Rate Of Urea Synthesis Research Articles

Pregnancy, insulin resistance and nitrogen accretion.

Metabolic adaptation to pregnancy in humans and animals is aimed at provision of nutrients for the growth and energy metabolism of the growing conceptus, as well as for the mother. Kinetic studies in human pregnancy have shown that fluxes of energy-yielding substrates, i.e. glucose, fatty acids and glycerol, increase in parallel with the increasing demands of the fetus and the mother. Resistance to insulin action, measured by hyperinsulinemic euglycemic clamp, appears early in gestation and is correlated with the infant's birth weight. Adaptive responses in nitrogen metabolism, decreased plasma urea concentration and decreased rate of urea synthesis, are apparent early in pregnancy, much before any significant increase in fetal demands. Recent studies of branched chain aminoacid (leucine) kinetics show a lower flux of leucine nitrogen and an unchanged flux of leucine carbon in gestation. A linear correlation between rate of deamination of leucine and rate of urea synthesis was observed in pregnant women. It is speculated that decreased anaplerotic carbon flux in the tricarboxylic acid cycle, as a consequence of insulin resistance, may have an important role in the down-regulation of transamination of leucine during pregnancy, and may contribute to the conservation and accretion of nitrogen by the mother and the fetus.

AN IN VITRO STUDY FOR METABOLIC CHARACTERIZATION OF HEPATOCYTE (HP) MULTIAGREGATES

414 The aim of our current study was to culture rat HP as multiagregate spheroids to provide an in vitro model for metabolic characterization prior to establish transplantation in in vivo studies. HP were isolated from Sprague Dawley rats using two-step enzyme dispersion method with collagenase and were later cultured in a continuous rotating culture system. The total number of cells isolated per animal was 2.5 − 3.0 × 103 cells and the viability was 78% (69-88 %). After 48 hours, 100% of the cultures were viables with a maximal duration of 144 hours in culture. Total DNA measurement was utilized to evaluate cell number, showing a slight decrease throughout the culture time, but differences were not statistically significant (n=4; p<0.005). HP viability in culture was evaluated by LDH leakage, results showed less than 2% release after 5 days in culture (n=5). Total protein/total DNA ratio increased 2 fold throughout the culture time (day 0: 18.8 ± 25.68 mg protein/mg DNA; day 5: 36.7 ± 21.22 mg protein/mg DNA; n=4) suggesting an active synthesis of protein by the HP in culture. Samples were incubated in media supplemented with NH4Cl and urea synthesis and amonium clereance was determined at different time-points, results showed a marked decrease in the rate of urea synthesis after one day in culture when comparing with freshly isolated HP and only a slight decrease thereafter. Moreover, amonium clearence was observed throughout the different time-points. Under electron microscopy, the isolated cells were spherical with 17−30 ∓ in diameter finding ocasional binucleated cells. HP were seeded at 1×106 cells/ml and after 24 hours in culture formed multiagregates with 150-600u in diameter. By 72 hours the multiagregates showed a compact structure where it was difficult to differenciate individual HP. By day 5 HP re-established cell to cell communication and resembled the architectural structure of the liver. Over 90% of HP were associated as spheroids. Enzyme activities were measured spectrophotometrically: Hexoquinase activity was very low (n=16; 1.6+0.35 umol/min/mg protein) corresponding to an in vivo conditions, whereas Glucoquinase activity was 4 fold higher (n=16; 6.24+0.29 umol/min/mg protein) than that observed for Hexoquinase. The induced Glucoquinase activity might be as a result of the aviability of insulin in the culture media. These findings suggest that the culture contains a highly pure and functional parenchymal cell preparation (HP). These results show an adequate way for in vitro metabolic evaluation of HP spheroids prior to in vivo studies in animal models with FHF.

Effect of liver disease and transplantation on urea synthesis in humans: relationship to acid-base status.

