Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios

Abstract

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.

Oxidation of glutamine in HeLa cells: Role and control of truncated TCA cycles in tumour mitochondria

“…These figures reflect the decline in relative specific activity, and the fact that the glutamine plus glutamate pools represent 63% of the total free amino acids (Table I). The decline in relative specific activity from glutamine to aspartate is to be expected and was very similar to that obtained when AS-30D hepatoma cells were incubated with [2,3-14 C]succinate [Kelleher et al, 1987]. In the latter experiments the HClO 4 -soluble cell fraction on addition to an anionexchange column gave rise to a succinate peak with 83% of the total radioactivity followed by malate, aspartate and alanine in descending order.…”

“…We propose that this arises from a modification of the truncated TCA cycle, in that alanine aminotransferase replaces aspartate aminotransferase as the truncating element and malic enzyme replaces malate dehydrogenase. This proposition is based on a study using AS-30D hepatoma cells, where [ 14 C]succinate was converted to [ 14 C]alanine [Kelleher et al, 1987].…”

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

“…Assuming that lactate, pyruvate, and alanine are all in labeling equilibrium (i.e., P3 = L3 = AL3) and can be treated as one pool, a label balance around the entire pool yields fsPE3 + f9M3 + fi3M3 = ( f l o + f l l + f12 + f27)L3 (22) A label balance around the glycolytic intermediates, accounting for the loss of label through the pentose phosphate shunt and setting GI = 1 (since the initial labeling is loo%),…”

Metabolic Fluxes and Metabolic Engineering

“…Isotopic labels have been used extensively in biochemistry for the elucidation of metabolic pathways (see, for example, Chance et al, 1983;Kelleher, 1985;Kelleher et al, 1987;Malloy et al, 1987Malloy et al, , 1988Malloy et al, , 1990). Here we focus on the systematic use of isotopic labels for the confirmation of flux estimates obtained by material balancing, as well as the extraction of additional information about pathway fluxes.…”