Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.     

Parallel labeling experiments with [U-(13)C]glucose validate E. coli metabolic network model for (13)C metabolic flux analysis

(13)C-metabolic flux analysis (MFA) is a widely used method for measuring intracellular metabolic fluxes in living cells. (13)C MFA relies on several key assumptions: (1) the assumed metabolic network model is complete, in that it accounts for all significant enzymatic and transport reactions; (2) (13)C-labeling measurements are accurate and precise; and (3) enzymes and transporters do not discriminate between (12)C- and (13)C-labeled metabolites. In this study, we tested these inherent assumptions of (13)C MFA for wild-type E. coli by parallel labeling experiments with [U-(13)C]glucose as tracer. Cells were grown in six parallel cultures in custom-constructed mini-bioreactors, starting from the same inoculum, on medium containing different mixtures of natural glucose and fully labeled [U-(13)C]glucose, ranging from 0% to 100% [U-(13)C]glucose. Macroscopic growth characteristics of E. coli showed no observable kinetic isotope effect. The cells grew equally well on natural glucose, 100% [U-(13)C]glucose, and mixtures thereof. (13)C MFA was then used to determine intracellular metabolic fluxes for several metabolic network models: an initial network model from literature; and extended network models that accounted for potential dilution effects of isotopic labeling. The initial network model did not give statistically acceptable fits and produced inconsistent flux results for the parallel labeling experiments. In contrast, an extended network model that accounted for dilution of intracellular CO(2) by exchange with extracellular CO(2) produced statistically acceptable fits, and the estimated metabolic fluxes were consistent for the parallel cultures. This study illustrates the importance of model validation for (13)C MFA. We show that an incomplete network model can produce statistically unacceptable fits, as determined by a chi-square test for goodness-of-fit, and return biased metabolic fluxes. The validated metabolic network model for E. coli from this study can be used in future investigations for unbiased metabolic flux measurements.     

Use of [2-14C]glucose and [5-14C]glucose for evaluating the mechanism and quantitative significance of the 'liver-cell' pentose cycle.

1. Expressions are derived for the steady-state measurement of the quantitative contribution of the liver-type pentose phosphate cycle to glucose metabolism by tissues. One method requires the metabolism of [5-14C]glucose followed by the isolation and degradation of glucose 6-phosphate. The second procedure involves the metabolism of [2-14C]glucose and the isolation and degradation of a triose phosphate derivative, usually lactate or glycerol. 2. Measurements of 14C in C-2 and C-5 of glucose 6-phosphate are required and the values of the C-2/C-5 ratios can be used to calculate the quantitative contribution of the L-type pentose cycle in all tissues. 3. The measurement of 14C in C-1, C-2 and C-3 of triose phosphate derivatives can be used to calculate the quantitative contribution of the L-type pentose cycle relative to glycolysis. 4. The effect of transaldolase and transketolase exchange reactions, reactions of gluconeogenesis and non-oxidative formation of pentose 5-phosphate, isotopic equilibration of triose phosphate pools and isotopic equilibration of fructose 6-phosphate and glucose 6-phosphate, which could interfere with a clear interpretation of the data using [2-14C]- and [5-14C]glucose are discussed.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

Measurement of exogenous carbohydrate oxidation: a comparison of [U-14C]glucose and [U-13C]glucose tracers

The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHO(EXO)). To accomplish this, seven healthy male subjects (24 +/- 3 yr, 74.8 +/- 2.1 kg, V(O2(max)) 62 +/- 4 ml x kg(-1) x min(-1)) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHO(EXO) to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO(2) in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHO(EXO) measured with [13C]glucose ([13C]CHO(EXO)) was significantly greater than CHO(EXO) measured with [14C]glucose ([14C]CHO(EXO)) from 30 to 120 min. There was a 15 +/- 4% difference between [13C]CHO(EXO) and [14C]CHO(EXO) such that the absolute difference increased with the magnitude of CHO(EXO). Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the "strength" of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285-294, 1977). Therefore, the explanation for the discrepancy in calculated CHO(EXO) remains to be fully understood.     

Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.     

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.     

Substrate cycles in the central metabolism of maize root tips under hypoxia

Substrate cycles, also called "futile" cycles, are ubiquitous and lead to a net consumption of ATP which, in the normoxic maize root, have been estimated at about 50% of the total ATP produced [Alonso, A.P., Vigeolas, H., Raymond, P., Rolin, D., Dieuaide-Noubhani, M., 2005. A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [(13)C] glucose and [(14)C] glucose. Plant Physiol. 138, 2220-2232]. To evaluate their role we studied the substrate cycles of maize root tips under an oxygen limitation of respiration (3% O(2)). Short-time labeling experiments with [U-(14)C]-Glc were performed to quantify the fluxes through sucrose and starch cycles of synthesis and degradation. Steady-state labeling with [1-(13)C]-Glc followed by (1)H NMR and (13)C NMR analysis of sugars and free alanine was used to quantify fluxes in the central metabolic pathways, including the Glc-P/Glc cycle and the fructose-P/triose-P cycle of glycolysis. Comparison with results previously obtained in normoxia [Alonso et al., as mentioned above] showed that 3% O(2) induced fermentation and reduced respiration, which led to a lesser amount of ATP produced. The rates of Glc consumption, glycolytic flux and all substrate cycles were lower, but the proportion of ATP consumed in the substrate cycles remained unchanged. These findings suggest that substrate cycles are not a luxury but an integral part of the organization of the plant central metabolism.     