It has been suggested that hepatic urea synthesis, which consumes HCO-3, plays an important role in acid-base homeostasis. This study measured urea synthesis rate (Ra urea) directly to assess its role in determining the acid-base status in patients with end-stage cirrhosis and after orthotopic liver transplantation (OLT). Cirrhotic patients were studied before surgery (n = 7) and on the second postoperative day (n = 11), using a 5-h primed-constant infusion of [15N2]urea. Six healthy volunteers served as controls. Ra urea was 5.05 +/- 0.40 (SE) and 3.11 +/- 0.51 micromol. kg-1. min-1, respectively, in controls and patients with cirrhosis (P < 0. 05). Arterial base excess was 0.6 +/- 0.3 meq/l in controls and -1.1 +/- 1.3 meq/l in cirrhotic patients (not different). After OLT, Ra urea was 15.05 +/- 1.73 micromol. kg-1. min-1, which accompanied an arterial base excess of 7.0 +/- 0.3 meq/l (P < 0.001). We conclude that impaired Ra urea in cirrhotic patients does not produce metabolic alkalosis. Concurrent postoperative metabolic alkalosis and increased Ra urea indicate that the alkalosis is not caused by impaired Ra urea. It is consistent with, but does not prove, the concept that the graft liver responds to metabolic alkalosis by augmenting Ra urea, thus increasing HCO-3 consumption and moderating the severity of metabolic alkalosis produced elsewhere.

Quantification of gluconeogenesis in cirrhosis: Response to glucagon

Background & Aims: Accelerated starvation and early recruitment of alternate fuels in cirrhosis have been attributed to reduced availability of hepatic glycogen. The aim of this study was to measure gluconeogenesis (as a marker of protein oxidation) in relation to total glucose production and glucagon-stimulated glycogenolysis. Methods: Glucose and urea production, gluconeogenesis, and glycogenolysis were calculated using stable isotope methods before and during glucagon infusion (3 ng · kg −1 · min −1) in 5 cirrhotic patients and 5 matched controls before and after glycogen repletion. Results: In the basal state, cirrhotic patients had a normal rate of glucose production, but the contribution of gluconeogenesis was increased (74.3% ± 4.1% vs. 55.6% ± 12.1%; P < 0.005). Glycogen repletion normalized the rate of gluconeogenesis. The glycemic response to glucagon (3 ng · kg −1 · min −1) was blunted in cirrhotic patients because of a lower rate of glycogenolysis (0.63 ± 0.23 vs. 1.22 ± 0.23 mg · kg −1 · min −1; P < 0.01) and was not affected by glycogen repletion. Despite increased gluconeogenesis, the simultaneously measured rate of urea synthesis was lower in cirrhotic patients (3.11 ± 1.02 vs. 5.0 ± 1.0 mg/kg; P < 0.05). Conclusions: These data show that in cirrhosis, glucose production is sustained by an increased rate of gluconeogenesis. The hepatic resistance to glucagon action is not caused by reduced glycogen stores. GASTROENTEROLOGY 1998;115:1530-1540

Oxygen consumption and biosynthetic function in perfused liver from rats at different stages of development.

Changes in mitochondrial function were studied in perfused liver from rats aged 24-365 days. Oxygen consumption together with the rates of gluconeogenesis, urea synthesis and ketogenesis were determined. Basal mitochondrial respiration as well as the ability of the liver to synthesize glucose, urea and ketone bodies declined from 24- to 365-day-old rats. On the other hand, on transition from 24 to 60 days the liver oxidation rate of hexanoate, sorbitol and glycerol is enhanced, but not of ketone bodies or palmitate. Our results show that the transition from weaning to middle age is accompanied by defined changes in hepatic substrate oxidation. From the observed time course of the decrease in basal and substrate-stimulated oxygen consumption, it is concluded that in rat liver cells a decline in respiratory chain function, long-chain fatty acid and ketone body metabolism, gluconeogenesis and ureogenesis occurs at a relatively early life stage.

Relation between transamination of branched-chain amino acids and urea synthesis: evidence from human pregnancy.

