Initial Urea Concentration Research Articles

Treatment of straw with urea. 1. Ureolysis in a low water medium

The aim of this work was to gain a better understanding of the reaction of ureolysis, with or without added ground dehulled soya bean, during the process of straw (wheat varieties 1,2,3) treatment by urea as an ammonia source in conditions limiting the addition of water. In Experiment 1 the reaction was made at 30 °C in an open liquid (without straw) medium at pH 9.0, avoiding product (NH 3) inhibition and in a closed medium (substrate and product inhibition) with and without straw 1 mixed with urea, soya bean and water to reach 30% moisture. Initial urea concentrations ( U i ) ranged from 2 to 400 g l −1. Soya bean concentrations ( S) were 10 g l −1 and 30g l −1 in open and closed media respectively. In open medium the kinetic parameters of ureolysis in dilute solutions (maximal specific initial rate of reaction V M = 16.6 μM urea g −1 soya protein s −1, Michaelis-Menten constant Km = 63.8 mM 1 −1) were close to those of pure urease. However, U i to reach V M (75 g l −1) was higher than for pure urease and beyond this value, the kinetics were different from that calculated on a basis of uncompetitive inhibition by the substrate. In a closed medium, the specific initial rates ( V i ) of the reaction were lower than in open medium beyond U i = 50 g l −1, and the reaction slowed down considerably beyond 200 g l −1. This may be an upper limit in conditions simulating a stack. In Experiment 2, it was shown that at U i = 75 g l −1 in open medium, the initial velocities (in μM s −1) were proportional to S, when S varied from 5 to 100 g l −1. Experiment 3 was designed to determine the effect of straw 3 on the development of the reaction. The treatments were made in tightly closed glass jars, with U i = 175 g l −1 (e.g. 8 g U per 100 g straw, 85% DM) and S = 30 g l −1, at 30 °C, with or withou'straw 3 (30% terminal moisture). The straw was either mixed with the urea-soya bean-water homogenate or placed on a sieve over it (indirect method). The accelerating effect of the straw was clear after the first hours of the reaction and was demonstrated better by the indirect method than by the mixing method. In Experiment 4, straws 1 and 2, with or without soya bean, were treated by the mixing method as above. With added urease, the reaction was nearly complete after 1 month for both straws, but without soya bean, it took 3 months to have total or only 77% ureolysis with straws 1 and 2, respectively. Because of the complex phenomena occurring during the ureolysis in a concentrated and heterogeneous medium, the kinetics can only be roughly predicted by V i when the same (i.e. mixing) method is compared. They are better characterized by other parameters, such as time to achieve 50 or 99% of ureolysis. It is concluded that in the process of straw treatment by urea, the addition of an external erude urease may be useful to reduce both the amount of water necessary and the treatment duration.

Suicidal destruction of Helicobacter pylori: metabolic consequence of intracellular accumulation of ammonia.

The role of pH, citrate buffer, and urea were investigated in the suicidal destruction of Helicobacter pylori, with particular reference to the organism's urea and ammonia metabolism. The median five minute survival of H pylori in the presence of 50 mmol/l urea in 0.2 M citrate buffer at pH 6.0 was only 14%, compared with 53% in the same solution at pH 7.0. The median amount of ammonium released into the incubating solution over five minutes was lower at pH 6.0 (9 mumol) than at pH 7.0 (18 mumol) despite similar uptake of urea. The median five minute survival of H pylori in 0.2 M citrate buffer, pH 6.0, decreased from 89% to 14% when the urea concentration was increased from 1 mmol/l to 50 mmol/l. Likewise, the recovery in the incubating solution of ammonia resulting from the hydrolysis of urea fell from 27% to 3% when the initial urea concentration was increased from 1 mmol/l to 50 mmol/l. Survival of H pylori in the presence of 30 mmol/l urea at pH 6.0 was compared in 0.2 M citrate, acetate, and phosphate buffers. The median five minute survival was less in the citrate buffer, at 29%, than in either the acetate buffer 80% or the phosphate buffer 100%. The percentage recovery of ammonia was similar in the three buffers. These findings indicate that the suicidal destruction of the bacterium may be explained by intracellular accumulation of ammonia due to production in excess of the rate of excretion.

