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Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism.

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO(2)) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from (15)N-labeled NO(2) taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen ( UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with (15)NO(2). Secondly, tobacco ( Nicotiana tabacum L.) plants fed with (15)N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen-oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.

Fumigation↗

Inaccuracy of nitrogen balance determinations in thermal injury with calculated total urinary nitrogen.

Many burn centers use nitrogen balance studies to estimate the adequacy of nutritional support. Nitrogen loss includes the sum of urinary urea nitrogen, nonurea urinary nitrogen, and losses from skin, wound, and stool. Urinary urea nitrogen is often used to calculate total urinary nitrogen by multiplying the urinary urea nitrogen by a factor of 1.25 to account for nonurea urinary nitrogen. This formula is appropriate when applied to a nonstressed individual who has fasted overnight but is not appropriate for patients who have undergone surgery or experienced trauma. We have undertaken this study to assess the predictability of this formula in patients with thermal injuries. Twenty-seven patients with major thermal injuries had random 24-hour urine collections, which were analyzed for both urinary urea nitrogen and total urinary nitrogen. In these patients with burns we found that urinary urea nitrogen represented approximately 65% of the directly measured total urinary nitrogen rather than 80% as assumed by the formula. This increase in the nonurea nitrogen loss is greater than that found after surgery or trauma. Individual measurements may underestimate losses by 20% to 60%. Directly measured total urinary nitrogen should replace calculated total urinary nitrogen as the index of urine nitrogen losses for nitrogen balance studies in patients with burns.

Burns↗

The nitrogen requirements and dietary nitrogen utilization for the gecarcinid land crab Gecarcoidea natalis.

The nitrogen requirements for tissue maintenance, moulting, and oogenesis were determined experimentally for the herbivorous land crab Gecarcoidea natalis. The maintenance nitrogen requirements for intermoult animals was very low (4.83+/-1.68 mmol N kg-1 dry body wt d-1), but during oogenesis the total requirement was much higher (8. 6 mmol N kg-1 dry body wt d-1). Gecarcoidea natalis could potentially assimilate enough nitrogen from rain forest leaf litter or leaves of Ficus or Erythrina to satisfy not only the maintenance nitrogen requirements but the observed rate of incorporation of nitrogen into the ovaries during oogenesis. The ovaries developed slowly over a period of 2 mo (mid-July to late September) and had a final nitrogen content of 359+/-15.9 (n=18) mmol kg-1 dry body wt. This was equivalent to 9.3%+/-0.4% of the total body nitrogen. A substantial nitrogen debt was incurred during ecdysis (658+/-126 mmol kg-1 dry body wt). This nitrogen debt could be satisfied slowly, from leaf litter, over a period of 1-3 mo. After ecdysis, the majority of the nitrogen and urate within the animal prior to moulting was retained within the soft crab (85.0%+/-1.2% total nitrogen, 82.0%+/-1.2% nonurate nitrogen and 99.56% urate), while only a minority was lost with the exuviae (18.0%+/-1.2% total nitrogen, 14.7%+/-1.2% nonurate nitrogen, and 0.4%+/-0.4% urate). The urate deposits in G. natalis were not mobilized as a source of nitrogen in animals maintained on a nitrogen-free diet.

Animals↗

Evaluating the effects of gross nitrogen mineralization, immobilization, and nitrification on nitrogen fertilizer availability in soil experimentally contaminated with diesel.

Sandy clay loam soil was contaminated with 5000 mg kg(-1) diesel, and amended with nitrogen (15.98 atom% (15)N) at 0, 250, 500, and 1000 mg kg(-1) to determine gross rates of nitrogen transformations during diesel biodegradation at varying soil water potentials. The observed water potential values were -0.20, -0.47, -0.85, and -1.50 MPa in the 0, 250, 500, and 1000 mg kg(-1) nitrogen treatments respectively. Highest microbial respiration occurred in the lowest nitrogen treatment suggesting an inhibitory osmotic effect from higher rates of nitrogen application. Microbial respiration rates of 185, 169, 131, and 116 mg O(2) kg(-1) soil day(-1) were observed in the 250, 500, control and 1000 mg kg(-1) nitrogen treatments, respectively. Gross nitrification was inversely related to water potential with rates of 0.2, 0.04, and 0.004 mg N kg(-1) soil day(-1) in the 250, 500, and 1000 mg kg(-1) nitrogen treatments, respectively. Reduction in water potential did not inhibit gross nitrogen immobilization or mineralization, with respective immobilization rates of 2.2, 1.8, and 1.8 mg N kg(-1) soil day(-1), and mineralization rates of 0.5, 0.3, and 0.3 mg N kg(-1) soil day(-1) in the 1000, 500, and 250 mg kg(-1) nitrogen treatments, respectively. Based on nitrogen transformation rates, the duration of fertilizer contribution to the inorganic nitrogen pool was estimated at 0.9, 1.9, and 3.2 years in the 250, 500, and 1000 mg kg(-1) nitrogen treatments, respectively. The estimation was conservative as ammonium fixation, gross nitrogen immobilization, and nitrification were considered losses of fertilizer with only gross mineralization of organic nitrogen contributing to the most active portion of the nitrogen pool.

