Rate Of Urea Synthesis Research Articles

Postprandial increases in nitrogenous excretion and urea synthesis in the giant mudskipperPeriophthalmodon schlosseri

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the giant mudskipper, Periophthalmodon schlosseri, with special emphasis on the role of urea synthesis in ammonia detoxification. The ammonia and urea excretion rates of P. schlosseri increased 1.70- and 1.92-fold, respectively, within the first 3 h after feeding on guppies. Simultaneously, there were significant decreases in ammonia levels in the plasma and the brain, and in urea contents in the muscle and liver, of P. schlosseri at 3 h post-feeding. Thus, it can be concluded that P. schlosseri was capable of unloading ammonia originally present in some of its tissues in anticipation of ammonia released from the catabolism of excess amino acids after feeding. Subsequently, there were significant increases in urea content in the muscle, liver and plasma (1.39-, 2.17- and 1.62-fold, respectively) at 6 h post-feeding, and the rate of urea synthesis apparently increased 5.8-fold between 3 h and 6 h. Increased urea synthesis might have occurred in the liver of P. schlosseri because the greatest increase in urea content was observed therein. The excess urea accumulated in the body at 6 h was completely excreted between 6 and 12 h, and the percentage of waste-N excreted as urea-N increased significantly to 26% during this period, but never exceeded 50%, the criterion for ureotely, meaning that P. schlosseri remained ammonotelic after feeding. By 24 h, 62.7% of the N ingested by P. schlosseri was excreted, out of which 22.6% was excreted as urea-N. This is the first report on the involvement of increased urea synthesis and excretion in defense against ammonia toxicity in the giant mudskipper, and our results suggest that an ample supply of energy resources, e.g. after feeding, is a prerequisite for the induction of urea synthesis. Together, increases in nitrogenous excretion and urea synthesis after feeding effectively prevented a postprandial surge of ammonia in the plasma of P. schlosseri as reported previously for other fish species. Consequently, contrary to previous reports, there were significant decreases in the ammonia content of the brain of P. schlosseri throughout the 24 h period post-feeding, accompanied by a significant decrease in brain glutamine content between 12 h and 24 h.

Effect of enteral glutamine or glycine on whole-body nitrogen kinetics in very-low-birth-weight infants

Glutamine is a critical amino acid for the metabolism of enterocytes, lymphocytes, and other proliferating cells. Although supplementation with glutamine has been suggested for growing infants, its effect on protein metabolism has not been examined. The objective was to examine the effect of enteral glutamine or glycine on whole-body kinetics of glutamine, phenylalanine, leucine, and urea in preterm infants. Infants at <32 wk of gestation were given formula supplemented with either glutamine (0.6 g. kg(-1). d(-1); n = 9) or isonitrogenous amounts of glycine (n = 9) for 5 d. Eight infants fed unsupplemented formula served as control subjects. Glutamine, phenylalanine, leucine nitrogen flux, leucine carbon flux, and urea kinetics were quantified during a basal fasting period and in response to nutrient intake. Growing preterm infants had a high weight-specific rate of appearance of glutamine, phenylalanine, and leucine nitrogen flux. When compared with the control treatment, enteral glutamine resulted in a high rate of urea synthesis, no change in the plasma glutamine concentration, and no change in the rate of appearance of glutamine. Glycine supplementation resulted in similar changes in nitrogen metabolism, but the magnitude of change was less than that in the glutamine group. In the nonsupplemented infants, the rate of appearance of leucine nitrogen flux was negatively correlated (rho = -0.72) with urea synthesis. In contrast, the correlation (rho = 0.75) was positive in the glutamine group. Enterally administered glutamine in growing preterm infants is entirely metabolized in the gut and does not have a discernable effect on whole-body protein and nitrogen kinetics.

Excretory nitrogen metabolism in the Chinese fire-belly newt Cynops orientalis in water, on land, or in high concentrations of environmental ammonia.

