Nitrate Metabolism Research Articles

PSXIV-16 Comparative in vitro effects of spray-dried and freshly-harvested Paenibacillus fortis strain 79R4 on rumen methane, nitrate, nitrite and ammonia metabolism

Abstract Nitrate supplementation into the ruminant diet can decrease ruminal methane emissions, but amounts needed to achieve appreciable decreases can risk ruminal accumulations of nitrite with potential for methemoglobinemia. A denitrifying rumen Paenibacillus fortis strain 79R4 (79R4) selected for enhanced nitrite-metabolizing ability has shown promise as a probiotic to decrease risks of nitrite toxicosis. Presently, a spray-dried prototype of this spore-forming, facultative anaerobe was tested during anaerobic culture (10 mL/tube) of rumen fluid freshly-collected from an alfalfa hay-fed cannulated Jersey cow. Cultures supplemented with 22 mM sodium nitrate and without or with inoculations of freshly-harvested (FH) cells or spray dried (SP) 79R4 spores (3 tubes/treatment; 108 cells/spores per tube) were cultured anaerobically (39oC for 24 h with 100% CO2). The FH- and SP- cells were grown aerobic, 72 h in tryptic soy broth, SP- cells were then processed and spray-dried. Nitrate-supplementation decreased (P = 0.0006; SEM = 0.31) methane production by the cultures, but this decrease was unaffected by 79R4 inoculation (2.57 µmol CH4/mL with no nitrate/no inoculum versus 0.15 µmol CH4/mL with nitrate/inoculum). Nitrate-metabolizing activity in nitrate-treated cultures were unaffected by 79R4 inoculations (P = 0.17; SEM = 0.15), rates being 0.95, 0.63 and 0.50 µmol nitrate/mL h-1 FH-, SP- and non-inoculated cultures, respectively. Nitrite accumulation rates (P = 0.10; SEM = 0.06) and peak nitrite concentrations (P = 0.06; SEM = 0.75) tended to be lower in SP- than in FH- and non-inoculated cultures (0.29, 0.47 and 0.47 µmol nitrite/mL h-1 and 3.33, 5.67 and 5.66 µmol/mL, respectively). Rates of ammonia accumulation were more rapid (P = 0.01; SEM = 0.01) in SP- and FH- than in non-inoculated cultures (0.16 and 0.15 versus 0.06 µmol/mL h-1, respectively). Results provide evidence that 79R4 prototype may aid rumen populations in detoxifying nitrite, therefore enhancing the abilities of high nitrate diets.

Nitrate Metabolism Decreases the Steroidal Alcohol Byproduct Compared with Ammonium in Biotransformation of Phytosterol to Androstenedione by Mycobacterium neoaurum.

In biotransformation of phytosterol to 4-androstene-3,17-dione (AD) by Mycobacterium, the steroidal alcohol (such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one, HBC) was a main byproduct. To weaken the accumulation of this byproduct in sterol biotransformation, ammonium was substituted by nitrate as nitrogen resource. The nitrate was utilized by Mycobacterium and led to metabolic flux shift towards AD production. The ratio of AD/HBC increased maximally from 2.1 to 5.5 and AD production increased correspondently. In the meanwhile, the nitrate metabolism resulted in the decreased intracellular redox level (NADH/NAD+) maximally by 59.5% and a slight descent tendency with the increase of the nitrate concentrations. It indicated that the nitrate utilization effectively decreased the steroidal alcohol production by regulating intracellular redox level in sterol biotransformation. These results gave an insight into the mechanism of the steroidal alcohol formation in sterol biodegradation and provided a simple strategy to regulate the metabolic distribution.

