scholarly journals Real-time study of the urea cycle using 15N n.m.r. in the isolated perfused rat liver

1992 ◽  
Vol 287 (3) ◽  
pp. 813-820 ◽  
Author(s):  
A Geissler ◽  
K Kanamori ◽  
B D Ross
Keyword(s):  
Rat Liver ◽  
Urea Cycle ◽  
Isolated Perfused Rat Liver ◽  
15N Recovery ◽  
Urea Synthesis ◽  
Perfused Liver ◽  
Biochemical Assays ◽  
Low Concentrations ◽  
15N Urea ◽  
Rate Limiting

1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega, omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.

Analysis of Regulatory Factors for Urea Synthesis by Isolated Perfused Rat Liver

1975 ◽  
Vol 77 (3) ◽  
pp. 671-678 ◽  
Author(s):  
Takeyori SAHEKI ◽  
Michio TSUDA ◽  
Tomi TANAKA ◽  
Nobuhiko KATUNUMA
Keyword(s):  
Rat Liver ◽  
Isolated Perfused Rat Liver ◽  
Urea Synthesis ◽  
Perfused Rat Liver ◽  
Regulatory Factors

Biliary excretion of dihydro-11-keto-progesterone-4-C14 by isolated perfused liver

1964 ◽  
Vol 207 (5) ◽  
pp. 1030-1034 ◽  
Author(s):  
G. F. Leong ◽  
D. M. Cazes ◽  
M. L. Berliner ◽  
D. L. Berliner
Keyword(s):  
Rat Liver ◽  
Biliary Excretion ◽  
Half Life ◽  
Water Soluble ◽  
Isolated Perfused Rat Liver ◽  
Perfused Liver ◽  
Rapid Rate ◽  
Perfused Rat Liver ◽  
Bile Formation ◽  
Entire Study

The rates of biliary excretion of dihydro-11-keto-progesterone-4-C14 and of its metabolites were studied in the isolated perfused rat liver. The half-life of this steroid in the perfusing blood was 2.5 min, and at 40 min about 75% of the injected steroid had been excreted in bile. Formation of water-soluble steroids (WS St) took place at a rapid rate and by 60 min 100% of the steroids in blood were found to be water soluble. During the entire study the steroids excreted in bile were water soluble and accounted for 97.2–100% (avg. 98.2%). No dihydro-11-keto-progesterone was found to be excreted in the bile. The rate of disappearance from the blood, excretion in the bile, and degree of formation of WS St of this compound when compared with corticosterone and cortisol shows the following pattern: dihydro-11-keto-progesterone > corticosterone > cortisol.

scholarly journals The effects of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on glycolysis and biosynthetic processes of the isolated perfused rat liver

1972 ◽  
Vol 128 (3) ◽  
pp. 711-720 ◽  
Author(s):  
J. F. Biebuyck ◽  
Patricia Lund ◽  
H. A. Krebs
Keyword(s):  
Rat Liver ◽  
Lactate Production ◽  
Fold Increase ◽  
Isolated Perfused Rat Liver ◽  
Urea Synthesis ◽  
Perfused Rat Liver ◽  
Anaesthetic Agents ◽  
Metabolic Functions ◽  
Tissue Content ◽  
Gluconeogenesis From Alanine

1. With reference to the post-operative dysfunction of the liver observed after halothane anaesthesia, the effects of the anaesthetic on some metabolic functions were studied in the isolated perfused rat liver. Oxygen uptake, glycolysis, gluconeogenesis and urea synthesis were affected by halothane at a concentration (2.5% of the gas phase) within the range used in clinical anaesthesia. 2. At this concentration of halothane uptake of oxygen was inhibited in livers from both fed and starved rats. 3. In livers from fed rats there was a 16-fold increase in lactate production. This was accompanied by a fivefold decrease in the tissue content of 2-oxoglutarate and a more than twofold decrease in citrate. The calculated [free NAD+]/[free NADH] ratio in both cytoplasm and mitochondria was lower in the halothane-exposed livers than in controls. 4. In livers of starved rats the rate of gluconeogenesis from lactate was decreased by halothane to 30% of the control rate. 5. Halothane inhibited gluconeogenesis from alanine and propionate to the same extent as from lactate, whereas glucose formation from dihydroxyacetone, glycerol, fructose and sorbitol was relatively unaffected. 6. During gluconeogenesis from 10mm-lactate the tissue content of ATP was decreased by 50%, glutamate by 50% and 2-oxoglutarate was decreased eightfold in the halothane-exposed livers. 7. Halothane decreased urea synthesis in the presence of 10mm-NH4Cl and 2mm-ornithine to 15% of the control rate. 8. The inhibitions of gluconeogenesis and urea synthesis were completely abolished within 15min of withdrawal of the anaesthetic. 9. The stimulation of uptake of oxygen brought about by the addition of lactate or precursors of urea was abolished by halothane. 10. Effects on gluconeogenesis similar to those of halothane occurred in livers exposed to the anaesthetic methoxyflurane, although normal rates were not restored on withdrawal of the drug. Other anaesthetic agents tested (ketamine–HCl and trichloroethylene) decreased gluconeogenesis to 66% of the control rate. 11. The inhibitory effects of halothane are consistent with an interference at the stage of the NADH dehydrogenase of the electron-transport chain.

