Biomedical subjects

H Brunengraber

Publications and source records attributed to H Brunengraber.

At least 19 recordsLinked to original sources

PubMed1995

Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis. Substrate cycling between glycerol and triose phosphates in liver.

Mass isotopomer distribution analysis allows studying the synthesis of polymeric biomolecules from 15N, 13C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. The mass isotopomer distribution of the polymer allows calculation of (i) the enrichment of the monomer and (ii) the dilution of the newly synthesized polymer by unlabeled polymer. We tested the conditions of validity of mass isotopomer distribution analysis of glucose labeled from [U-13C3]lactate, [U-13C3]glycerol, and [2-13C]glycerol to calculate the fraction of glucose production derived from gluconeogenesis. Experiments were conducted in perfused rat livers, live rats, and live monkeys. In all cases, [13C]glycerol yielded labeling patterns of glucose that are incompatible with glucose being formed from a single pool of triose phosphates of constant enrichment. We show evidence that variations in the enrichment of triose phosphates result from (i) the large fractional decrease in physiological glycerol concentration in a single pass through the liver and (ii) the release of unlabeled glycerol by the liver, presumably via lipase activity. This zonation of glycerol metabolism in liver results in the calculation of artifactually low contributions of gluconeogenesis to glucose production when the latter is labeled from [13C]glycerol. In contrast, [U-13C3]lactate appears to be a suitable tracer for mass isotopomer distribution analysis of gluconeogenesis in vivo, but not in the perfused liver. In other perfusion experiments with [2H5]glycerol, we showed that the rat liver releases glycerol molecules containing one to four 2H atoms. This indicates the operation of a substrate cycle between extracellular glycerol and liver triose phosphates, where 2H is lost in the reversible reactions catalyzed by alpha-glycerophosphate dehydrogenase, triose-phosphate isomerase, and glycolytic enzymes. This substrate cycle presumably involves alpha-glycerophosphate hydrolysis.

Animals↗

PubMed1995

Isotopomer analysis of citric acid cycle and gluconeogenesis in rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis.

We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.

ATP Citrate (pro-S)-Lyase↗

PubMed1995

Tracing hepatic gluconeogenesis relative to citric acid cycle activity in vitro and in vivo. Comparisons in the use of [3-13C]lactate, [2-13C]acetate, and alpha-keto[3-13C]isocaproate.

The validity of the use of a carbon tracer for investigating liver intermediary metabolism in vivo requires that the labeling pattern of liver metabolites not be influenced by metabolism of the tracer in other tissues. To identify such specific tracer, livers from 48-h starved rats were perfused with recirculating buffer containing [3-13C]lactate, [2-13C]acetate, or alpha-keto[3-13C]isocaproate. Conscious 48-h starved rats were infused with the same tracers for 5 h. The labeling patterns of liver glutamate and extracellular glucose were assayed by gas chromatography-mass spectrometry. In vivo data were corrected for 13CO2 reincorporation into C-1 of glutamate and C-3 and C-4 of glucose, using data from control rats infused with NaH13CO3. With [3-13C]lactate the labeling pattern of liver glutamate was the same in perfused organs and in vivo. In contrast, with [2-13C]acetate and alpha-keto[3-13C]isocaproate the labeling pattern of liver glutamate in vivo was clearly influenced by the expected labeling pattern of citric acid cycle intermediates formed in non-gluconeogenic organs, presumably glutamine made in muscle. Indeed, the labeling pattern of plasma glutamine and liver glutamate were similar in experiments with [3-13C]lactate but different in experiments with [2-13C]acetate and alpha-keto[3-13C]isocaproate. Similar conclusions were drawn from the labeling patterns of glucose. Therefore, labeled lactate appears as the best tracer for studies of liver intermediary metabolism in vivo. Our data also show that a substantial fraction of alpha-ketoisocaproate metabolism occurs in peripheral tissues.

Acetates↗

PubMed1995

Metabolism of R-beta-hydroxypentanoate and of beta-ketopentanoate in conscious dogs.