Nitrate uptake and metabolism in human skeletal muscle cell cultures

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Dietary nitrate metabolism and enteric methane mitigation in sheep consuming a protein-deficient diet

It was hypothesised that the inclusion of nitrate (NO3–) or cysteamine hydrochloride (CSH) in a protein deficient diet (4.8% crude protein; CP) would improve the productivity of sheep while reducing enteric methane (CH4) emissions. A complete randomised designed experiment was conducted with yearling Merino sheep (n = 24) consuming a protein-deficient wheaten chaff control diet (CON) alone or supplemented with 1.8% nitrate (NO3–; DM basis), 0.098% urea (Ur, DM basis) or 80 mg cysteamine hydrochloride/kg liveweight (CSH). Feed intake, CH4 emissions, volatile fatty acids (VFA), digesta kinetics and NO3–, nitrite (NO2–) and urea concentrations in plasma, saliva and urine samples were measured. There was no dietary effect on animal performance or digesta kinetics (P > 0.05), but adding NO3– to the CON diet reduced methane yield (MY) by 26% (P = 0.01). Nitrate supplementation increased blood MetHb, plasma NO3– and NO2– concentrations (P < 0.05), but there was no indication of NO2– toxicity. Overall, salivary NO3– concentration was greater than plasma NO3– (P < 0.05), indicating that NO3– was concentrated into saliva. Our results confirm the role of NO3– as an effective additive to reduce CH4 emissions, even in a highly protein-deficient diet and as a source of additional nitrogen (N) for microbial protein synthesis via N-recycling into saliva and the gut. The role of CSH as an additive in low quality diets for improving animal performance and reducing CH4 emissions is still unclear.

Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise.

Rodent skeletal muscle contains a large store of nitrate that can be augmented by the consumption of dietary nitrate. This muscle nitrate reservoir has been found to be an important source of nitrite and nitric oxide (NO) via its reduction by tissue xanthine oxidoreductase. To explore if this pathway is also active in human skeletal muscle during exercise, and if it is sensitive to local nitrate availability, we assessed exercise-induced changes in muscle nitrate and nitrite concentrations in young healthy humans, under baseline conditions and following dietary nitrate consumption. We found that baseline nitrate and nitrite concentrations were far higher in muscle than in plasma (~4-fold and ~29-fold, respectively), and that the consumption of a single bolus of dietary nitrate (12.8 mmol) significantly elevated nitrate concentration in both plasma (~19-fold) and muscle (~5-fold). Consistent with these observations, and with previous suggestions of active muscle nitrate transport, we present western blot data to show significant expression of the active nitrate/nitrite transporter sialin in human skeletal muscle. Furthermore, we report an exercise-induced reduction in human muscle nitrate concentration (by ~39%), but only in the presence of an increased muscle nitrate store. Our results indicate that human skeletal muscle nitrate stores are sensitive to dietary nitrate intake and may contribute to NO generation during exercise. Together, these findings suggest that skeletal muscle plays an important role in the transport, storage and metabolism of nitrate in humans.

Hybrid cluster proteins in a photosynthetic microalga.

Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron-sulfur-oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox-titrated samples. Both proteins contain a [4Fe-4S]-cluster as well as a [4Fe-2O-2S]-hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry-based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S-nitrosylation in bacteria.

Effects of Blue and Red Light On Growth And Nitrate Metabolism In Pakchoi

AbstractThis study investigated the effects of blue and red light on metabolites of nitrate, key enzymes, and the gene expression of key enzymes in pakchoi plants (Brassica campestris L. var. Suzhouqing). Plants were grown under three light quality treatments, namely, white light (W), red light (R) and blue light (B), at the same photosynthetic photon flux density (PPFD) of approximately 150 μmol m-2 s-1 for 48 hours of continuous illumination, and W was set as the control. The dynamics of net photosynthetic rate in pakchoi subjected to different light treatments were the same as the total chlorophyll contents: blue light > white light > red light. The nitrate reductase (NR) activity, nitrite reductase (NiR) activity, glutamine synthetase (GS) activity and glutamate synthase (GOGAT) activity were highest under blue light. Further, the expression levels of NR, NiR and GS genes were significantly higher under blue light. Under continuous illumination, the auxin content (IAA) in pakchoi leaves was highest under blue light, whereas the abscisic acid (ABA) content was highest under red light. In contrast, there was no significant effect for gibberellin (GA) under any type of light treatment.