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH3. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine-urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l(-1) NH4Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine-urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.

Differential utilization of systemic and enteral ammonia for urea synthesis in control subjects and ornithine transcarbamylase deficiency carriers

Female carriers of ornithine transcarbamylase deficiency (OTCD) have nearly normal rates of total urea synthesis, but they derive less urea from systemic glutamine amide nitrogen than do healthy persons. The objective of the study was to investigate whether females with symptomatic OTCD rely on alternative pathways to compensate for the reduced urea synthesis activity observed in this disorder. The 5-d study involved 6 control subjects (3 males, 3 females) and 6 female OTCD carriers who had a fixed energy intake of 133 kJ. kg(-)(1). d(-)(1) and a protein intake of 0.8 g. kg(-)(1). d(-)(1). They underwent two 12-h periods of isotopic tracer administration, separated by 2 d. On both occasions, [(18)O] or [(13)C]urea was infused intravenously, and the subjects consumed hourly meals. During the first period, [(15)N]NH(4)Cl was given intravenously; during the second period, the tracer was given as hourly oral doses. OTCD carriers produced less urea (P < 0.05) but had a higher (P < 0.05) mean ammonia appearance rate and plasma ammonia concentration than did control subjects. OTCD carriers incorporated a lower (P < 0.001) mean (+/- SE) proportion of the intravenous [(15)N]NH(4)Cl dose into circulating urea than did control subjects (16 +/- 1% compared with 36 +/- 2%), but there was no genotypic difference in the incorporation of orally administered tracer (81 +/- 4% compared with 72 +/- 4%, respectively). A good degree of dietary protein tolerance seemed to be retained in OTCD carriers by the maintenance of higher ammonia appearance rates, expansion of the plasma ammonia pool, and reliance on the ability of the perivenous hepatocytes to clear excess ammonia via glutamine synthesis.

Integrated analysis of glucose, lipid, and urea metabolism in patients with bladder cancer: Impact of tumor stage

Objective The aim of the study was to characterize the metabolic changes in non–weight-losing patients with cancer of the bladder and to investigate the effect of tumor stage. Methods The kinetics of glucose, glycerol, and urea metabolism were assessed in 22 weight-stable patients with non-metastatic bladder cancer (tumor stage p ≤ T2, n = 8; tumor stage p > T2, n = 14) and 10 patients with benign uterine myoma (controls). The kinetics of glucose, glycerol, and urea metabolism were assessed by [6,6- 2H 2]glucose, [1,1,2,3,3- 2H 5]glycerol, and [ 15N 2]urea. Plasma concentrations of glucose, glycerol, urea, lactate, free fatty acids, insulin, glucagon, cortisol, epinephrine, and norepinephrine also were determined. Results Plasma concentrations of glucose, urea, and insulin were higher in cancer patients than in controls ( P < 0.05). Whereas glucose production was similar in both groups, glucose clearance was lower in patients with bladder cancer ( P < 0.05). Glycerol turnover rate was comparable between groups. Whole-body urea synthesis rate was higher in the cancer group than in the control group ( P < 0.05), but there was no difference in urea synthesis when calculated per kilogram of fat-free body mass. Plasma concentrations of glycerol, lactate, free fatty acids, glucagon, cortisol, epinephrine, and norepinephrine were similar in both groups. There was no difference in any parameter between patients with an early tumor stage (p < T2) and patients with a later tumor stage (p > T2). Conclusion Patients with bladder cancer had a lower rate of glucose clearance than did control subjects. Lipid metabolism was not affected, whereas urea synthesis rate was elevated in cancer patients. However, when expressed per kilogram of fat-free body mass, no difference in protein breakdown could be observed. The tumor stage had no effect on glucose, lipid, or protein metabolism.