Algorithms↗

Dogmas and controversies in the handling of nitrogenous wastes: the effect of feeding and fasting on the excretion of ammonia, urea and other nitrogenous waste products in rainbow trout.

Ammonia and urea are the primary forms of nitrogen excretion in teleost fish. There exists, however, a discrepancy between the sum of ammonia plus urea nitrogen and total nitrogen, indicating that 'unknown' nitrogen end products may play an important role in nitrogen metabolism. The current study analysed a wide range of nitrogen end products in both fed and fasted juvenile rainbow trout. Ammonia-N (53-68%) and urea-N (6-10%) were confirmed as the most important forms of nitrogenous waste, but an interesting finding was the considerable excretion of nitrogen as amino acids (4-10%), via the gills, and as protein (3-11%), probably via the body mucus. Use of anal sutures delineated an important role for the gastrointestinal tract in the production of ammonia-N and urea-N in fed fish, but amino acid-N and protein-N output by this route were both negligible. Alternative nitrogen products - trimethylamine, trimethylamine oxide, uric acid, and nitrite + nitrate - were not excreted in detectable quantities. Creatine-N and creatinine-N outputs were detected but contributed only a small fraction to total nitrogen excretion (<1.4%). Despite the wide scope of nitrogenous end products investigated, a considerable proportion (12-20%) of nitrogen excretion remains unknown. Possible alternative end products and methodological considerations are proposed to explain this phenomenon. The findings described above were used to recalculate the nitrogen quotient (NQ=(N)/(O(2))) on trout that had been either fasted or fed various daily rations (1%, 3% or 5% dry food per unit wet body mass per day). Feeding increased oxygen consumption ((O(2))) and total-N excretion ((N)). The NQ is often used as a measure of protein utilisation in aerobic metabolism and assumes that all protein (and amino acid) fuels are converted by oxidation to nitrogenous waste products that are excreted. However, the results showed that calculation of the NQ based on total nitrogen excretion may overestimate protein utilisation in aerobic metabolism because of significant excretion of N in the form of proteins and amino acids, whereas the use of summed ammonia-N and urea-N excretion probably underestimates the contribution of protein towards aerobic metabolism. These errors increase as ration increases, because the discrepancy between total-N excretion and ammonia-N + urea-N excretion increases.

Amino Acids↗

Woodrat (Neotoma) herbivores maintain nitrogen balance on a low-nitrogen, high-phenolic forage, Juniperus monosperma.

The acquisition of adequate quantities of nitrogen is a challenge for herbivorous vertebrates because many plants are in low nitrogen and contain secondary metabolites that reduce nitrogen digestibility. To investigate whether herbivores maintain nitrogen balance on plant diets low in nitrogen and high in secondary compounds, we studied the effect of juniper (Juniperus monosperma) ingestion on the nitrogen balance of two species of herbivorous woodrats (Neotoma stephensi and N. albigula). These woodrat species feed on the foliage of juniper: N. stephensi is a juniper specialist, whereas N. albigula is a generalist that incorporates some juniper in its diet. Based on the nitrogen contents of the natural diets of these woodrats, we predicted that the generalist would be in negative nitrogen balance on a juniper diet whereas the specialist would not be affected. We found that both species of woodrat had low-nitrogen requirements (334.2 mg N/kg0.75/day) and that a diet of 50% juniper did not result in negative nitrogen balance for either species. However, excretion patterns of nitrogen were altered; on the 50% juniper diet, fecal nitrogen losses increased approximately 38% and urinary nitrogen losses were half that of the control diet. The results suggest that absorption and detoxification of juniper secondary compounds may be more important for restricting juniper intake by the generalist than nitrogen imbalance.

Analysis of Variance↗

Growth reduction of Sphagnum magellanicum subjected to high nitrogen deposition: the role of amino acid nitrogen concentration.

