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H Brunengraber

Publications and source records attributed to H Brunengraber.

At least 73 records · Page 4Linked to original sources
PubMed1990

One-stage hepatectomy in the dog.

This report describes a technique for total hepatectomy in the dog. The procedure maintains the integrity of the gastrointestinal tract and the pancreas in the normothermic, anesthetized animal. The technique involves (i) a mesentericocaval shunt, (ii) a temporary external bypass between the femoral and jugular veins, and (iii) a permanent internal bypass between the abdominal and thoracic inferior vena cava through the tendinous area of the diaphragm. This 60- to 70-min procedure yields a preparation useful for short-term metabolic studies in nonhepatic tissues.

Animals↗
PubMed1990

Pseudoketogenesis in hepatectomized dogs.

Overestimation of ketone body turnover in vivo, measured by tracer kinetics, could occur if specific activity or molar percent enrichment is diluted in extrahepatic tissues by label exchange via reversal of 3-oxoacid-CoA transferase, a process we call pseudoketogenesis. To test this hypothesis, euglycemic hepatectomized dogs were injected with a bolus of acetoacetate (0.8 mmol/kg), 32% enriched in [3,4-13C2]acetoacetate. Concentrations and labeling patterns of blood acetoacetate and R-3-hydroxybutyrate were measured by selected ion-monitoring gas chromatography-mass spectrometry. During the 60 min after bolus injection of [3,4-13C2]acetoacetate, the molar percent enrichment of blood [3,4-13C2]acetoacetate decreased to 73 +/- 3% (n = 5) in controls and to 11.5 +/- 0.8% (n = 3) during infusion of dichloroacetate, an activator of pyruvate dehydrogenase. The enrichment of R-3-hydroxy-[3,4-13C2]butyrate followed closely that of [3,4-13C2]acetoacetate. These dilutions occurred despite a net uptake of ketone bodies. Concomitantly, 10.6 +/- 2.2 (n = 5) and 6.0 +/- 2.9% (n = 3) of [13C]acetoacetate molecules were labeled on all four carbons in control and dichloroacetate-treated dogs, respectively. This uniformly labeled acetoacetate arises from partial equilibration between [3,4-13C2]acetoacetate and [1,2-13C2]acetyl-CoA via the reactions catalyzed by 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. Our data demonstrate the reversibility of the 3-oxoacid-CoA transferase in intact extrahepatic tissues and support the concept of pseudoketogenesis. This phenomenon has been quantitated by kinetic analysis of the data.

3-Hydroxybutyric Acid↗
PubMed1990

Interpretation of isotopomer patterns in tracing glycogen synthesis and glucose recycling using [13C6]glucose.

Tracing glycogen synthesis with [U-13C]glucose involves determination of isotopomer patterns in precursors and products. The contribution of the direct pathway to glycogen synthesis has been estimated from the ratio (R) of the number of glycogen glucose molecules having six 13C atoms to the total number of glucose molecules having one to six 13C atoms. Using theoretical examples, we show that the equation to calculate R underestimates the contribution of the direct pathway. We derive a correct stoichiometric expression for R. Also, we show how to correct these estimates for 1) recondensation of two M+3 three-carbon intermediates into M+6 glucose 6-phosphate, and 2) the increasing enrichment of arterial glucose in various isotopomers arising from the indirect pathway. Finally, using the developed formulas we recalculate data from the literature. Similar considerations apply to investigations of glucose recycling using [U-13C6]glucose.

Animals↗
PubMed1990

Metabolism of beta-methyl-heptadecanoic acid in the perfused rat heart and liver.

The metabolism of beta-methyl-[1-14C]heptadecanoic acid, a potential myocardial imaging agent, was investigated in perfused hearts and livers from rats. Hepatic uptake is approximately 4.5 times greater than cardiac uptake. In the heart, 66% of beta-methyl-heptadecanoic acid metabolism occurs via omega-oxidation, 33% by esterification and less than 1% via alpha-oxidation. In contrast, 53% of hepatic metabolism of beta-methyl-heptadecanoic acid occurs via alpha-oxidation, 27% via omega-oxidation, and 20% via esterification. Perfusion of hearts and livers with concentrations of beta-methyl-heptadecanoic acid 100 to 1000 times greater than that used for myocardial imaging does not alter any of the physiological and biochemical parameters measured. In the perfused liver, 3-methyl-[1-14C]glutarate was identified as the principal hydrosoluble catabolite of beta-methyl-heptadecanoic acid.