Protein and nitrogen (N) accretion by the mother is a major adaptive response to pregnancy in humans and animals to meet the demands of the growing conceptus. Quantitative changes in whole body N metabolism were examined during normal pregnancy by measuring the rates of leucine N (QN) and carbon (QC) kinetics with the use of [1-13C,15N]leucine. Rate of synthesis of urea was measured by [15N2]urea tracer. Pregnancy-related change in total body water was quantified by H2[18O] dilution, and respiratory calorimetry was performed to quantify substrate oxidation. A significant decrease in the rate of urea synthesis was evident in the 1st trimester (nonpregnant 4.69 +/- 1.14 vs. pregnant 3.44 +/- 1.11 micromol . kg-1 . min-1; means +/- SD, P < 0.05). The lower rate of urea synthesis was sustained through the 2nd and 3rd trimesters. QN was also lower in the 1st trimester during fasting; however, it reached a significant level only in the 3rd trimester (nonpregnant 166 +/- 35 vs. 3rd trimester 135 +/- 16 micromol . kg-1 . h-1; P < 0.05). There was no significant change in QC during pregnancy. A significant decrease in the rate of transamination of leucine was evident in the 3rd trimester both during fasting and in response to nutrient administration (P < 0.05). The rate of deamination of leucine was correlated with the rate of urea synthesis during fasting (r = 0.59, P = 0.001) and during feeding (r = 0.407, P = 0. 01). These data show that pregnancy-related adaptations in maternal N metabolism are evident early in gestation before any significant increase in fetal N accretion. It is speculated that the lower transamination of branched-chain amino acids may be due to decreased availability of N acceptors such as alpha-ketoglutarate as a consequence of resistance to insulin action evident in pregnancy.

Effects of xylitol on urea synthesis in patients with cirrhosis of the liver.

In individuals with cirrhosis the normal inhibiting effect of glucose on urea synthesis is lost, probably because of very high concentrations of glucagon. In agreement, glucose does not prevent the inducing effect of glucagon on urea synthesis in normal humans. In contrast, the sugar alcohol, xylitol, prevents the increasing effect of glucagon in normal humans. We, therefore, examined the effect of xylitol on urea synthesis in individuals with cirrhosis and hyperglucagonemia. Urea synthesis, calculated as urinary excretion rate corrected for accumulation in total body water and intestinal loss, was measured during infusion of alanine (2 mmol/[h x kg body wt]) and during infusion of alanine superimposed on infusion of xylitol (0.12 g/[h x kg body wt]) in 8 individuals with biopsy-proven alcoholic cirrhosis. The functional hepatic nitrogen clearance (FHNC), ie, urea synthesis expressed independent of changes in plasma amino acid concentration, was calculated as the slope of the linear relation between the urea synthesis rate and the plasma amino acid concentration. All individuals had elevated basal plasma glucagon concentration (261 +/- 61 ng/L; mean +/- SEM) and a markedly increased response to alanine infusion (1037 +/- 226 ng/L). This was not changed by xylitol. Neither the basal urea synthesis rate (13.2 +/- 2.5 mmol/h) nor the alanine-stimulated urea synthesis rate (76.8 +/- 3.64 mmol/h) was changed by xylitol. FHNC during the infusion of alanine alone was 10.5 +/- 0.9 L/h and did not change during the concomitant infusion of xylitol (10.1 +/- 1.1 L/h). Xylitol reduces neither urea synthesis nor FHNC. The data do not support an important role of xylitol as a nitrogen-sparing agent in cirrhosis.

Effects of shear stress on metabolic function of the co-culture system of hepatocyte/nonparenchymal cells for a bioartificial liver.

To improve the culture conditions of hepatocytes for use as a bioartificial liver, the effects of shear flow on the co-culture system of hepatocytes/nonparenchymal cells (NPC) were investigated. A flow chamber with a collagen coated rectangular glass plate, where hepatocytes (5 x 10(4) cell/cm2) and NPC (2 x 10(5) cell/cm2) were seeded, was used to attain a shear stress of 4.7 dyne/cm2. Concentrations of ammonia and urea in the medium were measured daily during the 2 week experiment. The metabolic activity of hepatocytes in the homotypic culture were lower than those of the co-culture, especially when the cultivation time exceeded 1 week. In addition, the applied shear flow promoted activity of the co-culture system. An enhancement in the rates of ammonium removal and urea synthesis was obtained in the perfusion systems. Morphologic observation revealed that aggregates of hepatocytes formed abundantly in the perfusion system and hepatocytes developed a cuboid shape. This suggested that perfusion affected the function and morphology of hepatocytes in the co-culture system. Shear flow could induce cell-cell interactions and secretion of extracellular matrix through the activation of NPC.

Control of oxidative phosphorylation, gluconeogenesis, ureagenesis and ATP turnover in isolated perfused rat liver analyzed by top-down metabolic control analysis.

