Rate Of Urea Synthesis Research Articles

No effect of ornithine alphaketoglutarate on nitrogen excretion or urea synthesis rate in healthy male subjects

Summary Background & aims Ornithine alphaketoglutarate (OKG) is known for its anabolic and anticatabolic effects. Little is known about its effect on proteolysis. We studied urea formation and nitrogen excretion rates following OKG supplementation in healthy volunteers. Methods Sixteen male subjects were kept on a diet stable in protein (1 g kg−1 d−1) and aimed at maintaining body weight. They received oral supplementation of OKG (10 g) or an isonitrogenous placebo ( l -alanine) from days 4 to 6 in single daily doses in a randomised, blinded fashion. Urea kinetics were studied on day 4 from plasma urea enrichments following an oral [13C]urea bolus. Nitrogen excretion was assessed from 24-h urine collections prior to supplementation, on day 4 and day 7. Results Mean urea rate of appearance was 380±86 and 353±120 μmol kg−1 h−1 in the OKG and placebo group, respectively (p=0.62). There were no significant differences in 24-h nitrogen excretion data between groups, neither at separate time points, in overall values (p=0.40) nor in development over time (p=0.53). Conclusions A single oral dose of OKG did not affect urea production rate differently than the alanine placebo. OKG supplementation led to a temporary increase in nitrogen excretion, as did administration of placebo.

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.

Effect of intravenous amino acids on protein kinetics in preterm infants

To summarize recent findings of the effects of intravenous amino acids on protein kinetics in low-birth-weight infants and to describe the potential cellular mechanism for these observations. Amino acids administered intravenously for 3-5 h in infants have been shown to suppress whole-body proteolysis. Recent data in low-birth-weight infants show that an increase in the dose of amino acid caused a suppression of proteolysis, and a decrease in the rate of glutamine and urea synthesis. These responses returned to basal state, however, when the amino acid infusion continued for 20-24 h. Supplementation with glutamine sustained the suppression of proteolysis after 3-5 days. Plasma insulin concentration did not change during the amino acid infusion. Data from studies in adults and from in vitro studies suggest that the amino acids impact protein breakdown and synthesis via the mammalian target of rapamycin pathway, stimulating initiation of translation and suppressing autophagic proteolysis. Intravenous amino acids, by increasing extracellular amino acid concentration, transiently stimulate protein synthesis and suppress protein breakdown. These effects return to basal state when the amino acid infusions are prolonged. The mechanism of this adaptive response remains to be determined.

Metabolic and neuroendocrine effects on diurnal urea excretion in the mangrove killifishRivulus marmoratus

In mangrove killifish Rivulus marmoratus, urea excretion (J(urea)) follows a distinct diurnal pattern with the highest rates between 12:00 h and 18:00 h. We investigated the regulating mechanisms that underlie temporal rhythms in J(urea) in R. marmoratus. We hypothesized that the daily pattern of J(urea) in R. marmoratus is (1) due to diurnal changes in urea synthesis rates and ultimately metabolic rate and/or (2) controlled by neuroendocrine messengers. Oxygen consumption and whole body urea content in R. marmoratus demonstrated a clear diurnal pattern with maximum rates for both parameters occurring at 12:00 h. A strong synchrony between diurnal patterns of oxygen consumption, whole body urea content and J(urea) implicated metabolic regulation of the diurnal J(urea) pattern. Ketanserin, a 5-HT(2) receptor antagonist, and RU-486, a cortisol receptor antagonist, were used to test the second hypothesis. Increasing antagonist concentrations of either ketanserin or RU-486 resulted in dose-dependent decreases in J(urea). Application of a single dose of either antagonist significantly decreases J(urea) for up to 12 and 6 h for ketanserin and RU-48, respectively. Repeated exposure to doses of either ketanserin or RU-486 did not abolish the diurnal pattern in J(urea); however, there was a significant decrease in the amplitude of the rates. Taken together, these findings indicate that the diurnal pattern of J(urea) in R. marmoratus are regulated by both metabolic and neuroendocrine factors. We propose that cortisol and 5-HT influence the absolute rate of urea excretion by altering the permeability of the gill membrane to urea and/or the rate of urea synthesis.

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, Pelodiscus sinensis

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9-, 3.5-, 2.6-, 2.9-, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0-fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.