Amino Acids as Regulators and Components of Nonproteinogenic Pathways

Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the rate of mitochondrial oxidation of glycolytic NADH. Glutamate also controls the rate of urea synthesis, not only as the precursor of ammonia and aspartate, but as substrate for synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase. This mechanism allows large variations in urea synthesis at relatively constant ammonia concentrations. Increases in intracellular amino acid concentration increase cell volume. Cell swelling per se has anabolic effects on protein, carbohydrate and lipid metabolism: enhanced synthesis of macromolecules compensates for increases in intracellular osmolarity. Mechanisms responsible for cell swelling-induced changes in pathway fluxes include changes in intracellular ion concentrations and in signal transduction. Specific amino acids (e.g., leucine) stimulate protein synthesis and inhibit (autophagic) protein degradation independent of changes in cell volume because they stimulate mTOR (mammalian target of rapamycin), a protein kinase, which is one of the components of a signal transduction pathway used by insulin. When the cellular energy state is low, stimulation of mTOR by amino acids is prevented by activation of AMP-dependent protein kinase. Amino acid-dependent signaling also promotes insulin production by beta-cells. This further adds to the anabolic properties of amino acids. It is concluded that amino acids are important regulators of major metabolic pathways.

The urea cycle in the liver of arthritic rats.

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (-32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.

UREA SYNTHESIS AND CYCLOSPORIN A BIOTRANSFORMATION IN A LABORATORY SCALE HEPATOCYTE BIOREACTOR MODEL

Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture. The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal. For the perifusion of cells, they must be immobilized to prepare a bioreactor model. The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture. Hepatocytes were isolated, immobilized and perifused with William’s E culture medium containing 1 mM NH 4Cl and CsA (20 μM). Hepatocytes in the culture were treated in the same way. CsA disappearance from the perifusion or culture media was determined by a HPLC method. Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5±0.05 mg h −1 vs 0.33±0.03 mg h −1, respectively). ALT leakage was lower in the bioreactor model (60 U l −1) as compared to hepatocyte culture (125 U l −1). The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively). The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture. Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes. In addition, the cellular model described here inherits economic and ethical potentials.

Effects of long-term oral misoprostol administration on hepatic amino acid–nitrogen metabolism in patients with cirrhosis

Background: The acute infusion of a Prostaglandin of E series 1 (PGE1) analogue results in nitrogen sparing in cirrhosis. Aims: To test the effects of long-term oral PGE1 on hepatic and whole-body nitrogen metabolism. Patients and methods: Ten patients with advanced cirrhosis were studied in paired experiments, before and 30–50 days after oral misoprostol therapy. α-Amino-nitrogen levels and urea-nitrogen synthesis rate were measured in the post-absorptive state and in response to continuous alanine infusion (2 mmol/kg per hour for 4.5 h). Data were used to compute the functional hepatic nitrogen clearance, i.e. the slope of the regression of α-amino-N levels to urea-N synthesis rate, and the apparent nitrogen exchange. Results: Misoprostol reduced urea-N synthesis rate (during fasting and in response to alanine), resulting in a positive nitrogen exchange. The functional hepatic nitrogen clearance slightly increased, and the regression line was rightwards shifted, indicating a reduced urea synthesis rate at any α-amino-N concentration. Amino acid- and ammonia-N did not accumulate in plasma. No systematic effects on insulin and glucagon were observed. Conclusions: Data are consistent with a nitrogen sparing mechanism of misoprostol, not mediated by hormone levels. These effects may be beneficial in clinical hepatology, and need to be tested in controlled trials.

Glutamine and leucine nitrogen kinetics and their relation to urea nitrogen in newborn infants.

Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.

Combined effect of oxygen and ammonia on the kinetics of ammonia elimination and oxygen consumption of adherent rat liver cells.

