15N Tracer Research Articles

Mature conifers assimilate nitrate as efficiently as ammonium from soils in four forest plantations.

Conifers are considered to prefer to take up ammonium (NH4+ ) over nitrate (NO3- ). However, this conclusion is mainly based on hydroponic experiments that separate roots from soils. It remains unclear to what extent mature conifers can use nitrate compared to ammonium under field conditions where both roots and soil microbes compete for nitrogen (N). We conducted an in situ whole mature tree nitrogen-15 (15 N) labeling experiment (15 NH4+ vs 15 NO3- ) over 15d to quantify ammonium and nitrate uptake and assimilation rates in four 40-yr-old monoculture coniferous plantations (Pinus koraiensis, Pinus sylvestris, Picea koraiensis and Larix olgensis, respectively). For the whole tree, 15 NO3- contributed 39% to 90% to total 15 N tracer uptake among four plantations during the study period. At day 3, the 15 NO3- accounted for 77%, 64%, 62% and 59% by Larix olgensis, Pinus koraiensis, Pinus sylvestris and Picea koraiensis, respectively. Our study indicates that mature coniferous trees assimilated nitrate as efficiently as ammonium from soils even at low soil nitrate concentration, in contrast to the results from hydroponic experiments showing that ammonium uptake dominated over nitrate. This implies that mature conifers can adapt to increasing availability of nitrate in soil, for example, under the context of globalization of N deposition and global warming.

Characteristics of uptake, residual and loss of nitrogen fertilizer in winter wheat after rice stubble

To promote the rational application of nitrogen fertilizer in winter wheat after rice stubble, the effects of nitrogen application rate (0, 150, 225, 300 kg·hm-2, expressed as N0, N150, N225, N300) on nitrogen recovery, residue, loss and grain yield were examined using field 15N tracer technology. The results showed that with the increases of application rate, nitrogen accumulation from different sources significantly increased while nitrogen recovery significantly decreased. The accumulation of basal nitrogen in plants reached the peak during overwintering stage to jointing stage, while the accumulation of topdressing nitrogen peaked between jointing to flowering stage. At maturity, nitrogen accumulation of the top fertilizer was higher than that of the base fertilizer. Plant nitrogen accumulation from soil under N150 was higher than that from nitrogen fertilizer, but with an opposite tendency under N225 and N300. With the increases of nitrogen application rate, the residual nitrogen in the 0-100 cm soil layer in the maturing stage significantly increased, while the residual nitrogen ratio in the 60-100 cm soil layer gradually increased. In the whole growth period of wheat, both nitrogen loss and loss ratio were positively correlated with nitrogen application rate. The nitrogen loss of base fertilizer reached the highest during sowing to overwintering stage, while the nitrogen loss of top fertilizer was at the peak from jointing to flowering period. Taking grain yield into consideration, N225 treatment was the proper application rate for winter wheat after rice stubble, with grain yield being 6735 kg·hm-2, and the nitrogen fertilizer recovery rate, soil residue rate and loss rate being 42.6%, 34.0% and 23.3%, respectively.

Retention of early-spring nitrogen in temperate grasslands: The dynamics of ammonium and nitrate nitrogen differ

In nitrogen (N)-limited temperate regions, winter is an important period of N accumulation. The accumulated N is released during snowmelt and thawing, and the availability peaks in early spring. However, the early-spring dynamics of specific N forms (i.e., ammonium NH4+ and nitrate NO3−) in temperate grasslands are still not fully understood. Here, we added 15NH4+ and 15NO3− (equivalent to 150 mg 15N m−2) to the soils of a meadow steppe and a typical steppe in northern China immediately after snowmelt, then quantified the retention dynamics of 15NH4+ and 15NO3− in soils, microbes, and plants over the subsequent growing season. Approximately 70% of the added 15N tracers were initially retained within the soil−microbe−plant systems in both temperate grasslands. In early spring, much 15N was immobilized in soils and microbes, while little had been taken up by plants. During the subsequent growing season, approximately 45% of the 15N was rapidly lost by the soils and microbes, but plant 15N acquisition gradually increased. Although soils and plants retained similar levels of 15NH4+ and 15NO3− during the growing season, soil microbes retained more 15NH4+ than 15NO3−. Different plant taxa had distinct 15N acquisition capacities: perennial grasses and forbs accumulated the 15N tracers rapidly, while annuals did not. Perennial grasses were effective immobilizers of the 15NH4+, whereas forbs were effective immobilizers of the 15NO3−. These findings provided evidence of the substantial retention of early-spring N over the following growing season in temperate grasslands, regardless of the vegetation type and N form. However, it was clear that the dynamics of early-spring 15NH4+ and 15NO3− differed within the soil−microbe−plant systems.