Membrane bioreactor using pig hepatocytes for in vitro evaluation of anti-inflammatory drugs

Membrane bioreactor using isolated hepatocytes is a good system for in vitro studies of cell drug metabolism. In this system cells are cultured on modified polyethersulfone membrane under homogeneous, stable and three-dimensional conditions in a similar in vivo microenvironment. The aim of this study was to evaluate liver specific functions of pig hepatocytes cultured in the bioreactor at increasing drug concentration of rofecoxib. Rofecoxib is a potent and highly inhibitor of cyclooxygenase-2 (COX-2), an inducible isoform of cyclooxygenase that plays a key role in inflammatory processes. The bioreactor performance was evaluated by assessing the ability of liver cells to eliminate rofecoxib. Biochemical activity of cells was assessed in terms of urea and albumin synthesis. Pig liver cells cultured in the membrane bioreactor exhibited viability and liver specific functions at higher levels than those of cells cultured in batch systems. The rate of rofecoxib elimination by cells increased with increasing rofecoxib concentration in the range 0–400 nM. Hepatocytes exhibited highest rates of albumin production and urea synthesis at 300–400 nM rofecoxib concentration.

Transamination of Leucine and Nitrogen Accretion in Human Pregnancy and the Newborn Infant1–3

Kinetics of leucine and its oxidation were determined in human pregnancy and in the newborn infant, using stable isotopic tracers, to quantify the dynamic aspects of protein metabolism. These data show that in human pregnancy there is a decrease in whole-body rate of leucine turnover compared with nonpregnant women. In addition, data in newborn infants show that leucine turnover expressed as per kg body weight is higher compared with adults. The administering of nutrients resulted in a suppression of the whole-body rate of proteolysis. Because nonessential amino nitrogen is an important component of nutritional nitrogen and can be limiting for growth under certain circumstances, and because BCAA are an important source of nonessential amino nitrogen, we have examined the relations among the transamination of leucine, leucine N kinetics, and urea synthesis and glutamine kinetics in human pregnancy and newborn infants. In human pregnancy, early in gestation, there is a significant decrease in urea synthesis in association with a decrease in the rate of transamination of leucine. A linear correlation was evident between the rate of leucine reamination and urea synthesis during fasting in pregnant and nonpregnant women. In healthy-term newborn and growing infants, although the reamination of leucine was positively related to glutamine flux, leucine reamination was negatively related to urea synthesis, suggesting a redirection of amino N toward protein accretion. The regulatory mechanism involved in this redirection of nitrogen from irreversible loss to accretion remains under investigation.

Nitrogen metabolism and excretion in the aquatic chinese soft-shelled turtle,Pelodiscus sinensis, exposed to a progressive increase in ambient salinity

This study aimed to determine effects of 6-day progressive increase in salinity from 1 per thousand to 15 per thousand on nitrogen metabolism and excretion in the soft-shelled turtle, Pelodiscus sinensis. For turtles exposed to 15 per thousand water on day 6, the plasma osmolality and concentrations of Na+, Cl- and urea increased significantly, which presumably decreased the osmotic loss of water. Simultaneously, there were significant increases in contents of urea, certain free amino acids (FAAs) and water-soluble proteins that were involved in cell volume regulation in various tissues. There was an apparent increase in proteolysis, releasing FAAs as osmolytes. In addition, there might be an increase in catabolism of certain amino acids, producing more ammonia. The excess ammonia was retained as indicated by a significant decrease in the rate of ammonia excretion on day 4 in 15 per thousand water, and a major portion of it was converted to urea. The rate of urea synthesis increased 1.4-fold during the 6-day period, although the capacity of the hepatic ornithine urea cycle remained unchanged. Urea was retained for osmoregulation because there was a significant decrease in urea excretion on day 4. Increased protein degradation and urea synthesis implies greater metabolic demands, and indeed turtles exposed to 15 per thousand water had significantly higher O2 consumption rate than the freshwater (FW) control. When turtles were returned from 15 per thousand water to FW on day 7, there were significant increases in ammonia (probably released through increased amino acid catabolism) and urea excretion, confirming that FAAs and urea were retained for osmoregulatory purposes in brackish water.

The effect of experimental bile-duct obstruction on critical biosynthetic functions of the liver.

