Intraocular penetration of toxic substances

PURPOSE: The seriousness of ocular alkali burns has been linked to the rapidity with which the alkali enters the eye. The authors report the results of an experimental study on intraocular ammonia penetration. MATERIALS AND METHODS: 23 eyes of New Zealand albino rabbits were burned by applying for one minute 100 ul of a solution titrating 15.3 percent of ammonia. A pH meter probe was inserted into the anterior chamber beforehand to permit pH measurements every 5 seconds. Experiments were carried out after 1,3,5,10 and 30 minutes. The ammonia concentration was measured at the end of the experiment by puncturing the anterior chamber. RESULTS: The pH increased 1 to 3 minutes after applying ammonia on the cornea, reaching a maximum (mean) of 10 five to six minutes later, before an exponential decrease. After 30 minutes, the pH was always more than the physiological pH. The penetration ratio of ammonia through the cornea is about 11 percent. At 30 minutes, the concentration of ammonia is low. The pH observed differs from the calculated pH. DISCUSSION: The difference between observed and calculated pH indicates increases with an intervening plateau, showing the existence of two successive acid-base chemical reactions between ammonia and two types of acid. In addition, the amount of protein destroyed by ammonia can be calculated. CONCLUSIONS: These data show for the time the possibility of calculating the density of proteins destroyed in vivo by a base penetrating the anterior chamber. This opens the possibilty of interesting research work, because it is possible to relate the density of proteins destroyed in the eye to the pK of this base, and to forecast the potential danger of a base of biological tissues.(AU)

Ocular ammonia burns: treatment

PURPOSE: The authors present the results of an experimental study to support the proposal for a delay in ocular bathing in the treatment of severe ocular burns due to ammonia. This study compares two solution of ocular wash: physiological serum and Diphoterine. MATERIALS AND METHODS: 23 eyes of New Zealand albino rabbits were burned for one minute by 100 ul of a solution containing 15.3 percent ammonia. Then, each eye was washed by an ocular wash of 250 ml of physiological serum, or of 250 ml of Diphoterine, after a delay of 1,3,5,10 and 30 minutes. The effects were appraised by measuring the anterior chamber, the concentration of ammonia in the anterior chamber, and by the cytopathological analysis of these burned corneas. RESULTS: An ocular wash with Diphoterine in the first few minutes following an ocular burn induces an inflexion of the pH graph, contrary to an ocular wash with physiological serum. On the other hand, there is no inflexion of the pH graph at 30 minutes, and the concentration of ammonia in the anterior chamber is low at that time. The cytopathological analysis shows stromal oedema with the ocular wash by physiological serum, but not when Diphoterine ocular wash is used. DISCUSSION AND CONCLUSIONS: This study proves the importance of ocular bathing in the first minutes following an ocular burn by ammonia. The efficacy of an external ocular wash by Diphoterine features; the importanve of sequelae has been linked to the initial stromal oedema.(AU)

Whole body protein turnover and resting metabolic rate in homozygous sickle cell disease

Whole body protein turnover and resting metabolic rate were measured in six adults with homozyguous sickle cell disease (genotype HbSS) and in six normal adults (genotype HbAA) of similar age. Turnover was measured with prime/intermittent oral doses of [15N]glycine over 18 h and resting energy expenditure was measured by indirect calorimetry. In HbSS, nitrogen flux (0.9 ñ 0.08 g day-1 kg-1), protein synthesis (6.0 ñ 0.5 g day-1 kg-1) and protein degradation (5.6 ñ 0.5 g day-1 kg-1) were significantly increased compared with HbAA nitrogen (flux 0.5 ñ 0.02g day-1 kg-1, protein synthesis 3.2 ñ 0.2 g day-1 kg-1 and protein degradation 2.8 ñ 0.2 g day-1 kg-1). Resting energy expenditure was significantly higher in HbSS compared with HbAA when expressed per unit of body weight (115 ñ 3 and 94 ñ 4 kj day-1 kg-1 respectively) or weight 0.75(317 ñ 6 and 269 ñ 8 kj day-1kg-0.75, respectively). The increase in protein turnover and energy expenditure suggest that patients with HbSS exist in a hypermetabolic state that requires greater dietary energy compared with HbAA. (AU)

