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Glucose metabolism, lactate, and ammonia production by the human placenta in vitro.

Fifteen human placentas were obtained at term. Placental fragments were incubated in a bicarbonate-buffered Earle's solution. Additions of glutamate (1 mM) or glutamine (1 or 2 mM) were made. All incubations showed a net utilization of glucose. There was a striking variability among placentas in the net glucose utilization rate (1.27 mumoles/g/hr-11.44 mumoles/g/hr, coefficient of variation = 62%). The intraplacental coefficient of variation in glucose utilization was only 14%. All placental incubations showed a net production of both lactate (mean = 7.5 mumoles/g placental wet weight/hr) and ammonia (mean = 3.5 mumoles/g placental wet weight/hr). There was no correlation between lactate of ammonia production and glucose utilization. The addition of sodium glutamate (1 mumole/ml) produced no change in glucose utilization or ammonia production. The addition of glutamine (1 and 2 mumoles/ml) produced a significant increase in ammonia production over that found in the controls, but no change in glucose utilization. Incubation with 2 mumoles glutamine/ml demonstrated an increase in lactate production. All incubations showed a striking increase in ammonia concentration after 45 min of incubation. Individual placentas may differ markedly in their ability to utilize glucose in an in vitro system. Ammonia production may be a normal metabolic endproduct in a tissue lacking an active urea cycle or a byproduct of the purine nucleotide cycle.

Ammonia↗

Exposure of brown trout Salmo trutta to a sublethal concentration of copper in soft acidic water: effects upon gas exchange and ammonia accumulation.

The present study was undertaken to answer two questions relating to the exposure of brown trout Salmo trutta to sublethal concentrations of copper and low pH (CLP) for 96 h. (1) What is the effect of these pollutants on the rate of oxygen consumption (M(O(2))) at different levels of exercise and (2) why does ammonia accumulate within these fish, when the low external pH should favour the diffusion of NH(3) across the gills? Mean M(O(2)) of fish in CLP and control (normal pH and no added copper) conditions were not significantly different from each other at any level of exercise. This suggests that exposure to CLP was not a 'loading' factor at any level of activity. However, both maximum M(O(2)) and critical swimming speed (U(crit)) were significantly lower in the CLP trout (5.5+/-1.6 mmol O(2) kg(-1) h(-1) and 1.12+/-0.06 BL s(-1), respectively) than in control fish (18.5+/-2.3 mmol O(2) kg(-1) h(-1) and 2.04+/-0.11 BL s(-1), respectively). There was no evidence from cardiovascular variables, such as heart rate and cardiac output, to suggest any changes in the oxygen transport system to compensate for any possible reduction in branchial gas exchange. Thus, it is suggested that oxygen exchange and transport do not limit the swimming performance of CLP trout, but that exposure to CLP reduces the maximum demand for O(2), i.e. it is a limiting factor. The accumulation of ammonia in the plasma and white muscles during exposure to CLP has already been implicated in reducing the swimming performance of brown trout. Inhibition of cortisol synthesis abolished a large proportion of the increases in both the accumulation and excretion of ammonia that occurred during the second 48 h of the exposure to CLP, but did not inhibit ammonia accumulation completely. It is suggested that CLP not only causes an increase in the rate of production of ammonia, which is enhanced when the level of cortisol starts to increase after 48 h, but that it also inhibits an excretory mechanism (most probably Na(+)/NH(4)(+) exchange) that is non-obligatory under 'normal' conditions (when passive diffusion is sufficient), but is required in order to respond to unusually high ammonia loads.

Ammonia↗

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

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

Amino Acids↗

Assessment of global and regional left ventricular function by electrocardiographic gated N-13 ammonia positron emission tomography in patients with coronary artery disease.

