Influence of dietary protein and gut microflora on endogenous synthesis of nitrate and N-nitrosamines in the rat.

Groups of four germ-free (GF) and conventional (CV) rats were given purified diets containing either 50 or 200 g lactalbumin/kg for 2 wk and their urinary excretion of nitrate was measured. Urinary excretion of N-nitrosoproline was also measured in one of the three experiments. Both GF and CV rats given the high-protein diet excreted significantly more nitrate and N-nitrosoproline than those given the low-protein diet. On both diets GF rats excreted more nitrate than their CV counterparts but N-nitrosoproline excretion was not affected by environment. Groups of 11 GF and CV rats given diets containing sesame meal with or without a supplement of lysine-HCl for 2 wk, excreted similar amounts of nitrate on both diets, but more nitrate was excreted by GF rats than by their CV counterparts. N-nitrosoproline excretion by rats given the lysine supplement was higher in both environments. It is concluded that endogenous synthesis of nitrate is mediated by mammalian tissues rather than microflora and that dietary protein is an important source of nitrogen for the synthesis, although surplus amino acids from an imbalanced protein source do not act as precursors of endogenously formed nitrate. Some of the synthesized nitrate or its precursors appears to be metabolized by the microflora in the CV rat.

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma.

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.

Pathway of oxidation of pyruvic oxime by a heterotrophic nitrifier of the genus Alcaligenes: evidence against nitroethane as an intermediate.

The role of nitroethane as an intermediate in the oxidation of pyruvic oxime to nitrate by an Alcaligenes sp. was examined. Unlike pyruvic oxime, which serves as a sole source of C and N for the bacterium, nitroethane was incapable of supporting the growth of the microbe. Nitroethane was metabolized and diauxic growth did occur, however, if the nitroethane medium was amended with yeast extract. Alcaligenes sp. resting cells and cell-free extracts were prepared from nitroethane-yeast extract grown cultures and the maximum rate of nitrite synthesis when nitroethane was the substrate was 6.8 nmol min-1 mg cell protein-1, a 10-fold lower rate than that previously noted for pyruvic oxime oxidation. These cell-free extracts were unable to metabolize pyruvic oxime. Resting cells and cell-free extracts prepared from Alcaligenes sp. cells grown in a pyruvic oxime medium were, conversely, incapable of metabolizing nitroethane. Collectively, these results indicate that nitroethane is not an intermediate in the pathway of pyruvic oxime oxidation and that two separate enzyme systems exist in the Alcaligenes sp. for the metabolism of pyruvic oxime and nitroethane.

Nitrate, nitrite balance, and de novo synthesis of nitrate in humans consuming cured meats.

Nitrate balance was measured in nine men consuming a fixed daily diet with constant nitrate (570 mumol/day), varying nitrite (18 to 150 mumol/day), and varying erythorbate levels. Nitrite and erythorbate were added to meat that was then cured and frozen until consumption. All diets were consumed by each subject for 17 days each. Average daily urinary nitrate excretion ranged from 959 to 2382 mumol/day. Subjects excreted significantly more nitrate in urine when fed nitrite cured meats with or without erythorbate than when fed uncured meat (1617 and 1577 versus 1430 mumol nitrate/day, respectively). The amount of nitrate excreted in urine consistently exceeded intakes of nitrate and nitrite by an average 870 mumol/day. This excess represented endogenous synthesis by subjects and was not due to unmeasured nitrate in the diet. The commonly used Greiss and xylenol procedures were unable to measure all nitrate in urine and in diets when compared to high performance liquid chromatographic analyses. The Greiss and xylenol analyses underestimated nitrate synthesis by 220 and 150 mumol/day, respectively when results were not adjusted by determining recovery of added nitrate.

Nitrate biosynthesis in the rat. Precursor-product relationships with respect to ammonia.

The endogenous biosynthesis of nitrate in rats was investigated by using 15NH3 administered as a continuous intravenous infusion for as long as 96 h. A comparison of the enrichment of 15N in urinary nitrate after a 24 h infusion revealed that it was 36% of the enrichment of plasma NH3 and about 50% of the enrichment of plasma urea and urinary NH3. Continuous infusion of 15NH3 for 96 h showed that a plateau for the incorporation of NH3 into nitrate is reached by 24 h, whereas the enrichment of urinary NH3 and urea increase during the 96 h. After the infusion of progressively larger doses of 15NH3, the concentration of nitrate synthesized de novo increased. Although there was a significant correlation between plasma 15NH3 concentration and 15NO3- appearance, a given change in plasma NH3 concentration does not produce a direct proportional change in nitrate synthesis. Our findings indicate that NH3 is a quantitatively significant nitrogen precursor for nitrate, but that approx. 50% of nitrate nitrogen is derived from other, as yet unidentified, sources.