Whole-body protein turnover in man determined in three hours with oral or intravenous 15N-glycine and enrichment in urinary ammonia

Studies were carried out in eight normal adults to simplify the continuous infusion-end product method for measuring whole-body protein turnover using 15 N-glycine. When a priming dose of label suitable for the urea pool was followed by intermittent oral doses of label, plateau enrichment was maintained in urinary urea and ammonia from 9 to 18 h, giving values for nitrogen flux. (18h) of 0.69ñ0.05 g N/kg/d with urea and 0.46ñ0.01 g N/kg/d with ammonia. With a priming dose appropriate for the ammonia pool, plateau was reached in urinary ammonia in less than 120 min an maintained for up to 6h. Nitrogen flux (3h) with oral 15N-glycine was 0.96ñ0.12 g N/kg/d, and with intravenous label was 0.61ñ0.13 g N/kg/d. There was a significant linear relationship between flux measured with oral and intravenous isotope. It is suggested that different components of protein turnover are measured with the different approaches, and that the short method in particular measures rapidly turning over proteins associated with the gastrointestinal tract.(AU)

Studies of whole body protein turnover within three hours in man using 15 N-glycine as tracer and urinary ammonia as end-product - abstract

We have been investigating ways in which to simplfy the method for measuring proteing turnover, by shortening the time required for reliable measurements. It has been possible, using 15N-Glycine and urinary ammonia as the end-product, to measure turnover in two to three hours in normal man. Turnover was calculated following either oral or continuous infusion of 15N-Glycine (0.005 15N/kg/h) in 8 adult men. Plataeu was reached within 120 minutes and was maintained for at least 4 to 6 hours. The results were as follows: AMMONIA ENRICHMENT: Oral dose - turnover (mgN/kg/D) 960ñ128 (SD), synthesis (gPr/kg/D) 4.7ñ0.7, breakdown (gPr/kg/D) 4.9ñ0.8; AMMONIA ENRICHMENT: I.V. dose turnover - (mgN/kg/D) 608ñ128, synthesis (gPr/kg/D) 3.1ñ0.8, breakdown (gPr/kg/D) 2.8ñ0.8. There was a significant linear relationship between ammonia enrichment (oral) and ammonia enrichment (I.V.) (R=0.82; p<0.05). In the same group, turnover measured from ammonia enrichment over 18 hours was 450 ñ 30mg N/kg/D. Turnover measured over 3 hours thus gave higher values than measurements over 18 hours; this is probably because more proteins with shorter half-lives are included in the 3 hour measurements. This would imply that previously reported measurements which used the longer measurement period underestimated turnover by at least 50 percent, and hence significantly underestimated the contribution of protein turnover to basal energy expenditure. The (3 hr) oral measurement appears to measure flux through a pool not included in the measurement intravenously. As this is of a similar magnitude to the pool into which hydrolysed urea nitrogen passess (Jackson et al, Hum. Nutr. Clin. Nutr., 38C, 339, 1984) it may represent a distinct enterohepatic pool of metabolic nitrogen (AU)

The use of 15 N-alanine as a tracer to measure whole body protein turnover (WBPT) in man - abstract

A preliminary study was designed to determine the most suitable metabolic condition (fed, fasted) and appropriate time (day, night) to measure whole body protein turnover. A primed intermittent oral dose of the tracer was given; urinary ammonia and urea were used as end-products. With feeding, an isotopic steady state was maintained but upon withdrawal of food it was disrupted. Also, as fasting progressed, the enrichment in urinary ammonia and urea kept increasing. This indicates that alanine did not seem to be a good tracer to measure WBPT under metabolic conditions, where the intake of food was not constant. In the fed state, rates of WBPT were measured after a primed, intermittent oral dose and after a primed, continuous intravenous infusion. Six subjects were used in the oral study and two of the six subjects were chosen for the intravenous study. During the oral administration, mean rate of WBPT as measured by the enrichment in urinary ammonia and urea, was found to be 2.25 ñ .2 gm protein/kg/day and 3.00 ñ .4 gm protein/kg/day respectively. The value obtained from the intravenous study was 1.87 gm protein/kg/day with ammonia and 4.57 gm protein/kg/day with urea. The results show that, within studies estimates of protein flux from ammonia and urea generally were not comparable, oral doses gave higher estimates with ammonia whereas the intravenous dose gave higher estimates with ureas. In contrast with the most commonly used tracer (15 N-glycine), 15N-alanine gave estimates that were lower when the respective technique and end product was considered. However, the rank order of these values for the different method was similar to those with 15 N-glycine (AU)