Interaction of cyanate uptake by rice seedlings with nitrate assimilation: gene expression analysis.

Cyanate (CNO-) has been produced in the environment through either natural or anthropogenic sources. However, due to industrialization, it has been led more over-loads. In this study, interaction of CNO- uptake by rice seedlings with nitrate assimilation was investigated using gene expression analysis after an acute phytotoxicity assay. Our results showed that CNO- exposure caused inhibition on relative growth rates of plants. CNO- analysis demonstrated that rice seedlings had higher potential for CNO- uptake and the removal rates showed a zero-order kinetic. PCR analysis exposed that OsCYN transcript was not significantly induced by CNO- treatments in rice tissues and CNO- exposure also repressed gene expression of the collaborative enzyme carbonic anhydrase (CA), suggesting that assimilation of CNO- initiated by the enzyme cyanase (CYN) in rice seedlings was an enzyme-limitation reaction. Gene expression of other enzymes involved in nitrate metabolism was tissue-specific under CNO- exposure, suggesting that rice seedlings were able to trigger its intrinsic regulative and responsive mechanisms to cope up with uneven N conditions. Significant upregulation of three OsGDH isogenes, except for OsGDH1 in roots, was detected in both rice materials with enhancing CNO- concentrations, suggesting that GDH may play a primary role to maintain the balance of C and N in plants under CNO- exposure. In conclusion, because the innate pool of CYN activity was non-sufficient to degrade exogenous CNO- by rice seedlings, CNO-derived ammonium only can serve as a supporting N source to support growth of rice seedling under non-effective doses of CNO- exposure.

Role of salicylic acid and hydrogen sulfide in promoting lead stress tolerance and regulating free amino acid composition in Zea mays L.

Lead toxicity is a threat to growth and production of crop plants. In this study, effect of salicylic acid and sodium hydrosulfide pretreatments was investigated on the alteration in the Pb accumulation, protein content, nitrate reductase activity and metabolism of selected free amino acids in maize plants subjected to lead stress. Maize seeds were soaked in 0.5 mM salicylic acid and sodium hydrosulfide for 12 h and then exposed to 2.5 mM Pb (NO3)2 for 9 days. Results demonstrated that lead stress significantly reduced the plant growth, shoot glutathione content and protein content, nitrate reductase activity in the shoots and roots while increased glutathione content in roots of maize plants. We also observed accumulation of most free amino acids composition except tyrosine and tryptophan under lead stress condition. Plants pre-treated with salicylic acid and sodium hydrosulfide exhibited improvement of plant growth, decrease of Pb accumulation, increase of protein and glutathione contents and nitrate reductase activity. Salicylic acid and sodium hydrosulfide pretreatments decreased most amino acids content in shoot of lead stressed plants. In case of roots, salicylic acid and sodium hydrosulfide had different effects on the content of amino acids. Pretreatment of sodium hydrosulfide resulted in greater levels of free amino acids in roots. These results indicate that moderate lead stress can be attributed to effect of salicylic acid and sodium hydrosulfide by improvement of nitrate reductase activity and glutathione content and regulation of amino acids metabolism.