We have analyzed the control exerted by the pathways of oxidative phosphorylation, gluconeogenesis, ureagenesis, and maintenance ATP consumption over each other's rates in isolated, perfused rat liver using top-down metabolic control analysis. The livers from fasted rats were perfused with 3-hydroxybutyrate as respiratory substrate, lactate as substrate for gluconeogenesis, and ammonium as substrate for urea synthesis, in conditions where these pathways were only linked by their common intermediates: ATP, ADP, and Pi. The rates of oxygen consumption, glucose and urea synthesis were measured continuously. The pathways were perturbed either by adding specific inhibitors or by adding new pathways that consumed ATP, and the relative changes in pathway rates were used to calculate the flux control coefficients of each pathway over all pathway rates. When the liver was in a relatively inactive metabolic state, where ATP was only being used by the maintenance ATP-consuming pathways, then essentially all the control over ATP production and consumption was located in the maintenance ATP consumers with ATP production having no control. Whereas, when the liver was in a highly active state using extra ATP for both glucose and urea synthesis, then ATP production (from oxidative phosphorylation) had strong control over its own rate and the rates of glucose and urea synthesis, but gluconeogenesis and ureagenesis still had strong control over their own rates and negative control over each others rates, i.e. they competed for the limited ATP supply. The rate of the maintenance ATP consumers is remarkably insensitive to changes in ATP production and consumption, but exerts considerable control over all other pathways. These results indicate that the general assumption that the rates of ATP production and consumption are controlled exclusively by ATP consumers is false under conditions where a significant amount of ATP is used for biosynthetic processes, such as glucose and urea synthesis, and indicate that the latter processes may be partly controlled by regulators of ATP production and by other ATP-consuming pathways.

Inhibitory effect of vanadium compounds on glutamate dehydrogenase activity in mitochondria and hepatocytes isolated from rabbit liver.

The effect of orthovanadate, vanadyl sulphate and vanadyl acetylacetonate on glutamate dehydrogenase activity was studied in liver mitochondria and isolated hepatocytes of rabbit. In permeabilized mitochondria with free access of substrates and drugs to glutamate dehydrogenase, orthovanadate and vanadyl sulphate at 200 microM concentrations decreased both glutamate synthesis and glutamate deamination by 80 and 50%, respectively, while vanadyl acetylacetonate was less potent. In view of kinetic data obtained at various ammonium concentrations, orthovanadate appeared to be a competitive inhibitor (Ki = 40 +/- 3 microM), while vanadyl sulphate was a non-competitive one (Ki = 147 +/- 10 microM). In contrast to orthovanadate, vanadyl sulphate augmented the inhibitory action of increased above 0.5 mM 2-oxoglutarate concentrations. All these effects on the enzyme activity were partially reversed in the presence of L-leucine and ADP, which are allosteric activators of glutamate dehydrogenase. Moreover, all compounds studied suppressed both glutamate formation and glutamate deamination in isolated hepatocytes incubated under various metabolic conditions, as concluded from decreased rates of glutamate and urea synthesis, respectively. In view of these observations it seems likely that vanadium-containing compounds may be potent inhibitors of glutamate metabolism in liver.

Effects of systemic prostaglandin E1 on hepatic amino acid-nitrogen metabolism in patients with cirrhosis.

Prostaglandins of the E (PGE) series have long been considered "catabolic" hormones, but recent data suggest that they may be secreted in critically ill patients to counteract stress hormones, stimulating protein synthesis. Their use is under scrutiny to improve hepatic microcirculation and as cytoprotective agents. We tested the effects of PGE1 on hepatic and whole-body nitrogen metabolism in eight patients with cirrhosis. Urea-nitrogen synthesis rate, alpha-amino-nitrogen levels, and nitrogen exchange were measured in the basal, postabsorptive state and in response to continuous alanine infusion, in paired experiments, during superinfusion of PGE1 or saline. Splanchnic and systemic hemodynamics were assessed by echo-Doppler at the beginning and at the end of each experiment. PGE1 produced a rapid fall in plasma amino acids and in urea-nitrogen synthesis rate, as well as a positive nitrogen exchange. The slope of the regression of alpha-amino-nitrogen levels on urea-nitrogen synthesis rate, a measure of liver cell metabolic activity, was not affected, but the regression line was shifted rightward, suggesting a nitrogen-sparing effect of PGE1. Mesenteric artery and portal flow were unchanged, whereas femoral artery flow increased by 30%. Insulin and glucagon levels were not systematically different. We conclude that PGE1 reduces hepatic urea synthesis rate, independent of hormones and/or hepatic flow, possibly acting at the peripheral level on amino acid transport, thus reducing amino acid supply to the liver. The resulting net nitrogen sparing might be the basis for the beneficial effect of PGE1 in clinical hepatology.