Whole-body protein turnover and nitrogen balance in young children at intakes of protein and energy in the region of maintenance

Nitrogen balance and whole-body protein turnover were measured in children aged about one year taking diets which provided 1.7 or 0.7 g milk protein/kg/d at three levels of metabolizable energy, 80, 90 and 100 kcal/kg/d. All the children were in positive nitrogen balance at all levels of energy intake on 1.7 g protein/ kg/d. Nitrogen equilibrium was maintained on 0.7 g protein/kg/d when the energy intake exceeded 90 kcal/kg/d, but on 80 kcal/kg/d nitrogen balance was negative. Whole-body protein turnover was measured from the enrichment in urinary ammonia following a continuous infusion of15N-glycine. The variation between individuals on the same diet was significantly greater than the variation within individuals at different levels of energy intake. For the group as a whole protein synthesis on 1.7 g protein/kg/d was 0.74, 0.75 and 0.87 g N/kg/d on 100, 90 and 80 kcal/kg/d respectively;whereas on 0.7 g protein/kg/d it was 0.37, 0.38 and 0.40 g N/kg/d. These results show that over this range of intakes protein synthesis decreased as dietary protein fell, but tended to increase as energy intake fell. (AU)

Studies of 15N-alanine metabolism in man: estimation of whole body protein turnover rates, alanine flux, de novo synthesis of alanine contribution to urinary ammonia

A primed intermittent oral dose and a primed continuous intravenous infusion of 15N-alanine was administered to healthy males to determine the rates of protein turnover, synthesis and breakdown. The subjects were fed an adequate intake throughout the day. Urine and blood samples were collected. Mean rate of protein flux as measured by the enrichment of urinary ammonia and urea were found to be 2.25ñ.21 and 3.00ñ.42 g. protein/kg./day respectively during the total administration. The values obtained from the intravenous study were 1.87 g. protein/kg./day with ammonia and 4.57 g. protein/kg./day with urea. When each end product and the respective technique was considered the mean rate obtained using 15N-alanine was lower than that reported using 15N-glycine. However, the rank order of the values for the different methods was similar to those with 15N-glycine. Mean plasma alanine flux after the oral and intravenous studies were 548 ñ128 and 281 æmol/kg./hr. respectively. Red cells alanine flux was different and higher than plasma flux in each study. During the studies a considerable amount of label was transferred to urinary ammonia and urea while glutamate and alanine had low enrichments. In the fed state, after the oral dose, de novo alanine synthesis was calculated to be 457.4 æmol/kg./hr. This accounted for eighty-three percent of plasma alanine flux, thus seventeen percent of alanine moving through the venous plasma compartment originated from performed alanine. Up to sixteen to twenty-two percent of urinary ammonia was derived from alanine (Summary)

Glutamine production rate and its contribution to urinary ammonia in normal man

Glutamine [15N]amide was infused at a steady rate of 33.34 micromoles/h into seven male adult volunteers who were in the fed state and normal acid-base status. Plasma glutamine amide N enrichment and urinary ammonia N enrichment rose to a constant value within 3h. The glutamine production rate was 51.8 ñ 7.9 millimoles/h. The total ammonia execretion rate was 0.87 millimoles/h. Of this excreted ammonia 62.6 ñ 9 percent was derived from the amide N atom of glutamine. The excreted glutamine amide N (0.53 millimoles/h) was only 1 percent of the glutamine production. If half the ammonia formed by the kidney is excreted in urine and half liberated into the renal vein in subjects with normal acid-base status [E. E. Owen & R. R. Robinson (1963) Journal of Clinical Investigation, 42, 263-276], then the kidney accounts for only 2 percent of glutamine disposal. Whole body protein turnover, measured from the urinary [15N]ammonia enrichment, was 30.3 ñ 7.7 g of N/day (2.8 g of protein/day/kg). (AU)