We tested the relationship between Sphagnum growth and the amount of nitrogen stored in free amino acids in a fertilisation experiment with intact peat monoliths in an open greenhouse in The Netherlands. Three nitrogen deposition scenarios were used: no nitrogen deposition, field conditions and a doubling of the latter, corresponding to 0, 40 and 80 kg N ha(-1 )year(-1). Growth of Sphagnum as expressed by height increment was reduced in the 80 kg N treatment, but showed no correlation with the total nitrogen tissue concentration or with the concentration of individual or pooled free amino acids. The amount of nitrogen stored in free amino acids increased concomitantly with deposition, although it lagged more and more behind the total nitrogen concentration, the latter pointing to the accumulation of unmeasured nitrogen compounds. Asparagine clearly acted as the major storage compound for nitrogen in Sphagnum stem tissue, whereas arginine fulfilled this function to a lesser extent in the capitulum. It appears that nitrogen-induced growth inhibition of Sphagnum is related to acclimation rather than to certain threshold concentrations of amino nitrogen or total nitrogen. We propose that when Sphagnum is exposed to a step increase of nitrogen, its nitrogen metabolism does not adapt fast enough to keep up with the enhanced uptake rate. This imbalance between nitrogen uptake and assimilation may lead to an accumulation of toxic NH(4)(+ )in the cell and a subsequent reduction in growth.

Asparagine↗

Protein and energy intake, nitrogen balance and nitrogen losses in patients treated with continuous ambulatory peritoneal dialysis.

The aim of this investigation was to analyze factors which influence the dietary protein intake (DPI), the energy intake and the utilization of ingested protein, and to determine the relationship between various types of nitrogen losses in stable continuous ambulatory peritoneal dialysis (CAPD) patients. We performed 23 nitrogen balance (NB) studies of 6 to 11 days duration in 12 CAPD patients. One study was performed in all patients 3.4 +/- 1.2 months after starting CAPD (early studies). The study was then repeated in nine patients after 12.1 +/- 2.6 months, and two of these patients were studied again after 16 and 24 months, respectively (late studies). Before each NB study, the dietary intakes prior to the study were assessed in diaries and interviews. During a few days preceding the NB periods and during the NB periods each patient received an individualized diet composed so as to resemble the patients' spontaneously chosen diet regarding DPI and dietary energy intake (DEI). Total nitrogen, protein, urea and creatinine were analyzed in the dialysate and urine collected daily. Total nitrogen was also analyzed in the feces, collected over the whole NB period. Total nitrogen appearance (TNA), non-protein nitrogen appearance (NPNA) and urea nitrogen appearance (UNA) were calculated by correcting total nitrogen output, non-protein nitrogen output, that is, TNA minus the total protein losses (PL) and urea nitrogen output for changes in total body urea nitrogen. Glucose was determined in the collected dialysate and the daily glucose absorption was calculated. DPI varied between 0.62 and 2.09 g/kg/day, DEI between 21 and 42 kcal/kg/day and the peritoneal energy (glucose) intake (PEI) between 4 and 13 kcal/kg/day. DPI (but not DEI) correlated with Kt/V(urea) and Kt/VCr and with total and renal clearances for urea and creatinine. NB (not corrected for "unmeasured" nitrogen losses) was positive in most studies, and it correlated with DPI and the total energy intake (TEI) in the early studies, but only with TEI in the late studies. DPI correlated with TNA, NPNA, UNA, non-protein-non-urea nitrogen loss and fecal nitrogen loss. UNA was highly correlated with TNA and NPNA (r = 0.95). We used data from 33 NB studies in CAPD patients (our present data combined with data from the literature) to calculate regression equations describing the relationship between TNA and NPNA, respectively, and UNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

The effect of stress level, amino acid formula, and nitrogen dose on nitrogen retention in traumatic and septic stress.

Eighty-seven patients were entered into a randomized, prospective, double-blind, six-center study to evaluate the effect of amino acid loading and a formula that was branched chain enriched (50%) on nitrogen retention in metabolic stress. The patients had varying levels of metabolic stress (0-3) after major surgery, polytrauma, or surgical sepsis. The study was isocaloric and isonitrogenous and lasted for 7 days. The patients received either a standard amino acid formula (SAA) (Travasol) or a 50% branched chain enriched formula that was equimolar, leucine, isoleucine, and valine (MAA) (Travasol + Branchamin concentrate) at a dose of 1.0-2.0 g/kg/day in a fixed ratio with 114 glucose calories per gram of nitrogen administered. The nitrogen retention was proportionate to the nitrogen (and, therefore, caloric) load in both groups. The MAA group, however, had better nitrogen retention, reached nitrogen equilibrium at a lower dose of amino acids, and had less urinary nitrogen excretion per gram of nitrogen administered. Since the groups were isonitrogenous and the calorie to nitrogen ratios were fixed, it appears that nitrogen equilibrium in surgical stress is proportionate to the amino acid load over a range of 0.05-0.4 g/kg/day of nitrogen; and that MAA are more efficient at inducing nitrogen retention and a reduction in urea excretion. These effects on nitrogen retention were more significant at level 2 stress or greater. At these higher stress levels, a dose of 2 +/- 0.2 g/kg/day of MAA seemed most efficient in promoting nitrogen retention.