BACKGROUND: Electrocardiographic gated 13N-ammonia positron emission tomography (PET) enables simultaneous assessment of myocardial blood flow and left ventricular (LV) function. The aim of this study was to assess the accuracy of gated 13N-ammonia PET for evaluating global and regional LV function in patients with coronary artery disease (CAD) in comparison with conventional left ventriculography (LVG). METHODS AND RESULTS: Fifty-four patients with CAD underwent gated 13N-ammonia PET and LVG. The LV end-diastolic and end-systolic volumes (LVEDV, LVESV) and ejection fraction (LVEF) by gated 13N-ammonia PET were calculated using Cedars-Sinai automated quantitative gated single photon emission computed tomography (QGS) and compared with those obtained by LVG. The regional wall motion (RWM) was visually scored, and compared with that on LVG. There were good correlations between the 2 methods for LVEF, LVEDV and LVESV (R=0.828, R=0.821 and R=0.874 respectively). The RWM assessed by gated 13N-ammonia PET also agreed well with that by LVG (complete agreement was 70.4%, kappa=0.58). CONCLUSIONS: Gated 13N-ammonia PET combined with QGS works reasonably well for the assessment of both global and regional LV function in CAD patients, although additional calibration may be necessary.

Adult↗

[Effects of lactulose on blood ammonia levels in beagles with end-to-side portacaval shunt].

The effects of lactulose on blood ammonia and fecal pH in beagles with an end-to-side portacaval shunt were investigated. Concentrations of blood ammonia before and two weeks after the operation were 4.65 +/- 0.34 micrograms/ml (N = 12) and 8.66 +/- 0.60 micrograms/ml (N = 6), respectively (P less than 0.01), while there was no significant difference in fecal pH values before and after the operation. The blood ammonia concentrations in the control and lactulose-treated (2.1 g/kg, p.o.) groups after administration of meat were 12.65 +/- 1.64 micrograms/ml (N = 8) and 8.45 +/- 0.90 micrograms/ml (N = 8), respectively (P less than 0.05). The fecal pH values in the control and lactulose-treated (2.1 g/kg, p.o.) beagles were 6.24 +/- 0.09 (N = 8) and 5.58 +/- 0.08 (N = 8), respectively (P less than 0.01). The lowering effect of lactulose on the blood ammonia levels could not be ascribed to its laxative action alone, because the oral administration of lactulose at doses less than 2.1 g/kg induced no fluid evacuation. The present studies in the beagle suggest that the action of lactulose produce a lowering of fecal pH, causing an increase in the concentration of less absorbable ammonium ions and a decrease in the production of toxic nitrogenous compounds such as ammonia, resulting in a reduction in blood ammonia levels.

Administration, Oral↗

Effects of ammonia on the gastric mucosal barrier in rats and dogs.

We examined the effect of ammonia on the gastric mucosal barrier by measuring the changes in transmucosal fluxes of H+, Na+ and K+. In rats, ammonia at concentrations of 0.1 to 0.5% increased the H+ loss from the lumen and 0.2 to 0.5% concentrations of ammonia increased both Na+ and K+ influxes into the lumen. In dogs, in an exactly similar manner to rats, ammonia at concentrations of 0.1 to 0.5% increased H+ loss, and ammonia at concentration of 0.5% increased both Na+ and K+ influxes into the lumen. These results suggest that ammonia breaks the gastric mucosal barrier.

Ammonia↗

Mechanism for lowering blood ammonia levels by lactitol.