Animals↗
PubMed1989

Reassessment of 14CO2 compartmentation and of [14C]formate oxidation in rat liver.

Our previous report (Marsolais, C., Huot, S., David, F., Garneau, M., and Brunengraber, H. (1987) J. Biol. Chem. 262, 2604-2607) had concluded that a fraction of [14C]formate oxidation in liver occurs in the mitochondrion. This conclusion was based on the labeling patterns of urea and acetoacetate labeled via 14CO2 generated from [14C]formate and other [14C]substrates. We reassessed our interpretation in experiments conducted in (i) perifused mitochondria and (ii) isolated livers perfused with buffer containing [14C]formate, [14C]gluconolactone, 14CO2, or NaH13CO3, in the absence and presence of acetazolamide, an inhibitor of carbonic anhydrase. Our data show that the cytosolic pools of bicarbonate and CO2 are not in isotopic equilibrium when 14CO2 is generated in the cytosol or is supplied as NaH14CO3. We retract our earlier suggestion of a mitochondrial site of [14C]formate oxidation.

Acetoacetates↗
PubMed1988

Pseudoketogenesis in the perfused rat heart.

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.

3-Hydroxybutyric Acid↗
PubMed1988

Interference of 3-hydroxyisobutyrate with measurements of ketone body concentration and isotopic enrichment by gas chromatography-mass spectrometry.

Concentrations and 13C2 molar percentage enrichments of blood R-3-hydroxybutyrate and acetoacetate are measured by selected ion monitoring gas chromatography-mass spectrometry. Samples are treated with NaB2H4 to reduce unlabeled and labeled acetoacetate to corresponding deuterium-labeled RS-3-hydroxybutyrate species. Only the gas chromatographic peak for the tert-butyldimethylsilyl derivative of 3-hydroxybutyrate needs to be monitored. The various compounds are quantitated using an internal standard of RS-3-hydroxy-[2,2,3,4,4,4-2H6]-butyrate. Concentrations of ketone bodies are obtained by monitoring the m/z 159 to 163 fragments of tert-butyldimethylsilyl derivatives of labeled and unlabeled 3-hydroxybutyrate species. High correlations were obtained between ketone body concentrations assayed (i) enzymatically with R-3-hydroxybutyrate dehydrogenase and (ii) by gas chromatography-mass spectrometry. The limit of detection is about 10 nmol of substrate in blood samples. The current practice of monitoring the m/z 275 to 281 fragments overestimates the concentration of endogenous R-3-hydroxybutyrate, due to co-elution of 3-hydroxyisobutyrate, a valine metabolite. The method presented is used to measure ketone body turnover in vivo in 24-h-fasted dogs.

3-Hydroxybutyric Acid↗
PubMed1988

Uptake of artificial model remnant lipoprotein emulsions by the perfused rat liver.

In comparison with their precursor lipoproteins, the remanants of the triacylglycerol-rich lipoproteins are reduced in contents of triacylglycerols and apolipoproteins AI and AIV, whereas the contents of cholesterol (free and esterified) and apolipoprotien E are increased. In this study, lipid emulsion models of remnant lipoproteins were used to explore which of these factors are necessary for physiological rates of remnant uptake by the perfused rat liver. Uptake rates of lipid emulsion models of remnant lipoproteins in the presence of apolipoprotein E were similar to in vivo uptake rates.

Animals↗
PubMed1988

Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats.