Impact of Exogenous Silicate Amendments on Nitrogen Metabolism in Wheat Seedlings Subjected to Arsenate Stress

We intended to investigate the response of arsenate on nitrogen metabolism in wheat seedlings and aimed to assess the efficacy of silicon amendments in modulating the metabolic disturbances caused by arsenate stress. The nitrogen metabolism of wheat cultivated in different levels of arsenate with or without silicate in a medium supplemented with modified Hoagland’s solution for 21 days was studied. Experimental design was completely randomized with different arsenate concentrations (0, 25, 50 and 100 μM) with or without 5 mM silicate. Arsenate treatment decreased growth along with decline in nitrate (NO3−) uptake and accumulation. Activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS) as well as glutamate synthase (GOGAT) were lowered in the test seedlings. Decline in nitrite (NO2−) and amino acid contents were also evident along with an enhancement in the accumulation of toxic ammonia. Silicate supplementation under arsenate stress however, improved growth, repaired the arsenate-induced effects leading to an enhancement in nitrate (NO3−) uptake and consequently improved nitrite (NO2−) and amino acid contents as well. The total and soluble nitrogen contents were enhanced along with enhancements in activities of enzymes associated with nitrate metabolism while ammonia accumulation was lowered. Results therefore, imply the involvement of exogenous silicon amendments in relieving the metabolic alterations in nitrogen metabolism caused by arsenate stress that enabled wheat seedlings to adapt under arsenate excess and eventually promoted plant growth.

Comparative study of Cu uptake and early transcriptome responses in the green microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii

Microalgae are widely used as representative primary producers in ecotoxicology, while macrophytes are much less studied. Here we compared the bioavailability and cellular toxicity pathways of 2 h-exposure to 10−6 mol L−1 Cu in the macrophyte Elodea nuttallii and the green microalga Chlamydomonas reinhardtii.Uptake rate was similar but faster in the algae than in the macrophyte, while RNA-Sequencing revealed a similar number of regulated genes. Early-regulated genes were congruent with expected adverse outcome pathways for Cu with Gene Ontology terms including gene regulation, energy metabolism, transport, cell processes, stress, antioxidant metabolism and development. However, the gene regulation level was higher in E. nuttallii than in C. reinhardtii and several categories were more represented in the macrophyte than in the microalga. Moreover, several categories including oxidative pentose phosphate pathway (OPP), nitrate metabolism and metal handling were only found for E. nuttallii, whereas categories such as cell motility, polyamine metabolism, mitochondrial electron transport and tricarboxylic acid cycle (TCA) were unique to C. reinhardtii. These differences were attributed to morphological and metabolic differences and highlighted dissimilarities between a sessile and a mobile species. Our results highlight the efficiency of transcriptomics to assess early molecular responses in biota, and the importance of studying more aquatic plants for a better understanding on the impact and fate of environmental contaminants.

The Molecular Mechanism of Nitrate Chemotaxis via Direct Ligand Binding to the PilJ Domain of McpN.

Chemotaxis and energy taxis permit directed bacterial movements in gradients of environmental cues. Nitrate is a final electron acceptor for anaerobic respiration and can also serve as a nitrogen source for aerobic growth. Previous studies indicated that bacterial nitrate taxis is mediated by energy taxis mechanisms, which are based on the cytosolic detection of consequences of nitrate metabolism. Here we show that Pseudomonas aeruginosa PAO1 mediates nitrate chemotaxis on the basis of specific nitrate sensing by the periplasmic PilJ domain of the PA2788/McpN chemoreceptor. The presence of nitrate reduced mcpN transcript levels, and McpN-mediated taxis occurred only under nitrate starvation conditions. In contrast to the NarX and NarQ sensor kinases, McpN bound nitrate specifically and showed no affinity for other ligands such as nitrite. We report the three-dimensional structure of the McpN ligand binding domain (LBD) at 1.3-Å resolution in complex with nitrate. Although structurally similar to 4-helix bundle domains, the ligand binding mode differs since a single nitrate molecule is bound to a site on the dimer symmetry axis. As for 4-helix bundle domains, ligand binding stabilized the McpN-LBD dimer. McpN homologues showed a wide phylogenetic distribution, indicating that nitrate chemotaxis is a widespread phenotype. These homologues were particularly abundant in bacteria that couple sulfide/sulfur oxidation with nitrate reduction. This work expands the range of known chemotaxis effectors and forms the basis for the exploration of nitrate chemotaxis in other bacteria and for the study of its physiological role.IMPORTANCE Nitrate is of central importance in bacterial physiology. Previous studies indicated that movements toward nitrate are due to energy taxis, which is based on the cytosolic sensing of consequences of nitrate metabolism. Here we present the first report on nitrate chemotaxis. This process is initiated by specific nitrate binding to the periplasmic ligand binding domain (LBD) of McpN. Nitrate chemotaxis is highly regulated and occurred only under nitrate starvation conditions, which is helpful information to explore nitrate chemotaxis in other bacteria. We present the three-dimensional structure of the McpN-LBD in complex with nitrate, which is the first structure of a chemoreceptor PilJ-type domain. This structure reveals striking similarities to that of the abundant 4-helix bundle domain but employs a different sensing mechanism. Since McpN homologues show a wide phylogenetic distribution, nitrate chemotaxis is likely a widespread phenomenon with importance for the life cycle of ecologically diverse bacteria.

