Variation of resorption rates in vivo of various bones in immature rats

The ³H-tetracycline method of measuring bone resorption in vivo was applied to the comparison of various whole bones in rats of two different ages. The rat was chosen because it grows via modelling processes and contains little, if any, cortical remodelling except for a small amount of trabecular remodelling. It was found that resorption rates in vivo are high and similar in almost all of the 18 bones measured between birth and 2 weeks of age. However, in weanling rats studied at 4–6 weeks of age, resorption rates in the skull and in the long bones had decreased significantly while remaining high in the vertebrae, scapula, sternum and pelvis. Bones of neonatal rats were quite alike in their rates of bone resorption, but the bones of the weanlings manifested significant heterogeneity in their rates.It is known that anatomic heterogeneity of metabolic turnover of various bones characterizes the mature state in humans and dogs as well. The present data are unique in that they reflect absolute resorption rates in vivo uncomplicated by the extensive re-utilization of calcium inherent in other isotopic or non-isotopic protocols.     

REPRODUCTION IN POLEMONIUM: PATTERNS AND IMPLICATIONS OF FLORAL NECTAR PRODUCTION AND STANDING CROPS

Patterns of floral nectar production and standing crop were measured in four populations of the herbaceous perennial plant species Polemonium foliosissimum. Contrary to prediction (Pleasants, 1983), individual flowers in this mass-flowering species were found to produce equivalent nectar volumes every day of their lives. Alternative methods of increasing the reward variability presented to pollinators are evaluated for P. foliosissimum and the relationship between that variability and risk-aversive foraging by pollinators is discussed. Significant spatial and temporal variability in rate of nectar production was found. Populations separated by approximately 200 m exhibited different rates. Nectar production declined significantly as a function of time of the flowering season in two populations but not in a third. In spite of such variability, individual plants showed consistency in production both within a single blooming season and across successive seasons. Because of the variability found in the present study, care should be taken to design appropriate sampling protocols in future nectar studies. Patterns of standing nectar crop were consistent with those expected if pollinators were using an area-restricted searching pattern.     

Investigation of metabolic variability observed in extended fed batch cell culture

A 13‐day fed‐batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell‐specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1519–1527, 2013     

Microbial production and uptake of nitric oxide in soil

Abstract Fluxes of NO from three different soils have been studied by a flow-through system in the laboratory as a function of gas flow rate, of NO mixing ratio, and of incubation conditions. The dependence of net NO fluxes on gas flow rates and on NO mixing ratios could be described by a simple model of simultaneous NO production and NO uptake. By using this model, rates of gross NO production, rate constants of NO uptake, and NO compensation mixing ratios could be determined as function of the soil type and the incubation condition. Gross NO production rates were one to two orders of magnitude larger under anaerobic than under aerobic conditions. NO uptake rate constants, on the other hand, were only 5–8 times larger so that the compensation mixing ratios of NO were in a range of about 1600–2200 ppbv under anaerobic and of about 50–600 ppbv under aerobic conditions. The different soils exhibited similar NO uptake rate constants, but the gross NO production rate and compensation mixing ratio was significantly higher in an acidic (pH 4.7) sandy clay loam than in other less acidic soils. Experiments with autoclaved soil samples showed that both NO production and NO uptake was mainly due to microbial metabolism.     

Chasing away accurate results: exhaustive chase protocols underestimate maximum metabolic rate estimates in European perch Perca fluviatilis

Metabolic rates are one of many measures that are used to explain species' response to environmental change. Static respirometry is used to calculate the standard metabolic rate (SMR) of fish, and when combined with exhaustive chase protocols it can be used to measure maximum metabolic rate (MMR) and aerobic scope (AS) as well. While these methods have been tested in comparison to swim tunnels and chambers with circular currents, they have not been tested in comparison with a no-chase control. We used a repeated-measures design to compare estimates of SMR, MMR and AS in European perch Perca fluviatilis following three protocols: (a) a no-chase control; (b) a 3-min exhaustive chase; and (c) a 3-min exhaustive chase followed by 1-min air exposure. We found that, contrary to expectations, exhaustive chase protocols underestimate MMR and AS at 18°C, compared to the no-chase control. This suggests that metabolic rates of other species with similar locomotorty modes or lifestyles could be similarly underestimated using chase protocols. These underestimates have implications for studies examining metabolic performance and responses to climate change scenarios. To prevent underestimates, future experiments measuring metabolic rates should include a pilot with a no-chase control or, when appropriate, an adjusted methodology in which trials end with the exhaustive chase instead of beginning with it.     