Klebsiella pneumoniae bacteraemia at an urban general hospital

Of 47 patients with Klebsiella pneumoniae bacteraemia admitted to the Hillbrow Hospital, Johannesburg during a period of 18 months, 31 were males and 16 were females. Features predisposing to illness were found in 89.4% patients, chronic alcoholism, neoplastic disease and diabetes mellitus being the most common. Twenty-five infections were acquired in hospital and 22 in the community. Most patients (59.6%) had pneumonia. All isolates of K. pneumoniae were resistant to ampicillin (100%); several (42.6%) were resistant to other antibiotics also. The overall mortality rate was 55.3%. A higher mean initial blood pressure and lower concentrations of serum urea and bilirubin were found in survivors. None of the 28 patients, surviving more than 48 h who received combined therapy with an aminoglycoside and a beta-lactam antibiotic (to which the organism was susceptible) died. Among the remaining patients treated with either an appropriate beta-lactam agent alone, an appropriate aminoglycoside alone or ciprofloxacin the combined mortality rate was 83.3% (P = 0.007).

Recovery of urea nitrogen from seawater for measurement of 15N abundance in urea regeneration studies using the isotope-dilution approach

Two procedures for extracting urea nitrogen from seawater for determination of its 15N abundance in urea production measurements are described. They involve hydrolysis of urea in the presence of urease and subsequent concentration of the liberated ammonium by either vacuum distillation with diffusion (distillation-diffusion method) or solely by diffusion (diffusion method). Recovery tests, both for total nitrogen and 15N content, showed that these methods are suitable for application to isotope-dilution experiments. Sufficient nitrogen for mass spectrometric analysis is recovered from 300-ml filtrate samples with low initial urea concentrations by adding ∼ 6 μmol of 14N-urea carrier. Comparisons between theoretically expected and measured isotopic compositions of urea-nitrogen standards gave a near-1: 1 correspondence for abundances ranging from 0.32 to 86.0 at.% excess 15N. The best precision obtained for isotopic determination enables the detection of differences in enrichment of 0.28 at.% excess 15N for samples with an initial enrichment (before 15N-urea carrier addition) of ∼ 4.0 at.% excess 15N. Results from an application of one of the two procedures (diffusion method) to a field study (time-course experiment) indicated that (1) urea uptake and production are strongly time dependent, (2) ignoring 15N-isotope dilution resulting from urea production leads to underestimates in calculated uptake rates of up to 25%, and (3) during the early stage of incubation (< 1 hh) up to 88% of 15N leaving the dissolved phase is not recovered in the particulate phase. A possible mechanism responsible for the apparent missing 15N suggests efflux of urea as 15N-labelled ammonia/ammonium from algal cells.

Urea-induced unfolding of the alpha subunit of tryptophan synthase: evidence for a multistate process.

The urea-induced unfolding of the alpha subunit of tryptophan synthase from E. coli was monitored by optical spectroscopy and by urea-gradient gel electrophoresis. Three independent lines of evidence support the conclusion that one or more stable intermediates are present in this process: (i) Satisfactory fits of the equilibrium unfolding transitions obtained from difference spectroscopy at 286 nm and circular dichroism spectroscopy at 222 nm require a model which involves a stable intermediate in addition to the native and unfolded forms. (ii) Kinetic studies of the change in the extinction coefficient at 286 nm show that while the unfolding is well described by a single exponential change the refolding kinetics are complex. The nature of the dependence of the refolding kinetics on the initial concentration of urea supports the conclusion that at least one stable intermediate exists. (iii) The patterns obtained from urea-gradient gel electrophoresis experiments on the alpha subunit show that at least one and possibly two stable intermediates are involved; the intermediates have markedly different degrees of compactness. A kinetic model for the folding of the alpha subunit, consistent with all of these results, can be formulated.