Fencing enclosure alters nitrogen distribution patterns and tradeoff strategies in an alpine meadow on the Qinghai-Tibetan Plateau

The grasslands of Qinghai-Tibetan Plateau have become extremely degraded, resulting in widespread deficiency of soil N. In efforts to restore degraded lands, fencing enclosure has been used extensively. However, the effect of fencing on N allocation patterns and nutritional strategy of alpine plants are equivocal. In this study, we used 15N tracer (CO (15NH2)2, 10 g N m−2) to examine the allocation and distribution of N in plants and soil in grasslands either grazed heavily by livestock or fenced for three years in an alpine meadow of Northern Tibet. The 15N recovery (15Nrec) in shoots of the fenced enclosure increased by 207% in grasses, decreased by 103% in forbs, and did not change in sedges when compared to the grazed meadow. The 15Nrec in shoots accounted for only 1.97% and 4.65% of the total N in the grazed and fenced meadows, respectively. Fencing increased soil 15N content at 0–5 cm depth by 6.9%, but decreased the content at 5–10 cm depth by 11.7%. The results demonstrated that fencing altered the soil N distribution by increasing 15Nrec in top soil and by decreasing 15Nrec in subsurface soil. In addition, fencing had no impact on root 15N storage (33%–39%), 15N losses (9.6%–12.5%) and soil available 15Nrec (NH4+-N, NO3–-N and light fraction organic N), but decreased root:shoot 15Nrec ratio by 49.8% (16.9:1 in grazed and 8.4:1 in fenced grassland). Fencing increased soil organic carbon, total N and NO3–-N concentrations, which indicated that the strategy of the plants was to allocate relatively more N to roots in nutrient-poor soil (grazed) but relatively more N to shoots in nutrient-rich soil (fenced).

Pseudomonas mendocina LYX: A novel aerobic bacterium with advantage of removing nitrate high effectively by assimilation and dissimilation simultaneously

The problem of nitrate accumulation in aerobic tank and total nitrogen excessive discharge in effluent was very common in traditional livestock and poultry farming wastewater treatment systems owing to the lengthy process flow and low process control level. A strain LYX of aerobic bacterium was isolated from the activated sludge of a wastewater treatment system in a pig farm, which could remove nitrate effectively in aerobic tank and was identified Pseudomonas mendocina by 16S rRNA sequencing. Under the condition of nitrate as the sole nitrogen source, this strain removed over 90% of NO3−-N with an initial concentration of 110 mg/L under aerobic conditions within 48 hours. Among them, 37.9% of NO3− -N was assimilated into Bio-N, about 51.9% was reduced to gaseous nitrogen and less than 0.5% of nitrogen was replaced by NO3−-N and NH4+-N, 9.7% NO3−-N remained in the effluent at the end. At the same time, four key genes (napA, nirK, norB and nosZ) related to nitrate nitrogen removal were expressed during the denitrification process of P. mendocina LYX, in which the transcription level of the indicator genes of this aerobic denitrifying bacterium (napA) was the highest. In addition, it was found with the 15N tracer technique that inoculation of this strain on sludge increased the amount of nitrogen loss from 9.26 nmol N/(g·h) to 23.835 nmol N/(g·h). Therefore, P. medocina LYX is a potential bioagent for advanced nitrogen removal by assimilating and reducing nitrate simultaneously in aerobic tanks.

Potential of Alginate and Mesoporous Carbon to Improve the Fertilizer Value of Urea

ABSTRACT Mesoporous C (MC) has potential for controlling NH3 volatilization from fertilizer urea because of a high capacity for sorption of ions and gases, and so does sodium alginate (SA), a natural superabsorbent that has received considerable attention for developing slow-release fertilizers. Following stirred-flow experiments to evaluate both amendments for slowing urea release in water, novel studies were conducted that determined their impact on transformations of urea N during aerobic incubation of a sand and silt loam using 15N tracer techniques to unambiguously differentiate fertilizer from soil N. Neither amendment showed appreciable slow-release behavior nor had any significant effect on urea N transformations in the silt loam; however, NH3 volatilization from the sand was significantly decreased by up to 47% with SA and by up to 38% with MC, while SA also significantly increased NO3 − production by 42–114%. Taken together with the inherent advantages of a natural material that is widely available, economical, and biodegradable, the results reported indicate that SA has more potential than MC for enhancing the fertilizer value of urea.