Production of ketone bodies and their contribution to lipogenesis were measured in isolated livers from normal and streptozocin-induced diabetic (STZ-D) rats perfused with tracer amounts of 3H2O and (R)-3-hydroxy[3-14C]butyrate. Diabetes decreased by 80-95% the total rates of fatty acid and 3-beta-hydroxysterol synthesis in perfused livers and livers of live rats. The activity of cytosolic acetoacetyl-CoA synthetase was slightly (17%) decreased in livers from STZ-D rats. The incorporation of ketone bodies into fatty acids and sterols was markedly inhibited in perfused livers from STZ-D rats despite the stimulation of ketogenesis by diabetes and the presence of oleate. Treatment of the rats with insulin before liver perfusion led to a normalization of the rates of ketogenesis and fatty acid synthesis. The rates of sterol synthesis were only partially normalized by insulin treatment. We conclude that in STZ-D, ketosis does not stimulate hepatic lipogenesis via cytosolic activation of acetoacetate.

3-Hydroxybutyric Acid↗
PubMed1987

Contribution of the shunt pathway of mevalonate metabolism to the regulation of cholesterol synthesis in rat liver.

The modulation of the shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in livers from fed, starved, and diabetic rats perfused with a physiological concentration (300 nM) of [5-14C] + [5-3H]mevalonate. Shunt activity was measured by (i) production of 14CO2 (corrected for loss of label by exchange reactions) and (ii) production of 3H2O. Contribution of exogenous mevalonate to total mevalonate production (0.06-0.11%) was assessed in parallel experiments by the incorporation of 3H2O into sterols. Inhibition of non-saponifiable lipid synthesis by starvation and diabetes is not associated with an inhibition of mevalonate production but with a major increase in shunting (7-34%) of sterol-bound mevalonate. The shunt pathway of mevalonate metabolism appears to participate in the regulation of cholesterol synthesis.

Animals↗
PubMed1987

Metabolism of S-3-hydroxybutyrate in the perfused rat liver.

The metabolism of millimolar concentrations of S-3-hydroxybutyrate (the unnatural enantiomer) has been studied in perfused livers from fed and starved rats. Protocols were designed to test whether S-3-hydroxybutyrate is metabolized in the cytosol or in the mitochondria via a racemase, a dehydrogenase, or a ligase. Our data show that only a minor fraction of S-3-hydroxybutyrate metabolism could occur via L-3-hydroxyacid dehydrogenase. Most of the metabolism of S-3-hydroxybutyrate proceeds via mitochondrial activation. In rat liver, S-3-hydroxybutyrate is converted to physiological ketone bodies (i.e., R-3-hydroxybutyrate, acetoacetate, acetone), lipids, and CO2. Carbons from S-3-hydroxybutyrate are transferred from the mitochondria to the cytosol mostly via citrate and the citrate cleavage pathway.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1987

Production of acetone and conversion of acetone to acetate in the perfused rat liver.

The utilization of millimolar concentrations of [2-14C]acetone and the production of acetone from acetoacetate were studied in perfused livers from 48-h starved rats. We devised a procedure for determining, in a perfused liver system, the first-order rate constant for the decarboxylation of acetoacetate (0.29 +/- 0.09 h-1, S.E., n = 8). After perfusion of livers with [2-14C]acetone, labeled acetate was isolated from the perfusion medium and characterized as [1-14C]acetate. No radioactivity was found in lactate or 3-hydroxybutyrate. After 90 min of perfusion with [2-14C]acetone, the specific activity of acetate was 30 +/- 4% (n = 13) of the initial specific activity of acetone. We conclude that, in perfused livers from 2-day starved rats, acetone metabolism occurs for the most part via free acetate.

Acetates↗
PubMed1987

Compartmentation of 14CO2 in the perfused rat liver.

The specific activity of the mitochondrial CO2 + bicarbonate system has been measured in perfused livers using the specific activities of urea and acetoacetate derived from 2-ketoisocaproate catabolism. Label was supplied either as NaH14CO3, 2-keto[1-14C]isocaproate, [1-14C]pyruvate, [1-14C]glutamine, or [14C]formate. With labeled bicarbonate, pyruvate, or 2-ketoisocaproate, the specific activities of effluent bicarbonate, urea, and acetoacetate were equal (acetoacetate was labeled only on C-1). In the presence of [14C]formate, the specific activity of acetoacetate was double that of urea. Acetazolamide (0.2 mM), an inhibitor of carbonic anhydrase, decreased the specific activities of urea and acetoacetate labeled from NaH14CO3 and increased the specific activities of urea and acetoacetate labeled from the other tracers. We conclude that: acetoacetate derived from 2-ketoisocaproate is, like urea, an index of the specific activity of mitochondrial CO2 in liver, carbonic anhydrase activity equalizes the specific activities of the CO2 + bicarbonate system on both sides of the mitochondrial membrane, and a fraction of [14C] formate-derived 14CO2 appears to be generated in a mitochondrial compartment, in the close vicinity of methylcrotonyl-CoA carboxylase.