scholarly journals Rates of ketone-body formation in the perfused rat liver

1969 ◽  
Vol 112 (5) ◽  
pp. 595-600 ◽  
Author(s):  
H. A. Krebs ◽  
Patricia G. Wallace ◽  
R. Hems ◽  
R. A. Freedland
Keyword(s):  
Fatty Acids ◽  
Rat Liver ◽  
Ketone Bodies ◽  
Short Chain Fatty Acids ◽  
Diabetic Rats ◽  
Isolated Perfused Rat Liver ◽  
Perfused Liver ◽  
Perfused Rat Liver ◽  
Normal Range ◽  
Physiological Value

1. The rates of formation of acetoacetate and β-hydroxybutyrate by the isolated perfused rat liver were measured under various conditions. 2. The rates found after addition of butyrate, octanoate, oleate and linoleate were about 100μmoles/hr./g. wet wt. in the liver of starved rats. These rates are much higher than those found with rat liver slices. 3. The differences between the rates given by slices and by the perfused organ were much higher with the long-chain than with short-chain fatty acids. The increments caused by oleate and linoleate were 12 and 16 times as large in the perfused organ as in the slices, whereas the increments caused by butyrate and octanoate were about four times as large. 4. The rates of ketogenesis in the unsupplemented perfused liver of well-fed rats, and the increments caused by the addition of fatty acids, were about half of those in the liver from starved rats. 5. The value of the [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was raised by octanoate, oleate and linoleate. 6. Carnitine did not significantly accelerate ketogenesis from fatty acids. 7. Oleate formed up to 82% of the expected yield of ketone bodies. 8. In the liver of alloxan-diabetic rats the endogenous rates of ketogenesis were raised, in some cases as high as in the liver from starved rats, after addition of oleate. 9. On addition of either β-hydroxybutyrate or acetoacetate to the perfusion medium the liver gradually adjusted the [β-hydroxybutyrate]/[acetoacetate] ratio towards the normal range. 10. The [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was about 0·4 when slices were incubated, but near the physiological value of 2 when the liver was perfused. 11. The experiments demonstrate that for the study of ketogenesis slices are in many ways grossly inferior to the perfused liver.

Sources of arginine for induced nitric oxide synthesis in the isolated perfused liver

1995 ◽  
Vol 269 (6) ◽  
pp. G861-G866 ◽  
Author(s):  
C. M. Pastor ◽  
S. M. Morris ◽  
T. R. Billiar
Keyword(s):  
Nitric Oxide ◽  
Urea Cycle ◽  
Inos Expression ◽  
Urea Synthesis ◽  
Perfused Liver ◽  
Nitric Oxide Synthesis ◽  
Bicarbonate Buffer ◽  
Oxide Synthase ◽  
Rat Livers ◽  
Inducible Nitric Oxide

Hepatocytes can be stimulated to express high levels of inducible nitric oxide synthase (iNOS), which utilizes arginine for nitric oxide (NO) synthesis. Hepatocytes also synthesize and catabolize arginine, an intermediate in the urea cycle, raising the possibility that the urea pathway may provide substrate for hepatic NO synthesis. To identify the sources of arginine for iNOS, we measured the release of NO-2 + NO-3 and urea in isolated rat livers perfused in a recirculation model with a Krebs-Henseleit-bicarbonate buffer containing either no added amino acid, arginine, or precursors for urea synthesis. To induce iNOS expression, rats were injected with killed Corynebacterium parvum (C. parvum) or with endotoxin. In livers from C. parvum- and endotoxin-treated rats, we found that 1) an intracellular source of arginine exists that provides substrate to iNOS; 2) additional exogenous arginine increase NO synthesis, demonstrating that endogenous arginine is insufficient for maximal NO synthesis; and 3) an increase in the rate of endogenous arginine synthesis within the urea cycle is inefficient in increasing NO synthesis, demonstrating the independence of the two pathways in the liver.