Lactitol has been reported to decrease the blood ammonia concentration in various experimental hyperammonemia models such as portacaval shunted rats. The mechanism responsible for this lowering of blood ammonia levels was investigated in rats. When lactitol was given orally twice a day for 7 days at doses of 3 and 10 g/kg/day and at half that daily dose on day 8, it significantly decreased the ammonium concentration of the portal blood by 27.3-43.2%, cecal ammonia contents by 49.2-57.6% and the pH of the cecal contents from 6.52 to 5.92-5.54, 4 hr after the final administration. Lactitol also inhibited increases in the portal ammonia concentration induced by the intracecal administration of ammonium acetate (300 mg/kg) 4 hr after the final administration. When lactitol was given orally at bolus doses of 1 and 3 g/kg simultaneously with a charcoal meal, lactitol significantly facilitated small intestinal transit by 12-13%. At a bolus dose of 3 g/kg, given 1 hr before the administration of a charcoal meal into the proximal colon, lactitol significantly facilitated colonic transit by 29.5%. These effects of lactitol were similar to those of lactulose. These findings suggest that lactitol decreases blood ammonia concentration by inhibiting both the production and the absorption of ammonia through reducing intestinal pH and shortening the residence time of intestinal contents in the intestinal tract.

Acetates↗

Effect of temperature, duration of storage and sampling procedure on ammonia concentration in equine blood plasma.

The effect of storage duration at different storage temperatures on the plasma ammonia concentration of equine EDTA whole blood, EDTA plasma and heparin plasma samples was investigated. Further, the effect of jugular vein compression before and during blood sampling on the plasma ammonia values was evaluated. In EDTA whole blood kept at 4 degrees C there was no significant increase of ammonia content after 6 hours of storage, whereas the increase was already significant 3 hours after collection if the EDTA whole blood was kept at 20-22 degrees C. EDTA plasma samples stored at 20-22 degrees C, 4 degrees C and -20 degrees C showed mean ammonia formation rates of 26.8 mumol/l, 6.6 mumol/l and 0.03 mumol/l per day, respectively. There were no differences between ammonia values of EDTA and heparin plasma samples immediately after sampling and following 3 days of storage at 4 degrees C and 3 and 90 days of storage at -20 degrees C. Although significant, the absolute differences between the mean plasma ammonia contents of EDTA blood sampled with and without compression of the jugular vein were small (20.2 +/- 4.1 mumol/l and 23.4 +/- 4.3 mumol/l resp.).

Ammonia↗

Structural elements in IGP synthase exclude water to optimize ammonia transfer.

In the complex pathway of histidine biosynthesis, a key branch point linking amino acid and purine biosynthesis is catalyzed by the bifunctional enzyme imidazole glycerol phosphate (IGP) synthase. The first domain of IGP synthase, a triad glutamine amidotransferase, hydrolyzes glutamine to form glutamate and ammonia. Its activity is tightly regulated by the binding of the substrate PRFAR to its partner synthase domain. Recent crystal structures and molecular dynamics simulations strongly suggest that the synthase domain, a (beta/alpha)(8) barrel protein, mediates the insertion of ammonia and ring formation in IGP by channeling ammonia from one remote active site to the other. Here, we combine both mutagenesis experiments and computational investigations to gain insight into the transfer of ammonia and the mechanism of conduction. We discover an alternate route for the entrance of ammonia into the (beta/alpha)(8) barrel and argue that water acts as both agonist and antagonist to the enzymatic function. Our results indicate that the architecture of the two subdomains, most notably the strict conservation of key residues at the interface and within the (beta/alpha)(8) barrel, has been optimized to allow the efficient passage of ammonia, and not water, between the two remote active sites.

Amino Acid Sequence↗

Diurnal fluctuation of blood ammonia levels in adult-type citrullinemia.

A 48-year-old man, who was diagnosed as adult-type citrullinemia by quantitative estimation of urea cycle enzymes in the liver, showed a regular nocturnal rise of blood ammonia level. In order to elucidate the mechanism of diurnal fluctuation of blood ammonia level, the patient was put into fasting state for four days. The blood ammonia level rose at the night of the first fasting day even though no food was taken, then it decreased gradually and reached the lowest level in the morning of the third fasting day. Intravenous administration of amino acids mixture under the same starved condition gave rise to a significant elevation of blood ammonia level. Based on these results, it was concluded that hyperammonemia of adult-type citrullinemia could result from the accumulation of free ammonia which was produced from the catabolism of amino acids absorbed from the small intestine and surpassed the urea synthesis of defective urea cycle to flood into the blood. Furthermore, the rise of blood ammonia level at the night of the first fasting day suggested that the circadian rhythm of amino acid-carbohydrate metabolism might superimpose on the process mentioned above.