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-¹³C]glucose (2.5 mM) and lactate (1 mM) or [U-¹³C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-¹³C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. ¹³C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-¹³C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-¹³C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-¹³C]glucose and [U-¹³C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-¹³C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized. Special issue dedicated to John P. Blass.     

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants

Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C-13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

In vitro oxidation of [U-14C] palmitate, [1-14C] and [6-14C] glucose in newborn and adult rats and pigs

Studies have been made on the intensity of oxidation of [U-14C]-palmitate, [1-14C]- and [6-14C]-glucose by slices of the liver and skeletal muscles of new-born, 1-day, 5-day and adult Wistar rats and domestic pigs. It was found that the level of 14CO2 production from these substrates is higher in tissues of rats than in those of pigs. At early stages of ontogenesis, in tissues of both species intensive oxidation of glucose is observed together with oxidation of fatty acids. In the course of ontogenetic development, the intensity of glucose utilization significantly decreases, whereas the level of fatty acid catabolism remains relatively unaffected.     

Biochemical evidence for a secretory role of the pineal body. Oxidation of [1-14C]- and [6-14C]glucose in vitro by pineal body, pituitary, and brain from young and adult animals

The conversion of [1-¹⁴C] label from glucose to ¹⁴CO₂in vitro by bovine pineal bodies was 7-24 times as great as that of [6-¹⁴C]. These values for C-1/C-6 oxidation ratios are similar to those found for all known endocrine tissues and in contrast to those for brain which range from 1.0 to 1.4. Total glucose oxidation, both C-1 and C-6, and C-1/C-6 ratios were lower in pineal bodies from adult (3-8 years) than from young (5-10 months) animals. Total glucose oxidation by the posterior pituitary was lower in the adult than in the young, generally lower in the anterior pituitary of the adult, and higher in the brain of the adult. Epinephrine, 10^?4m , increased the oxidation by pineal tissue of [1-¹⁴C] by 170 per cent and of [6-¹⁴C] by 46 per cent. The relatively high C-1/C-6 ratios found for pineal tissue are indicative of an operative hexosemonophosphate pathway, which we have previously suggested to be correlated with hormone secretion and/or storage. The present findings provide biochemical support for the hypothesis that the pineal body has an endocrine function in mammals.     

Metabolic interactions between glucose, glycerol, alanine and acetate in Leishmania braziliensis panamensis promastigotes

13C-nuclear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeled substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-1/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [1,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of [14C]glucose from UDP[14C]glucose into a phosphoglycolipid by cell-free particulate systems of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423.

A glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme preparation from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP[U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol.     

Glucose isotope, carbon recycling, and gluconeogenesis using [U-13C]glucose and mass isotopomer analysis

Experimental determinations of glucose carbon recycling using 14C or 13C glucose tracer often underestimate true Cori cycle activity because of dilution and exchange of isotope tracer through the tricarboxylic acid (TCA) cycle. The term glucose isotope recycling therefore is used to distinguish recycling of isotope from recycling of glucose carbon, the actual quantity of circulating glucose recycled. Recently, per-labeled glucose ([U-13C6]glucose) has been used to estimate glucose appearance rate and glucose isotope recycling. Chemical structural information determined by mass isotopomer analysis has been used to correct for dilution of isotope through the TCA cycle. In this report, we present experiments in the study of glucose turnover and recycling using [U-13C6]glucose. Methods of single injection and continuous infusion of [U-13C6]glucose are compared. A formula for the calculation of a dilution factor using TCA cycle parameters is derived. In this study of six rabbits, glucose turnover rate ranged from 3.4 to 8.8 mg/kg/min, and glucose m + 3 mass isotopomer recycling from 7 to 12%. The rate of pyruvate carboxylation (Y) was comparable to that of citrate synthetase, having an average relative flux of 0.89. Applying the correction factor for tracer dilution to the observed mass isotopomer recycling, we determined glucose carbon recycling (or Cori cycle activity) to be 22-35% of hepatic glucose output.     

Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.     

The distribution of glucose and [14C]glucose between erythrocytes and plasma in the rat

1. Of the glucose in rat blood 79.8+/-3.3% (s.d.) was in the plasma. The variance was mostly due to differences between rats. 2. The concentration of glucose in erythrocyte water was 51+/-8% (s.d.) of that in plasma water. 3. The ratio (specific radioactivity in plasma)/(specific radioactivity in whole blood), i.e. the P/B ratio, was estimated for glucose at intervals after intravenous injection of [U-(14)C]glucose and [U-(14)C]fructose. The ratio differed from unity by more than the standard error of a single determination of the specific radioactivity of blood or plasma glucose except from 10 to 17min. after injection of [(14)C]glucose and from 22 to 30min. after injection of [(14)C]fructose. At all other times specific radioactivities in blood had to be corrected to give specific radioactivities in plasma. How to do so is described. 4. The P/B ratios were accounted for by a turnover of glucose in erythrocytes of 0.14mumole/min./ml. of erythrocytes. 5. Metabolism of glucose in rat erythrocytes is unlikely to be a major source of lactate.