The Fate of [15N]Ammonium and [15N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract

A ¹⁵N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [¹⁵N]ammonium and [¹⁵N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha^?1 with 11 water m^?2. To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994.

One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994.

In July 1994, three months after tracer application, 40% of the [¹⁵N]ammonium label and 29% of the [¹⁵N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Uptake of [(15)N] Ammonium and [(15)N]Nitrate in a 140-Year-Old Spruce Stand (Picea abies) in the Fichtelgebirge (NE Bavaria)

Abstract In April 1994 a (15)N tracer pulse study was started in a 140-year-old spruce stand (Picea abies [L.] Karst.) located at the Fichtelgebirge (NE Bavaria). Highly enriched (98%) [(15)N]ammonium and [(15)N]nitrate were applied simulating wet deposition. For two growing seasons the pathways and dynamics of the tracer were followed in all compartments of spruce (needles and twigs of all age classes, stem wood and bark, roots) and understorey vegetation and in soils of the organic (L/Of and Oh) and mineral horizons (A(0-5) and A(5-10)). By variations of the application time on different plots within the growing season (spring, summer and autumn) a seasonal effect of labelling on uptake and distribution patterns was tested. First results of this tracer study indicate that young and old spruce stands do not differ basically in pattern of uptake and distribution of mineral nitrogen. There are indications that spruce uses preferentially ammonium versus nitrate and that the ratio of ammonium/nitrate which is being consumed depends on the ammonium/nitrate ratio in the soil solution. The uptake rates decrease within the growing season.     

The Influence of Ammonium on Nitrate Uptake and Assimilation in 2-Year-Old Ash and Oak Trees - A Tracer-Study with 15N

Abstract

Interactions between ammonium and nitrate as competitive N sources depend on various biotic and abiotic factors. The preference for one of these N sources and the influence of ammonium on nitrate uptake and nitrate reductase activity was investigated in a ¹⁵N labelling experiment using 2-year-old potted plants of ash (Fraxinus excelsior L.) and oak (Quercus robur L.) under greenhouse conditions.

Seedlings of both tree species use ammonium and nitrate in equal amounts when both N forms are supplied in a 1:1 ratio (1.5 mM NH₄ ⁺ + 1.5 mM NO₃ ^?), although there is a slight tendency that ammonium is preferred. In both species total N uptake is higher if ammonium and nitrate are supplied simultaneously when compared with uptake of nitrate alone (3 mM nitrate). If nitrate is the sole N source N uptake is only half as high as if ammonium and nitrate are supplied in a ratio of 1:1.

The distribution of nitrate reductase between shoot and roots is not influenced by the N-form: nitrate reductase activity is always highest in the roots of both species under the conditions of this experiment.

Xylem sap analyses showed that both species transport higher concentrations of amino acids than of nitrate from the roots to the shoot. The amino acid composition is independent of the type of N source. Furthermore, ash trees contain more nitrate in the xylem sap than oak trees, reflecting the higher N uptake and the higher nitrate reductase activity in the leaves of this species.     

The Relationship of Nitrate Reductase Activity to Uptake and Assimilation of Atmospheric 15NO2-Nitrogen in Needles of Norway Spruce (Picea abies [L.] Karst.)

Abstract

Using ¹⁵NO₂, relations between nitrate reductase activity and stomatal conductance, ¹⁵N-uptake and ¹⁵N-glutamate were studied in the two youngest needles flushes of potted Norway spruce (Picea abies [L.] Karst.). There were linear correlations between the stomatal conductance and the ¹⁵N-uptake and between the ¹⁵N-uptake and nitrate reductase (E.C. 1.6.6.1/1.6.6.2) activity. The ¹⁵N labelling of free glutamate shows the assimilation of NO₂ from the atmosphere in addition to the nitrogen from the soil. The portion of glutamate originating from ¹⁵NO₂ was linearly related to nitrate reductase activity in spring experiments. This indicates that this enzyme activity reflected the rate of NO₂-assimilation.     

Improved measurement of (15)N-[(1)H] NOEs in the presence of H(N)-water proton chemical exchange.

A simple method is presented to accurately determine (15)N-[(1)H] NOEs in biomolecules in the presence of H(N)-water proton chemical exchange. Three measurements are required: one with nonselective proton saturation and two with different water saturation conditions to determine the equilibrium value of the (15)N signal. This approach is exemplified with data on two peptides, one helix-forming 17-mer and one compactly folded 56-mer. Results indicate that (15)N-[(1)H] NOEs determined using the standard approach with short recycle times (3 to 4 s) can be significantly in error when H(N)-water proton chemical exchange is relatively rapid, water proton relaxation is relatively slow, and (15)N-[(1)H] NOEs are away from the value of -1. This new method avoids such inaccuracies resulting from the use of short recycle times.     