Dissimilatory nitrate ammonification and N2 fixation helps maintain nitrogen nutrition in resource-limited rice paddies

Un-fertilized rice paddies have shown maintained soil nitrogen (N) status, stable N supply to the rice plant and sustained rice yields at moderate levels for hundreds of years. Microbial N2 fixation is known to contribute N to un-fertilized paddies, but it cannot fully explain the maintained N nutrition, where favourable conditions exist for N loss by denitrification. We used 15N tracer, 15N2 uptake, acetylene reduction assay and qPCR to simultaneously investigate N2 fixation, dissimilatory nitrate reduction to ammonium (DNRA), denitrification and related microbial gene abundances in long-term low (or no) and high N input rice paddies of Myanmar. We also determined how varying soil organic carbon-to-nitrate (SOC/NO3−) ratios affect nitrate partitioning between DNRA and denitrification by manipulating these ratios through labile organic carbon addition. We observed more than 2.5 times higher N2 fixation (1.49–2.08 μg N g−1 soil day−1) and significantly higher N2 fixing gene (nifH) abundance in low compared with high N input paddies. Up to 60% of the soil nitrate (1.51–2.67 μg NO3−-N g−1 soil day−1) was ammonified through DNRA, and only 15% was lost as N2 through denitrification in low N input paddies, whereas denitrification exceeded DNRA in high N input paddies. The microbial gene related to DNRA activity (nrfA) was also higher in low input than in high input rice paddies. We found that nitrate retention can be improved in high N input rice paddies by maintaining a higher soil organic carbon-to-nitrate ratio. Our findings highlight the unique microbial N-cycling strategies in resource-limited paddies which support maintained N nutrition of the paddy system.

Effect of urea fertilization on growth of broad bean (Vicia faba L.) under various nickel (Ni) levels with or without acetic acid addition, using 15N-labeled fertilizer.

Although nickel (Ni) has direct relationship with nitrogen metabolism of plants, the high dose of Ni fertilizer in broad bean plants may affect the nitrogen use efficiency (NUE), impair plant development and even cause Ni pollution in soil. Thus, a pot experiment was set up to study the effect of urea fertilization on N-uptake, root and shoots' Ni content as well as growth of broad bean plants under different levels of Ni, using 15N tracer technique. 15N-labeled urea (5% 15N atom excess) was added at three doses (0, 30 and 60mgN kg-1 soil). Nickel sulfate (NiSO4) was also applied at three levels (0, 50 and 100mgNi kg-1 soil). The experiment was laid out with or without acetic acid in randomized complete block design in three replicates. Treatment with the addition of 60mg N + 50mg Ni showed the highest values in dry weights of root and shoots, N-uptake by shoots, nitrogen derived from fertilizer (Ndff %) and NUE % by shoots in both with or without acetic acid solution. Higher rate of Ni addition can decrease shoot and root biomass by inhibiting the ability of the plant to uptake the nitrogen efficiently. However, addition of acetic acid solution induced the improvement of NUE % and Ndff % by shoot and root of broad bean plants. This study provides insight into how to improve plant yield without damaging the soil health and will be helpful to create a better world with sustainable agriculture.

Temporal and Vertical Oxygen Gradients Modulate Nitrous Oxide Production in a Seasonally Anoxic Fjord: Saanich Inlet, British Columbia

AbstractNitrous oxide (N2O) is a strong greenhouse gas and an ozone depleting agent. In marine environments, N2O is produced biologically via ammonium oxidation, nitrite, and nitrate reduction. The relative importance of these principle production pathways is strongly influenced by oxygen availability. We conducted 15N tracer experiments of N2O production in parallel with measurements of N2O concentration and natural abundance isotopes/isotopomers in Saanich Inlet, a seasonally anoxic fjord, to investigate how temporal and vertical oxygen gradients regulate N2O production pathways and rates. In April, June, and August 2018, the depth of the oxic‐anoxic interface (dissolved oxygen = 2.5 μmol L−1 isoline) progressively deepened from 110 to 160 m. Within the oxygenated and suboxic water column, N2O supersaturation coincided with peak ammonium oxidation activity. Conditions in the anoxic deep water were potentially favorable to N2O production from nitrate and nitrite reduction, but N2O undersaturation was observed indicating that N2O consumption exceeded rates of production. In October, tidal mixing introduced oxygenated water from outside the inlet, displacing the suboxic and anoxic deep water. This oxygenation event stimulated N2O production from ammonium oxidation and increased water column N2O supersaturation while inhibiting nitrate and nitrite reduction to N2O. Results from 15N tracer incubation experiments and natural abundance isotopomer measurements both implicated ammonium oxidation as the dominant N2O production pathway in Saanich Inlet, fueled by high ammonium fluxes (0.6–3.5 nmol m−2 s−1) from the anoxic depths. Partial denitrification contributed little to water column N2O production because of low availability of nitrate and nitrite.

