Levels Of Oxaloacetate Research Articles

Studies of gluconeogenic mitochondrial enzymes: III. The conversion of α-ketoglutarate to glutamate by bovine liver mitochondrial glutamate dehydrogenase and glutamate-oxaloacetate transaminase

The rate of conversion of α-ketoglutarate to glutamate by the bovine liver mitochondria glutamate dehydrogenase and glutamate-oxaloacetate transaminase has been studied. In arsenate buffer, initial velocity studies (in the absence of added product) are consistent with the concept that the order of addition of substrate to glutamate dehydrogenase is sequential with DPNH adding first followed by α-ketoglutarate and then ammonium ions. The order of addition with respect to ammonium ions and α-ketoglutarate is opposite to that found in Tris-acetate buffer and could be the result of the higher Michaelis constant of ammonium ions in arsenate buffer. The value of this constant was found to be high, compared to physiological levels under all of the experimental conditions employed. Neither of the products of this reaction (DPN and glutamate) were significant inhibitors. Therefore these results suggest that ammonium ions are important regulators of this reaction. The glutamate-oxaloacetate transaminase reaction (when assayed in a coupled assay with malate dehydrogenase and DPNH) seems to react in a typical transaminase or ping-pong manner. α-Ketoglutarate has a much lower Michaelis constant for glutamate dehydrogenase than glutamate-oxaloacetate transaminase and is a substrate inhibitor of glutamate dehydrogenase. Consequently, the ratio of activity of glutamate dehydrogenase to glutamate-oxaloacetate transaminase would be higher at low (0.4 m m) than at high (4 m m) concentrations of α-ketoglutarate. One of the main regulators of the glutamate-oxaloacetate transaminase reaction would be the level of oxaloacetate since this compound is a very potent inhibitor of this reaction.

Effects of ethanol infusion on the redox state and metabolite levels in rat liver in vivo.

Hepatic levels of several metabolites of the Embden‐Meyerhof pathway, the citric acid cycle and oxidized and reduced substrate pairs of the lactate, α‐glycerophosphate, β‐hydroxybutyrate, malate, and glutamate dehydrogenase systems were measured in freeze clamped livers from fed male rats, before and after ethanol infusion. Ethanol infusion in fed male rats after 30 min resulted in an increase in the levels of hexosemonophosphates, triosephosphates, fructose 1,6 diphosphate, lactate, α‐glycerophosphate, malate, β‐hydroxybutyrate, and glutamate. [Lactate]/[pyruvate] ratio increased about 3 fold, [α‐glycerophosphate]/[dihydroxyacetonephosphate] 1.5 fold, [malate]/[oxaloacetate], [β‐hydroxybutyrate]/[acetoacetate], and [glutamate]/[2‐oxoglutarate] × [NH4+] about 2 fold. The levels of pyruvate, acetyl CoA, citrate, and ATP were decreased during ethãnol treatment. The hepatic levels of glycogen, oxaloacetate, 2‐oxoglutarate, ammonia, aspartate, and acetoacetate remained unchanged, 30 min after ethanol infusion, as compared to values before ethanol infusion. Hepatic glucose 6‐phosphatase activity was significantly increased after 15 min of ethanol infusion (1.5 g/kg body weight). The glucose 6‐phosphatase activity showed a maximum 15 min after ethanol infusion, the fructose 1,6 diphosphatase activity did not change significantly 15 and 30 min after ethanol infusion. Hepatic acid and alkaline phosphatase activities measured with β‐glycerophosphate as substrate showed no significant changes compared to values from normal rat livers. The presence of ethanol (8 mM) in the incubation medium showed no significant effect on the [6‐14C]glucose incorporation into glycogen by the liver slices.