R-beta-Hydroxypentanoate and beta-ketopentanoate are homologues of physiological ketone bodies R-beta-hydroxybutyrate and acetoacetate. They derive from the oxidation in liver of the R-moiety of R,S-1,3-pentanediol, a potential nutrient. This report documents the metabolism of R-beta-hydroxypentanoate and beta-ketopentanoate in conscious dogs. Whether administered by bolus or constant infusion, the two substrates are interconverted and rapidly metabolized. When beta-ketopentanoate was infused at a rate corresponding to 75% of the dog's caloric requirement, the steady-state total plasma concentration of the two substrates was only 1.3 mM. Because the substrates are precursors of propionyl-CoA, we assayed the urinary concentrations of markers of propionic acidemia. Their accumulation was minor compared with what is observed in patients suffering from propionic acidemia. We conclude that, at least during short-term experiments, R-beta-hydroxypentanoate and beta-ketopentanoate are well metabolized in the dog without apparent intolerance to a large supply of propionyl-CoA.

Acyl Coenzyme A↗

PubMed1995

Metabolism of (R,S)-1,3-butanediol acetoacetate esters, potential parenteral and enteral nutrients in conscious pigs.

The (R,S)-1,3-butanediol-acetoacetate monoesters and diester are nonionized sodium-free precursors of ketone bodies (beta-hydroxybutyrate and acetoacetate). They represent a convenient form of ketone body administration for parenteral and enteral nutrition. We have studied the metabolism of the esters in the conscious pig, an animal in which ketogenesis is congenitally impaired. Some pigs were infused for 3 h, intravenously or intragastrically, with the esters or with (R,S)-1,3-butanediol at 30% of the hourly caloric requirement. Other pigs were given intragastric boluses of esters or of (R,S)-1,3-butanediol at 15% of the daily caloric requirement. Our data show that continuous infusion of the esters at 30% of the caloric requirement leads to low concentrations of (R,S)-1,3-butanediol (0.1 mM) and total ketone bodies (0.5 mM). In pigs given intragastric boluses of esters at 15% of the daily caloric requirement, concentrations of (R,S)-1,3-butanediol and total ketone bodies peaked briefly at 2-3 and 5 mM, respectively. No deleterious side effects were observed in any group, including no hypoglycemia and no acidosis. Thus the (R,S)-1,3-butanediol acetoacetate esters appears to be well utilized as a nutrient by the pig despite its impaired ketogenesis.

3-Hydroxybutyric Acid↗

PubMed1995

Lactate and pyruvate isotopic enrichments in plasma and tissues of postabsorptive and starved rats.

It has been proposed that plasma pyruvate isotopic enrichment (IE) during infusion of labeled lactate could be used to estimate the intracellular IE of lactate and pyruvate and thus to calculate their turnover rate. We determined the relations of plasma and tissue IE of lactate and pyruvate in anesthetized rats infused with [3-13C]lactate in an artery and sampled from a vein (A-V mode) or infused in a vein and sampled from an artery (V-A mode). In both groups of rats, the ratio of tissue to plasma lactate IE was < 1 with large differences between tissues: the highest ratio was observed in heart and the lowest in soleus. With the exception of liver, this ratio was higher in the A-V than in the V-A mode. Pyruvate IE was lower than lactate IE in tissues, with a few exceptions, and in plasma. This ratio of pyruvate to lactate IE was approximately 0.70 in plasma in A-V and V-A modes. Moreover pyruvate IE was also always higher in plasma than in tissues. This seemingly surprising result could be explained by the production of labeled pyruvate from labeled lactate inside the circulation by erythrocytes, because we observed a rapid isotopic equilibrium between lactate and pyruvate in blood "in vitro." Apparent lactate turnover was higher in the A-V than in the V-A mode when it was calculated using lactate as well as pyruvate IE. Therefore plasma pyruvate IE cannot be used in rats to estimate tissue IE and did not reconcile turnover rates measured using the A-V or V-A mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

A limitation in the use of mass isotopomer distributions to measure gluconeogenesis in fasting humans.

