The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guine... more The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. 14C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [14C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.
Livers from 125- to 150-g rats were perfused and the effect of fructose metabolism on liver energ... more Livers from 125- to 150-g rats were perfused and the effect of fructose metabolism on liver energetics was observed using 31P nuclear magnetic resonance (NMR). Livers were exposed to fructose in a nonrecirculating manner for approximately 15 min and monitored for recovery for 30 min. At concentrations ranging from 1 to 25 mM, fructose exposure led to depletion of the nucleoside triphosphate level. Subsequently, the nucleoside triphosphate level recovered fully at a fructose concentration of 1 mM; however, recovery was only 65% of the initial values if the fructose concentration was 10 mM and 55% if the concentration was 25 mM. Adenine was found to protect nucleoside triphosphate levels against the adverse effects of fructose by blocking the deamination of AMP. In the presence of adenine, AMP accumulation was observed in the NMR spectra upon exposure of the liver to fructose.
The biochemistry of hepatic injury and recovery from preservation for transplantation was studied... more The biochemistry of hepatic injury and recovery from preservation for transplantation was studied in rat liver perfused in vitro with erythrocytes. ATP and its metabolites, inorganic phosphate (Pi) and pH were quantitated as often as every 2.5 min by 31P NMR spectroscopy during preservation and recovery. Release of the hepatocellular enzymes, lactate dehydrogenase V (LDV) and aspartate aminotransferase (AST) were also measured. The duration of preservation with Collins' solution, the standard clinical preservative, affected the rate of recovery of ATP and monophosphate esters (MP), which include AMP + IMP, and the final recovery of Pi, but not of ATP. The difference between Collins' and Ringer's lactate solution, a poor preservative, became more apparent as preservation time increased. The differences included (1) pH at the end of preservative infusion; (2) pH between 0 and 2.5 min of reperfusion; (3) the MP increase (AMP + IMP) at the end of 13 h of preservation; (4) rate of recovery of ATP after preservation; (5) final ATP recovery during reperfusion; (6) LDV after 13h of preservation. These biochemical differences between good and poor preservation form a rational basis for prediction of liver failure after transplantation and for tests of the quality of new preservatives.
The phosphorus NMR spectra of isolated perfused rat liver displays a prominent inorganic phosphat... more The phosphorus NMR spectra of isolated perfused rat liver displays a prominent inorganic phosphate peak at 2.76 +/- 0.05 ppm relative to liver glycerolphosphocholine at 0.49 ppm. From titration curves of phosphorus-containing compounds this corresponds to a pH of 7.4. The spectra also display a shoulder on the prominent inorganic phosphate peak at 2.22 +/- 0.55 ppm corresponding to a pH of 7.0. Fructose is phosphorylated in the C1 position by the liver and the resulting fructose-1-phosphate is located in the cytosol. From the titration curves, this compound was at pH 7.0. When inorganic phosphate was added to the perfusate, 13 of 25 livers showed two inorganic phosphate peaks resolved in the difference spectra, one originating from the perfusate, the other at pH 7.0. When large (50 mM) fructose doses were administered to the liver, the prominent peak decreased allowing two peaks to be resolved. FCCP treatment of the liver caused the two peaks to coalesce with the final pH of both the fructose-1-phosphate and inorganic phosphate being the same. 19F NMR of difluoromethylalanine gave an intracellular pH of 7.4 for the isolated perfused liver. The data presented do not lend themselves to satisfactory interpretation, and call into question the correctness of the traditional assignment of liver inorganic phosphate being cytosolic in origin.
The broad phosphorus resonance of isolated perfused liver can be removed by selective presaturati... more The broad phosphorus resonance of isolated perfused liver can be removed by selective presaturation with a DANTE pulse train. The use of the DANTE pulse allows the broad resonance to be removed without the use of multiple RF channels or the ability to switch the observation channel from low to high power.