Oxygen is essential for the survival of isolated liver cells and its concentration is known to affect their viability and function. Recent reports have also shown that ammonia is eliminated at a rate depending on its concentration and that high ammonia concentrations may be cytotoxic to rat liver cells. Nonetheless, little quantitative information on the effect of either metabolite on liver cell reaction kinetics is available although important to the design of bioreactors for bioartificial livers (BALs). In this investigation, we characterized the dependence of the rate of oxygen consumption (OCR), ammonia elimination (AER) and urea synthesis (USR) on ammonia concentration at physiological (i.e., 43 and 72 mmHg) and supra-physiological (i.e., 134 mmHg) dissolved oxygen tensions. To this purpose, isolated rat liver cells were cultured in adhesion on collagen in a continuous-flow bioreactor optimised for the kinetic characterisation of liver cell metabolic reactions. Rates of the investigated reactions generally increased with increasing ammonia concentrations. OCR and USR significantly increased with increasing dissolved oxygen tensions, particularly at high ammonia concentrations. The actual dissolved oxygen tension significantly influenced also OCR and USR dependence on ammonia concentration. The best-fit rate equations were used to show that, at the beginning of the treatment with a bioreactor packed with primary liver cells, high ammonia concentration in the blood may cause large hypoxic zones in the bioreactor as a result of its effect on OCR. This suggests that plasma (or blood) detoxification prior to entering the bioreactor might enhance BAL efficacy by preserving a large fraction of the available cell activity for longer times.

Urea synthesis in patients with chronic pancreatitis: relation to glucagon secretion and dietary protein intake

Background & Aims: Up-regulation of urea synthesis by amino acids and dietary protein intake may be impaired in patients with chronic pancreatitis (CP) due to the reduced glucagon secretion. Conversely, urea synthesis may be increased as a result of the chronic inflammation. The aims of the study were to determine urea synthesis kinetics in CP patients in relation to glucagon secretion (study I) and during an increase in protein intake (study II).Methods:In study I, urea synthesis rate, calculated as urinary excretion rate corrected for accumulation in total body water and intestinal loss, was measured during infusion of alanine in 7 CP patients and 5 control subjects on spontaneous protein intake. The functional hepatic nitrogen clearance (FHNC), i.e. urea synthesis expressed independent of changes in plasma amino acid concentration, was calculated as the slope of the linear relation between urea synthesis rate and plasma α -amino nitrogen concentration. In study II, 6 of the patients of study I had urea synthesis and FHNC determined before and after a period of 14 days of supplementation with a protein-enriched liquid (dietary sequence randomized).Results: Study I: Alanine infusion increased urea synthesis rate by a factor of 10 in the control subjects, and by a factor of 5 in the CP patients (P<0.01). FHNC was 31.9±2.4 l/h in the control subjects and 16.5±2.0 l/h (P<0.05) in the CP patients. The glucagon response to alanine infusion (AUC) was reduced by 75 % in the CP patients. The reduction in FHNC paralleled the reduced glucagon response (r2=0.55, P<0.01). Study II: The spontaneous protein intake was 0.75±0.14 g/(kg · day) and increased during the high protein period to 1.77±0.12 g/(kg · day). This increased alanine stimulated urea synthesis by a factor of 1.3 (P<0.05), FHNC from 13.5±2.6 l/h to 19.4±3.1 l/h (P<0.01), and the glucagon response to alanine infusion (AUC) by a factor of 1.8 (P<0.05).Conclusions: Urea synthesis rate and FHNC are markedly reduced in CP patients. This is associated with, and probably a result of, impaired glucagon secretion, and predicts a lower than normal postprandial hepatic loss of amino nitrogen. An increase in dietary protein intake increases alanine stimulated urea synthesis and FHNC by a mechanism that involves an increase in glucagon. This indicates that the low FHNC during spontaneous protein intake included an adaptation to the low protein intake, effectuated by a further decrease in glucagon secretion.