Possible dual role of nitric oxide in oxidative stress injury: A study in perfused hepatocytes

Nitric oxide (NO) is a multifunctional messenger in many vertebrates. In the liver, NO was found to play an important but controversial role in injury produced by toxins or sepsis. The purpose of the present investigation was to further characterize the role of NO in hepatocyte oxidative injury. A cellular system formed of immobilized and perfused rat hepatocytes was used to test the ability of the latter to produce endogenous NO after lipopolysaccharide administration in vivo (LPS, 20 mg/kg i.p.) and how hepatocyte functionality competence is modified according to NO level. This cellular system also was used to delineate a relationship between exogenously delivered NO to the perfusion medium as produced by the NO donor, sodium nitroprusside (2.0 and 0.2 mM), and any alteration in the degree of injury as evoked by anoxia/reoxygenation or cumene hydroperoxide (1.0 mM and 0.2 mM). Rat hepatocytes were immobilized in low-gelling agarose and perfused with Williams E medium. Endogenous or exogenous NO was evaluated by measuring the end products of NO (NO 2 − + NO 3 −) in the perfusion medium. Functional integrity of hepatocytes was evaluated from lactate dehydrogenase (LD) leakage, urea synthesis in the perfusion medium and lipid peroxides (LP) formation. Normal, anoxia/reoxygenation or cumene hydroperoxide injured hepatocytes did not exhibit measurable NO while LPS-treated hepatocytes produced NO. Apparently, within the present experimental conditions, it seems that there was an inverse relation between the rate of NO produced after LPS administration and the rate of lipid peroxides formed in the hepatocytes. Low concentration of sodium nitroprusside (as NO donor) significantly decreased LD leakage, increased the rate of urea synthesis and increased trypan blue exclusion by hepatocytes in anoxia/reoxygenation or cumene hydroperoxide injured (0.2 mM) cells. Lipid peroxides were decreased by NO in cumene hydroperoxide injured hepatocytes. The present data suggest that NO endogenously produced, or exogenously delivered, has an ameliorative role in mild oxidative liver injury models, but not in severe cases and that inside hepatocytes, there is a very delicate balance between the rate of NO production and its consumption. The disturbance in this balance may be responsible for injury due to the formation of more toxic oxygen species.

Hepatic amino nitrogen conversion and organ N-contents in hypothyroidism, with thyroxine replacement, and in hypethyroid rats

Background/Aims: The role of thyroid hormones in the regulation of hepatic conversion of amino nitrogen to urea is unresolved. The present study was designed to assess ureagenesis in rats with experimentally well-established hypo- and hyperthyroidism. The possible role of propylthiuracil (PTU), used for induction of hypothyroidism, was ascertained during thyroxine replacement of PTU treated hypothyroid rats. Methods: Basal blood amino nitrogen concentrations (AAN), the urea nitrogen synthesis rate (UNSR) and the maximal hepatic capacity for urea nitrogen synthesis (CUNS) obtained during alanine infusion were determined together with N-contents in the soleus muscle and kidneys in experimentally hypothyroid rats( n=19), upon thyroxine replacement ( n=14) and in experimentally hyperthyroid rats ( n=19). Hypothyroidism was induced by adding propylthiouracil (0.05%) to the drinking water for 5 weeks. Hyperthyroidism was induced by thyroxine 100 μg/100 g body weight. Results: During hyperthyroidism, T 3 fell to less than 10%, food intake was halved, and body weight fell by 13%. Basal blood AAN fell by 25% ( p<0.01), UNSR more than doubled ( p<0.01), and CUNS rose by 45% ( p<0.05). N-contents of the soleus muscle fell by 13% and by 20% in kidneys, respectively ( p<0.05). Thyroxine replacement normalized AAN, UNSR, CUNS and reduced N-loss to 7% in the soleus muscle (NS) and kidneys ( p<0.05), respectively. During hypethyroidism, T 3 rose fivefold, food intake rose by two thirds, and body weight fell by 10%. Basal AAN rose by 20% ( p<0.05), UNSR doubled ( p<0.01), and CUNS rose by 25% ( p<0.05). N-contents of the soleus muscle decreased by 19%, whreas kidney N-contents increased by 25% ( p<0.05). Overall liver function assessed by galactose elimination capacity did not differ among groups. Both conditions increased the rate of urea synthesis; in the hypothyroid state the hepatic waste of amino-N was limited by low blood concentration of amino-N, probably due to lower proteolysis. In the hyperthyroid state hepatic amino-N loss was aggravated by higher blood concentration of amino-N, probably due to higher proteolysis. This difference may explain the markedly different dietary nitrogen economy between the two groups. Conclusions: The findings suggest that distinct hepatic acceleration of urea synthesis may contribute to the protein loss seen in both myxedema and in thyrotoxicosis in humans.