Amino Acids↗

Influence of dietary factors on the pH and ammonia emission of slurry from growing-finishing pigs.

We investigated the effects of dietary factors on the pH and the ammonia emission from slurry of growing-finishing pigs. Sixteen male hybrid pigs (80 to 90 kg BW) were allotted to one of four diets based on barley-wheat, tapioca, barley-tapioca, and sugar beet pulp. Diets were formulated to have similar NE and CP contents and a similar lysine:NE ratio. Diets differed in nonstarch polysaccharide content (NSP) and dietary electrolyte balance (dEB). Urine and feces were daily collected quantitatively in metabolism cages and mixed as a slurry at the end of the collection period. After mixing, the pH and the ammonia emission from the slurry were measured daily in a laboratory setup for 7 d at 20 degrees C. The type of diet affected the pH of the slurry and the ammonia emission (P < .001). The pH of the slurry from pigs fed the sugar beet pulp-based diet was .8 unit lower and ammonia emission was 52 to 53% lower than that of the other three diets. The low dEB and high NPS sugar beet pulp-based diet increased the VFA concentration and reduced the pH and ammonia emission from the slurry. We conclude that dietary NSP and dEB influence the pH and ammonia emission from slurry of growing-finishing pigs.

Ammonia↗

Changes in ruminal concentrations of microbial ammonia and amino acids due to monensin and time.

The effect of monensin (33 mg/kg) and no monensin on distribution of ammonia and free amino acids in fluid and particulate phases of ruminal contents was studied. Two Jersey cows and two Angus-Hereford steers, weighing approximately 350 kg and each containing a ruminal cannula were meal fed a 65% concentrate diet for two periods. Feed was available for 30 min; any feed not eaten was removed after this time. Whole ruminal contents were sampled before feeding, immediately after feed removal and 1, 2, 3, 4, 6, 8, 10 and 12 h after feed removal. A crossover design with split-plot in time was used with four observations per time point per treatment. Extracellular (E) ammonia and amino acids were determined on high speed supernatants of strained ruminal fluid. Total (T) ammonia and amino acids were obtained by lysing microbial cells with 1% cetyltrimethylammonium bromide (CTAB) and one freeze treatment before centrifugation. Intracellular (I) amino acids and ammonia were determined as T minus E. Squeezed particles were suspended in .1 N HCl containing 1% CTAB. Total and E ammonia were higher throughout the study for no monensin vs monensin (P less than .05). Intracellular ammonia was low (.03 to 1 mM) and did not change (P greater than .10) across time for either dietary treatment. Intracellular amino acids ranged from 1.2 to 4.5 mM and were higher (P less than .05) than E amino acids (.9 to 2.2 mM) for both diets immediately after feeding. Monensin did not significantly lower (P greater than .10) T amino acids compared with no monensin throughout sampling (2.6 and 3.1 mM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Microencapsulated genetically engineered E. coli DH5 cells for plasma urea and ammonia removal based on: 1. Column bioreactor and 2. Oral administration in uremic rats.

We report a novel approach for plasma urea and ammonia removal using artificial cells microencapsulated genetically engineered bacteria E. coli DH5 cells. This has been evaluated for use in a column bioreactor for removing plasma urea and ammonia. It has also been evaluated in uremic rats for urea removal by oral administration. In 30 minutes, microencapsulated E. coli DH5 in a column bioreactor in-vitro lowered plasma urea to 10.47 +/- 3.45 mg/dl from 45.85 +/- 2.98 mg/dl and lowered plasma ammonia concentration to 46.00 +/- 4.00 microM from 679 +/- 32 microM/1. The efficiency of this bioreactor for plasma urea and ammonia removal is much higher than any other available methods. Initial plasma urea and ammonia concentration does not affect the plasma urea and ammonia removal efficiency of the column bioreactor. In in-vivo studies of oral administration to uremic rats, both free and encapsulated bacteria both lowered systemic urea from the initial 52.08 (S.D.2.37) mg/dl to 10.58 (S.D.0.85) mg/dl. Unlike free bacteria, microencapsulated bacteria was not retained in the intestine.