15N Distribution in Wheat and Chemical Fractionation of Root-Borne 15N in the Soil

Abstract

Spring wheat plants were grown in split-root containers and labelled with ¹⁵N by fertilizing one compartment of the container with ¹⁵NH₄ ¹⁵NO₃ (95 at.-% ¹⁵N exc.). After the harvest, approx. 90% of the ¹⁵N incorporated by the plants were found in the shoots and 3% in the roots; approx. 7% had been released into the soil of the unlabelled compartment. The ¹⁵N in the soil of the unlabelled compartment was extracted with KCl and hydrolysed with HCl. Approx. 60% of the ¹⁵N was found in the hydrolysable organic N pool of the soil and 9% in the fraction of the soluble and exchangeable inorganic nitrogen.     

Determination of internucleotide (h)J(HN) couplings by the modified 2D J(NN)-correlated [(15)N, (1)H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.     

The role of (15)N CSA and CSA/dipole cross-correlation in (15)N relaxation in solid proteins

The influence of the (15)N CSA on (15)N longitudinal relaxation is investigated for an amide group in solid proteins in powder form under MAS. This contribution is determined to be typically 20-33% of the overall longitudinal relaxation rate, at 11.74 and 16.45 T, respectively. The improved treatment is used to analyze the internal dynamics in the protein Crh, in the frame of a motional model of diffusion in a cone, using the explicit average sum approach. Significant variations with respect to the determined dynamics parameters are observed when properly accounting for the contribution of (15)N CSA fluctuations. In general, the fit of experimental data including CSA led to the determination of diffusion times (tau(w)) which are longer than when considering only an (15)N-(1)H dipolar relaxation mechanism. CSA-Dipole cross-correlation is shown to play little or no role in protonated solids, in direct contrast to the liquid state case.     

Rapid, Sensitive and Highly Selective (15)N Analysis of (15)N Enriched Nitrite in Water Samples and Soil Extracts by Nitric Oxide Production and CF-QMS Measurement

Abstract Nitrite is a very important intermediate in many microbiological N transformations in soils and water. The stable isotope (15)N is often used to investigate these processes. The determination of (15)N in low concentrations of nitrite in the presence of large concentrations of nitrate is very difficult. Methods used so far for the isotope analysis of nitrite are unsatisfactory, because the nitrite must be calculated as the difference between nitrate plus nitrite and nitrate alone. More useful are mehods by which the nitrite is selectively converted into a chemical form that is suitable for (15)N analysis and that is free from interference from other N species, particularly nitrate. Using this principle in the present study we developed a method where the nitrite is reduced to nitric oxide by iodide in acid medium. This reaction is fast and quantitative, and the (15)N abundance of NO can be precisely measured by continuous flow mass spectrometry. This method is used for samples from tracer experiments with artificially enriched nitrogen 15. Therefore, the use of simple quadrupole mass spectrometers directly linked to the reaction unit is possible with sufficient precision (Reaction-Continuous Flow Quadrupole Mass Spektrometry-RCFQMS). Using the technique developed sample volumes up to 10ml containing at least 1.0 μg nitrite-N (0, 1 μg/ml) with a (15)N abundance of ? 0.42 at.% gave a precision of RSD ? ± 3%.     

2,3-二羟基萘钼多酸有机衍生物的合成及1H NMR和IR谱学研究

本文对二种新合成的2，3－二羟基萘二钼和四钼多酸有机衍生物[n-Bu)4N]2[Mo2O5(OC10H6O)2](Ⅰ）和[n-Bu)4N]2[Mo4O10(OC10H6O)2(OCH3)2](Ⅱ)进行了红外光谱与核磁共振波谱研究，发现[Mo2O5]^2 中钼氧多桥键的红外振动频率较[Mo4O10(OCH3)2]^2 中钼氧多桥键的红外振动频率红移，而在配合物Ⅱ中2，3－二羟基中芳环的^1H化学位移较配合物Ⅰ中向低场移动。同时还发现含二钼配位中心[Mo2O5]^2 的[Mo2O5(OC10H6O)2]^2-与含四钼配位中心[Mo4O10(OCH3)2]^2 的[Mo4O10(OC10H6O)2(OCH3)2]^2-生成条件的差异仅仅只在反应体系的pH值的微小变化，说明钼多酸有机衍生物阴离子是对体系酸碱度极为敏感的物质。     

Estimation of urea production rate with [(15)n(2)]urea and [(13)c]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 (13)C and with doubly (15)N 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 ((14)N(14)N), 190 ((14)N(15)N) and 191 ((15)N(15)N) urea are monitored to estimate the [(15)N(2)]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% [(15)N(2)]urea. The interassay coefficient of variation amounted to < 10% for a [(15)N(2)]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.     