Nitrogen metabolism in the rat

The intermediary metabolism of 6 isotopic amino acids, 15N-aspartic acid, 15N-glutamic acid, 15N-alanine, 15N-glutamine (amide-15N), 15N-glycine and 15N-lysine (O-15N), and 15N-ammonium chloride were investigated. The aim of this study was to establish the precursor-product relationships existing between these amino acids, ammonia and urea in the amino-N pools of the major organs and tissue beds of the normal postprandial rat with the specific objective of following the movement of nitrogen to urea synthesis. It was hoped to ascertain whether glutamic acid played a central role in providing nitrogen for urea synthesis and whether there existed any relationship between 15N distribution patterns of the different isotopes and WBTP rates calculated from hepatic and renal urea-N enrichments. The method employed involved the administration of tracer quantities of the isotopes by the constant infusion technique and measuring the 15N excess of ammonia-N, glutamine amide-N, alanine-N, glutamate-N, aspartate-N and urea-N. It was found that nitrogen from 15N-alanine, 15N-aspartic acid and 15N-glutamic acid was distributed evenly in most of the amino-N pools studied. Nitrogen from the other four isotopes was distributed unevenly, preferentially to ammonia, glutamine amide and urea. 15N-glycine and 15N-lysine were only sparingly metabolised. WBPT rates obtained from urea-N enrichments were not affected by the nitrogen distribution patterns of the isotopes but by the extent to which they metabolised. WBPT rates calculated from ammonia-N enrichments were unduly affected by the extent to which each isotope contributed nitrogen to ammoniagenesis. Glutamic acid does not seem to be the precursor of both nitrogens used for urea synthesis. It supplies only one nitrogen. It is possible that urea is synthesised from an amino-N received via the glutamate to aspartate pathway and an amide-N received via the glutamine to ammonia to carbamyl phosphate pathway. Free ammonia entering the liver is first fixed as glutamine amide before being used for urea synthesis. (AU)

Glutamine metabolism in metabolic acidosis

In chronic metabolic acidosis in the rat, there is increased ammoniagenesis, gluconeogenesis and renal extraction of gluatmine with induction of renal phosphate-dependent glutaninase (PDG). Because the stimulus for these changes is not yet clear and also because acute acidosis is the more common clinical problem, the present study deals mainly with the metabolism of glutamine in acute metabolic acidosis. When acute metabolic acidosis is produced in rats by administration of mineral acid or by causing them to swim, thus inducing a severe lactic acidosis, a factor is found in the plasma which stimulates renal glutamine uptake and ammoniagenesis in vivo as well as in vitro. Acute acidosis dose not induce synthesis of PDG in the kidney but causes a change in enzyme kinetics. The plasma factor not only enhances glutamine entry into cells, but apparently causes a conformational change in PDG, as shown by an increase in V1.0mM/Vmax. Intestinal metabolism of glutamine is also stimulated in vivo and in vitro by the plasma factor of acute acidosis. (AU)