The use of distributions of mass isotopomers in glucose from [U-13C]glycerol to estimate fractional rates of gluconeogenesis was examined. [U-13C]glycerol was infused into normal subjects who ingested acetaminophen and fasted for 60 h. Isotopomer distributions were measured by mass spectrometry in blood glucose and in glucuronic acid from urinary acetaminophen glucuronide. The distributions are incompatible with glucose production solely via gluconeogenesis from a single pool of triose phosphates. Rather, with the assumption of a single enriched triose phosphate pool, the distributions indicate, despite the 60 h of fasting, about as much glucose formation from an unlabeled glucose source as from that pool. Therefore the data indicate cellular heterogeneity in glycerol's metabolism, so that two or more pools with significantly different enrichments were the source of the glucose and glucuronic acid. This heterogeneity is related to much greater concentrations of glycerol in periportal than in pericentral zones of the liver lobule. Beyond evidence for heterogeneity, the findings emphasize a limitation in applying analyses of mass isotopomer distributions to measure polymer biosynthesis in the presence of heterogeneity in the precursor pool.

Acetaminophen↗

PubMed1994

Reversibility of the mitochondrial isocitrate dehydrogenase reaction in the perfused rat liver. Evidence from isotopomer analysis of citric acid cycle intermediates.

The reversal of the mitochondrial isocitrate dehydrogenase reaction was investigated in rat livers perfused with [U-13C5]glutamate or [U-13C5]glutamine. The mass isotopomer distribution of citric acid cycle intermediates extracted from the livers was determined by gas chromatography-mass spectrometry. Citrate was enriched in an isotopomer containing five 13C. The formation of this isotopomer can only be explained by the reversal of the isocitrate dehydrogenase reaction. Calculation of kinetic parameters from the mass isotopomer data reveals a rapid interconversion of isocitrate and alpha-ketoglutarate. This interconversion results in an isotopic exchange between carbon 6 of citrate and mitochondrial CO2 that can affect the calculation of citric acid cycle kinetic parameters. Thus, the reversal of the isocitrate dehydrogenase reaction should be included in isotope labeling models of the citric acid cycle.

Animals↗

PubMed1994

Nonhomogeneous labeling of liver extra-mitochondrial acetyl-CoA. Implications for the probing of lipogenic acetyl-CoA via drug acetylation and for the production of acetate by the liver.

The labeling of liver extra-mitochondrial acetyl-CoA was investigated in isolated rat livers perfused with [2-(13)C]acetate, [1-(13)C]octanoate, or [1,2,3,4-(13)C4]docosanoate and with drugs that undergo acetylation (phenylaminobutyrate, paraaminobenzoate, and sulfamethoxazole; singly or in combination). The 13C enrichment of mitochondrial acetyl-CoA was probed by the enrichment of R-beta-hydroxybutyrate. The latter was not enriched from [1,2,3,4-(13)C4]docosanoate, thus excluding mitochondrial beta-oxidation of docosanoate. The 13C enrichment of extra-mitochondrial acetyl-CoA was probed by the enrichments of acetylated drugs and of free acetate. In most cases, the four probes yielded different enrichments. Thus, extra-mitochondrial acetyl-CoA appears nonhomogeneous. Competition between drugs alters the labeling of individual acetyl-CoA sub-pools. The labeling pattern of acetylated drugs suggests the existence of more than the two N-acetyltransferases identified so far by others. Our data question the possibility of probing the pool of lipogenic acetyl-CoA via drug acetylation.

4-Aminobenzoic Acid↗

PubMed1994

Determination of (13C) urea enrichment by gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry.

We present gas chromatographic/mass spectrometric and gas chromatographic/isotope ratio mass spectrometric assays of the 13C enrichment of plasma urea converted to its dimethylaminomethylene derivative. The limits of sensitivity of the two techniques are 0.2% and 0.02%, respectively. The techniques were tested in rats and humans infused with (13C)urea or (3-13C)lactate. (13C)Urea enrichment during the infusion of (3-13 C)lactate in humans was not detectable by gas chromatography/mass spectrometry but was easily measured by gas chromatography/isotope ratio mass spectrometry. These assays should be useful for clinical investigations, in which the incorporation of a (13C)gluconeogenic substrate into glucose must be corrected for the incorporation of 13CO2 derived from the oxidation of the substrate. This correction involves measuring the low-level 13C enrichment of urea.