Nitrate reduction pathways and interactions with iron in the drainage water infiltration zone of a riparian wetland soil

Eutrophication of natural water bodies is moderated by transformation of nitrate (NO3−) in riparian wetlands, which serve as filters of infiltrating drain water from upland agricultural areas. The present study comprised field observations, laboratory experiments and metagenomic studies to describe NO3− removing transformation pathways and interactions with the cycling of iron (Fe) in a temperate riparian wetland soil profile down to 1 m depth. Water samples from piezometers showed a distinct plume of NO3− in the subsurface soil where agricultural drain water was infiltrating. However, within a distance of few meters in the water flow direction, NO3− was depleted from the percolating water. Sampling and analyses of soil from the active zone of the biogeochemical NO3− removal showed that denitrifying enzyme activity was ~ tenfold higher in the upper 0–25 cm than in the lower 25–100 cm. Yet, net transformation of NO3− was substantial also at 25–100 cm when assayed with relatively undisturbed soil samples and by 15N tracer techniques in soil slurries. Transformation pathways of dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation were identified, but were quantitatively minor as compared to denitrification. Heterotrophic denitrification and denitrification mediated by oxidation of ferrous iron, Fe(II), were identified as important processes in the wetland soil. The latter was substantiated by geochemical observations, by rates of NO3− depletion in slurry incubations with added FeCl2, and by identification of microorganisms with known capacity of NO3− reduction coupled to Fe(II) oxidation (Acidovorax sp.). The transformation pathway of iron-mediated NO3− reduction could involve biotic and abiotic reactions, and N2O, which is a potent greenhouse gas, was a major product of the process. It remains to be seen under field conditions if N2O emission hotspots are linked to specific sites of dynamic NO3− reduction coupled to Fe(II) oxidation.

Nitrogen Recoveries and Nitrogen Use Efficiencies of Organic Fertilizers with Different C/N Ratios in Maize Cultivation with Low-Fertile Soil by 15N Method

The rising cost of inorganic fertilizers, coupled with their adverse effects on soil conditions, has resulted in increasing interest in organic amendments. The objective of the present study was to evaluate the effect of organic amendments (OAs) with different C/N ratios on nitrogen use efficiency (NUE) and recovery rate, as well as on the growth and yield of Zea mays and soil properties. A precise pot experiment was conducted on a low-fertile, sandy-loam soil, and the dynamics of nitrogen (N) were also analyzed by the A-value method, using 15N tracer. The plant height of the treatment groups decreased in the following order: inorganic fertilizer (IF) = rapeseed waste (RW) > chicken manure (CnM) > bamboo tealeaf (BTL) > cow manure (CwM) > bamboo compost (BC). Furthermore, the maize fertilized with RW only took up half of the N in IF, despite producing the same yield, which indicates that the physiological nitrogen efficiency (PUE) of RW was twice as high as that of IF. RW and CnM were regarded as valuable fertilizers that could be used to replace inorganic fertilizers. A linear relationship between the N mineralization of the OAs was obtained by an incubation test and the pot experiments, estimating the effect of OA application on the maize. Maize plants mainly absorbed N derived from fertilizers; however, for the both sources of N (fertilizer and soil), N was mainly accumulated in grains followed by the leaves, stem, and root, suggesting that studies should be conducted to improve soil N use efficiency.