Adult↗

Influence of dry matter and nitrogen intakes on fecal nitrogen losses in cattle.

Dietary factors influencing loss of fecal nitrogen in Holstein steers have been examined in data on metabolism from forage diets. Two factors- total nitrogen and dry matter intake, accounted for a large part of the variation in fecal nitrogen. Nitrogen intak expressed as grams per kilogram dry matter intake more accurately accounted for variability in total fecal nitrogen than did nitrogen intake expressed as grams per animal per day. Data were from trials on 68 forages covering a range of nitrogen intakes and of nitrogen concentration in dry matter of forage. Correlations were .93 between total fecal nitrogen and dry matter intake, .90 between total fecal nitrogen and total nitrogen intake, and .95 between total fecal nitrogen and total fecal dry matter. However, at high and low nitrogen intake there was evidence of departure from a linear relationship. The relative usefulness of three regression analyses to estimate metabolic fecal nitrogen is discussed.

Animal Feed↗

Can the foliar nitrogen concentration of upland vegetation be used for predicting atmospheric nitrogen deposition? Evidence from field surveys.

The deposition of atmospheric nitrogen can be enhanced at high altitude sites as a consequence of cloud droplet deposition and orographic enhancement of wet deposition on hills. The degree to which the increased deposition of nitrogen influences foliar nitrogen concentration in a range of upland plant species was studied in a series of field surveys in northern Britain. A range of upland plant species sampled along altitudinal transects at sites of known atmospheric nitrogen deposition showed marked increases in foliar nitrogen concentration with increasing nitrogen deposition and altitude (and hence with decreasing temperature). For Nardus stricta L., Deschampsia flexuosa (L.) Trin., Calluna vulgaris (L.) Hull, Erica cinerea L. and Hylocomium splendens (Hedw.) Br. Eur. on an unpolluted hill, foliar nitrogen increased by 0.07, 0.12, 0.15, 0.08 and 0.04% dry weight respectively for each 1 kg ha(-1) year(-1) increase in nitrogen deposition. Most species showed an approximately linear relationship between foliar nitrogen concentration and altitude but no trend with altitude for foliar phosphorus concentration. This provided evidence that the tissue nutrient status of upland plants reflects nutrient availability rather than the direct effects of climate on growth. However, differences in the relationship between foliar nitrogen concentration and atmospheric nitrogen deposition for N. stricta sampled on hills in contrasting pollution climates show that the possibility of temperature-mediated growth effects on foliar nitrogen concentration should not be ignored. Thus, there is potential to use upland plant species as biomonitors of nitrogen deposition, but the response of different species to nitrogen addition, in combination with climatic effects on growth, must be well characterised.

Journal Article↗

Abomasal nitrogen flow affects the relationship between dietary nitrogen and insulin-like growth factor-I in growing lambs.

Twelve abomasally cannulated wether lambs were fed isocaloric diets containing 9, 12 or 15% crude protein to determine insulin-like growth factor-I (IGF-I) responses to altered abomasal nitrogen flow and nitrogen status. Lambs were offered 1100 g/d of their respective diets. Voluntary feed consumption was not affected by nitrogen intake. Ruminal and total tract digestibilities of dry matter, organic matter and nitrogen increased linearly (P < 0.05) with increased dietary nitrogen. Abomasal flows of total, bacterial and rumen escape nitrogen increased (linear, P < 0.01), whereas dry matter and organic matter flows decreased (linear, P < 0.01). Total amino acid flow was greater (linear, P < 0.01) in lambs fed additional nitrogen due to increased (linear, P < 0.01) flows of essential and nonessential amino acids. Nitrogen retention and blood urea nitrogen increased linearly (P < 0.01). Serum IGF-I concentrations and relative hybridization intensity of hepatic IGF-I mRNA increased (linear, P < 0.05) as lambs consumed more nitrogen. Serum IGF-I and hepatic IGF-I mRNA were correlated positively (P < 0.05) with nitrogen intake and abomasal flows of nitrogen and various amino acids. These data provide evidence of a relationship between abomasal amino acid flow, as influenced by nitrogen intake, and hepatic gene expression and serum concentrations of IGF-I in growing lambs.