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.

Interactive effect of nitrogen nutrition, nitrate reduction and seasonal variation on oxalate synthesis in leaves of Napier-bajra hybrid (Pennisetum purpureum × P. glaucum)

Oxalate may cause hypocalcaemia or formation of urinary calculi in animals with prolonged grazing of Napier grass (Pennisetum purpureum) × pearl millet (bajra, P. glaucum) hybrid (NBH). We investigated the influence of nitrate metabolism, nitrogen (N) nutrition, N forms and seasonal variation on oxalate accumulation in leaves of NBH in a field experiment in Ludhiana, India. The experiment was a randomised block design with three N sources (nitrate, amide and ammonium), three application rates (50, 75 and 100 kg N/ha), four seasons (summer, monsoon, autumn, pre-winter) and three replicates. Applied N nutrition induced oxalate synthesis and activities of nitrate reductase (NR) and nitrite reductase (NiR) enzymes. A positive association of N nutrition with both oxalate accumulation and nitrate-reducing enzymes was found. Nitrate-N increased oxalate accumulation and NiR activity more than ammonium and amide. A differential effect of seasons on NR and NiR activities, as well as on oxalate accumulation, was observed. Among different harvest seasons, NR and NiR activities were positively associated with oxalate accumulation in summer and the monsoon season. These results suggest that N fertilisation, particularly in nitrate form, is associated with upregulation of nitrate-reducing enzymes, leading to oxalate accumulation in NBH leaves.

Trichoderma asperellum T42 induces local defense against Xanthomonas oryzae pv. oryzae under nitrate and ammonium nutrients in tobacco.

Trichoderma has been explored and found to play a vital role in the defense mechanism of plants. However, its effects on host disease management in the presence of N nutrients remains elusive. The present study aimed to assess the latent effects of Trichoderma asperellum T42 on oxidative burst-mediated defense mechanisms against Xanthomonas oryzae pv. oryzae (Xoo) in tobacco plants fed 10 mM NO3− and 3 mM NH4+ nutrients. The nitrate-fed tobacco plants displayed an increased HR when Xoo infected, which was enhanced in the Trichoderma-treated plants. This mechanism was enhanced by the involvement of Trichoderma, which elicited NO production and enhanced the expression pattern of NO-modulating genes (NR, NOA and ARC). The real-time NO fluorescence intensity was alleviated in the NH4+-fed tobacco plants compared to that fed NO3− nutrient, suggesting the significant role of Trichoderma-elicited NO. The nitrite content and NR activity demonstration further confirmed that nitrate metabolism led to NO generation. The production of ROS (H2O2) in the plant leaves well-corroborated that the disease resistance was mediated through the oxidative burst mechanism. Nitrate application resulted in greater ROS production compared to NH4+ nutrient after Xoo infection at 12 h post-infection (hpi). Additionally, the mechanism of enhanced plant defense under NO3− and NH4+ nutrients mediated by Trichoderma involved NO, ROS production and induction of PR1a MEK3 and antioxidant enzyme transcription level. Moreover, the use of sodium nitroprusside (100 μM) with Xoo suspension in the leaves matched the disease resistance mediated via NO burst. Altogether, this study provides novel insights into the fundamental mechanism behind the role of Trichoderma in the activation of plant defense against non-host pathogens under N nutrients.

