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.     

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.     

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.     

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.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

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.     

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.     

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.     

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.     

Metabolism of [1-(13)C)glucose and [2-(13)C]acetate in the hypoxic rat brain.

The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.     

Brain cholesterol XVIII: EFFECt of methylphenidate (Ritalin) on [U-14C] glucose and [2-3H] acetate incorporation.

The effect of a single injection of methylphenidate (Ritalin, 4 mg/kg) on precursor ([2-3H]acetate and [U-14C]glucose) incorporation into brain cholesterol was studied. The drug caused a steady decrease in the concentration of brain cholesterol during the 24-hr period examined. Incorporation studies during this time with [U-14C]glucose indicated higher than normal incorporation for all time periods studied. The most significant incorporation increases took place 2 and 4 hr after drug injection. Experiments using [2-3H]acetate as the sterol precursor gave incorporation values which tended (not significantly) to be lower than control values at 2 and 4 h. The values after 12 hr were less than normal, while the 24-hr group indicated an increase to or slightly higher than normal values. These data suggest that the pharmacological effect of methylphenidate may be due to lowering of brain cholesterol levels directly or on some more basic metabolic process leading to a decreased level of membrane sterols.     

Metabolism of [U-14C, 2-3H] glucose in rat liver during non-recirculating perfusion: effects of insulin

Livers from fed male rats were perfused in a non-recirculating manner with undiluted blood containing either 6 or 13 mM [U-14C,2-3H] glucose. At the lower concentration there was a small output of glucose which was unaffected by insulin whereas at the high concentration there was a substantial uptake of glucose which was significantly increased by the hormone. The rate of metabolism of [2-3H] glucose was greater than that of [U-14C] glucose in all experiments indicating an active substrate cycle between glucose:glucose 6-phosphate. Cycling was unaffected by insulin at the lower glucose concentration but was increased by perfusion with 13 mM glucose, the latter increase being abolished by insulin. These data show that although the perfused liver acts to autoregulate blood glucose, this is not achieved solely at the substrate cycle glucose:glucose 6-phosphate.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

Hexose monophosphate shunt measurement in cultured cells with [1-13C]glucose: correction for endogenous carbon sources using [6-13C] glucose

Hexose monophosphate shunt (HMPS) activity can be measured with 1H nuclear magnetic resonance spectroscopy or gas chromatography--mass spectrometry by monitoring the differential production of [3-13C]lactate and [3-12C]lactate from the degradation of [1-13C]-glucose. Errors in measurement of HMPS activity can arise from unlabeled lactate precursors, by recycling of HMPS products, and by incomplete fractional enrichment of labeled glucose. A method utilizing cultured cells incubated with [1-13C]glucose in parallel with incubations using [6-13C]glucose to correct for all these problems is presented. In cultured rat C6 glioma and 9L gliosarcoma cells, failure to apply this correction results in an approximately twofold overestimation of HMPS activity.     

Mathematical modelling for the generation of L-[3-2H,3-13C]lactic acid isotopomers by erythrocytes exposed to either D-[1-13C]glucose or D-[6-13C]glucose in the presence of2H2O

Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48–60 kD. From Lineweaver-Burk plots apparent K_m's of 23M for inosine, 42 M for deoxyinosine, 40 M for phosphate, 2 M for hypoxanthine, and 163 M for ribose-1-phosphate were calculated. Both 8-aminoguanosine (K_i=0.5 M) and araG (K_i=381 M) are inhibitors of the enzyme. The protein's isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent K_m's calculated for the mitochondrial enzyme are,with the exception of that using hypoxanthine, within the range commonly associated with K_m's from the cytosolic species. The mitochondrial enzyme's molecular weight and pI are less than normally described. The enzyme's isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.