Administration, Oral↗

Effect of adding alum or zeolite to dairy slurry on ammonia volatilization and chemical composition.

Development of cost-effective amendments for treating dairy slurry has become a critical problem as the number of cows on farms continues to grow and the acreage available for manure spreading continues to shrink. To determine effectiveness and optimal rates of addition of either alum or zeolite to dairy slurry, we measured ammonia emissions and resulting chemical changes in the slurry in response to the addition of amendments at 0.4, 1.0, 2.5, and 6.25% by weight. Ammonia volatilization over 96 h was measured with six small wind tunnels with gas scrubbing bottles at the inlets and outlets. Manure samples from the start and end of trials were analyzed for total nitrogen and phosphorus, and were extracted with 0.01 M CaCl2, 1.0 M KCl, and water with the extracts analyzed for ammonium nitrogen, phosphorous, aluminum, and pH. The addition of 6.25% zeolite or 2.5% alum to dairy slurry reduced ammonia emissions by nearly 50 and 60%, respectively. Alum treatment retained ammonia by reducing the slurry pH to 5 or less. In contrast, zeolite, being a cation exchange medium, adsorbed ammonium and reduced dissolved ammonia gas. In addition, alum essentially eliminated soluble phosphorous. Zeolite also reduced soluble phosphorous by over half, but the mechanism for this reduction is unclear. Alum must be carefully added to slurry to avoid effervescence and excess additions, which can increase soluble aluminum in the slurry. The use of alum or zeolites as on-farm amendment to dairy slurry offers the potential for reducing ammonia emissions and soluble phosphorus in dairy slurry.

Alum Compounds↗

Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media.

Catalyzed phenol-hypochlorite and ninhydrin colorimetric procedures were adapted to the Technicon AutoAnalyzer for simultaneous determination of ammonia and total amino acids in ruminal fluid or ruminal in vitro media. The manifold developed was compatible with a sampling rate of 40/h without significant sample-to-sample carryover. With proper storage, reagents for both the phenol-hypochlorite and the air-stable ninhydrin systems were stable for 8 mo or more. Response of individual amino acids in the phenol-hypochlorite system were generally 1% or less than equimolar amounts of ammonia. Certain amino acids inhibited ammonia color yield 10 to 15% when with equimolar amounts of ammonia; however, the inhibitory effect of casein amino acids was only 2 to 3%. Although ninhydrin response, relative to leucine, of individual alpha-amino acids ranged from 62 (valine) to 151% (histidine), recoveries of casein amino acids from ruminal fluid had coefficients of variation of 1% or less. Coefficients of variation for ammonia recoveries from ruminal fluid by the phenol-hypochlorite procedure were about half of those for the Conway microdiffusion technique. Intraclass correlations for the adapted procedures indicated high degrees of accuracy and precision for both ammonia and amino acid analyses.

Amino Acids↗

Effect of rumen ammonia concentration on in situ rate and extent of digestion of feedstuffs.

