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

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.     

Dynamic aspects of radiorespirometry during the cell cycle of Candida utilis: Some observations in phased culture under carbon-, nitrogen-, and phosphorus-limited growth

Many changes that occur in a cell during the cell cycle can be demonstrated in synchronous cultures and can reveal dimensions of cell metabolism not attainable by the study of balanced growth of asynchronous populations in batch cultures or the steady state in chemostat cultures. The release of ¹⁴CO₂ from specifically labeled glucose by phased (continuously synchronized) cultures follows a characteristic pattern (profile) that depends upon the stage in the cell cycle and the period of labeling used. Successive profiles throughout a cycle showed differences that were altered under different nutrient-limiting growth conditions. Profiles obtained with glucose-1-¹⁴C, glucose-2-¹⁴C, glucose-3,4-¹⁴C, and glucose-6-¹⁴C and phased cells of Candida utilis under N-, P-, and C-limited growth demonstrated the variable character of the metabolic activity that occurred in the cells while contour changes within the profiles across the cycle indicated possible correlations with activities of the hexose monophosphate, Embden-Meyerhof-Parnas, and tricarboxylic acid cycle pathways during the cell cycle. The basis of these changes and their use as elementary parameters for study of problems of physiological changes in vivo are considered.     

Glucose metabolism in proliferating epithelial cells from the rat colon.

1. The effects of fasting and fasting followed by refeeding on the activities of the oxidative pentose pathway (OPP) and the tricarboxylic acid cycle (TCA) in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]glucose and [6-14C]glucose, respectively. 2. Refeeding after a fast induced a 2-3-fold increase in glucose flux through the OPP and TCA cycle and the degree of change was similar in colonocytes from the proximal and distal colon. 3. Butyrate at a concentration of 40 mM inhibited the OPP by 20-30% (P less than 0.05) but had no effect on the activity of the TCA cycle. Glutamine at a concentration of 2 mM decreased the glucose flux through both the OPP and the TCA cycle by 30-50% (P less than 0.05). 4. Production of 14CO2 from the oxidation of butyrate or glucose indicated that the former was 5-7 times more active in colonocytes from fasted rats. After refeeding, however, butyrate utilization was similar to fasting values in the proximal colon but significantly lower (P less than 0.05) in the distal colon.     

Changes in pattern of respiration and glucose utilisation in Candida utilis during the cell cycle: some variations with growth rate.

The release of 14CO2 from 14C-labelled glucose(G-1(-14)C, G-3,4(-14)C, G-6(-14)C) was followed in phased cultures of Candida utilis grown in a glucose- mineral salts medium under altered conditions of carbon:nitrogen limitation at doubling times of 2,4 and 6, h. Changes in oxygen uptake and CO2 evolution were observed and respirometric studies showed that the relative contributions of the Embden-Meyerhof-Parnas and hexose monophosphate pathways varied over the cell cycle and changed with growth rate. The results are discussed in relation to the growth metabolism of the cells.     

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.     

Carbon dioxide production by red skeletal muscle of goldfish (Carassius auratus L.) aerobic and anaerobic metabolism of glucose and glutamate

Oxygen uptake and metabolic CO2 production by lateral red muscle of goldfish have been measured in vitro. Added glucose 6-phosphate depresses the rate of oxygen uptake by minced red muscle (Crabtree effect). Total CO2 production is stimulated resulting in a respiratory quotient which is considerably greater than one. 14CO2 release from [U-14C] glucose 6-phosphate and [U-14C] glutamate continues during anoxia. No activity of the hexose monophosphate shunt was observed. The results suggest that both aerobic and anaerobic CO2 production is of mitochondrial origin and, at least partially, derived from TCA cycle reactions.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hexose metabolism in pancreatic islets: unequal oxidation of the two carbons of glucose-derived acetyl residues.