Abomasum↗

Nitrogen partitioning in Heterorhabditis bacteriophora-infected hosts and the effects of nitrogen on attraction/repulsion.

Entomopathogenic nematode behavior is affected by the condition of their infected hosts. We hypothesized that nitrogen compounds released from infected hosts may be one factor affecting entomopathogenic nematode host-finding and infection behaviors. Our objectives were to (1) investigate the partitioning of nitrogen in Galleria mellonella (L.) infected by Heterorhabditis bacteriophora Poinar and (2) determine attraction and repulsion of H. bacteriophora to various quantities of nitrogen (ammonia). Volatile (ammonia), organic, and inorganic nitrogen forms were monitored during the course of infection. Approximately 0.052 mg of nitrogen was released from a single infected host as volatile ammonia. Most of the ammonia release was detected within the first 3 days postinoculation. Organic nitrogen increased during the course of infection, whereas inorganic nitrogen decreased. The net nitrogen change in the infected host consisted of a loss of approximately 47 mg, most of which was lost within the 1st week of infection. Accelerated loss of nitrogen early in the infection process was likely correlated with activity and growth of bacterial symbionts. Increased organic nitrogen was likely associated with nematode reproduction within the host. Attraction or repulsion of H. bacteriophora to nitrogen (ammonium hydroxide) was measured on agar quadrant plates. Nematodes were attracted to 16 and 160 microg of nitrogen and repelled by concentrations of 1600 and 8000 microg. Our data indicate that nitrogen released from H. bacteriophora-infected hosts attracts nematodes at lower levels (early in the infection) and repels them at higher concentrations (later in the infection process).

Animals↗

[Effect of the ruminal amino nitrogen level on the nitrogen passage into the isolated rumen of sheep].

Eight trials were performed with two sheep to study the passage of ammonia nitrogen, urea nitrogen, and amino nitrogen into an isolated rumen at different amino nitrogen levels in rumen after a single intraruminal application of enzymatic casein hydrolyzate. After casein hydrolyzate application the level of amino nitrogen in rumen increased; consequently, the passage of ammonia to the isolated rumen rapidly decreased. The passage of nitrogen as urea and amino nitrogen to the isolated rumen is the same, both at a higher and a lower level of amino nitrogen in the rumen. The total quantity of urea and ammonia nitrogen (N-NH3 + N-urea) and amino nitrogen (N-NH3 + N-urea + amino-N) present in the isolated rumen shows a highly significant correlation with ammonia passage. This quantity reaches its maximum before the application of casein hydrolyzate to the rumen. An intensive drop occurs within one and two hours after application. The results of our study testify to the fact that the chemical composition of rumen content plays an important role in the ruminohepatic circulation of nitrogen, particularly when endogenic nitrogen passes into the rumen through rumen wall, and that the passage of nitrogen compounds from blood to the rumen is influenced not only by the concentration ratio between blood and rumen content but also by neurohumoral effects.

Amino Acids↗

Effects of nitrogen allocation and photosynthetic proteins response in peanut leaves on photosynthesis under conditions of water scarcity and nitrogen deficiency.

Leaf nitrogen allocation and photosynthetic proteins response can affect net photosynthetic rate (Pn), ultimately influencing crop yield under diverse environmental stresses. However, the internal relationship between Pn with leaf nitrogen allocation and photosynthetic proteins response under nitrogen or water scarcity in peanut (Arachis hypogaea L.) remains elusive. Here, comprehensive physiological property and proteomic analyses of peanut were conducted, revealing that both nitrogen and water scarcity remarkably impeded leaf growth and reduced Pn. Nitrogen deficiency significantly reduced the total nitrogen content per unit leaf area (Narea), chlorophyll content, and Pn, whereas drought stress caused a greater decline in photosynthetic nitrogen use efficiency (PNUE). The allocation of leaf nitrogen to photosynthetic components, including the carboxylation system and electron transport system in leaves, was significantly reduced when subjected to individual or combined deficiency. Proteomic analyses exhibited that several key photosynthetic proteins underwent a decrease under both single and combined water and nitrogen deficiency conditions. Thereby, Pn may decline due to the disruption of nitrogen allocation and down-regulated expression of photosynthetic proteins under these stress conditions. Our findings establish a benchmark for future research exploring the roles of leaf nitrogen allocation and photosynthetic proteins in the plant's response to nitrogen or water deficiency.

Nitrogen↗