Effects of pH and urea concentration on urea removal from dialysate by enzymatic conversion and gas absorption using a capillary hemodialyzer

This paper reports the effects of initial urea concentrations and pH values on the performance of a capillary hemodialyzer for the removal of free ammonia (NH3) formed by enzymatic hydrolysis of urea. Air was chosen as the gas phase. NaOH was used for alkalinization of dialysate. It was shown that one of the most important parameters was free ammonia concentration. The degrees of alkalinization and the initial urea concentration, were also important. Although even without the addition of NaOH, about 42% of the initial urea can be removed in one hour.

Tracing the Transformations of Urea Fertilizer during Leaching

AbstractThe transport and transformation of urea, NH4+, and NO3‐ in soil were mathematically described as functions of depth and time, subject to either steady feed or pulse application of N, using relationships derived by assuming diffusion and mass transfer as transport processes and first‐order kinetics as a transformational mechanism. The mathematical model was used to study the enzymatic hydrolysis of urea, nitrification, and denitrification in laboratory soil columns. Urea hydrolysis was found to be independent of initial urea concentration and the O2 concentration of the soil atmosphere. The rate of nitrification under transient N conditions was found to be one order of magnitude greater than the rate of denitrification when there was 20% O2 in the soil atmosphere. Populations of nitrifiers were found to increase in response to successive applications of N.

The effect of growth and urea concentration on ammonia production by a urea-hydrolysing mycoplasma (Ureaplasma urealyticum).

The rate of accumulation of ammonium ion in cultures of Ureaplasma urealyticum was independent of the growth rate and of the initial urea concentration above 0-025% in the medium, although the quantity of ammonium ion accumulating did depend on the initial urea concentration. Ammonium ions accumulated at a similar rate in U. urealyticum cultures of both rapidly and slowly growing organisms. Viable but non-growing ureaplasmas also produced ammonia in complete medium at a lower temperature than usual (25 degrees C) or in an inadequate growth medium at 37 degrees C. The rate of ammonium ion accumulation in a dying culture depended on the number of viable organisms present; this is relevant to diagnostic methods for ureaplasms which depend on detecting ammonia colorimetrically.

Dialysis culture of T-strain mycoplasmas.

Using dialyzing cultures of T-strain mycoplasmas, it was possible to make some observations relevant to the growth and metabolism of these organisms which would not be possible in nondialyzing cultures due to growth inhibition of the organisms by elevated pH and increased ammonium ion concentration in media containing urea. The rate of ammonia accumulation was found to be related to the initial urea concentration in the medium and could not be accounted for by any change in the multiplication rate of the organisms. More ammonia was generated than could be accounted for by the added urea alone, suggesting that an ammonia-producing activity other than urease may be present in T-strain mycoplasmas. Titers above 10(7) color change units per ml were achieved in dialysis cultures of a T-strain mycoplasma in the presence of urea, and such titers were maintained for approximately 60 h during dialysis culture in the absence of added urea.

Effect of urea concentration on its utilization, on the pH and the formation of ammonia and carbon dioxide in a human salivary sediment system

Experiments were initiated to study the metabolism of urea in a salivary sediment system in which the sediment concentration was 16.7 per cent (v/v). The pH, utilization of urea and formation of NH 3 and CO 2 at each of several urea concentrations, between 0 and 10 per cent (w/v), were measured before and during incubation of the system for 4 hr at 37 °C. At low levels of urea, the urea was completely utilized before the end of the 4-hr experimental period, and the pH rose and subsequently fell. At higher levels, the urea was not used up and the pH rapidly rose to an asymptote. Ammonia and CO 2 formation both increased with increase in the initial urea concentration. Both at low and high substrate levels, NH 3 and CO 2 progressively accumulated, the latter much more than the former since most of the urea-N was “stored” whereas most of the urea-C was not. At the low urea levels, nitrogen storage occurred as the urea was utilized but N was released as NH 3 after the urea had disappeared from the medium. No urease activity was observed in the system and it was postulated that urea breakdown occurred by a reverse ornithine cycle. Radioautographs of two-dimensional paper chromatograms of alcohol extracts of sediment showed the formation and depletion of an unidentified 14C-labelled compound during the early part of the 4-hr incubation and the formation of some lactic and succinic acid during the later part. The formation of these two acids indicated that pathways of urea catabolism are linked to pathways involved in the catabolism of carbohydrate.