Abstract Using biosynthetic activities which have a maximum energy requirement, the effect of obstruction of the bile-duct has been studied in isolated perfused rat livers, and compared with controls undergoing “sham” ligation and with normal animals. The maximum rate of gluconeogenesis from lactate was inhibited by 67 per cent after bile-duct ligation when compared with the normal value. The maximum rate of urea synthesis which occurred in the presence of ornithine was the same in the jaundiced and in the normal animals. ‘Sham’ operation, without ligation of the bile-duct, inhibited gluconeogenesis by less than 20 per cent but doubled the maximum rate of urea synthesis. It is concluded that complete biliary obstruction seriously impairs gluconeogenesis and prevents the normal increase of urea synthesis which occurs in response to trauma and that these changes develop earlier than might be expected after the onset of obstruction. Some possible mechanisms of these effects are discussed. If similar abnormalities of hepatocyte function are demonstrated in man it is suggested that surgical relief of obstructive jaundice is indicated at an early stage.

Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period

Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

Oxygenation during hypothermic rat liver preservation: An in vitro slice study to demonstrate beneficial or toxic oxygenation effects

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea-synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia-reperfusion injury. Furthermore, expression of uncoupling protein-2 (UCP-2) mRNA was assessed since UCP-2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW-MP) with 0%, 21%, or 95% oxygen at 0-4 degrees C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW-MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea-synthesis rate as well as lower ATP levels. mRNA upregulation of UCP-2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW-MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP-2. We conclude that saturation of UW-MP with 21% oxygen allows optimal preservation results.

Casein and Soy Protein Meals Differentially Affect Whole-Body and Splanchnic Protein Metabolism in Healthy Humans

Dietary protein quality is considered to be dependent on the degree and velocity with which protein is digested, absorbed as amino acids, and retained in the gut as newly synthesized protein. Metabolic animal studies suggest that the quality of soy protein is inferior to that of casein protein, but confirmatory studies in humans are lacking. The study objective was to assess the quality of casein and soy protein by comparing their metabolic effects in healthy human subjects. Whole-body protein kinetics, splanchnic leucine extraction, and urea production rates were measured in the postabsorptive state and during 8-h enteral intakes of isonitrogenous [0.42 g protein/(kg body weight . 8 h)] protein-based test meals, which contained either casein (CAPM; n = 12) or soy protein (SOPM; n = 10) in 2 separate groups. Stable isotope techniques were used to study metabolic effects. With enteral food intake, protein metabolism changed from net protein breakdown to net protein synthesis. Net protein synthesis was greater in the CAPM group than in the SOPM group [52 +/- 14 and 17 +/- 14 nmol/(kg fat-free mass (FFM) . min), respectively; P < 0.02]. Urea synthesis rates decreased during consumption of both enteral meals, but the decrease tended to be greater in the subjects that consumed CAPM (P = 0.07). Absolute splanchnic extraction of leucine was higher in the subjects that consumed CAPM [306 +/- 31 nmol/(kg FFM . min)] vs. those that consumed SOPM [235 +/- 29 nmol/(kg FFM . min); P < 0.01]. In conclusion, a significantly larger portion of soy protein is degraded to urea, whereas casein protein likely contributes to splanchnic utilization (probably protein synthesis) to a greater extent. The biological value of soy protein must be considered inferior to that of casein protein in humans.

Microfabricated grooved substrates as platforms for bioartificial liver reactors

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

Ornithine‐urea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days

The objectives of this study were (1) to determine the type of carbamoyl phosphate synthetase (CPS) present, and the compartmentalization of arginase, in the livers of the African lungfishes, Protopterus aethiopicus and Protopterus annectens, and (2) to elucidate if these two lungfishes were capable of increasing the rates of urea synthesis and capacities of the ornithine-urea cycle (OUC) during 6 days of aerial exposure without undergoing aestivation. Like another African lungfish, Protopterus dolloi, reported elsewhere, the CPS activities from the livers of P. aethiopicus and P. annectens had properties similar to that of the marine ray (Taeniura lymma), but dissimilar to that of the mouse (Mus musculus). Hence, they possessed CPS III, and not CPS I as reported previously. CPS III was present exclusively in the liver mitochondria of both lungfishes, but the majority of the arginase activities were present in the cytosolic fractions of their livers. Glutamine synthetase (GS) activity was also detected in the hepatic mitochondria of both specimens. Therefore, our results suggest that the evolution of CPS III to CPS I might not have occurred before the evolution of extant lungfishes as suggested previously, prompting an examination of the current view on the evolution of CPS and OUC in vertebrates. Aerial exposure led to significant decreases in rates of ammonia excretion in P. aethiopicus and P. annectens, but there were no accumulations of ammonia in their tissues. However, urea contents in their tissues increased significantly after 6 days of aerial exposure. The estimated rates of urea synthesis in P. aethiopicus and P. annectens increased 1.2- and 1.47-fold, respectively, which were smaller than that in P. dolloi (8.6-fold) reported elsewhere. In addition, unlike P. dolloi, 6 days of aerial exposure had no significant effects on the hepatic CPS III activities of P. aethiopicus and P. annectens. In contrast, aerial exposure induced relatively greater degrees of reductions in ammonia production in P. aethiopicus (34%) and P. annectens (37%) than P. dolloi (28%) as previously reported. Thus, our results suggest that various species of African lungfishes respond to aerial exposure differently with respect to nitrogen metabolism and excretion, and it can be concluded that P. aethiopicus and P. annectens depended more on reductions in ammonia production than on increases in urea synthesis to ameliorate ammonia toxicity when exposed to terrestrial conditions.

Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfishProtopterus dolloi

The slender African lungfish, Protopterus dolloi, does not aestivate in a subterranean mud cocoon, but is capable of aestivating inside a layer of dried mucus on land during drought. In this study, we aimed to elucidate if a slight increase in salinity in association with changes in the ionic composition could act as signals for P. dolloi to decrease endogenous ammonia production, in preparation for aestivation when the external medium dries up. Specimens of P. dolloi exposed to 3 per thousand water for 6 days exhibited consistently lower daily urea excretion rate than the freshwater control. This led to significant decreases in the cumulative total nitrogenous wastes excreted on days 3, 5 and 6. On day 6, there were decreases in urea contents in various tissues and organs. Taken together, these results suggest that there was a decrease in the rate of urea synthesis, the magnitude of which was greater than the decrease in the rate of urea excretion, and therefore resulted in decreases in internal urea contents. A decrease in the rate of urea synthesis should result in a decrease in the rate of glutamine utilization, and subsequently led to the accumulations of glutamine and/or ammonia. However, there were no changes in contents of glutamine and ammonia in various tissues and organs in the experimental animals. A logical explanation for this is that there must be a simultaneous reduction in ammonia production; if not, ammonia would accumulate due to the decrease in rate of urea synthesis. Since fish were unfed during the experiment, endogenous ammonia must be derived mainly from amino acid catabolism. Therefore, these results suggest that a suppression of amino acid catabolism occurred in specimens exposed to 3 per thousand for 6 days. The differences in effects of freshwater and 3 per thousand water on endogenous ammonia production could not be due to food deprivation because both groups of fish were fasted for the same period. Because control and experimental fish were kept in water and because there were no changes in the wet mass of the fish and blood osmolality before and after the experiment, dehydration did not occur. Furthermore, both groups of fish have comparable blood pH, pO2 and pCO2 on day 6 as they had free access to air, and therefore CO2 retention could be eliminated as the initiating factor of suppressed endogenous ammonia production. In conclusion, our results suggest that P. dolloi could respond to increases in salinity and changes in ionic composition in the external medium by suppressing ammonia production in preparation for aestivation when the water dries up.

Increases in urea synthesis and the ornithine-urea cycle capacity in the giant African snail,Achatina fulica, during fasting or aestivation, or after the injection with ammonium chloride

