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.     

Metabolic response to [13C]glucose and [13C]fructose ingestion during exercise

Seven healthy male volunteers exercised on a cycle ergometer at 50 +/- 5% VO2max for 180 min, on three occasions during which they ingested either water only (W), [13C]glucose (G), or [13C]fructose (F) (140 +/- 12 g, diluted at 7% in water, and evenly distributed over the exercise period). Blood glucose concentration (in mM) significantly decreased during exercise with W (5.1 +/- 0.4 to 4.2 +/- 0.1) but remained stable with G (5.0 +/- 0.4 to 5.3 +/- 0.6) or F ingestion (5.4 +/- 0.5 to 5.1 +/- 0.4). Decreases in plasma insulin concentration (microU/ml) were greater (P less than 0.05) with W (11 +/- 3 to 3 +/- 1) and F (12 +/- 4 to 5 +/- 1) than with G ingestion (11 +/- 2 to 9 +/- 5), and fat utilization was greater with F (103 +/- 11 g) than with G ingestion (82 +/- 9 g) and lower than with W ingestion (132 +/- 14 g). However F was less readily available for combustion than G; over the 3-h period 75% (106 +/- 11 g) of ingested G was oxidized, compared with 56% (79 +/- 8 g) of ingested fructose. As a consequence, carbohydrate store utilizations were similar in the two conditions (G, 174 +/- 20 g; F, 173 +/- 17 g; vs. W, 193 +/- 22 g). These observations suggest that, during prolonged moderate exercise, F ingestion maintains blood glucose as well as G ingestion, and increases fat utilization when compared to G ingestion. However, due to a slower rate of utilization of F, carbohydrate store sparing is similar with G and F ingestions.     

Oxidation of an oral [13C]glucose load at rest and prolonged exercise in trained and sedentary subjects

The purpose of this study was to compare the oxidation of[¹³C]glucose (100 g)ingested at rest or during exercise in six trained (TS) and sixsedentary (SS) male subjects. The oxidation of plasma glucose was alsocomputed from the volume of¹³CO₂and¹³C/¹²Cin plasma glucose to compute the oxidation rate of glucose released from the liver and from glycogen stores in periphery (mainly muscle glycogen stores during exercise). At rest, oxidative disposal of bothexogenous (8.3 ± 0.3 vs. 6.6 ± 0.8 g/h) and liver glucose (4.4 ± 0.5 vs. 2.6 ± 0.4 g/h) was higher in TS than in SS.This could contribute to the better glucose tolerance observed at rest in TS. During exercise, for the same absolute workload [140 ± 5 W: TS = 47 ± 2.5; SS = 68 ± 3 %maximal oxygen uptake(O_{2 max})], [¹³C]glucose oxidationwas higher in TS than in SS (39.0 ± 2.6 vs. 33.6 ± 1.2 g/h),whereas both liver glucose (16.8 ± 2.4 vs. 24.0 ± 1.8 g/h) and muscle glycogen oxidation (36.0 ± 3.0 vs. 51.0 ± 5.4 g/h) were lower. For the same relative workload (68 ± 3% O_{2 max}:TS = 3.13 ± 0.96; SS = 2.34 ± 0.60 lO₂/min), exogenous glucose(44.4 ± 1.8 vs. 33.6 ± 1.2 g/h) and muscle glycogen oxidation (73.8 ± 7.2 vs. 51.0 ± 5.4 g/h) were higher in TS. However,despite a higher energy expenditure in TS, liver glucose oxidation was similar in both groups (22.2 ± 3.0 vs. 24.0 ± 1.8 g/h). Thus exogenous glucose oxidation was selectively favored in TSduring exercise, reducing both liver glucose and muscle glycogen oxidation.

     

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.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

Muscle glycogen oxidation during prolonged exercise measured with oral [13C]glucose: comparison with changes in muscle glycogen content.

Plasma glucose and muscle glycogen oxidation during prolonged exercise [75-min at 48 and 76% maximal O(2) uptake (Vo(2 max))] were measured in eight well-trained male subjects [Vo(2 max) = 4.50 l/min (SD 0.63)] using a simplified tracer technique in which a small amount of glucose highly enriched in (13)C was ingested: plasma glucose oxidation was computed from (13)C/(12)C in plasma glucose (which was stable beginning at minute 30 and minute 15 during exercise at 48 and 76% Vo(2 max), respectively) and (13)CO(2) production, and muscle glycogen oxidation was estimated by subtracting plasma glucose oxidation from total carbohydrate oxidation. Consistent data from the literature suggest that this small dose of exogenous glucose does not modify muscle glycogen oxidation and has little effect, if any, on plasma glucose oxidation. The percent contributions of plasma glucose and muscle glycogen oxidation to the energy yield at 48% Vo(2 max) [15.1% (SD 3.8) and 45.9% (SD 5.8)] and at 76% Vo(2 max) [15.4% (SD 3.6) and 59.8% (SD 9.2)] were well in line with data previously reported for similar work loads and exercise durations using conventional tracer techniques. The significant reduction in glycogen concentration measured from pre- and postexercise vastus lateralis muscle biopsies paralleled muscle glycogen oxidation calculated using the tracer technique and was larger at 76% than at 48% Vo(2 max). However, the correlation coefficients between these two estimates of muscle glycogen utilization were not different from zero at each of the two work loads. The simplified tracer technique used in the present experiment appears to be a valid alternative approach to the traditional tracer techniques for computing plasma glucose and muscle glycogen oxidation during prolonged exercise.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

The biosynthesis of trichothecin from acetate- [1,2- 13C2]

The coupling pattern of trichothecin biosynthesized from acetate-[1,2-¹³C₂] is in accord with previous enrichment studies. Multiple labelling was observed. Exogenous acetate has been shown to inhibit the utilization of glucose and the incorporation of radioactivity from pyruvate-[2-¹⁴C] and citrate-[1,5-¹⁴C] into the metabolites. Two pairs of ¹³C NMR assignments are interchanged.     