Adult↗

PubMed1994

Assay of the concentration and 13C enrichment of acetate and acetyl-CoA by gas chromatography-mass spectrometry.

We present two techniques for determining the concentration and 13C enrichment of acetate in biological fluids. After the sample has been spiked with an internal standard of [2,2,2,2H3,1-13C]acetate, acetate is first enzymatically converted to acetyl-coenzyme A, which is chemically converted to acetylglycine. The latter is analyzed by gas chromatography-mass spectrometry, either as a methyl ester by positive chemical ionization or as a pentafluorobenzyl ester by negative chemical ionization. The mole percentage enrichment of tissue acetyl-CoA can also be assayed after conversion to acetylglycine pentafluorobenzyl ester.

Acetates↗

PubMed1994

Use of [6,6-2H2]glucose and of low-enrichment [U-13C6]-glucose for sequential or simultaneous measurements of glucose turnover by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometric methods for assaying the enrichment of 99 at.% [6,6-2H2]glucose and 30 at.% [U-13C6]glucose, although both tracers are mostly M + 2. 13C enrichment is determined either by the C-1 to C-5 fragment of glucose aldonitrile pentaacetate or by oxidation of glucose to glucarate. 2H enrichment is assayed as the difference between the 13C enrichment of glucarate and the 2H + 13C enrichment of glucose. The techniques, which were validated in in vivo experiments, are applicable to the determination of simultaneous or sequential measurements of the rate of glucose appearance before and after an intervention. They could also be applied to the simultaneous determination of (i) gluconeogenesis by incorporation of a 13C-labeled precursor into glucose and (ii) the rate of glucose appearance by [6,6-2H2]glucose infusion.

Animals↗

PubMed1994

Assay of the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and the corresponding hydroxyacids by gas chromatography-mass spectrometry.

We developed gas chromatographic-mass spectrometric assays for the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and their corresponding hydroxyacids, lactate, beta-hydroxybutyrate, and beta-hydroxypentanoate (3-hydroxyvalerate) in biological fluids. The corresponding ketoacids, acetoacetate and beta-ketopentanoate, can be assayed simultaneously by pretreating the samples with NaB2H4. The assays involve spiking the samples with deuterated internal standards, deproteinization, ether extraction, and derivatization of the carboxyl groups with (R,S)-2-butanol/HCl and of the hydroxyl groups with chiral (S)-(+)-2-phenylbutyryl chloride. Mass spectrometric analysis is conducted under ammonia positive chemical ionization. We used these assays to follow the metabolism of diol enantiomers in dogs. For (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol, the uptakes from dog plasma of the R and S enantiomer of each diol were identical. In contrast, the metabolism of (S)-1,2-propanediol was faster than that of (R)-1,2-propanediol. (R)-1,2-Propanediol is formed during acetone metabolism, while (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol are potential nutrients. The assays developed will allow further investigations of the metabolisms of acetone, (R)-lactate, and artificial nutrients derived from the 1,3-butanediol and 1,3-pentanediol enantiomers.

3-Hydroxybutyric Acid↗

PubMed1994

Assay of the concentration and 13C-labeling pattern of phenylacetylglutamine by nuclear magnetic resonance.

Phenylacetate, derived from phenylalanine, is converted in human and primate liver to phenylacetylglutamine. The latter, which is excreted in urine, has been used to probe noninvasively the labeling pattern of liver citric acid cycle intermediates. We present nuclear magnetic resonance assays for the urinary concentration of phenylacetylglutamine and for the 13C-labeling pattern of its glutamine moiety. The concentration of phenylacetylglutamine is calculated from the natural 13C signals of all carbons of its benzene ring and C-2 of its acetyl moiety. The limit of detection is 13 mumol of unlabeled phenylacetylglutamine. The minimum amount of phenylacetylglutamine needed to determine a 1% enrichment of one of its carbons is 26 mumol. The technique was tested by analyzing phenylacetylglutamine in the urine from monkeys infused with various 13C tracers. The labeling patterns obtained agreed with theoretical calculations and patterns reported in phenylacetylglutamine and glutamine labeled from 14C and 13C tracers, respectively.