Acetazolamide↗
PubMed1987

Lipogenesis from ketone bodies in the perfused rat liver: effects of acetate and ethanol.

The interactions between acetate or ethanol metabolism, lipogenesis, and ketone body utilization have been studied in isolated livers from fed rats perfused with 15 mM glucose and 10 mM acetate or ethanol. The contribution of acetate to ketogenesis is constant; on the other hand, the contribution of ethanol to ketogenesis increases with time, presumably because of the accumulation of acetate in the perfusate. Ketogenesis is decreased in the presence of ethanol (but not acetate), while ketone body utilization is not affected by ethanol or acetate. Acetate contributes one third and ethanol contributes one half of the carbon incorporated into fatty acids and 3-beta-hydroxysterols. Only a small fraction (less than 5%) of the incorporation of acetate or ethanol into fatty acids and sterols occurs via transient incorporation into ketone bodies.

Acetates↗
PubMed1987

Energy metabolism and blood perfusion in a mouse mammary adenocarcinoma during growth and following X irradiation.

Biochemical and blood perfusion changes in a mouse tumor system (MDAH MCaIV) were studied relative to normal tissues under conditions of normal blood flow and clamped blood supply. Further studies were performed during tumor growth and after local X irradiation. The biochemical profiles of three untreated human soft tissue sarcomas were also investigated. Animal tumors were irradiated in situ with either a single or fractionated regime to total doses of 20 or 49 Gy. Assays of lactate, pyruvate, AMP, ADP, and ATP were made on freeze-clamped tissue following authentic or sham treatments. Blood perfusion to tumors treated in the same way was measured using iv injection of 201Tl. The human tumors were found to have a lower lactate to pyruvate ratio (L/P) than the MCaIV tumors; their ATP levels were also lower. L/P was much higher in the MCaIV tumors than in normal liver, kidney, and muscle in the mouse. Occlusion of the blood supplies of the normal kidney and the MCaIV tumor caused an increase in the lactate and L/P levels in both cases. However, whereas the ATP level in the kidney fell, the level in the tumor was maintained. There was some evidence that the adenine nucleotides were not in equilibrium via the adenyl kinase catalyzed reaction. In addition, tumors were found to contain the enzyme creatine kinase. These results suggest that energy charge calculations cannot be computed in a meaningful manner because the creatine kinase catalyzed phosphorylation of ADP would maintain a higher than normal ATP level. Lactate and L/P ratio was found to increase during tumor growth and decrease following X irradiation. The total adenine nucleotides (AMP + ADP + ATP) exhibited a trend toward lower values with increasing tumor size. There was no significant change in total adenine nucleotides after a single 20-Gy dose; however, fractionated radiation caused some fall in total nucleotides. It is concluded that, in this tumor system, lactate level is a sensitive index of radiation-induced biochemical changes which are likely to reflect changes in tumor oxygenation.

Adenine Nucleotides↗
PubMed1986

Determination of the concentration and specific activity of acetone in biological fluids.

The concentration of acetone dissolved in liver perfusion medium was determined by injection of the sample into a gas chromatograph equipped with a Carbopack/Carbowax-packed glass column. Interference from labile acetoacetate which readily decomposes to acetone was eliminated by treating the samples with NaBH4 prior to the analysis. Acetone was detected and quantified as 2-propanol. Separation of labeled 2-propanol in the sample by high-performance liquid chromatography allowed the determination of its specific activity. These methods make possible the convenient and rapid determination of acetone concentration and specific activity in biological samples.

1-Propanol↗