Restriction of hepatic gluconeogenesis and ureogenesis from threonine when at low concentrations

1975 ◽  
Vol 229 (6) ◽  
pp. 1718-1723 ◽  
Author(s):  
DL Bloxam
Keyword(s):  
Essential Amino Acid ◽  
Hepatic Uptake ◽  
Point Of View ◽  
Perfused Liver ◽  
Short Supply ◽  
Low Concentrations ◽  
Urea Production ◽  
Rate Limiting ◽  
Parallel Fashion ◽  
Stimulation Of

Experiments were carried out with the isolated perfused liver of the overnight-starved rat to study the control of the conversion of the essential amino acid threonine to glucose and urea from the point of view of its conservation when in short supply. The relationships between the concentration of added L-threonine and the rate of glucose and urea production showed that both pathways have considerable capacity and were saturated at a high (15 mM) concentration of threonine. However, these concentration-rate relationships were sigmoidal, so that at low concentrations the rates of conversion were disproportionately low. Thus at physiologic levels of threonine, no measurable stimulation of glucose or urea output was observed. Hepatic uptake of threonine was similarly disproportionately reduced at near-physiologic levels. Glucagon stimulated glucose and urea outputs in parallel fashion and stimulated the uptake and inward membrane transport of threonine at both saturating and low concentrations. This and the changes in intracellular and extracellular concentrations of threonine indicate the transport is rate limiting for both pathways. If this is so, the apparent restrictive property probably resides at the plasma membrane. Since the liver is the end point of threonine metabolism, this property would effectively limit the utilization of threonine when in short supply.

scholarly journals Effect of 3-mercaptopicolinic acid on gluconeogenesis and gluconeogenic metabolite concentrations in the isolated perfused rat liver

1975 ◽  
Vol 150 (1) ◽  
pp. 137-139 ◽  
Author(s):  
M N Goodman
Keyword(s):  
Rat Liver ◽  
Metabolic Regulation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Ideal Agent ◽  
Low Concentrations ◽  
Metabolite Concentrations

3-Mercaptopicolinic acid inhibited gluconeogenesis from lactate and alanine, but not dihydroxyacetone, in the perfused rat liver. Hepatic metabolite concentrations suggested that gluconeogenesis was inhibited at phosphoenolpyruvate carboxykinase. The compound is very effective at low concentrations, and seems an ideal agent for use in studying metabolic regulation involving gluconeogenesis and anaplerotic mechanisms.

The utilization of ethanol by the isolated perfused rat liver

1968 ◽  
Vol 46 (4) ◽  
pp. 609-616 ◽  
Author(s):  
Ellen R. Gordon
Keyword(s):  
Rat Liver ◽  
Ethanol Consumption ◽  
Isolated Perfused Rat Liver ◽  
Gas Mixture ◽  
Perfused Liver ◽  
Perfused Rat Liver ◽  
Isolated Perfused Liver ◽  
Partial Pressures ◽  
Series Of Experiments ◽  
Lactic Acids

A series of experiments has shown that the mechanisms by which the metabolism of ethanol occurs are strongly influenced by the partial pressures of the oxygen present in the aerating medium. For the most frequently used gas mixture, 95% O2 + 5% CO2, the ethanol consumption was abnormally large, and proportional to the concentration of ethanol in the perfusate. However, when the system was aerated with 18% O2 + 5% CO2 + 77% N2 the consumption of ethanol was similar to that found in the intact rat. The perfusate concentration of glucose, acetone, and acetic, pyruvic, and lactic acids was measured in all experiments. When ethanol was consumed at these two different rates, increases in the lactic: pyruvic ratio and acetate levels of the perfusate were noted in both types of experiments. However, the utilization of glucose by isolated perfused liver was inhibited, and acetone levels increased markedly, when ethanol was consumed at abnormally rapid rates, although no effect was noted in the perfusate levels of these metabolites when ethanol was consumed at normal rates.

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