Animals↗

PubMed1993

Metabolism of 2,3-butanediol stereoisomers in the perfused rat liver.

The identification of 2,3-butanediol in sera of alcoholics led to the hypothesis that it may be a specific marker of alcohol abuse. We have investigated the metabolism of the individual isomers of 2,3-butanediol (2R,3R-, 2S,3S-, meso-2,3-butanediol and racemic 2,3-butanediol) in perfused livers from fed rats. Rates of uptake of the isomers decrease in the order (i) 2R,3R-, (ii) meso-, (iii) 2S,3S-2,3-butanediol. We observed interconversion of isomers and oxidation to acetoin with 2R,3R- and meso- but not with 2S,3S-2,3-butanediol. In perfusions conducted in deuterium oxide, interconversion of isomers was accompanied by incorporation of deuterium. Thus, interconversion of isomers occurs via a reversible oxidation to acetoin with incorporation of hydrogen from water. In perfusions with either 2R,3R- or meso-[2-14C]2,3-butanediol, the substrates were converted to labeled acetate, R-3-hydroxybutyrate and CO2, suggesting that 2,3-butanediol is oxidized to acetyl-CoA via acetoin.

3-Hydroxybutyric Acid↗

PubMed1993

Assay of the acetyl-CoA probe acetyl-sulfamethoxazole and of sulfamethoxazole by gas chromatography-mass spectrometry.

We present gas chromatographic-mass spectrometric assays for (i) the concentration of sulfamethoxazole and (ii) the concentration and molar percentage enrichment of acetyl-sulfamethoxazole in biological fluids. The compounds are extracted with ethyl acetate, derivatized with either diazomethane or pentafluorobenzyl bromide, and analyzed by gas chromatography-mass spectrometry. Quantitation is achieved using internal standards, [2H4]sulfamethoxazole and acetyl-[2H4]sulfamethoxazole. Limits of detection are 200 nmol for the methyl derivatives and 2 nmol for the pentafluorobenzyl derivatives. The high sensitivity of the assay with the pentafluorobenzyl derivatives allows measuring in plasma and urine (i) the pharmacokinetics of sulfamethoxazole and acetyl-sulfamethoxazole and (ii) the stable isotope enrichment of the acetyl moiety of acetyl-sulfamethoxazole. The latter is used as a probe for the noninvasive chemical biopsy of liver extramitochondrial acetyl-CoA.

Acetyl Coenzyme A↗

PubMed1993

Determination of the 13C-labeling pattern of glucose by gas chromatography-mass spectrometry.

We developed a gas chromatography-mass spectrometric method which allows to determine the complete 13C-labeling pattern of glucose. The method uses four derivatives of glucose (methyloxime trimethylsilyl, bisbutylboronate acetate, aldonitrile pentaacetate, and permethyl) and selective analysis of fragment ions retaining specific carbon atoms. The technique was tested by analyzing glucose from rat livers perfused with various 13C tracers. The labeling patterns agree with theoretical calculations and with literature reports where [14C]glucose was analyzed by degradation and [13C]glucose was analyzed by NMR.

Animals↗

PubMed1993

Determination of the 13C-labeling pattern of glutamate by gas chromatography-mass spectrometry.

We present a simple technique for determining the 13C-labeling pattern of glutamate by gas chromatography-mass spectrometry. Glutamate is derivatized with dimethylformamide dimethyl acetal (Methyl-8R). The dimethylaminomethylene methyl ester derivative of glutamate yields fragment ions that allow calculation of 13C enrichment on each carbon. The technique was tested by analyzing glutamate from rat livers perfused with various 13C tracers. The labeling patterns obtained agreed with theoretical calculations or patterns reported with 14C and 13C tracers.

Animals↗