Retention of intravenously infused [13C]bicarbonate is transiently increased during recovery from hard exercise.

The effects of exercise on energy substrate metabolism persist into the postexercise recovery period. We sought to derive bicarbonate retention factors (k) to correct for carbon tracer oxidized, but retained from pulmonary excretion before, during, and after exercise. Ten men and nine women received a primed-continuous infusion of [(13)C]bicarbonate (sodium salt) under three different conditions: 1) before, during, and 3 h after 90 min of exercise at 45% peak oxygen consumption (Vo(2peak)); 2) before, during, and 3 h after 60 min of exercise at 65% Vo(2peak); and 3) during a time-matched resting control trial, with breath samples collected for determination of (13)CO(2) excretion rates. Throughout the resting control trial, k was stable and averaged 0.83 in men and women. During exercise, average k in men was 0.93 at 45% Vo(2peak) and 0.94 at 65% Vo(2peak), and in women k was 0.91 at 45% Vo(2peak) and 0.92 at 65% Vo(2peak), with no significant differences between intensities or sexes. After exercise at 45% Vo(2peak), k returned rapidly to control values in men and women, but following exercise at 65% Vo(2peak), k was significantly less than control at 30 and 60 min postexercise in men (0.74 and 0.72, respectively, P < 0.05) and women (0.75 and 0.76, respectively, P < 0.05) with no significant postexercise differences between men and women. We conclude that bicarbonate/CO(2) retention is transiently increased in men and women for the first hour of postexercise recovery following endurance exercise bouts of hard but not moderate intensity.     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

Oral [13C]glucose oxidation during prolonged exercise after high- and low-carbohydrate diets

The effect of a diet either high or low in carbohydrates (CHO)on exogenous ¹³C-labeled glucoseoxidation (200 g) during exercise (ergocycle: 120 min at 64.0 ± 0.5% maximal oxygen uptake) was studied in six subjects. Between 40 and 80 min, exogenous glucose oxidation was significantly higher afterthe diet low in CHO (0.63 ± 0.05 vs. 0.52 ± 0.04 g/min), butthis difference disappeared between 80 and 120 min (0.71 ± 0.03 vs.0.69 ± 0.04 g/min). The oxidation rate of plasma glucose, computedfrom the volume of¹³CO₂produced the¹³C-to-¹²Cratio in plasma glucose at 80 min, and of glucose released from theliver, computed from the difference between plasma glucose andexogenous glucose oxidation, was higher after the diet low in CHO (1.68 ± 0.26 vs. 1.41 ± 0.17 and 1.02 ± 0.20 vs. 0.81 ± 0.14 g/min, respectively). In contrast the oxidation rate ofglucose plus lactate from muscle glycogen (computed from the difference between total CHO oxidation and plasma glucose oxidation) was lower(0.31 ± 0.35 vs. 1.59 ± 0.20 g/min). After a diet low in CHO,the oxidation of exogenous glucose and of glucose released from theliver is increased and partly compensates for the reduction in muscleglycogen availability and oxidation.

     

Incorporation and utilization of [3-13C]lactate and [1,2-13C]acetate by rat skeletal muscle.

Skeletal muscle can utilize many different substrates, and traditional methodologies allow only indirect discrimination between oxidative and nonoxidative uptake of substrate, possibly with contamination by metabolism of other internal organs. Our goal was to apply 1H- and 13C-nuclear magnetic resonance spectroscopy to monitor the patterns of [3-13C]lactate and [1,2-13C]acetate (model of simple carbohydrates and fats, respectively) utilization in resting vs. contracting muscle extracts of the isolated perfused rat hindquarter. Total metabolite concentrations were measured by using NADH-linked fluorometric assays. Fractional oxidation of [3-13C]lactate was unchanged by contraction despite vascular endogenous lactate accumulation. Although label accumulated in several citric acid cycle (CAC) intermediates, contraction did not increase the concentration of CAC intermediates in any muscle extracts. We conclude that 1) the isolated rat hindquarter is a viable, well-controlled model for measuring skeletal muscle 13C-labeled substrate utilization; 2) lactate is readily oxidized even during contractile activity; 3) entry and exit from the CAC, via oxidative and nonoxidative pathways, is a component of normal muscle metabolism and function; and 4) there are possible differences between gastrocnemius and soleus muscles in utilization of nonoxidative pathways.     

Respiratory metabolism of D[U-14C]glucose in hens and cocks during prolonged treadmill exercise

1. The specific activity of expired 14CO2 was measured at rest and during 90 min treadmill exercise following an initial intravenous injection of D[U-14C]glucose. 2. The rate of CO2 production rose 4.5-fold during exercise in cocks but only 2.5-fold in females. The mean respiratory quotient was close to unity at rest and during exercise. 3. Estimated glucose turnover rate rose approximately 3.5-fold during exercise in cocks. Turnover rate did not increase in hens but the fraction of the glucose turnover oxidized to provide energy for the working muscles was increased. 4. It is concluded that carbohydrate sources account for the major fraction of energy expenditure during exercise of this magnitude and duration.     

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.