The mechanisms that underlie the balance between the consumption and oxidative generation of ATP ... more The mechanisms that underlie the balance between the consumption and oxidative generation of ATP in the intact cell are not well-defined. Cytosolic inorganic phosphate (Pi) and ADP levels, the cytosolic ATP/ADP ratio, and the cytosolic phosphorylation potential (PP) have all been proposed as major regulatory variables, the latter as a component of a "near-equilibrium" thermodynamic regulatory scheme. Therefore, the potential regulatory roles of these variables in the intact cell were evaluated with 31P NMR and Langendorff perfused rat hearts; in this preparation, the tissue oxygen consumption rate (MVO2) can be varied over a wide range. When the exogenous carbon source was varied, none of the proposed regulatory parameters, i.e., the ATP/ADP ratio, PP, or cytosolic ADP level, were found to be uniquely related to MVO2. Rather, ADP levels at a given MVO2 decreased progressively for the exogenous carbon sources in the following order: glucose, glucose + insulin, palmitate + glucose, lactate, pyruvate + glucose, and octanoate + glucose. In the octanoate and pyruvate groups, MVO2(-1) was linearly dependent upon [ADP]-1 with apparent Km values being in the range previously observed in isolated mitochondria. A similar trend was observed in the MVO2-[Pi] relationship. The present findings suggest that exogenous carbon sources which effectuate deregulation of intramitochondrial NADH generation lower cytosolic ADP and Pi to levels which are limiting to the rate of oxidative phosphorylation. For other carbon sources, the processes controlling the rate of NADH generation also participate in determining the rate of oxidative ATP synthesis. However, this control must be exerted kinetically rather than through a near-equilibrium thermodynamic mechanism as indicated by the present data and prior kinetic studies of the ATP synthetic process in both isolated mitochondria and intact myocardium [La Noue, K. F., et al. (1986) Biochemistry 25, 7667-7675; Kingsley-Hickman, P., et al. (1987) Biochemistry 26, 7501-7510].
Biochimica et Biophysica Acta (BBA) - General Subjects, 1984
The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guine... more The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. 14C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [14C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.
The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guine... more The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. 14C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [14C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.
Livers from 125- to 150-g rats were perfused and the effect of fructose metabolism on liver energ... more Livers from 125- to 150-g rats were perfused and the effect of fructose metabolism on liver energetics was observed using 31P nuclear magnetic resonance (NMR). Livers were exposed to fructose in a nonrecirculating manner for approximately 15 min and monitored for recovery for 30 min. At concentrations ranging from 1 to 25 mM, fructose exposure led to depletion of the nucleoside triphosphate level. Subsequently, the nucleoside triphosphate level recovered fully at a fructose concentration of 1 mM; however, recovery was only 65% of the initial values if the fructose concentration was 10 mM and 55% if the concentration was 25 mM. Adenine was found to protect nucleoside triphosphate levels against the adverse effects of fructose by blocking the deamination of AMP. In the presence of adenine, AMP accumulation was observed in the NMR spectra upon exposure of the liver to fructose.
The biochemistry of hepatic injury and recovery from preservation for transplantation was studied... more The biochemistry of hepatic injury and recovery from preservation for transplantation was studied in rat liver perfused in vitro with erythrocytes. ATP and its metabolites, inorganic phosphate (Pi) and pH were quantitated as often as every 2.5 min by 31P NMR spectroscopy during preservation and recovery. Release of the hepatocellular enzymes, lactate dehydrogenase V (LDV) and aspartate aminotransferase (AST) were also measured. The duration of preservation with Collins' solution, the standard clinical preservative, affected the rate of recovery of ATP and monophosphate esters (MP), which include AMP + IMP, and the final recovery of Pi, but not of ATP. The difference between Collins' and Ringer's lactate solution, a poor preservative, became more apparent as preservation time increased. The differences included (1) pH at the end of preservative infusion; (2) pH between 0 and 2.5 min of reperfusion; (3) the MP increase (AMP + IMP) at the end of 13 h of preservation; (4) rate of recovery of ATP after preservation; (5) final ATP recovery during reperfusion; (6) LDV after 13h of preservation. These biochemical differences between good and poor preservation form a rational basis for prediction of liver failure after transplantation and for tests of the quality of new preservatives.