Relationship between flow rate and NO production in postnatal mesenteric arteries

We studied mesenteric arterial arcades from 3- and 35-day-old swine to determine the relationship between perfusate flow rate and release of nitric oxide (NO) into mesenteric effluent. Mesenteric arterial arcades were perfused under controlled-flow conditions with a peristaltic pump using warm oxygenated Krebs buffer. Basal rates of NO production were 43.6 +/- 4.2 vs. 12.1 +/- 2.5 nmol/min in 3- vs. 35-day-old mesentery during perfusion at in vivo flow rates (9 vs. 20 ml/min, respectively). Rate of NO production was directly related to flow rate over a wide range of flows (5-40 ml/min) in 3- but not 35-day-old mesentery. Both age groups demonstrated a brisk, albeit brief, increase in NO production in response to infusion of NO-dependent vasodilator substance P (10(-8) M/min). Tyrosine kinase inhibitor herbimycin A and L-arginine analog L-NMMA significantly attenuated flow-induced increase in NO production, and phosphatase inhibitor phenylarsine oxide increased magnitude of flow-induced increase in NO production in 3-day-olds. Removal of extracellular Ca(2+) and depletion of intracellular Ca(2+) stores (Ca(2+)-free Krebs with EGTA plus thapsigargin) had no effect on NO production in either group. Thus, basal rate of NO production is greater in mesenteric arterial arcades from 3- than from 35-day old swine, a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-olds, and phosphorylation events are necessary for this interaction to occur.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity

The capacity of isolated porcine heart mitochondria to produce nitric oxide (NO) via mitochondrial NO synthase (NOS) was evaluated. The mitochondrial NOS content and activity (0.2 nmol NO x mg mitochondrial protein(-1) x min(-1)) were approximately 10 times lower than previously reported for the rat liver. No evidence for mitochondrial NOS-generated NO was found in mitochondrial suspensions based on the lack of NO production and the lack of effect of either L-arginine or NOS inhibitors on the rate of respiration. The reason that even the low mitochondrial NOS activity did not result in net NO production and metabolic effects is because the mitochondrial metabolic breakdown of NO (1-4 nmol NO x mg mitochondrial protein(-1) x min(-1)) was greater than the maximum rate of NO production measured in homogenates. These data suggest that NO production at the mitochondria via NOS is not a significant source of NO in the intact heart and does not regulate cardiac oxidative phosphorylation.     

Citrate production from n-alkane by Candida lipolytica in reference to carbon fluxes in vivo

Summary Citrate production by Candida lipolytica from n-alkane as sole carbon source was studied with a NH ₄ ⁺ -limited chemostat culture. This organism produced citrate and isocitrate simultaneously, the ratio of these acids remaining nearly unchanged within the range of dilution rates used. However, the specific production rates of these acids were maximal around a particular dilution rate. For each steady-state culture, specific activities of some key enzymes in tricarboxylate and glyoxylate cycles were measured in vitro. In order to correlate the enzyme activites with the change in specific production rate of each acid, the intracellular carbon fluxes were assessed by solving algebraic equations derived from carbon balance with respect to a simplified metabolic map of n-alkane metabolism. By comparing the fluxes assessed in vivo with the enzyme activities measured in vitro, the metabolic pathway(s) most likely to control the production of acids and cell material were investigated.     

Free nitric oxide diffusion in the bronchial microcirculation

Theoretical mass transfer rates and concentration distributions were determined for transient diffusion of free nitric oxide (NO) generated in vivo from vascular endothelial cells. Our analytical framework is typical of the bronchial circulation in the human pulmonary system but is applicable to the microvascular circulation in general. We characterized mass transfer rates in terms of the fractional mass flux across a boundary relative to the total endothelial NO production rate. NO concentration in the tissue surrounding blood vessels was expressed in terms of fractional soluble guanylate cyclase (sGC) activity. Our results suggest that endothelium-derived free NO is capable of vascular smooth muscle dilation despite its rapid consumption by hemoglobin in blood. An optimal blood vessel radius of 20 microm was estimated for NO signaling. We hypothesize intermittent generation of endothelial NO as a possible mechanism for sGC activation in vascular smooth muscle. This mechanism enhances the efficacy of NO-modulated vascular smooth muscle dilation while minimizing NO losses to blood and surrounding tissue.     

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the L-arginine/NO pathway in health and disease

Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid L-arginine by the action of nitric oxide synthases (NOS) in what is called the L-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, L-arginine and its simultaneously generated product, L-citrulline. Considering only a fraction of the endogenous L-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of L-arginine and/or L-citrulline alone. Instead, to estimate NO-related changes in the L-arginine and/or L-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of L-arginine to NO and L-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.     