15N tracer enrichment in response to winter soil temperature manipulation differs between canopy trees and juveniles

Juveniles and canopy trees may not exhibit similar nitrogen acquisition responses to soil temperature change caused by variation in snow cover over winter. The use of 15N tracer is a powerful tool for tracking the effects of variation in soil frost on plant nitrogen acquisition. While the responses of juvenile trees to environmental change are often used to infer the responses of canopy trees, the 15N enrichment responses of juveniles and mature canopy trees may not be comparable. We conducted a winter soil temperature manipulation study (snow exclusion, ambient snow or soil insulation) in a lowland Fagus sylvatica forest. 15N tracer was applied the following spring and the 15N enrichments of soil, juvenile and mature canopy trees were examined in late fall. Within canopy trees and juveniles, the relative treatment effects on 15N enrichment were consistent among all sampled tissues (roots, stem cores, leaves, buds and the current year’s shoot growth). For juveniles, 15N enrichment was highest under snow exclusion (coldest soil) and lowest under soil insulation (warmest soil), and lower 15N enrichment occurred under ambient conditions than under snow exclusion. For canopy trees, 15N enrichment also was highest under snow exclusion and lowest under soil insulation, but there was no difference in 15N enrichment between ambient conditions and the snow exclusion treatment. Therefore, our results indicate that sampling of juveniles may overestimate the nitrogen acquisition responses of mature trees to winter temperature variation.

Effects of exotic Spartina alterniflora on saltmarsh nitrogen removal in the Yangtze River Estuary, China

Salt marshes play an important role in removing excess nitrogen through microbial processes such as denitrification and anaerobic ammonia oxidation (anammox), and one of the major factors known to influence the nitrogen purification capacity of salt marshes worldwide is the invasion of alien species. Previous studies on the effects of invasive plants on nitrogen removal have tended to focus on the upper soils, whereas only a few have sought to examine the effects of exotic species on nitrogen removal in deep soils. This disparity may thus have led to unrepresentative evaluations when attempting to make integrated assessments of the ecosystem services provided by exotic species. Here, using the 15N tracer technique, we investigated the processes of denitrification and anammox in sediments of four habitat types (bare mudflat and marshlands dominated by the native plant species Scirpus mariqueter and Phragmites australis and the invasive plant Spartina alterniflora) on Chongming Island, located in the Yangtze River Estuary, China. Measurements were taken in May and October 2018 from six different depth ranges (0–5, 5–10, 10–20, 20–30, 30–50, and 50–100 cm) within the soil profile. With respect to the entire soil profile, we found that S. alterniflora did not facilitate effective nitrogen removal compared with the native plants S. mariqueter and P. australis. However, S. alterniflora did significantly increase nitrogen removal in the upper 50 cm of soil, whereas a slight reduction in nitrogen removal was observed in the deeper 50–100 cm soil layer. These results indicate that nitrogen removal was significantly increased only in the upper soils subsequent to S. alterniflora invasion, which contributes to our existing knowledge regarding the effects of invasive species on the nitrogen cycle in salt marshes. Furthermore, we established that in the upper soil layers between 0 and 50 cm, denitrification is primarily regulated by NO3− and the C/N ratio, whereas anammox is predominantly regulated by bulk density. Contrastingly, in the deeper soil layers between 50 and 100 cm, both these processes are largely regulated by redox potential and pH. These results serve to highlight that when evaluating the ecosystem services provided by S. alterniflora in salt marshes, it is necessary to estimate nitrogen removal in both the upper and deeper soil layers.

N2O emissions and product ratios of nitrification and denitrification are altered by K fertilizer in acidic agricultural soils

Potassium (K) fertilizer plays an important role in increasing crop yield, quality, and nitrogen use efficiency. However, little is known about its environmental impacts, such as its effects on emissions of the greenhouse gas nitrous oxide (N2O). A nitrogen-15 (15N) tracer laboratory experiment was therefore performed in an acidic agricultural soil in the suburbs of Wuhan, central China, to determine the effects of K fertilizer on N2O emissions and nitrification/denitrification product ratios under N fertilization. During 15-d incubation periods with a fixed initial N concentration (80 mg kg−1), K application increased average N2O emission rates significantly (1.6–10.8-fold) compared to the control treatment. N2O emissions derived from nitrification and denitrification both increased in K-treated soil, and denitrification contributed more to the increase; its contribution ratio rose from 32% without K fertilizer to 53% with 300 mg kg−1 of K applied. The increase in N2O emissions under K fertilization is probably due to an increase in the activity of denitrifying microorganisms and acid-resistant nitrifying microorganisms caused by higher K+ concentrations and lower soil pH. Combined treatment with potassium chloride (KCl) and N fertilizer produced lower N2O emissions than combined treatment with potassium sulfate (K2SO4) and N fertilizer during 15-d incubation periods. Our results imply that there are significant interaction effects between N fertilizers and K fertilizers on N2O emissions. In particular, combining N fertilizers with fertilizers that reduce soil acidity or contain Cl or K ions may significantly affect agricultural N2O emissions.