The phosphorus NMR spectra of isolated perfused rat liver displays a prominent inorganic phosphat... more The phosphorus NMR spectra of isolated perfused rat liver displays a prominent inorganic phosphate peak at 2.76 +/- 0.05 ppm relative to liver glycerolphosphocholine at 0.49 ppm. From titration curves of phosphorus-containing compounds this corresponds to a pH of 7.4. The spectra also display a shoulder on the prominent inorganic phosphate peak at 2.22 +/- 0.55 ppm corresponding to a pH of 7.0. Fructose is phosphorylated in the C1 position by the liver and the resulting fructose-1-phosphate is located in the cytosol. From the titration curves, this compound was at pH 7.0. When inorganic phosphate was added to the perfusate, 13 of 25 livers showed two inorganic phosphate peaks resolved in the difference spectra, one originating from the perfusate, the other at pH 7.0. When large (50 mM) fructose doses were administered to the liver, the prominent peak decreased allowing two peaks to be resolved. FCCP treatment of the liver caused the two peaks to coalesce with the final pH of both the fructose-1-phosphate and inorganic phosphate being the same. 19F NMR of difluoromethylalanine gave an intracellular pH of 7.4 for the isolated perfused liver. The data presented do not lend themselves to satisfactory interpretation, and call into question the correctness of the traditional assignment of liver inorganic phosphate being cytosolic in origin.
The broad phosphorus resonance of isolated perfused liver can be removed by selective presaturati... more The broad phosphorus resonance of isolated perfused liver can be removed by selective presaturation with a DANTE pulse train. The use of the DANTE pulse allows the broad resonance to be removed without the use of multiple RF channels or the ability to switch the observation channel from low to high power.
The mechanisms that underlie the balance between the consumption and oxidative generation of ATP ... more The mechanisms that underlie the balance between the consumption and oxidative generation of ATP in the intact cell are not well-defined. Cytosolic inorganic phosphate (Pi) and ADP levels, the cytosolic ATP/ADP ratio, and the cytosolic phosphorylation potential (PP) have all been proposed as major regulatory variables, the latter as a component of a "near-equilibrium" thermodynamic regulatory scheme. Therefore, the potential regulatory roles of these variables in the intact cell were evaluated with 31P NMR and Langendorff perfused rat hearts; in this preparation, the tissue oxygen consumption rate (MVO2) can be varied over a wide range. When the exogenous carbon source was varied, none of the proposed regulatory parameters, i.e., the ATP/ADP ratio, PP, or cytosolic ADP level, were found to be uniquely related to MVO2. Rather, ADP levels at a given MVO2 decreased progressively for the exogenous carbon sources in the following order: glucose, glucose + insulin, palmitate + glucose, lactate, pyruvate + glucose, and octanoate + glucose. In the octanoate and pyruvate groups, MVO2(-1) was linearly dependent upon [ADP]-1 with apparent Km values being in the range previously observed in isolated mitochondria. A similar trend was observed in the MVO2-[Pi] relationship. The present findings suggest that exogenous carbon sources which effectuate deregulation of intramitochondrial NADH generation lower cytosolic ADP and Pi to levels which are limiting to the rate of oxidative phosphorylation. For other carbon sources, the processes controlling the rate of NADH generation also participate in determining the rate of oxidative ATP synthesis. However, this control must be exerted kinetically rather than through a near-equilibrium thermodynamic mechanism as indicated by the present data and prior kinetic studies of the ATP synthetic process in both isolated mitochondria and intact myocardium [La Noue, K. F., et al. (1986) Biochemistry 25, 7667-7675; Kingsley-Hickman, P., et al. (1987) Biochemistry 26, 7501-7510].
Biochimica et Biophysica Acta (BBA) - General Subjects, 1984
The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guine... more The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. 14C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [14C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.
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