Metabolic Changes in Liver Associated with Spontaneous Ketosis and Starvation in Cows

Although some information is available concerning the physiological alterations in vivo associated with spontaneous bovine ketosis, a broad study of pathways and possible changes of enzyme activities has not been carried out. To this aim, liver samples were taken from spontaneously ketotic cows, from the same cows when normal, and when starved for 96 hours. Measurements were made to ascertain the degree of ketosis and to compare changes in lipid synthesis, substrate oxidation, enzyme activities and the level of several metabolic intermediates. This research was particularly directed to a comparison of differences and similarities between the normal and spontaneously ketotic animal and between spontaneous ketosis and starvation ketosis. The liver of the spontaneously ketotic cow had a lower activity of mitochondrial NAD-malate dehydrogenase than the normal, higher levels of lactate, pyruvate, acetoacetate and β-hydroxybutyrate, and a substantially lower concentration of citrate. Livers from the spontaneously ketotic cows, when compared to with those of the starved animal, had a lower activity of mitochondrial pyruvate carboxylase, lower levels of NADH and higher levels of citrate, β-hydroxybutyrate and acetoacetate. Many other parameters of carbohydrate and lipid metabolism were measured and found to be unchanged from the normal in the two ketotic conditions. Notable among these was the lack of any alterations in the activity of phosphoenolpyruvate carboxykinase or levels of oxaloacetate. These findings conflict with the oxaloacetate shortage theory of bovine ketosis as generally stated.

THE EFFECT OF NEUTRAL SALTS ON PARTICLE PREPARATIONS FROM RAT LIVER. I. THE EFFECT OF NEUTRAL SALT ON PYRUVATE OXIDATION.

The effect of neutral salts on the utilization of pyruvate by saline-washed particles from rat liver has been investigated in the presence of ATP, Mg++, and fluoride. Addition of increasing quantities of NaCl, KCl, LiCl, or an osmolar equivalent of sucrose to particle preparations metabolizing pyruvate produced a pronounced inhibition of oxygen consumption, which was characterized by a sharp threshold of onset. In the presence of relatively high concentrations of salt, lactate formation is reduced but ketone body synthesis is only slightly impaired. No protection from salt inhibition was afforded by pretreatment of the particles with pyruvate. Fluoride appears to sustain the inhibition produced by the osmotic activity of NaCl. The inhibition is associated with a lack of utilization of pyruvate but produces no change in oxaloacetate levels. The oxidation of D-(−)-β-hydroxybutyrate under these conditions seems to be unimpaired. Within limits, the elevation of the osmolarity appeared to be without effect on oxidative phosphorylation, regardless of the substrate, but was associated with certain well-defined morphological changes in the mitochondrion.

Oxaloacetate Levels in Normal and Ketotic Pregnant Sheep

SUMMARY Signs of pregnancy toxaemia have been induced by starvation, in Clun Forest ewes pregnant with twins. Oxaloacetate and acetoacetate levels in liver and muscle have been compared with the levels of these constituents in liver and muscle of normal ewes pregnant with twins. Differences between the 2 sets of values were not significant. Oxaloacetate levels in sheep liver were appreciably higher than those reported in the literature for the rat, and acetoacetate does not increase in the liver of starved sheep as it does in the rat.

Absence of a relation between lipogenesis and ketogenesis in vivo.

IN the normal liver acetyl-coenzyme A is formed from glucose, fatty acids and the keto-acids produced by deamination and transamination of amino-acids. It is removed by oxidation, by fatty acid synthesis and, to a small extent, by ketone body formation (Fig. 1). Lipogenesis is defective in the livers of diabetic and fasting animals, a block occurring in the path of conversion of acetyl-coenzyme A to fatty acids1. Formerly, many workers related the development of ketosis to a diminished rate of oxidation of acetyl-coenzyme A, due to a possible lowering in the level of oxaloacetate. Lately, as a result of experiments in vitro, increased ketogenesis has been attributed to a diversion in the utilization of acetyl-coenzyme A (or acetoacetyl-coenzyme A) from fatty acid synthesis towards the formation of ketone bodies2–4. Stadie4 has advanced the hypothesis that the supply of reduced triphosphopyridine nucleotide, which depends upon the rate of glucose metabolism via the hexosemonophosphate ‘shunt’, regulates fatty acid synthesis and, consequently, ketogenesis.