The biodiversity - N cycle relationship: a 15N tracer experiment with soil from plant mixtures of varying diversity to model N pool sizes and transformation rates

We conducted a 15N tracer experiment in laboratory microcosms with field-fresh soil samples from a biodiversity experiment to evaluate the relationship between grassland biodiversity and N cycling. To embrace the complexity of the N cycle, we determined N exchange between five soil N pools (labile and recalcitrant organic N, dissolved NH4+ and NO3− in soil solution, and exchangeable NH4+) and eight N transformations (gross N mineralization from labile and recalcitrant organic N, NH4+ immobilization into labile and recalcitrant organic N, autotrophic nitrification, heterotrophic nitrification, NO3− immobilization, adsorption of NH4+) expected in aerobic soils with the help of the N-cycle model Ntrace. We used grassland soil of the Jena Experiment, which includes plant mixtures with 1 to 60 species and 1 to 4 functional groups (legumes, grasses, tall herbs, small herbs). The 19 soil samples of one block of the Jena Experiment were labeled with either 15NH4+ or 15NO3- or both. In the presence of legumes, gross N mineralization and autotrophic nitrification increased significantly because of higher soil N concentrations in legume-containing plots and high microbial activity. Similarly, the presence of grasses significantly increased the soil NH4+ pool, gross N mineralization, and NH4+ immobilization, likely because of enhanced microbial biomass and activity by providing large amounts of rhizodeposits through their dense root systems. In our experiment, previously reported plant species richness effects on the N cycle, observed in a larger-scale field experiment within the Jena Experiment, were not seen. However, specific plant functional groups had a significant positive impact on the N cycling in the incubated soil samples.

Effect of different fertilization regimes on the vertical distribution of anaerobic ammonium oxidation in paddy soils

Fertilization is a necessary practice for paddy fields to achieve high crop yields, and this practice could have an important impact on soil nitrogen cycle. Anammox (anaerobic ammonium oxidation) is an important process in the nitrogen cycle of paddy ecosystems. However, until now, the influence of nitrogen fertilization on anammox, is not well understood. Here, the vertical distribution of the activity, abundance and community composition of anammox bacteria was investigated in paddy soils (0–50 cm) under three different fertilizer treatments: chemical fertilizer (CF), organic fertilizer (OF) and unfertilized control (CK). The 15N tracer experiments confirmed anammox activity in different depths of paddy soils across all treatments, with the highest activity in the surface soil layer (0–10 cm) under OF treatment. The potential anammox rates were 0.76–1.82, 0.95–2.39 and 1.19–3.62 nmol N g−1 (dry soil) h−1, respectively, under CK, CF and OF treatments. The abundance of anammox bacterial hzsA genes in upper 30 cm layers was significantly higher under CF (3.1 × 106-6.5 × 106 copies g−1 dry soil) and OF (3.4 × 106-7.1 × 106 copies g−1 dry soil) treatments than CK treatment (2.1 × 106-3.5 × 106 copies g−1 dry soil). Similar community composition of anammox bacteria was observed in soils under different fertilizer treatments, with Candidatus Brocadia being the single dominant genus. Overall, our results demonstrated that the input of inorganic or organic fertilizers significantly increased the anammox bacterial abundance and activity in both surface and subsurface paddy soils, while the anammox community remained relatively stable.

1910-P: Effects of Glucagon Concentrations on Splanchnic and Leg Glucagon Extraction in Healthy Humans

We have recently developed a novel isotope dilution method using nonradioactive, stable human Glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate the effects of increasing circulating glucagon concentrations, spanning the physiological range, on splanchnic and leg glucagon extraction. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. After a 90 min period for basal sampling, exogenous glucagon was infused at 0.65 ng/kg/min for 60 minutes (low) followed by 1.5 ng/kg/min (high) for another 60 minutes. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of Glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Arterial glucagon concentrations were 45.2±9.6 pg/ml (basal), 56.3±13.3 pg/ml (low) and 81.4±15.5 pg/ml (high). While fractional splanchnic glucagon extraction remained relatively unchanged (24.0±12.5% vs. 27.7±15.6% vs. 23.5±10.1%: basal vs. low vs. high), fractional leg glucagon extraction tended to increase (13.5±3.8% vs. 16.0±7.2% vs. 18.5±9.4%: basal vs. low vs. high) with increasing glucagon concentrations. These initial data suggest differing mechanisms of glucagon clearance across the splanchnic and leg tissues in humans. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