Measurement of human plasma proteome dynamics with (2)H(2)O and liquid chromatography tandem mass spectrometry

Dysfunction of protein turnover is a feature of many human diseases, and proteins are substrates in important biological processes. Currently, no method exists for the measurement of global protein turnover (i.e., proteome dynamics) that can be applied in humans. Here we describe the use of metabolic labeling with deuterium ((2)H) from (2)H(2)O and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of mass isotopomer patterns to measure protein turnover. We show that the positions available for (2)H label incorporation in vivo can be calculated using peptide sequence. The isotopic incorporation values calculated by combinatorial analysis of mass isotopomer patterns in peptides correlate very closely with values established for individual amino acids. Inpatient and outpatient heavy water labeling protocols resulted in (2)H label incorporation sufficient for reproducible quantitation in humans. Replacement rates were similar for peptides deriving from the same protein. Using a kinetic model to account for the time course of each individual's (2)H(2)O enrichment curves, dynamics of approximately 100 proteins with half-lives ranging from 0.4 to 40 days were measured using 8 μl of plasma. The measured rates were consistent with literature values. This method can be used to measure in vivo proteome homeostasis in humans in disease and during therapeutic interventions.     

Regional heterogeneities in the production of uric acid from adenosine in the bivascularly perfused rat liver

The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 M) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 M adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.     

Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion.     

Endogenous superoxide production and the nitrite/nitrate ratio control the concentration of bioavailable free nitric oxide in leaves

We have quantitatively measured nitric oxide production in the leaves of Arabidopsis thaliana and Vicia faba by adapting ferrous dithiocarbamate spin tapping methods previously used in animal systems. Hydrophobic diethyldithiocarbamate complexes were used to measure NO interacting with membranes, and hydrophilic N-methyl-d-glucamine dithiocarbamate was used to measure NO released into the external solution. Both complexes were able to trap levels of NO, readily detectable by EPR spectroscopy. Basal rates of NO production (in the order of 1 nmol g(-) (1) h(-1)) agreed with previous studies. However, use of methodologies that corrected for the removal of free NO by endogenously produced superoxide resulted in a significant increase in trapped NO (up to 18 nmol g(-) (1) h(-1)). Basal NO production in leaves is therefore much higher than previously thought, but this is masked by significant superoxide production. The effects of nitrite (increased rate) and nitrate (decreased rate) are consistent with a role for nitrate reductase as the source of this basal NO production. However, rates under physiologically achievable nitrite concentrations never approach that reported following pathogen induction of plant nitric-oxide synthase. In Hibiscus rosa sinensis, the addition of exogenous nitrite generated sufficient NO such that EPR could be used to detect its production using endogenous spin traps (forming paramagnetic dinitrosyl iron complexes). Indeed the levels of this nitrosylated iron pool are sufficiently high that they may represent a method of maintaining bioavailable iron levels under conditions of iron starvation, thus explaining the previously observed role of NO in preventing chlorosis under these conditions.     

Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release

Endothelium-derived nitric oxide (NO) is synthesized in response to chemical and physical stimuli. Here, we investigated a possible role of the endothelial cell glycocalyx as a biomechanical sensor that triggers endothelial NO production by transmitting flow-related shear forces to the endothelial membrane. Isolated canine femoral arteries were perfused with a Krebs-Henseleit solution at a wide range of perfusion rates with and without pretreatment with hyaluronidase to degrade hyaluronic acid glycosaminoglycans within the glycocalyx layer. NO production rate was evaluated as the product of nitrite concentration in the perfusate and steady-state perfusion rate. The slope that correlates the linear relation between perfusion rate and NO production rate was taken as a measure for flow-induced NO production. Hyaluronidase treatment significantly decreased flow-induced NO production to 19 +/- 9% of control (mean +/- SD; P < 0.0001 vs. control; n = 11), whereas it did not affect acetylcholine-induced NO production (88 +/- 17% of pretreatment level, P = not significant; n = 10). We conclude that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in detecting and amplifying the shear force of flowing blood that triggers endothelium-derived NO production in isolated canine femoral arteries.     

The modulation of oxygen radical production by nitric oxide in mitochondria

Biological systems that produce or are exposed to nitric oxide (NO radical) exhibit changes in the rate of oxygen free radical production. Considering that mitochondria are the main intracellular source of oxygen radicals, and based on the recently documented production of NO(radical) by intact mitochondria, we investigated whether NO(radical), produced by the mitochondrial nitric-oxide synthase, could affect the generation of oxygen radicals. Toward this end, changes in H(2)O(2) production by rat liver mitochondria were monitored at different rates of endogenous NO(radical) production. The observed changes in H(2)O(2) production indicated that NO(radical) affected the rate of oxygen radical production by modulating the rate of O(2) consumption at the cytochrome oxidase level. This mechanism was supported by these three experimental proofs: 1) the reciprocal correlation between H(2)O(2) production and respiratory rates under different conditions of NO(radical) production; 2) the pattern of oxidized/reduced carriers in the presence of NO(radical), which pointed to cytochrome oxidase as the crossover point; and 3) the reversibility of these effects, evidenced in the presence of oxymyoglobin, which excluded a significant role for other NO(radical)-derived species such as peroxynitrite. Other sources of H(2)O(2) investigated, such as the aerobic formation of nitrosoglutathione and the GSH-mediated decay of nitrosoglutathione, were found quantitatively negligible compared with the total rate of H(2)O(2) production.     