Inhibition of endotoxemia-induced nitric oxide synthase expression by cyclosporin A enhances hepatocyte injury in rats: amelioration by NO donors

The goals of the present study were to provide information into the controversy about nitric oxide (NO) status of the liver during endotoxemia and to assess the role of the phosphatase inhibitor cyclosporin A (CsA) during the insult. Rats were injected with saline, lipopolysaccharide (LPS, 10 mg/kg i.p.) or cyclosporin A (CsA, 5 mg/kg. i.p.)+LPS, S-nitroso- N-acetyl penicillamine (SNAP, 0.1 mM/kg)+CsA+LPS or molsidomine (molsid, 0.2 mg/kg)+CsA+LPS. Rat hepatocytes were isolated and tested for metabolic competence by the rate of urea synthesis and for lipid peroxidation. Hepatocytes were cultured under various treatments as LPS or cytokine mixture (CM, TNF-α 500 U/ml, INF-γ 100 U/ml, IL-1β 200 U/ml) with or without CsA and iNOS expression was evaluated by NO productivity and by RT-PCR. Twenty-four hours after LPS dosing in vivo, the mortality rate was 15%, while CsA pretreatment increased mortality rate to 30% and reduced hepatocyte viability, increased ALT leakage and reduced urea synthesis. SNAP and Molsid resulted in complete survival of rats, increased urea synthesis, increased cell viability and reduced alanine aminotransferase leakage. LPS or CM increased iNOS expression while CsA pretreatment reduced iNOS expression. There was no correlation between lipid peroxide levels in hepatocytes and functional status of hepatocytes under various treatments. This study demonstrates that NO produced during endotoxemia and under the present conditions is protective to the liver and may function as an adaptive mechanism and that the inhibition of iNOS by compounds like CsA produce unfavorable effects.

Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor.

The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.

Acetyl-L-carnitine treatment stimulates oxygen consumption and biosynthetic function in perfused liver of young and old rats.

The effect of treatment with acetyl-L-carnitine on hepatic mitochondrial respiration and biosynthetic function in perfused liver from young (90 days) and old (22-24 months) rats was studied. Rats were given a 1.5% (w/v) solution of acetyl-L-carnitine in their drinking water for 1 month and oxygen consumption together with the rate of gluconeogenesis, urea synthesis, and ketogenesis with and without added substrates were measured in perfused liver. Mitochondrial oxygen consumption was also assessed in liver homogenate and isolated mitochondria to determine the maximal capacity for oxidative phosphorylation. Acetyl-L-carnitine treatment almost completely restored the age-dependent decline in oxygen consumption, gluconeogenesis, urea synthesis, and ketogenesis found in perfused liver of old rats to the levels found in young rats. In addition, acetyl-L-carnitine treatment increased oxygen consumption and biosynthetic function in perfused liver from young rats. After acetyl-L-carnitine treatment, we found detectable 3-oxoacyl-CoA-transferase activity associated with a consumption of ketone bodies in young and old rats. Finally, oxygen consumption measured in homogenate and isolated mitochondria did not change with age and acetyl-L-carnitine treatment. Our results show that in perfused liver, acetyl-L-carnitine treatment slows the age-associated decline in mitochondrial respiration and biosynthetic function. In addition, treatment of young rats with acetyl-L-carnitine has a stimulating effect on liver metabolism, probably through an increase in ATP production.