152-OR: Postabsorptive Splanchnic and Leg Glucagon Metabolism in Healthy Subjects: Use of 13C 15N Glucagon

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a novel isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate splanchnic and leg glucagon extraction and uptake. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Fractional splanchnic glucagon extraction was 24.0±12.5 % while total splanchnic glucagon uptake was 214.3±56.1 ng/kg total body weight/min. Net splanchnic glucagon balance was -34.4±10.0 ng/kg total body weight/min implying net splanchnic glucagon production. Fractional leg glucagon extraction was 13.5±3.8% while total leg glucagon uptake was 53.0±21.2 pg/kg total body weight/min. To the best of our knowledge, these initial data provide the first direct assessment of regional glucagon metabolism in healthy humans in the post absorptive fasting state. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

1916-P: Postprandial Glucagon Metabolism in Healthy Subjects: Use of 13C 15N Glucagon

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a new isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon fluxes. To estimate postprandial glucagon turnover using this novel technique, we present data from the first 5 healthy subjects (3 males, age 26.2±7.9 years, BMI 25.4±2.6 kg/m2, fasting plasma glucose 4.7±0.2 mM, HbA1c 5.1±0.2 %) done thus far. After IRB approval and informed consent, subjects presented to the clinical research unit in the morning after an overnight fast. A catheter was inserted in a forearm vein for glucagon tracer infusion and another catheter placed in the contralateral hand and the hand kept in a heated box for periodic draws of arterialized venous samples for measurements of glucagon tracer (tandem mass spectrometry), glucagon and glucose concentrations during the study. A mixed meal (75 grams carb, 15% protein, 35% fat; 8 kcal/kg) was ingested at time 0 and the glucagon tracer infusion rate adjusted to mimic the anticipated changes in postprandial circulating glucagon concentrations for 6 hours. Systemic glucagon appearance rate (Ra glucagon) was calculated by the isotope dilution method. Fig 1 shows the Ra glucagon in all subjects. These initial data show that postprandial glucagon turnover can be calculated in humans applying this novel technique. Disclosure F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. M. Schiavon: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

Assessing the Systematic Effects of the Concentration of Nitrogen Supplied to Dual-Root Systems of Soybean Plants on Nodulation and Nitrogen Fixation

The specific mechanisms by which nitrogen affects the nodulation and nitrogen fixation of soybean plants are unclear. Investigating the relationship between nitrogen, nodulation and nitrogen fixation can provide new insights for the rational and proper use of nitrogen fertilizer in soybean plants. In this study, we grafted soybean roots to construct a dual-root system with a single nodulated side. Experiment I was performed at the third trifoliate leaf to initial seed filling (V3-R3) growth stages (for 30 days) for long-term nitrogen supply, and Experiment II was performed at the third trifoliate leaf to fourth trifoliate leaf (V3-V4) growth stages (for 5 days) for short-term nitrogen supply. For the two experiments, a nutrient solution providing 15NH415NO3 or NH4NO3 as the nitrogen source was added to the non-nodulated side, while a nitrogen-free nutrient solution was added to the nodulated side. The concentrations of nitrogen supplied were 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L. The results showed the following: (1) Short-term nitrogen supply systematically regulated the specific nitrogenase activity (SNA), thereby inhibiting the acetylene reduction activity (ARA). Under long-term nitrogen supply, the recovery of SNA was generally consistent across treatments, and the concentration of nitrogen supplied systematically regulated the growth of root nodules, thereby inhibiting the ARA. (2) Using the 15N tracer method, the concentration of fertilizer nitrogen was positively correlated with the amount of nitrogen redistributed to other organs. Although the percentage of nitrogen derived from the atmosphere (Ndfa%) decreased significantly with increasing concentrations of nitrogen supplied, the effect on the accumulation of nitrogen fixed by nodules (Naccumulation of nodules) was not significant. By establishing the relationships between the ARA (measured by the acetylene reduction method), Ndfa% (based on 15N calculations), and Naccumulation nodules (based on 15N calculations), it was found that the ARA reliably reflected the Ndfa% but not the Naccumulation of nodules.