The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements.

We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 µmoles of 15N-ammonium, 1.8 to 420 µmoles of 15N-nitrate/nitrite, and ≥21 µmoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained ≥11 µmoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L(-1) d(-1), to 530 nmoles N L(-1) d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2 gas must be ensured prior to use in future N2 fixation rate determinations.

Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo.

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

PET Imaging of cerebral astrocytoma with 13N-ammonia.

UNLABELLED: We performed this study in order to assess the clinical potential of (13)N-ammonia PET in patients with cerebral astrocytoma. METHODS: Dynamic 13N-ammonia PET was performed in 25 patients with suspected cerebral gliomas or recurrent cerebral astrocytomas (19 male and 6 female patients; age range 18-64 years) detected by MRI. The histopathological diagnoses were made for all cases either by biopsy or craniotomy, except for one patient with brain infarction and one patient with brain radiation necrosis confirmed by repeated MRI imaging. PET images were visually inspected, and the tumor-to-white matter count (T/W) ratios and the perfusion index (PI) of the tumors were determined. RESULTS: Six out of nine cases of low-grade gliomas were detected with 13N-ammonia PET, and three non-astrocytoma low-grade gliomas were not detected with 13N-ammonia PET. All 11 high-grade astrocytomas exhibited markedly increased uptake of 13N-ammonia. The five non-neoplastic lesions exhibited low uptake, low T/W ratios and low PI. The significant differences were observed between high-grade and low-grade gliomas with respect to both the T/W ratios and PI (T/W ratios: 5.92+/-2.27, n=11 vs. 1.66+/-0.61, n=9, P<0.01; PI: 5.22+/-1.67, n=11 vs. 1.60+/-0.54, n=9, P<0.01). There were the significant differences between the T/W ratios and PI in low-grade astrocytomas and that in non-neoplastic lesions (T/W ratios: 2.00+/-0.42, n=6 vs. 0.97+/-0.11, n=5, P<0.01; PI: 1.89+/-0.37, n=6 vs. 0.99+/-0.03, n=5, P<0.01). CONCLUSIONS: There is a substantial uptake of 13N-ammonia in cerebral astrocytomas. 13N-ammonia PET may enable differentiation between low- and high-grade astrocytomas, and has the potential to enable differentiation between low-grade astrocytomas and non-neoplastic lesions.

Oxygen and nitrogen isotopic fractionations during human respiration.

Both oxygen and nitrogen isotope compositions (delta18O and delta15N) of exhaled air from 10 individuals were measured. Results show linear relations between isotope variation and the fraction of O2 used during the respiration process. The isotopic influence of physiological parameters such as smoking habits, age, haemoglobin count, oxygen fixation rate or physical exercise was assessed. Among them, only smoking habits do not have any effect on delta18O. Delta15N differences between inhaled and exhaled air may indicate an active (but minor) role for nitrogen during the human respiration process. Nevertheless, nitrogen fractionation is homogenous among all the individuals, which is coherent with the fact that nitrogen metabolism is controlled by the intestinal bacterial activity.

Fate of orally administered 15N-labeled polyamines in rats bearing solid tumors.

We studied absorption, distribution, metabolism, and excretion of polyamines (putrescine, spermidine, and spermine) in the gastrointestinal tract using (15)N-labeled polyamines as tracers and ionspray ionization mass spectrometry (IS-MS). The relatively simple protocol using rats bearing solid tumors provided useful information. Three (15)N-labeled polyamines that were simultaneously administered were absorbed equally from gastrointestinal tract, and distributed within tissues at various concentrations. The uptake of (15)N-spermidine seemed preferential to that of (15)N-spermine since the concentrations of (15)N-spermidine in the liver and tumors were higher, whereas those of (15)N-spermine were higher in the kidney, probably due to the excretion of excess extracellular spermine. Most of the absorbed (15)N-putrescine seemed to be lost, suggesting blood and tissue diamine oxidase degradation. Concentrations of (15)N-spermidine and (15)N-spermine in the tumor were low. We also describe the findings from two rats that were administered with (15)N-spermine. The tissue concentrations of (15)N-spermine were unusually high, and significant levels of (15)N-spermidine were derived from (15)N-spermine in these animals.

Low-abundance plasma and urinary [(15)N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry.

We report a method for determining plasma und urinary [(15)N]urea enrichments in an abundance range between 0.37 and 0.52 (15)N atom% (0-0.15 atom% excess (APE) (15)N) using a dimethylaminomethylene derivative. Compared with conventional off-line preparation and (15)N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 microl of urine). The (15)N/(14)N ratio of urea derivatives was measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two peaks were separated; one was identified by gas chromatography/mass spectrometry (GC/MS) as the complete derivatized urea. Calibration of the complete urea derivative was performed by linear regression of enrichment values of known standard mixtures. Replicate standard (6-465 per thousand delta(15)N) derivatizations showed a relative standard deviation ranging from 0.1 to 7%. In order to test the feasibility of the method, human subjects and rats ingested a single meal containing either 200 mg of [(15)N]glycine (95 AP (15)N) or 0.4 mg of [(15)N]-alpha-lysine (95 AP (15)N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After (15)N glycine intake, maximum urinary urea (15)N enrichments were 330 and 430 per thousand delta(15)N (0.12 and 0.16 APE (15)N) measured by GC/C/IRMS, whereas plasma [(15)N]glycine enrichments were 2.5 and 3.3 APE (15)N in the two human subjects 2 h after the meal. (15)N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [(15)N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N-containing substances (e.g. creatinine). In rats plasma urea (15)N enrichments were 15-20 times higher than those in urinary urea (10-20 per thousand delta(15)N). The different [(15)N]urea enrichments observed after ingestion of [(15)N]-labeled glycine and lysine confirm known differences in the metabolism of these amino acids.