A 13C Mass Isotopomer Study of Anaplerotic Pyruvate Carboxylation in Perfused Rat Hearts

Abstract

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13 C 3 ]lactate, and [U-13 C 3 ]pyruvate. Also, a fatty acid, [1-13 C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13 C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by ␤-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13 C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

“…An`isotopomer' is described by a specific number of 13 C atoms in specific positions of the molecule. For a compound with n carbon atoms, 2 n isotopomers are possible.…”

“…The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-13 C glucose or with mixtures of 13 C 6 / 12 C 6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway.…”

“…Keywords: MALDI-TOF mass spectrometry; metabolic flux analysis; Corynebacterium glutamicum; lysine; 13 C tracer.…”

“…Therefore their consideration in flux analysis can cause errors or uncertainties in the obtained results. To circumvent this problem, additional information can be provided by tracer studies with 13 C-labeled substrates. Recently, different research groups have quantified carbon fluxes in the central metabolism of Corynebacteria by isotopic tracer studies with NMR [6,7] which has provided valuable insights into the cellular metabolism.…”

“…In the present work, a novel approach comprising 13 C tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was applied to determine the entire carbon flux distribution in the central metabolism of lysine producing C. glutamicum during batch culture. MALDI-TOF MS was hereby applied for labeling measurements of the extracellular products lysine, alanine and trehalose.…”