Nitrate balance and biosynthesis in the ferret.

The net biosynthesis of nitrate in the ferret was demonstrated to be 8.89 to 10.3 mumol/kg bw/day. Nitrate balance studies indicated that, because of metabolism, excretion of nitrate was lower than ingestion when oral doses were higher than 6.3 mumol/day. At nitrate ingestion levels of less than 6.3 mumol/day, excretion exceeded intake. Studies with 15N-labeled nitrate indicated that only 36% of the oral dose was recoverable as [15N]nitrate from urine. Oral doses of 15N-labeled ammonia resulted in the incorporation of the 15N label into nitrate in urine and feces. This demonstrates that ammonia can serve as a precursor to biosynthesized nitrate. An increase in nitrate excretion was seen when animals were treated with neomycin sulfate and mycostatin to reduce gut microflora populations. This increase was as large as 200%. Short-term studies indicated that dietary protein quality had little effect on nitrate excretion.

Source of the oxygen atoms of nitrate in the oxidation of nitrite by Nitrobacter agilis and evidence against a P-O-N anhydride mechanism in oxidative phosphorylation.

15N, 18O Tracer studies were applied to the aerobic oxidation of nitrite to nitrate by the chemolithotrophic bacterium, Nitrobacter agilis. It was established that, in conversion of nitrite to nitrate, one oxygen atom of nitrate arose from water and none from O2 or inorganic phosphate. This result confirms that of Kumar et al. [(1983) FEBS Lett. 152, 71-74]. Oxygen exchange between water and inorganic phosphate was small and that between water and nitrite or nitrate or any reaction intermediates between these two was not detected. Oxidation of nitrite was, therefore, effectively irreversible under the conditions employed. The uptake of extracellular phosphate was sufficient to allow significant transfer of 18O from phosphate to nitrate if oxidative phosphorylation had occurred by way of a P-O-N anhydride between phosphate (or ADP) and nitrate. The results are, therefore, inconsistent with the occurrence of a reaction of this type during nitrite oxidation.

Use of nitrifying activity measurements for describing the effect of salinity on nitrification in the Scheldt estuary.

Nitrifying activity measurements, carried out on freshly collected samples from an estuarine environment, show that the composition of the nitrifier population undergoes a progressive modification during the mixing of freshwater masses in seawater, with increasing tolerance to salt. As a result, the overall effect of increasing salinity on nitrification is much less severe than the direct effect of salt on the freshwater nitrifying population.

[Nitrification in soil columns subjected to continuous flow]. / Nitrificación en columnas de suelo sometidas a flujo continuo.

Nitrification in columns of soil under continuous flow of substrate was studied. The soil was previously diluted with sterile sand (1 part of soil: 9 parts of sand; w : w) and admixed with 2% CaCO3. Soil columns, 15 cm high, were contained in glass cylinders with a cross sectional area of 39.6 cm2. In the main experiment, a soil column was subjected to the continuous flow of a KNO2 solution (100 ppm NO2- - N) at a flow rate of 46.0 cm3 h-1. An exponential increase of nitrate concentration in the column effluent was observed during the first 4 days (Fig. 1), suggesting an exponential growth of NO2- oxidizers in the soil column, with an apparent generation time of 1, 2 days. At the end of this phase, an almost complete conversion of nitrite to nitrate was reached followed by an important decrease in conversion and by a partial recovery with stabilization at 69 ppm NO3- - N in the column effluent. This phenomenon was presumably due to a rapid rate of O2 demand by the microorganisms which exceeded the supply, and to the subsequent adaptation of the NO2- oxidizers to a situation in which the O2 concentration was the limiting factor. At the end of the experiment, the average population density of NO2- oxidizers in the column was 1.5. 10(7) cells cm-3. In a preliminary experiment, a column of the same soil was continuously perfused with an (NH4)2SO4 solution with 140 ppm NH+4 - N, at a constant rate of 40.8 cm3 h-1 (Fig. 2.).(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrate biosynthesis in man.

Nitrate metabolism was investigated in long-term metabolic studies in healthy young men. Under conditions of constant low ingestion of nitrate (less than 180 mumol/day per subject), the amount of nitrate excreted in urine was an average of 4-fold greater than the amount ingested. Balance studies with 15NO3- showed that the source of the excess nitrate in urine was the endogenous biosynthesis of nitrate, rather than the emptying of a body pool. Nitrate biosynthesis occurred when nitrate ingestion was high as well as low, and the amounts synthesized appeared to be independent of intake and comparable to the amounts ingested from normal diets. Analysis of the 15NO3- data also revealed that half of ingested nitrate was recovered as urinary nitrate. Because nitrate in urine is the net result of (i) intake, (ii) endogenous synthesis, and (iii) metabolic losses, the magnitude of the losses is such that, despite ongoing synthesis, the amount of nitrate in the urine of people consuming most diets will be less than the amount ingested.