IN VITRO PERFORMANCE OF A FLAT PLATE BIOREACTOR USING LIVER SLICES

These studies were performed to determine the efficiency of liver slices to metabolize lidocaine and ammonia. Rabbit liver slices 1cm in diameter with an average weight of 50mg were cultured in a newly designed flat plate bioreactor. Fifty slices were placed on top of wire mesh stretched over each of four metal plates (5 ± 10cm). Plates were seated at a 45° angle in a sealed chamber at 37°C. Media pumped to the top of the plate perfused the tissue by gravity flow. Two hundred ml of Williams E media with 10%FCS was circulated through the chamber and a reservoir at 400ml/min. A mixture of 95% oxygen, 5% CO2 at +1 ATM was maintained in the chamber throughout the study. The gas mixture was exchanged every 12 minutes. Over a three day period, bolus doses of lidocaine and ammonia were injected. A 2mg bolus dose of Lidocaine was cleared at a rate of 3.6ug/g liver/min. The lidocaine metabolite DMX (dimethyl xylidine) was produced at a rate of 33ug/g Hver/Hr. In response to a 60mg dose of Lidocaine, DMX was produced at the rate of 196ug/g liver/Hr. Ammonium chloride was delivered in bolus doses ranging between 2 and 100mg. The rate of urea synthesis increased with the dose (Table 1).TableClearance and metabolism rates were consistent throughout the three day perfusion period. The results of this study demonstrate that liver slices in a oxygenated, flat-plate perfusion system have the potential to be used in a bioartificial liver device.

Morphology and metabolism of hepatocytes cultured in Petri dishes on films and in non-woven fabrics of hyaluronic acid esters

Polymers of hyaluronic acid (Hyal) esters exhibit good tissue compatibility and are available in various geometrical configurations. These properties can be exploited for the design of innovative bioartificial liver support devices (BALSDs) using primary hepatocytes. In this paper, we report a preliminary investigation of the polymer feasibility of the ethyl and the benzyl Hyal ester in the form of films and non-woven fabrics for the in vitro culture of primary rat hepatocytes. Cell function was evaluated daily in Petri dishes with respect to the rate of ammonia elimination (AER) and urea synthesis (USR). Cells cultured in non-woven fabrics of the ethyl ester of Hyal (HYAFF7 nw) exhibited an initial AER about 32% lower and synthesised urea 33% faster than that of cells on collagen films. After a week in culture, cells on collagen films retained only a minor fraction of their initial rates. Cells cultured in non-woven fabrics of HYAFF7 nw retained about 62 and 44% of their initial AER and USR, respectively, and exhibited an AER approximately equal to and a USR 3.6 times greater than those of cells adherent to collagen. These results suggest that non-woven fabrics of HYAFF7 nw are promising substrata for hepatocyte culture in BALSDs.

Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma.

Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbecco's Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.

Protein metabolism in pregnancy

Adaptation to pregnancy involves major changes in maternal metabolism to provide for the growing demands of the conceptus. Although changes in glucose metabolism, and possibly in fatty acid metabolism, occur in parallel with the increasing energy demands of the mother and the fetus, adaptation of protein metabolism appears to be in anticipation of maternal and fetal needs. During pregnancy, there is an excess of maternal nitrogen in the form of lean body mass over that deposited in the fetus and the products of conception; there is also a pregnancy-induced hypoaminoacidemia and a diminished amino acid response to protein intake, suggesting an increased uptake of amino acids in the splanchnic compartment. With the use of stable-isotope-labeled tracers, it was shown that there is a decreased rate of urea synthesis during pregnancy that is evident early in gestation. Kinetic studies of leucine metabolism showed no significant change in leucine carbon turnover but a significantly lower rate of leucine nitrogen turnover, suggesting a lower rate of leucine transamination. These data suggest an integral regulation of whole-body protein and nitrogen metabolism starting early in gestation and aimed at conservation and accretion of nitrogen by the mother and the fetus.

A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions.

When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells. The common goal is to achieve high cell density culture and to design the bioreactor for full-scale primary liver cell cultures under adequate mass transfer conditions. An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high-density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo-like microenvironment. In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen-permeable flat-sheet membranes. In this investigation we used a novel scaled-up prototype consisting of up to 20 modules in a parallel mode. Each module was seeded with 2 x 10(8) cells. Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo. Cell concentration increased in the first days of culture, as indicated by DNA measurements. The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism. The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture. Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture. The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant. Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period. Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam. The production of such metabolites was sustained until 18 days of culture. These results demonstrated that the scale-up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver-specific tissue architecture supported the expression of liver-specific functions of primary pig liver cells.