Renal transport and metabolism of glutamine

The experiments described in this thesis were designed to investigate the mechanism of glutamine transport and the effect of acute acidosis on glutamine transport and metabolism in the rat kidney. The experiments done with kidney slices investigated the effect of several amino acids chosen from different structural groups on uptake of glutamine. Proline (an ammino acid) and glycine (at inhibitory concentrations) were found to inhibit ammonia production and glutamine uptake significantly. When a sodium free incubation buffer was used the inhibitory effect of proline and glycine on glutamine uptake disappeared, indicating that the inhibition by proline was sodium dependent. In similar experiments with isolated kidney proximal tubules in a sodium containing buffer, proline also caused a decrease in glutamine uptake and ammonia production. Intracellular/extracellular glutamine distributions ratios in tubules however, showed a marked increase in the presence of proline. The explanation for this is not that proline shares a transport site with glutamine but rather that it is metabolised to glutamate which causes an inhibition of phosphate dependent glutaminase and hence decreased glutamine utilisation. This decreased utilisation leads to an accumulation of glutamine within the renal cell and subsequently increased intracellular/extracellular glutamine distribution ratios. The inhibitory effect of proline on glutamine uptake was much less in experiments with kidney slices from chronically acidotic rats. Isolated tubules from acutely acidotic rats showed increased intracellular/extracellular glutamine distribution ratios, increased ammonia production and glutamine uptake in tubules. When tubules from normal rats were incubated in sera from acutely acidotic rats there was a similar increase in intracellular/extracellular glutamine distribution ratios, ammonia production and glutamine uptake. The effect of the serum however, was not due to the accumulation of glutamate because glutamate levels in tubules were unchanged after incubation in sera from the acutely acidotic rats (AU)

[15N]Glycine metabolism in normal man: the metabolic O-amino-nitrogen pool

[15N]Glycine was infused into fed, fasted or acidotic humans. In all metabolic states there was considerable transfer of labelled nitrogen to urea and amonia, but alanine and glutamate did not become enriched. The findings show that free exchange of nitrogen between all amino acids does not take place. Glycine, serine, threonine, lysine and histidine cannot be considered part of the a-amino-nitrogen pool as classically conceived, although they form up to one-half of that pool. (AU)

Comparison of 15N-labelled glycine, aspartate, valine and leucine for measurement of whole-body protein turnover

Whole-body protein turnover was measured in rats by constant infusion of 15N-labelled glycine, aspartate, valine and leucine and measuring the enrichment of hepatic and renal urea and ammonia nitrogen. The values obtained with [15N] glycine were comparable with values reported with methods based on different assumptions. [15N] Aspartate gave rise to an increased enrichment of urea and ammonia and hence to lower protein-turnover rates. [15N] Valine and [15N] leucine gave low enrichments of nitrogenous end products and hence to high protein-turnover rates. All 15N-labelled amino acids are not equally suitable for measuring whole-body protein turnover by the end-product method. The relative amounts of 15N going to the end products can be prodicted from the known individual metabolism of aspartate and the branched-chain amino acids (AU)

The effect of acidosis on the renal metabolism of glutamine in the rat

Glutamine is the major source of urinary ammonia and this study examines the metabolism of that amino acid in rats under varying acid-base conditions. Other workers have shown that in acidosis there is enhanced renal oxidation of glutamine, which requires increased amounts of acetyl CoA. The present study has shown that during acidosis there was no change in the activity of oxaloacetate decarboxylase and malic enzyme, two enzymes capable of increasing the formation of acetyl CoA. This study also showed that 3-mercaptopicolinic acid inhibited PEPCK - the rate limiting enzyme of gluconeogenesis - and thereby inhibited ammoniagenesis from glutamine mainly by inhibiting deamination. Metabolic acidosis was induced with NH4C1 or HC1. In rats given NH4C1 there was an immediate increase in blood ammonia levels and urinary excretion of ammonia, but this did not occur with rats fed HC1 which showed no change in urinary ammonia but a decrease in urea excretion. Rats fed either NH4C1 or HC1 had similar increases in the plasma concentrations of glutamine, renal PEPCK activity, ammonia and glucose production by renal cortical slices. The time course of changes in metabolic intermediates was measured in rats given NH4C1 or HC1. NH4C1 caused a striking decrease in renal levels of malate, 2-oxoglutarate and phosphoenol pyruvate. Similar changes were observed with HC1, but in addition the levels of glucose and glucose-6-phosphate were elevated. The results of the studies with 3-mercaptopicolinic acid and the metabolite profile in response to acidosis are both constant with the theory that displacement of the glutamic dehydrogenase equilibrium is an important mechanism in the control of glutamine utilisation and the ammoniagenic response to acidosis (AU)

Ammonia production by the small intestine of the rat

Slices of duodenum and jejunum produce ammonia from glutamine in vitro. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. Gut contains glutamine 1 as well as ç-glutamyl transpeptidase. These enzymes do not show any increase during starvation (SUMMARY)