Three dry Holstein cows fitted with rumen fistula were fed a 7.4% crude protein diet consisting of 47.4% corn, 50% cottonseed hulls, and 2.6% minerals and vitamins during a 44-d experiment. Treatments consisted of rumen infusion, 0, 33, 67, and 100 g/d of urea nitrogen applied in a four period Youden Square design. Increasing urea infusion increased rumen fluid ammonia nitrogen from 4.3 to 25.0 mg/dl. Estimated effective dry matter degradation based on in situ rates of digestion were increased from 67.9 to 74.4% for corn and 77.5 to 80.3% for soybean meal with maximums at 100 g/d infused urea nitrogen. Up to 67 g/d infused nitrogen increased dry matter degradation of corn gluten feed from 67.0 to 71.4% and cottonseed meal dry matter degradation from 56.7 to 60.1%. Alfalfa hay dry matter and neutral detergent fiber degradation were not increased by urea infusion. Minimum rumen ammonia required in feeds in this trial were pooled with literature data and suggest that lowest ammonia concentrations required for maximal digestion was a function of the rumen fermentability of the diet or feed. The equation: minimum ammonia concentration (mg/dl) = .452 fermentability % - 15.71, accounted for 50% of the variation in minimum ammonia requirements. We conclude that rumen ammonia concentrations required for maximum digestion are not constant but rather are a function of fermentability of the diet.

Ammonia↗

Effects of dietary nitrogen manipulation on ammonia volatilization from manure from Holstein heifers.

Decomposition of livestock manure produces gaseous ammonia. Dietary manipulation is one means to reduce N in manure and ammonia volatilization. The effects of dietary crude protein concentration on N intake, N and urinary urea-N excretion, and ammonia volatilization were measured. Eight Holstein heifers (body weight = 260 to 488 kg) were fed a total mixed ration containing either 9.6 or 11.0% crude protein in a crossover design. Oatlage and concentrate were fed at 77:23 (dry matter basis), and soybean meal was used to alter total dietary crude protein. Seven-day adjustment periods preceded 5-d collection periods. Indwelling urinary catheters were inserted 2 d prior to the collection periods. Daily feces and acidified urine were collected, stirred, and subsampled for total Kjeldahl N, urinary urea N, dry matter, P, K, and ash. Urine collection tubes were split during period 2 to allow for collection of unacidified samples for urea N and total N determinations. Unacidified urine and fecal samples were combined (1:1.3) for collection of volatilized ammonia. Remaining slurries were extracted for total and urea N. Increased dietary crude protein concentration increased N intake, N excretion, urea-N excretion, and N excreted in the urine by the heifers. Dietary manipulation of N intake by reduction of 14.0% (dry matter basis) resulted in a 28.1% decrease in ammonia emission and decreases in the urea N, total N, and percentage N excreted in the urine of 29.6, 19.8, and 7.4%, respectively. Ammonia volatilization was dependent on N quantity and form in the urine.

Ammonia↗

Plasma ammonia response to sprint swimming.

BACKGROUND: To study the plasma ammonia response after sprint crawl swimming. METHODS: Nine sprinters (S) and ten non-sprinters (NS) completed a 15-, a 25- and a 50-m crawl at maximal intensity with a 10-min and a 15-min resting period in-between. Capillary blood samples were collected before and at regular intervals after each effort for plasma ammonia determination. RESULTS: Ammonia kinetics differed among distances, but not between groups, with peak values (observed 2-8 min postexercise) being higher after 50 m as compared to shorter distances. Significant differences between S and NS were found in peak ammonia after 50 m (124.5 +/- 58.2 vs 98.7 +/- 6.3 mumoL-1) and in the change of ammonia relative to swim time (delta NH3/delta t) after 25 m (2.66 +/- 1.87 vs 1.49 +/- 0.84 mumol L-1 s-1) and 50 m (1.87 +/- 1.33 vs 1.01 +/- 0.49 mumol L-1 s-1). delta NH3/delta t was highest after 15 m (3.33 +/- 2.53 in S, 3.92 +/- 1.67 mumol L-1 s-1 in NS). CONCLUSIONS: These differences in the plasma ammonia response to sprint swimming according to duration and athlete seem to be connected to distinctions in muscle fiber profile and energy providing processes.

Adult↗