸样品在溶液中的¹⁵N化学位移δ_N及偶合常数J_N-P,J_N-H. 实验表明：对于¹⁵N天然丰度样品,这是一种快速有效的实验方法. 研究发现：N-酰化后的氨基酸,其δ_N以及与氮原子直接相连的质子¹H的化学位移均发生十分明显的高场位移,而偶合常数¹J_N-P,¹J_N-H的变化与化合物构型相关联 .     

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.     

Human and climate impact on 15N natural abundance of plants and soils in high-mountain ecosystems: a short review and two examples from the Eastern Pamirs and Mt. Kilimanjaro

Population pressure increasingly endangers high-mountain ecosystems such as the pastures in the Eastern Pamirs and the mountain forests on Mt. Kilimanjaro. At the same time, these ecosystems constitute the economic basis for millions of people living there. In our study, we, therefore, aimed at characterising the land-use effects on soil degradation and N-cycling by determining the natural abundance of ¹⁵N. A short review displays that δ¹⁵N of plant–soil systems may often serve as an integrated indicator of N-cycles with more positive δ¹⁵N values pointing towards N-losses. Results for the high-mountain pastures in the Eastern Pamirs show that intensively grazed pastures are significantly enriched in ¹⁵N compared to the less-exploited pastures by 3.5 ‰, on average. This can be attributed to soil organic matter degradation, volatile nitrogen losses, nitrogen leaching and a general opening of the N-cycle. Similarly, the intensively degraded savanna soils, the cultivated soils and the soils under disturbed forests on the foothill of Mt. Kilimanjaro reveal very positive δ¹⁵N values around 6.5 ‰. In contrast, the undisturbed forest soils in the montane zone are more depleted in ¹⁵N, indicating that here the N-cycle is relatively closed. However, significantly higher δ¹⁵N values characterise the upper montane forest zone at the transition to the subalpine zone. We suggest that this reflects N-losses by the recently monitored and climate change and antropogenically induced increasing fire frequency pushing the upper montane rainforest boundary rapidly downhill. Overall, we conclude that the analysis of the ¹⁵N natural abundance in high-mountain ecosystems is a purposeful tool for detecting land-use- or climate change-induced soil degradation and N-cycle opening.