The objectives of this study are to determine whether a full complement of ornithine-urea cycle (OUC) enzymes is present in the hepatopancreas of the giant African snail Achatina fulica, and to investigate whether the rate of urea synthesis and the OUC capacity can be up-regulated during 23 days of fasting or aestivation, or 24 hr post-injection with NH(4)Cl (10 micromol g(-1) snail) into the foot muscle. A. fulica is ureotelic and a full complement of OUC enzymes, including carbamoyl phosphate synthetase III (CPS III), was detected from its hepatopancreas. There were significant increases in the excretion of NH(4)(+), NH(3) and urea in fasting A. fulica. Fasting had no significant effect on the tissue ammonia contents, but led to a progressive accumulation of urea, which was associated with an 18-fold increase in the rate of urea synthesis. Because fasting took place in the presence of water and because there was no change in water contents in the foot muscle and hepatopancreas, it can be concluded that the function of urea accumulation in fasting A. fulica was unrelated to water retention. Aestivation in arid conditions led to a non-progressive accumulation of urea in A. fulica. During the first 4 days and the last 3 days of the 23-day aestivation period, experimental snails exhibited significantly greater rates of urea synthesis compared with fasted snails. These increases were associated with significant increases in activities of various OUC enzymes, except CPS III, in the hepatopancreas. However, the overall urea accumulation in snails aestivated and snails fasted for 23 days were comparable. Therefore, the classical hypothesis that urea accumulation occurred to prevent water loss through evaporation during aestivation in terrestrial pulmonates may not be valid. Surprisingly, there were no accumulations of ammonia in the foot muscle and hepatopancreas of A. fulica 12 or 24 hr after NH(4)Cl was injected into the foot muscle. In contrast, the urea content in the foot muscle of A. fulica increased 4.5- and 33-fold at hour 12 and hour 24, respectively, and the respective increases in the hepatopancreas were 4.9- and 32-fold. The exogenous ammonia injected into A. fulica was apparently detoxified completely to urea. The urea synthesis rate increased 148-fold within the 24-hr experimental period, which could be the greatest increase known among animals. Simultaneously, there were significant increases in activities of glutamine synthetase (2.5-fold), CPS III (3.1-fold), ornithine transcarbamoylase (2.3-fold), argininosuccinate synthetase+lyase (13.6-fold) and arginase (3.5-fold) in the hepatopancreas 12 hr after the injection of NH(4)Cl. Taken altogether, our results support the view that the primary function of urea synthesis through the OUC in A. fulica is to defend against ammonia toxicity, but suggest that urea may have more than an excretory role in terrestrial pulmonates capable of aestivation.

The interplay of increased urea synthesis and reduced ammonia production in the African lungfishProtopterus aethiopicus during 46 days of aestivation in a mucus cocoon

This study was undertaken to test the hypothesis that the rate of urea synthesis in Protopterus aethiopicus was up-regulated to detoxify ammonia during the initial phase of aestivation in air (day 1-day 12), and that a profound suppression of ammonia production occurred at a later phase of aestivation (day 35-day 46) which eliminated the need to sustain the increased rate of urea synthesis. Fasting apparently led to a greater rate of nitrogenous waste excretion in P. aethiopicus in water, which is an indication of increases in production of endogenous ammonia and urea probably as a result of increased proteolysis and amino acid catabolism for energy production. However, 46 days of fasting had no significant effects on the ammonia or urea contents in the muscle, liver, plasma and brain. In contrast, there were significant decreases in the muscle ammonia content in fish after 12, 34 or 46 days of aestivation in air when compared with fish fasting in water. Ammonia was apparently detoxified to urea because urea contents in the muscle, liver, plasma and brain of P. aethiopicus aestivated for 12, 34 or 46 days were significantly greater than the corresponding fasting control; the greatest increases in urea contents occurred during the initial 12 days. There were also significant increases in activities of some of the hepatic ornithine-urea cycle enzymes from fish aestivated for 12 or 46 days. Therefore, contrary to a previous report on P. aethiopicus, our results demonstrated an increase in the estimated rate of urea synthesis (2.8-fold greater than the day 0 fish) in this lungfish during the initial 12 days of aestivation. However, the estimated rate of urea synthesis decreased significantly during the next 34 days. Between day 35 and day 46 (12 days), urea synthesis apparently decreased to 42% of the day 0 control value, and this is the first report of such a phenomenon in African lungfish undergoing aestivation. On the other hand, the estimated rate of ammonia production in P. aethiopicus increased slightly (14.7%) during the initial 12 days of aestivation as compared with that in the day 0 fish. By contrast, the estimated rate of ammonia production decreased by 84% during the final 12 days of aestivation (day 35-day 46) compared with the day 0 value. Therefore, it can be concluded that P. aethiopicus depended mainly on increased urea synthesis to ameliorate ammonia toxicity during the initial phase of aestivation, but during prolonged aestivation, it suppressed ammonia production profoundly, eliminating the need to increase urea synthesis which is energy-intensive.