The fate of the C1 and C2 of glucose-derived acetyl residues was examined in rat pancreatic islets. The production of 14CO2 from D-[2-14C]glucose exceeded that from D-[6-14C]glucose, in the same manner as the oxidation of [1-14C]acetate exceeded that of [2-14C]acetate. The difference in 14CO2 output from D-[2-14C]glucose and D-[6-14C]glucose was matched by complementary differences in the generation of 14C-labeled acidic metabolites and amino acids. Even the production of 14C-labeled L-lactate was somewhat higher in the case of D-[6-14C]glucose than D-[2-14C]glucose. The ratio between D-[2-14C]glucose and D-[6-14C]glucose oxidation progressively decreased at increasing concentrations of the hexose (2.8, 7.0, and 16.7 mM), was higher after 30 than 120 min incubation, and was decreased in the presence of a nonmetabolized analogue of L-leucine. These findings are consistent with the view that the difference between D-[6-14C]glucose and D-[2-14C]glucose oxidation is mainly attributable to the inflow into the Krebs cycle of unlabeled metabolites generated from endogenous nutrients, this being compensated by the exit of partially labeled metabolites from the same cycle. The present results also indicate that the oxidation of glucose-derived acetyl residues relative to their generation in the reaction catalyzed by pyruvate dehydrogenase is higher than that estimated from the ratio between D-[6-14C]glucose and D-[3,4-14C]glucose conversion to 14CO2.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Negligible glucose-6-phosphatase activity in cultured astroglia

2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG-6-P) dephosphorylation and glucose-6-phosphatase (G-6-Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG-6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 microM 2-deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2-[14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2-[14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carried out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U-14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.     

Pyruvate sparing by butyrate and propionate in proliferating colonic epithelium

1. The effects of fasting and fasting followed by refeeding on the relative activities of the pyruvate dehydrogenase (PDH) complex and the tricarboxylic acid (TCA) cycle in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]pyruvate and [3-14C]pyruvate, respectively. 2. Decarboxylation of pyruvate by the PDH complex exceeded that by the TCA cycle in both fasted and fasted/refed colonocytes, was higher in distal than in proximal colon, and was stimulated by refeeding following a fast. 3. Oxidation of pyruvate by both the PDH complex and the TCA cycle was inhibited by butyrate. 4. Propionate alone had no effect, but synergized with butyrate to further reduce pyruvate decarboxylation by the TCA cycle. 5. Preferential utilization of butyrate by proliferating colonic epithelial cells is postulated to maximize the energy yield and spare pyruvate and its precursors for alternative synthetic roles necessary for active cell division.     

Galactose and glucose metabolism in galactokinase deficient, galactose-1-P-uridyl transferase deficient and normal human fibroblasts.

Despite the genetic interruption of the Leloir pathway both galactosemic patients and galactosemic fibroblasts can convert galactose to CO2 and TCA precipitable products, although at less than the normal rate. These observations stimulated investigations into the identity of the alternative metabolic routes which allows for galactose metabolism in the absence of in vitro galactose-1-P-uridyl transferase. Four lines of galactosemic cells, each without detectable gal-transferase, produced 14CO2 from [1-14C]-galactose (0.094 mumoles in 20 cc of medium) at approximately 39% +/- 16% the rate of transferase positive cells over a 48-hour period. However, galactokinase deficient fibroblasts produced 14CO2 and TCA precipitable products from [1-14C]-galactose or [U-14C]-galactose at only 3% to 9% the rate of normal fibroblasts. Therefore it seems likely that gal-transferase deficient fibroblasts must first synthesize galactose-1-P for further metabolism of galactose.     

Anomeric specificity of glucose metabolism in the pentose cycle

The production of 3H2O from alpha- and beta-D-[5-3H]glucose and that of 14CO2 from either alpha- and beta-D-[1-14C] or alpha- and beta-D-[6-14C]glucose were measured in rat pancreatic islets and tumoral insulin-producing cells incubated at 7 degrees C. The ratio in 14CO2 output from D-[1-14C]glucose/D-[6-14C]glucose, the fraction of glucose metabolism occurring through the pentose cycle, and the flow rate through such a cycle were always higher in the presence of beta- than alpha-D-glucose. This indicates that the anomeric specificity of glucose-6-phosphate dehydrogenase is operative in intact islet cells.     