Nitrate synthesis in the germfree and conventional rat.

Metabolic balance studies show that germfree and conventional Sprague-Dawley rats synthesize nitrate. Equivalent results for germfree and conventional rats eliminate the microflora as obligatory components of nitrate production. Nitrate synthesis appears to be a mammalian process.

Effect of temperature on nitrification intensity in soil.

The temperature dependence of nitrification can be expressed by the Arrhenius equation while the time course of nitrate production can be expressed by the Gomperz function. These two findings served as a basis for a mathematical model which makes it possible to calculate nitrate production in the soil even when the temperature changes once or more times during the incubation.

Death associated with nitrite ingestion: report of a case.

The tissue concentrations of nitrite and nitrate found at autopsy of a case of intentional ingestion of nitrite salts have been reported. The percentage of methemoglobin and the serum nitrite concentrations are consistent with those reported for acute overdoses. We conclude that noth nitrite and nitrate salts may be identified in tissues from persons ingesting only nitrite salts and that a significant conversion to nitrate may result from oxidation of nitrite during the conversion of heme iron to Fe3+.

Method for in situ measurement of nitrification in a stream.

A method is described in which the oxidation of NH(3)-N to NO(2)-N and NO(3)-N in a stream was measured in situ by use of an equilibration chamber. The conversion was stoichiometric.

Autecological study of the chemoautotroph Nitrobacter by immunofluorescence.

Fluorescent antibodies (FA) prepared for Nitrobacter agilis and N. winogradskyi were highly reactive in homologous staining. Low-level cross-reactions between the two species were removed by adsorption. All 15 pure-culture isolates of Nitrobacter tested reacted strongly with either N. agilis FA or N. winogradskyi FA. All pure-culture isolates from soils were determined to be N. winogradskyi; those from Mammoth Cave sediments and a cattle waste oxidation ditch were N. agilis. No cross-reaction was found in extensive tests that included five isolates of Nitrosomonas europaea and 668 heterotrophic aerobic and anaerobic bacteria isolated from soil, sewage, and cave sites. The FA preparations were used to detect Nitrobacter species in Mammoth Cave sediments, in a cattle waste oxidation ditch, and in surface waters and sediments of a river and to observe that N. winogradskyi can outgrow N. agilis in enrichment culture.

Mechanism of nitrification by Arthrobacter sp.

Resting cells of Arthrobacter sp. excrete as much as 60 mug of hydroxylamine-nitrogen per ml when supplied with ammonium. An organic carbon source in abundant supply is necessary for the oxidation. Resting cells oxidize hydroxylamine to nitrite and 1-nitrosoethanol, the former accumulating only when an exogenous carbon source is available. Cell-free extracts contain an enzyme catalyzing the formation of hydroxylamine from acetohydroxamic acid, a hydroxylamine-nitrite oxido-reductase, and an enzyme producing nitrite and nitrate from various primary nitro compounds. Nitrite is not produced from hydroxylamine by the extracts, but 1-nitrosoethanol is formed from hydroxylamine in the presence of acetate. 1-Nitrosoethanol is also produced from acetohydroxamic acid by these preparations. Nitrite was formed from hydroxylamine, however, by extracellular enzymes excreted by the bacterium.

Heterotrophic nitrifiction by Arthrobacter sp.

Arthrobacter sp. isolated from sewage oxidized ammonium to hydroxylamine, a bound hydroxylamine compound, a hydroxamic acid, a substance presumed to be a primary nitro compound, nitrite, and nitrate. The concentration of free hydroxylamine-nitrogen reached 15 mug/ml. The identification of hydroxylamine was verified by mass spectrometric analysis of its benzophenone oxime derivative. The bound hydroxylamine was tentatively identified as 1-nitrosoethanol on the basis of its mass spectrum, chemical reactions, and infrared and ultraviolet spectra. Hydroxylamine formation by growing cells was relatively independent of pH, but the accumulation of nitrite was strongly favored in alkaline solutions. The formation of hydroxylamine but not nitrite was regulated by the carbon to nitrogen ratio of the medium. The hydroxamic acid was the dominant product of nitrification in iron-deficient media, but hydroxylamine, nitrite, and 1-nitrosoethanol formation was favored in iron-rich solutions. Heterotrophic nitrification by Arthrobacter sp. was not inhibited by several compounds at concentrations which totally inhibited autotrophic nitrification.