Kinetics of the formation of biuret from urea in 2-methoxyethanol

Abstract The formation of biuret from urea has been studied kinetically in 2-methoxyethanol at 120° by estimating the formed biuret with visible spectrophotometry. The apparent second-order rate constant decreases with proceeding of the reaction and with increasing the initial concentration of urea and ammonia. The addition of ammonia is effective in improving the constancy of apparent second-order rate constants. The reaction is subject to the base catalysis, where imidazole is more effective than triethylamine (a stronger base) or N-methylimidazole. The kinetic data are explicable by a mechanism involving the decomposition of urea to ammonia and isocyanic acid followed by the base-catalysed addition of the latter to urea.

The reversibility of the mercuric chloride inhibition of urease at varied initial urea concentrations

1. 1. The change in “titratable—SH” during urease activity has been found by the inactivation-reactivation method to pass through a maximum value when determined at varied initial urea substrate concentrations. 2. 2. The observed change in “titratable—SH” during urease activity is in agreement with results obtained by the direct colorimetric method (9). 3. 3. The concentration of thiol compound required for the reactivation of urease after mercuric chloride inhibition also varied according to the varied initial urea substrate concentration. 4. 4. In agreement with the results of Hellerman et al. when activity measurements were made at a fixed urea substrate concentration, the constancy of the—SH groups in urease was demonstrated.

An approximative colorimetric method for the determination of urea, with an application to the detection and quantitative estimation of arginase

The urease method for the determination of urea consists essentially of two distinct operations: first, the enzymatic conversion of the urea into its equivalent of ammonium carbonate, and, secondly, the quantitative determination of the latter. Each of these operations may be accomplished in a variety of ways. In the second, for instance, no less than five different analytical procedures have hitherto been made use of. Either the alkaline ammonium carbonate has been titrated directly with acid (1); or its ammonia has been liberated by a stronger base, conducted into an excess of standard acid, and determined by titration (2); or the ammonia has been estimated colorimetrically by Nesslerization (3); or a measured amount of acid has been added to the alkaline reaction product, and the uncombined excess determined iodometrically (4); or, finally, the ammonium carbonate has been estimated by gasometric determination of its carbon dioxide component (5). One other procedure at least is theoretically possible. The transformation of a molecule of neutral urea into a molecule of ammonium carbonate involves a corresponding reduction in hydrogen ion concentration of the medium in which the change takes place. If, therefore, that medium contains an appropriate indicator, it should be possible to deduce from its change of colour the amount or concentration of the urea hydrolysed. Upon this principle we have devised a method which, although hardly a method of precision, has proved itself in certain special circumstances exceedingly useful. Our procedure is in its essential features identical with one already described by Kay (6), but achieves, through certain refinements, a much closer approach to accuracy than Kay was concerned to attain. The first step in the elaboration of the method was to ascertain what effect upon hydrogen ion concentration is produced by the ammonium carbonate derivable from known quantities of urea. Obviously this must depend upon three factors, of which the concentration of the urea itself is only the first. The others are (i) the original p H of the urea-urease mixture, and (ii) the nature and concentration of the buffer salts present. Only when these two conditions are maintained invariable will there be a fixed correspondence between initial concentration of urea and final concentration of hydrogen ions. It is further evident that if the buffer value of the medium chosen be large, the change of p H effected in a given volume by a given addition of ammonium carbonate will be relatively small; and vice versa . With any selected buffer, therefore, the sensitivity of the reaction is controlled by the concentration in which that buffer is present, and may accordingly be varied to suit the nature of the material, the quantities of urea to be dealt with, and other pertinent circumstances. For the purposes we have had in view the most suitable buffer has been a phosphate mixture of M/20 concentration and of initial p H 6·8. The effect upon this mixture of the enzymatic transformation of known quantities or concentrations of urea was determined in the following way:— 1. A series of about thirty Pyrex test-tubes, 1 x 10 cms. in size and of as nearly as possible equal internal diameter, was graduated at 5 c. c.