Precise nitrogen topdressing upregulates nitrogen metabolism and improves soybean (Glycine max) grain yield

Soybean (Glycine max. (L.) Merr.) is a symbiotic nitrogen-fixing crop. In order to increase grain yield, it is important to know how soybean plants respond to nitrogen topdressing for the improvement of nitrogen utilisation. We used two soybean cultivars with different grain yield potentials and applied 13 nitrogen topdressing treatments to determine optimal topdressing time and nitrogen metabolism. Nitrogen treatments included a base fertiliser and single topdressings at different times, in 10-day intervals from 10 to 120 days after emergence (DAE). Among the nitrogen treatments, the optimal times for topdressing were at 40 DAE or 90 DAE to increase grain yield, and both soybean cultivars also had higher nitrate reductase (NR) and glutamine synthetase (GS) activities with topdressing at these times. Higher expression of the NR2 gene was associated with upregulated NR activity in leaves of both cultivars at the early-mature stage. With topdressing at 90 DAE, higher GS1 expression and GS activity were found in the leaves of the higher yielding cultivar at the full-seed stage and the early-mature stage. With topdressing at 90 DAE, the higher yielding cultivar had a higher nitrate metabolism capacity at the late reproductive stages than the lower (common) yielding cultivar.

Nitrate reductase activity in high-mountain plants: a test across species, growth form and habitat type

For many terrestrial plants, nitrate is the most important form of available soil nitrogen for growth. However, many plant species, which grow on acidic, ammonium-dominated soils, exhibit a constantly low level of nitrate reductase activity (NRA). Little is known about NRA in high-mountain vascular plants in similar conditions. We tested the hypothesis that high-mountain vascular plants in acidic and ammonium-dominated habitats have low levels of NRA. Twenty-six plant species of the families Asteraceae, Ericaceae, Poaceae, Polygonaceae, Salicaceae, Pinaceae, Ranunculaceae, Woodsiaceae, Cyperaceae, Juncaceae, Rosaceae and Urticaceae representing different growth forms were investigated in seven native and anthropogenic habitats of subalpine and alpine belts of the Karkonosze (Hercynian middle-mountains, Central Europe), with respect to leaf NRA and mineral nitrogen forms in soil. NRA was measured by an in vivo assay for the first time directly in the field using portable water bath. An NRA study of vascular plants from high mountains is presented for the first time, and most of the studied subalpine and alpine species were subjected to field measurements for the first time ever. Differences among species, families, growth forms and habitats for NRA were found. These differences reflected mainly the taxonomical position and in part ecological preferences. PERMANOVA analysis confirmed that variance component showed that enzyme activities were mostly explained by plant species and habitat. Overall, the subalpine and alpine species from their native habitats are characterized by very low and low ability for nitrate metabolism. We also compared NRA with Ellenberg’s N values for the studied plants. Using a regression equation, N values of some species were calculated for the first time and corrected for the others.