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.     

Elucidating the key member of a linuron-mineralizing bacterial community by PCR and reverse transcription-PCR denaturing gradient gel electrophoresis 16S rRNA gene fingerprinting and cultivation

A bacterial community from Danish agricultural soil was enriched with linuron [N-(3,4-dichlorophenyl)-N'-methoxy-N'-methylurea] as the sole carbon and nitrogen source. The community mineralized [ring-U-14C]linuron completely to 14CO2 and 14C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a Variovorax sp. was responsible for the mineralization activity.     

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-¹⁴C]-glucose,[u-¹⁴C]G-1-P, [u-¹⁴C]G-6-P, [2-¹⁴C]pyruvate and [1-¹⁴C]acetate.Little was incorporated from [1-¹⁴C]pyruvate into LFAs, butmuch into ¹⁴CO_a. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO₂ output and LFAsynthesis from [1-¹⁴C]glucose, [6-¹⁴C]glucose and [u-¹⁴C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )     

Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species.

1. The patterns of 14CO2 evolution from specifically labeled glucose substrates by washed bull, ram, boar, rabbit, dog, rooster and turkey spermatozoa were similar and indicated the Embden-Meyerhof and Kreb's cycle pathways as the major route of energy metabolism. 2. Honey bee spermatozoa metabolized glucose-3,4-[14C], glucose-[U-14C] or fructose-[U-14C], but not glucose-1-[14C], glucose-2-[14C]or glucose-6-[14C], indicating the presence of the glycolytic pathway, but the absence of respiration via the Kreb's cycle. 3. The rate of glycolysis exceeded the rate of respiration in the spermatozoa of all the species studied. 4. A preferential utilization of glucose-1-[14C] over glucose-6-[14C] was evident in some sperm samples, but no consistent indication of pentose cycle metabolism was observed, due to considerable variability between samples within each group. 5. Fructose metabolism was greater than glucose metabolism in the rooster, less in the dog, boar and turkey, and similar in the spermatozoa from the other species examined. 6. Only ram and bull spermatozoa metabolized acetate-1-[14C] to any extent.     

Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose.

The purpose of the present experiment was to compare 13CO2 recovery at the mouth, and the corresponding exogenous glucose oxidation computed, during a 100-min exercise at 63 +/- 3% maximal O2 uptake with ingestion of glucose (1.75 g/kg) in six active male subjects, by use of [U-13C] and [1,2-13C]glucose. We hypothesized that 13C recovery and exogenous glucose oxidation could be lower with [1,2-13C] than [U-13C]glucose because both tracers provide [13C]acetate, with possible loss of 13C in the tricarboxylic acid (TCA) cycle, but decarboxylation of pyruvate from [U-13C]glucose also provides 13CO2, which is entirely recovered at the mouth during exercise. The recovery of 13C (25.8 +/- 2.3 and 27.4 +/- 1.2% over the exercise period) and the amounts of exogenous glucose oxidized computed were not significantly different with [1,2-13C] and [U-13C]glucose (28.9 +/- 2.6 and 30.7 +/- 1.3 g, between minutes 40 and 100), suggesting that no significant loss of 13C occurred in the TCA cycle. This stems from the fact that, during exercise, the rate of exogenous glucose oxidation is probably much larger than the flux of the metabolic pathways fueled from TCA cycle intermediates. It is thus unlikely that a significant portion of the 13C entering the TCA cycle could be diverted to these pathways. From a methodological standpoint, this result indicates that when a large amount of [13C]glucose is ingested and oxidized during exercise, 13CO2 production at the mouth accurately reflects the rate of glucose entry in the TCA cycle and that no correction factor is needed to compute the oxidative flux of exogenous glucose.     

Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.