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.     

New (15)N NMR exchange experiments for the unambiguous assignment of (1)H(N)/(15)N resonances of proteins in complexes in slow chemical exchange with free form

The potentialities of a 2D proton-detected heteronuclear exchange experiment to assign the nitrogen and amide proton resonances in a uniformly (15)N-enriched macromolecule involved in a complex, starting from the free form assignments, are demonstrated on a protein-DNA complex. This 2D experiment is further extended to a 3D experiment in the case of severe superpositions.     

The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]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.     

15N(p,po)15N and the levels of 16O for Ex = 14.8–18.6 MeV

Angular distributions of cross section and analyzing power for elastic scattering of protons from ¹⁵N have been measured for E_p = 2.7–7.0 MeV. A phase-shift analysis of the data yields spin-parity assignments and level parameters for seventeen states of ¹⁶O in the excitation energy region 14.8–18.6MeV. Three of the resonances have not previously been identified, among them being a broad J^π = 2^? level at E_p = 6.1 MeV which is almost certainly the analog of the 2^? 1p1h state with configuration ($\text{d}{}_{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}\text{,}\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}{}^{\mathrm{?1}}$) at E_x ∽ 5.0 MeV in ¹⁶N. The broad level previously reported near E_p = 5.0 MeV has been observed and its parameters determined. A resonance analysis of the phase shifts yielded values of E_r, Γ and Γ_p for all of the levels. The J^π assignments are in agreement with previously reported values. For resonances having J = l, the data can usually be fit with a resonant phase shift corresponding to either J = l + 1 or J = l ? 1, in addition to the phase shift for J = l. Which of the two spurious-J solutions occurs seems to depend on whether the partial wave through which the resonant state is formed is $\text{J = l +}\text{1}\text{2}\text{or}\text{J = l ?}\text{1}\text{2}$.     

Mechanism of the 12C(11B,15N)8Be reaction and 8Be + 15N optical-model potential

Angular distributions of the ¹²C( ¹¹B, ¹⁵N) ⁸Be reaction were measured at the energy E_lab(¹¹B) = 49 MeV for the transitions to the ground and 2.94 MeV (2⁺) excited state of ⁸Be and to the ground and 5.270 MeV (5/2⁺) + 5.299 MeV (1/2⁺), 6.324 MeV (3/2^-), 7.155 MeV (5/2⁺) + 7.301 MeV (3/2⁺), 7.567 MeV (7/2⁺) excited states of ¹⁵N. The data were analyzed by the coupled-reaction-channel method. The elastic, inelastic scattering and one- and two-step transfers were included in the coupling scheme. The data of the ¹²C( ¹¹B, ⁸Be) ¹⁵N reaction at E_cm = 9.4-17.8 MeV known from the literature, were also included in the analysis. The mechanism of the ¹²C( ¹¹B, ¹⁵N) ⁸Be reaction and the optical-model potential parameters for the ¹⁵N + ⁸Be channel were deduced. The energy dependence of the optical-model parameters for the ¹⁵N + ⁸Be channel was obtained.     

Spin alignment and polarisation in the (16O,15N) transfer reactions

The spin alignment of the¹⁵N ₃ ^2/* (6.33 MeV) state was measured in the⁸⁸Sr(¹⁶O,¹⁵N)⁸⁹Y reaction atE _L =96 MeV using theγ-recoil method. Angular distributions of excited states in¹⁵N and⁸⁹Y were measured with high accuracy. The analysis in terms of DWBA shows that the spin alignment is correctly described by the usual reaction models. The polarisation of the outgoing¹⁵N_1/2(GS) and¹⁵N ₃ ^2/* (6.33 MeV) is discussed. It is shown that cross section differences for transitions to final states with different configurations are sensitive to a spin-orbit potential of¹⁵N. The strength and sign of the spin-orbit potential for¹⁵N is determined.