310 - Urinary nitrates in relation to glomerular filtrate rate (GFR) and albumin-creatinine rate (ACR)

Background Intervention studies have shown that dietary nitrate supplementation significantly increased circulating nitrates and inhibited platelet aggregation, improved vascular function and lowered blood pressure. Thus, studying physiological functions that are associated with circulating nitrates may help us to identify a population who are likely to have lower circulating nitrates and may need supplementation. Circulating nitrates reflect cumulative influences of production of nitric oxide and nitrate metabolism. Animal studies have shown that the production of nitric oxide (NO) is reduced in chronic kidney disease (CKD) and is associated with the progression of CKD. Further, nitrate metabolism can be significantly influenced by gut microbiome composition. Compared to people with normal renal function, individuals with reduced renal function have different gut microbiome composition. Until now, no studies have examined the association between circulating nitrates and renal function among healthy individuals who have not been diagnosed with CKD. Glomerular filtrate rate (GFR) and albumin-creatinine rate (ACR) have been used to assess renal function. Our study aimed to determine if there was an association between urinary nitrates and GFR or ACR. Methods Using a cross-sectional design, we analyzed data obtained from 5,071 adults, ages 18-85, enrolled in the 2005-2006 National Health and Nutrition Examination Survey (NHANES). Demographic data were obtained and urinary nitrates, creatinine, GFR and ACR ratio were assessed among these individuals. We classified GFR into three categories: ≥90 mL/min/1.73 m2 (normal renal function), 60-90 mL/min/1.73 m2 (renal insufficiency) and Results We have found that urinary nitrates were inversely associated with renal function.

229 - Red meat, white meat and vegetable intakes and circulating nitrates

Background Intervention studies have shown that dietary nitrate supplementation significantly increased circulating nitrates and inhibited platelet aggregation, improved vascular function and lowered blood pressure. Thus, studying factors that may influence circulating nitrates will help us design strategies to prevent or improve these conditions associated with chronic diseases. Nitrate metabolism in humans are significantly influenced by oral and gut microbiome composition. Meat and vegetable intakes can influence human microbiome composition; thus, determine the associations between consumption of these foods and circulating nitrates may shed light on the interrelationship between meat, nitrate and microbiomes. Objective Determine the associations between red, white meat and vegetable consumption and circulating nitrates in two large observational studies. Methods Using a cross-sectional design, we analyzed data collected from 5,058 adults (men and women) in the National Health and Nutrition Examination Survey (NHANES) and from 1,260 men in the Health Professionals Follow-up Study (HPFS). The non-quantitative food frequency questionnaire (NFFQ) was collected, and urinary nitrates were measured in the NHANES. Plasma nitrates were measured, and a semi-quantitative FFQ (SFFQ) was collected in the HPFS. Results We found a similar trend when assessing the associations of vegetables and red meat with circulating (plasma and urinary) nitrate across two studies (NHANES and HPFS). Both urinary and plasma nitrates were positively associated with vegetables (p Conclusion Our results suggest that increased consumption of vegetable intakes are associated with increased circulating nitrates and increased consumption of processed and unprocessed red meat are associated with reduced circulating nitrates. Our study prompts new studies to determine the interrelationship between red meat, white meat, microbiome and circulating

LED lighting for reduced nitrate contents in green vegetables

Green leafy vegetables occupy a very important place in the human diet, however, they also represent a group of plants that tend to accumulate nitrates in their tissues. In recent years, nitrate problems have been revived in closed environment intense cultivation systems. In such systems, it is possible to control nitrate metabolism by tailoring controllable environmental parameters. In this study we review the effects of light-emitting diode (LED) lighting spectrum and photosynthetic photon flux density (PPFD) on nitrate assimilation in green vegetables. Experiments were performed in closed environment growth chambers (21/17°C day/night temperature, ~65% relative humidity). Green and red leaf lettuces (Lactuca sativa ‘Lobjoits green cos’ and ‘Red cos’) and tatsoi (Brassica rapa var. rosularis ‘Rosetto’ F(1)) were cultivated under combinations of red (660, 640 nm), blue (445 nm) and far-red (731 nm) LED lighting under a PPFD of 100-300 µmol m(‑2) s(‑1). The activities of nitrate, nitrite, nitrate reductase and nitrite reductase, as well as total protein content were evaluated. Both spectral differences and light intensity have remarkable, species-specific effects on nitrate contents and activities of reducing enzymes.