The African Lungfish,Protopterus dolloi, Detoxifies Ammonia to Urea during Environmental Ammonia Exposure

The African lungfish, Protopterus dolloi, was able to maintain a low level of blood plasma ammonia during exposure to high concentrations of environmental ammonia. After 6 d of exposure to 30 or 100 mM NH(4)Cl, the total ammonia concentrations in the blood plasma were 0.288 and 0.289 mM, respectively, which were only 1.7-fold greater than the control value of 0.163 mM. In addition, accumulation of ammonia occurred only in the muscle, but not in the liver. This was achieved in part through urea synthesis, as reflected by significant increases in urea contents in the muscle, liver, and plasma of the experimental animals. In contrast with plasma ammonia, the plasma urea concentrations of specimens exposed to 30 or 100 mM NH(4)Cl for 6 d increased 15.4-fold and 18.8-fold, respectively. Taken together, these results suggest that P. dolloi upregulated the rate of urea synthesis to detoxify ammonia during environmental ammonia exposure and that the increased rate of urea synthesis was fast enough to compensate for the rate of endogenous ammonia production plus the net influx of exogenous ammonia in these experimental animals. Simultaneously, there were increases in the rates of urea excretion in the experimental animals between day 2 and day 6 of environmental ammonia exposure. Interestingly, the rates of urea excretion in specimens exposed to 100 mM NH(4)Cl were lower than those exposed to 30 mM NH(4)Cl, despite the presumably greater load of ammonia to be detoxified to urea in the former situation. It would appear that P. dolloi was regulating the rate of urea excretion during ammonia exposure to retain urea, which might have some physiological functions under environmental stresses yet to be determined. There were decreases in the contents of glutamate, glutamine, and total free amino acids in the liver of the experimental animals, which indirectly suggest that a reduction in the rate of proteolysis and/or amino acid catabolism would have occurred that might lead to a decrease in ammonia production. Our results suggest that, unlike marine elasmobranchs and coelacanths, which synthesize and retain urea for osmoregulatory purposes, the ureogenic P. dolloi was adapted to synthesizing and excreting urea for the purpose of ammonia detoxification.

Insulin Concentration during Preconditioning Mediates the Regulation of Urea Synthesis during Exposure to Amino Acid-Supplemented Plasma

Understanding the effects of preconditioning on cellular metabolism is important in providing insight into how cells and tissues may behave when confronted with alterations in their environment. Previously, elevated accumulation of lipids and a correspondingly reduced rate of urea synthesis were found in primary heptocytes preconditioned in culture medium containing high levels of insulin and then exposed to plasma. Subsequent studies found that preconditioning primary hepatocytes in medium containing low levels of insulin before exposure to plasma supplemented with amino acids was able to confer resistance to the lipid-accumulating effect of plasma exposure and restored urea synthesis. In the current study, we investigated the effects of insulin preconditioning and amino acid supplementation on the gene expression profile of the urea cycle and fatty acid metabolism enzymes. We found that insulin preconditioning mediates the effects of amino acids in the plasma exposure period on urea synthesis. Urea synthesis is regulated by insulin and amino acids through both short-term and long-term control. Possible mechanisms of long-term control through transcriptional regulation of urea synthesis by insulin, amino acids, and enzymes and transcription factors associated with lipid metabolism are discussed.

The ammonotelic African lungfish, Protopterus dolloi, increases the rate of urea synthesis and becomes ureotelic after feeding.

This study aimed to elucidate the role of urea synthesis in the slender African lungfish Protopterus dolloi in detoxifying ammonia after feeding. There were significant increases in the rate of ammonia excretion in P. dolloi between hours 6 and 15 after feeding. Simultaneously, there were significant increases in urea excretion rates between hours 3 and 18. Consequently, the percentage of total nitrogen (N) excreted as urea N increased to approximately 60% between hours 12 and 21 post-feeding. Hence, after feeding, the normally ammonotelic P. dolloi became ureotelic. Approximately 41% of the N intake from food was excreted within 24 h by P. dolloi, 55% of which was in the form of urea N. At hour 12 post-feeding, the accumulation of urea N was greater than the accumulation of ammonia N in various tissues, which indicates that feeding led to an increase in the rate of urea synthesis. This is contrary to results reported previously on the infusion of ammonia into the peritoneal cavity of the marine dogfish shark, in which a significant portion of the exogenous ammonia was excreted as ammonia. In contrast, feeding is more likely to induce urea synthesis, which is energy intensive, because feeding provides an ample supply of energy resources and leads to the production of ammonia intracellularly in the liver. The capacity of P. dolloi to synthesize urea effectively prevented a postprandial surge in the plasma ammonia level as reported elsewhere for other non-ureogenic teleosts. However, there was a significant increase in the glutamine content in the brain at hour 24, indicating that the brain had to defend against ammonia toxicity after feeding.