In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction ...in coronary flow on these processes and on the tissue content of CAC intermediates.
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.
▪ Abstract Investigations into regulating metabolic pathways with stable isotopes have, over the past decade, undergone major development with the use of nuclear magnetic resonance and mass ...spectrometry in studying labeling patterns of newly synthesized biomolecules. In this review, we concentrate on investigations of mass isotopomer distribution (MID) measured by mass spectrometry. We review the applications of MID to analytical problems, in particular the possibility of amplifying the measurement of low isotopic enrichments by incorporating multiple molecules or atoms of a primary analyte into the molecule of a secondary analyte, the MID of which is assayed. We also review new information on the regulation of intermediary metabolism gathered from the analysis of MID patterns of synthesized compounds. Lastly, we review the applications of MID to the synthesis of polymeric molecules, with emphasis on the validity of these techniques. A number of these techniques are applicable to investigations of nutrient metabolism in health and disease.
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Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
The assay of oxaloacetate and α-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological ...concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of U-13C4oxaloacetate and U-13C5α-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the M-57+ ion in electron impact. Although the internal standards of U-13C4oxaloacetate and U-13C5α-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting 1,2,3,6-13C4citrate with citrate lyase, and L-U-13C5glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of 1,2,3,6-13C4citrate is prepared from L-U-13C4aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and α-ketoglutarate in human blood and rat liver.
The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts ...by infusing U-(13)C3lactate and/or U-(13)C3 pyruvate into the left anterior descending (LAD) coronary artery.
Measurement of specific aldehydes such as malondialdehyde, hexanal, or 4-hydroxynonenal provides a method to evaluate the extent of lipid peroxidation. However, assay of aldehydes is complicated by ...their high reactivity toward other molecules and their chemical instability. In the proposed method, aldehydes are reduced to stable alcohols. Except for malondialdehyde, this is readily accomplished under neutral conditions at room temperature or at 4 degrees C with either sodium borodeuteride (NaB2H4) or sodium borohydride (NaBH4). Complete reduction of malondialdehyde requires a more powerful reducing agent, borane trimethylamine. After ether extraction and evaporation of the organic phase, the alcohols are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry in the positive chemical ionization mode for malondialdehyde and 4-hydroxynonenal and electron impact mode for all other aldehydes. Quantitation is achieved using internal standards of 1,3-2H8propanediol for MDA, of 4-2-2Hhydroxynonenal for 4-hydroxynonenal, and of the corresponding saturated per-deuterated alcohols for all saturated aldehydes, for example and 2H13hexanol for hexanal. Saturated per-deuterated alcohols also serve as external standards for their unsaturated aldehydes with same chain length. The detection limit for individual aldehydes is 0.5 nmol in a given sample. In addition to being highly sensitive and selective, the present method allows one to verify the identity of each aldehyde by observing a mass shift when aldehydes are reduced with either NaB2H4 or NaBH4. The usefulness of our method is demonstrated by measuring aldehyde accumulation in autoxidized arachidonic acid solutions and in heart homogenates before and after induction of peroxidation.
The results of a study show that, in a model of hypoxia-reoxygenation, the cardioprotective effects of fumarate in rats were associated with its predominant metabolism to succinate through the ...reductive pathway.
Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of ...radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.