Does habitat fragmentation promote climate‐resilient phenotypes?

Understanding how individual differences in physiological performance modify behavioral responses to environmental variability and its fitness consequences is key to predicting the vulnerability of species and populations to environmental change. For many species, summit metabolic rate (M_SUM; the upper limit to heat production) and basal metabolic rate (BMR; the lower limit related to energy acquisition and processing) often constrain aspects of physiological performance and behavioral activity. We examined the relationship between metabolic phenotypes, foraging behavior, and survival in overwintering black‐capped chickadees Poecile atricapillus inhabiting contiguous and fragmented forested landscapes. We found that birds with lower summit metabolic rates were generally more sensitive to winter weather and increased their use of supplemental feeding stations as ambient temperatures decreased. In highly fragmented forests, this relationship may have incurred strong survival consequences, as birds with lower summit metabolic rates were less likely to survive the winter season. Additionally, we found that chickadee populations persisting in fragmented landscapes were characterized by slightly higher thermogenic capacity (M_SUM) and lower maintenance metabolic costs (BMR). We suggest that habitat loss and fragmentation present unique selection pressures that alter the relationships between environmental variability, behavior and physiology, and result in context‐specific fitness consequences.     

Leishmania amazonensis infection does not inhibit systemic nitric oxide levels elicited by lipopolysaccharide in vivo

Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Downregulation of nitric oxide (NO) synthesis by the parasite infecting the macrophages has been proposed to be an important evading mechanism based on in vitro studies. We confirmed inhibition of NO release by macrophages infected with Leishmania amazonensis in vitro. To examine the role of the parasite in regulating NO production in vivo, we monitored systemic NO levels elicited by challenging naive and L. amazonensis-infected BALB/c mice with lipopolysaccharide (LPS). Animals were challenged after 1, 2, 6, and 9 wk of infection. NO production was monitored by electron paramagnetic resonance spectroscopy as the levels of hemoglobin nitrosyl complexes (HbNO) present in the animal's blood. No significant differences in HbNO levels were observed between LPS-treated naive and inoculated mice at any time during infection. To control for increased macrophage numbers in infected mice, naive mice were injected with a macrophage cell line before LPS challenge; this treatment did not increase produced NO levels. The results argue against a major role for the parasite in downregulating NO production in vivo.     

Role of nitrate and nitrite for production and consumption of nitric oxide during denitrification in soil

Abstract Anaerobic production and consumption of NO was measured in a calcic cambisol (KBE; pH 7.3) and a forest luvisol (PBE; pH 4.4) which were incubated at 80% water-holding capacity and continuously flushed with N₂. Both NO production and NO consumption were negligibly low when nitrate and nitrite concentrations in the soil were exhausted. Addition of glucose alone had no effect, but addition of nitrate ± glucose greatly stimulated both NO production and NO consumption. NO consumption followed an apparent first-order reaction at low NO mixing ratios (1–3 ppmv), but a higher NO mixing ratios it followed Michaelis-Menten kinetics. In PBE the apparent K _m was 980 ppbv NO (1.92 nM in soil water). During reduction of nitrate, nitrite intermediately accumulated and simultaneously, production rates of NO and N₂O were at the maximum. Production rates of NO plus N₂O amounted to 20% and 34% of the nitrate reduction rate in KBE and PBE, respectively. NO production was hyperbolically related to the nitrite concentration, indicating an apparent K_m of 1.6 μg nitrite-N g⁻¹ d.w. soil (equivalent to 172 μM nitrite in soil solution) for the reduction of nitrite to NO in KBE. Under nitrate and nitrite-limiting conditions, 62–76% and 93–97% of the consumed NO-N were recovered as N₂O-N in KBE and PBE, respectively. Gassing of nitrate plus nitrite-depretsu KBE with increasing mixing ratios of NO₂ resulted in increasing rates of NO₂ uptake and presumably in the formation of low concentrations of nitrite and nitrate. This NO₂ uptake resulted in increasing rates of both NO production and NO consumption indicating that nitrite or nitrate was limiting for both reactions.