In vitro ammonia utilization and urea synthesis by rat liver after portacaval shunt.

The objective of this study was to study in vitro ammonia utilization and urea synthesis by the rat liver after portacaval shunt (PCS) in comparison to controls. This study is part of a series of experiments to investigate metabolic derangements in the liver following PCS. Sixteen male Wistar rats weighing 150-230 g were used. PCS was performed on 8 and the rest served as sham-operated controls. The experiments were performed 4 weeks postoperatively. The usually monitored postoperative parameters after PCS like body weight, rise in ammonia levels in the peripheral blood, atrophy of mesenteric fat, atrophy of the liver, etc., were comparable to other PCS series. Liver slices (1 x 2 x 1 mm) were prepared from each rat liver and incubated in Krebs-Henseleit buffer. After incubation, ammonia utilization in the controls was 162.88+/-20.12 micromol/g dry weight/h while in PCS rats it was 81.35+/-2.64 micromol/g dry weight/h (p < 0.0001). The rate of urea synthesis in controls was 122.54+/-12.93 micromol/g dry weight/h whereas in the PCS group it was 14.99+/-2.21 micromol/g dry weight/h (p < 0.0001). These results indicate a significant decline in the capacity of the liver for ammonia utilization as well as urea synthesis after PCS.

Extracorporeal Plasma Perfusion of Cultured Hepatocytes: Effect of Intermittent Perfusion on Hepatocyte Function and Morphology

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.

In Vivo activation by diets and in vitro activation by hormones are additive for urea synthesis in the rat liver

The basal rate of urea or glucose synthesis, set in vivo by different diets, was challenged by addition of hormones to isolated hepatocytes. Basal rate of urea synthesis ranged from 7.1 to 23.5 and 27.8 nmol/mg wet weight cells/hr in hepatocytes prepared from rats with a protein free diet or with two distinct but complete protein diets. Maximum difference in the basal rate of glucose synthesis from lactate was only 26.7% for the same diets. Depending on the basal rate of urea synthesis fixed in vivo by the diets, significant in vivo activations superimposed on these, either low or high, basal rates with a remarkable constancy: 2.4-fold with glucagon, 1.9-fold with epinephrine and 1.4-to 1.8-fold with adenosine. Likewise, the basal rate of glucose synthesis from lactate, established by these diets, superimposed 3.7-fold with glucagon, 2.4-fold with epinephrine, 2.1-to 2.4-fold with adenosine and 2.0-to 2.5-fold with inosine. The stimulation of hepatocytes in vivo showed a direct relationship in the increased rates of urea and glucose synthesis. The short-term activation in urea and glucose synthesis superimposed in proportion to the long-term status of urea or glucose formation set in the liver by the diet.

Decreased ureagenesis from alanine, but not from ammonia and glutamine, in the perfused rat liver after partial hepatectomy

Ureagenesis from ammonia, alanine, and glutamine in the liver after partial hepatectomy (PH) was determined by using the liver-perfusion system. The maximum rate of ureagenesis from ammonium chloride (10 mmol/L) in hepatectomized (HX) rats at 24 hours after surgery was obtained in the presence of ornithine, lactate, and pyruvate, and it was almost identical to that in sham-operated (SO) rats. The rate of urea production from glutamine (1 mmol/L or 10 mmol/L) in HX rats was significantly lower than that of SO rats with a concomitant decrease in hepatic glutaminase activities. However, the rate of urea synthesis from glutamine (1 mmol/L) in the presence of added ammonia (0.5 mmol/L) was accelerated approximately 10-fold, and the significant difference in the rate of urea formation between HX and SO rats was abolished. This result indicates that there is enough glutaminase to generate ammonia from glutamine in the liver of HX rats. The rate of urea production from alanine (1 mmol/L or 10 mmol/L) in HX rats was significantly decreased at 24 hours following surgery, while that of SO rats was increased. The decreased formation of urea from alanine was not seen at 72 and 120 hours after the operation. These results suggest that during the proliferation phase of liver regeneration, a reduction of ureagenesis from alanine facilitates the remnant liver to make nonessential amino acids such as aspartate. This metabolic alteration might be related to the proliferation of liver cells. (Hepatology 1996 Jun;23(6):1584-90)

