Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers

Abstract

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.

Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13 C magnetic resonance

“…These 13 C spectra were acquired at 14.1 T using a Agilent VNMRS system (Agilent Inc.) using a 45°pulse and a 2.5-s delay time between pulses. Relative peak areas of each glutamate multiplet were obtained by line fitting in ACD software (Advanced Chemistry Development) and the multiplets were input into the tcaCALC software available through University of Texas Southwestern (17).…”

“…To determine the relative contribution of PDH and pyruvate carboxylase pathways to the incorporation of pyruvate carbons into the TCA cycle, the source of the [ 13 C] bicarbonate signal was evaluated using classic steady-state 13 C isotopomer analysis (16). Livers from fed animals were perfused to steady state with [U- 13 (17) indicated the ratio of pyruvate carboxylase/PDH flux was 7.6 ± 2.0, demonstrating that the great majority of pyruvate entry into the TCA cycle was via pyruvate carboxylase rather than PDH. A small flux of [U-13 C]pyruvate through PDH was detected; the fraction of the acetyl-CoA pool derived from [U- 13 C]pyruvate ([1,2-13 C]acetyl CoA) was 7 ± 3%.…”

13C NMR Isotopomer Analysis of Anaplerotic Pathways in INS-1 Cells

“…Steady-state flux rates, relative to Kreb's cycle flux, were calculated from the isotopomers of glutamate observed at carbon positions 2, 3, and 4 in the 13 C NMR spectra, and modeled using the program "tcacalc" (15).…”

“…Using the nomenclature adopted by the authors for tcacalc, the model was used to provide the best fit to our NMR data by calculating the relative fluxes of ACS, lactate dehydrogenase, PDH, PC, pyruvate kinase (PK), anaplerosis leading to succinyl-CoA (YS), and enrichments of "lactate" and "fat" (15). Fat includes all metabolite inputs into acetylCoA other than glucose (e.g.…”

Direct observation of lactate and alanine by proton double quantum spectroscopy in rat hearts supplied with [3‐13C]pyruvate

“…The results are pre-.~ented as [~ ~C]lactate/total lactate and [~SC]alanino/total alanine ratJc).~ in Table I. High resolution ~sC NMR spectra were also obtained on thes¢ same extract sampies and the glutamate multiplets were dcconvoluted and that data applied to a steady-state isotopomer analysis [13]. The fractional contribution made by [3J~C]pyruvate to the acetyl-CoA which entered the TCA cycle in each heart is also tabulated.…”