Decreased Ureagenesis From Alanine, But Not From Ammonia and Glutamine, in the Perfused Rat Liver After Partial Hepatectomy

Ureagenesis from ammonia, alanine, and glutamine in the liver after partial hepatectomy (PH) was determined by using the liver–perfusion system. The maximum rate of ureagenesis from ammonium chloride (10 mmol/L) in hepatectomized (HX) rats at 24 hours after surgery was obtained in the presence of ornithine, lactate, and pyruvate, and it was almost identical to that in sham–operated (SO) rats. The rate of urea production from glutamine (1 mmol/L or 10 mmol/L) in HX rats was significantly lower than that of SO rats with a concomitant decrease in hepatic glutaminase activities. However, the rate of urea synthesis from glutamine (1 mmol/L) in the presence of added ammonia (0.5 mmol/L) was accelerated approximately 10–fold, and the significant difference in the rate of urea formation between HX and SO rats was abolished. This result indicates that there is enough glutaminase to generate ammonia from glutamine in the liver of HX rats. The rate of urea production from alanine (1 mmol/L or 10 mmol/L) in HX rats was significantly decreased at 24 hours following surgery, while that of SO rats was increased. The decreased formation of urea from alanine was not seen at 72 and 120 hours after the operation. These results suggest that during the proliferation phase of liver regeneration, a reduction of ureagenesis from alanine facilitates the remnant liver to make nonessential amino acids such as aspartate. This metabolic alteration might be related to the proliferation of liver cells.

Actions of Selected Hepatotoxicants on Freshly Isolated and Cryopreserved Rat Hepatocytes

The present study compares the actions of the hepatotoxic agents allyl alcohol, acetaminophen, and carbon tetrachloride on energy metabolism in freshly isolated and cryopreserved rat hepatocytes. After 30 min incubation of freshly isolated hepatocytes at 37°C to allow metabolic equilibration, hepatocytes were supplemented with cryoprotectants and cooled in a stepwise manner to liquid nitrogen temperature. Hepatocytes stored in liquid nitrogen for 2 weeks to 6 months were thawed and centrifuged through Percoll to remove damaged cells. Despite similarities in energy status as indexed by ATP content and the rate of urea synthesis in freshly isolated and cryopreserved hepatocytes, cryopreserved hepatocytes were more sensitive to hepatotoxicants. All three hepatotoxicants caused ATP and rates of urea synthesis to decline to a greater extent in cryopreserved than in freshly isolated hepatocytes. Rates of oxygen uptake were higher in cryopreserved cells than in freshly isolated hepatocytes and declined in cryopreserved cells but not in freshly isolated cells during the initial period of incubation. Rates of mitochondrial respiration stimulated with site-specific substrates were comparable in freshly isolated and cryopreserved cells permeabilized with digitonin. Allyl alcohol and acetaminophen inhibited site-specific respiration to the same extent in both groups of cells. Collectively, these results suggest that increased sensitivity to hepatotoxic agents and elevated oxygen consumption in cryopreserved hepatocytes recovered after storage in liquid nitrogen are related to higher demand for energy in these cells rather than to permanent injury caused by cryopreservation and irreversible uncoupling of oxidative phosphorylation.

ESTIMATES OF PROTEIN OXIDATION IN LOW BIRTH WEIGHT INFANTS DURING PARENTERAL NUTRITION: COMPARISON OF UREA SYNTHESIS, NITROGEN EXCRETION AND ESSENTIAL AMINO ACID OXIDATION. † 1912

Rates of protein oxidation are quantified in order to obtain estimates of nitrogen accretion and balance in nutritional studies, using rates of urea synthesis, nitrogen excretion or oxidation of essential amino acids, e.g. leucine and phenylalanine.