14C Glucose Research Articles

Fate of 14C from glucose and the herbicide metsulfuron–methyl in a plant–soil microcosm system

The effects of weeds and soil nutrient additions on the fate of 14C-labelled glucose and metsulfuron–methyl (metsulfuron) herbicide were determined in a growth-chamber soil–plant microcosm study. Nodding thistle (Carduus nutans) was used as a test weed species to which these compounds were applied. The experiment consisted of a full factorial design of N addition, P addition, planting with C. nutans, and addition of 14C-labelled compounds (glucose and herbicide metsulfuron). The experiment ran for 131 days following addition of the 14C-labelled compounds. There was a significant positive effect of C. nutans plants on soil microbial biomass-C and basal respiration. The addition of nutrients did not consistently affect any of the parameters measured in this study. The fate of 14C-labelled compounds was determined by quantifying the relative amounts of 14C absorbed by plants, incorporated into soil, taken up into microbial biomass and respired by soil organisms. Two days after application of the 14C compounds, 7–8% of the 14C was absorbed by the C. nutans (weed) canopy. By day 131, approximately 2–3% of the applied 14C was still detected in the tissues of dead thistle plants. The amounts and pattern of decomposition differed considerably between metsulfuron and glucose. Almost all (99%) of the applied 14C glucose was either respired by the microbes or incorporated into microbial biomass by day 131 while only 38–42% of the applied metsulfuron was respired or incorporated into microbes by this time. Greater amounts of metsulfuron pesticide were degraded in microcosms containing C. nutans plants than in those without plants. By day 131, 27.4% of 14C added from metsulfuron was respired in the planted treatment and only 22.8% from the non-planted treatment. This would suggest that the presence of decomposing weed litter (dying weed plants) has the potential to enhance the decomposition of added xenobiotics such as herbicides.

Metabolism of [1- 13C]glucose and [2- 13C]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- 13C]glucose and [2- 13C]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 13C 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- 13C]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.

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System.

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10-11 to 10-12 m s-1) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20-30 ka BP), a disturbed interfacial zone (20-30 ka BP), and a Cretaceous clay aquitard (71-72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and the narH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (14C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.

Relative efficiencies of carbon transfer from bacteria and algae to zooplankton in a subtropical lake

Direct measurements were made of carbon transfer efficiency from bacteria and algae to zooplankton in subtropical Lake Okeechobee, Florida, USA. A radio-tracer method was used to determine carbon transfer efficiency to the >200 μm size class, comparing results from treatments where the label was added as 14C glucose or 14C-bicarbonate. The studies were done at a near-shore and an offshore site, and repeated on five occasions between July 1998 and 1999. The results displayed little variation with location, month, or type of tracer used. After 4 h incubations, between 0.1 and 1.0% of the particulate activity occurred in the >200 μm size class. Low efficiencies suggest a considerable loss of carbon from the copepod- and cyanobacteria-dominated food web; this seems to be typical of highly eutrophic lakes.

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO 2 production from (U- 14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO 2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO 2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO 2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO 2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.

Degradation of 13C–U–Glucose in Sphagnum majus Litter Responses to Redox, pH, and Temperature

We studied the utilization of 13C–U–glucose by the microbial community in shallow Sphagnum majus (Russ.) C. Jens. ssp. norvegicum Flatb. litter and its regulation by pH, temperature, and redox conditions. The transformation of 13C–glucose was monitored by solution‐ and solid‐state 13C–nuclear magnetic resonance (NMR) spectroscopy. The aerobic microbial community used the glucose C for respiration and, to a lesser degree, for storage as mannitol, triglycerides, and polysaccharides. Under both aerobic and anaerobic conditions, the allocation of glucose C for storage was greater at pH 6.8 than at 4.3; however, the amount of C used for building new biomass was the same at both pH settings. At 15°C, 15 to 18% of the utilized C under aerobic conditions was found in new microbial biomass: less than the previously reported values of 40 to 72%. This indicates that peat soils may promote significantly different microbial growth patterns from other minerogenic and moor humus soils. The production of mannitol and triglycerides suggests that fungi dominated the microbial community and utilized the glucose under aerobic conditions. Using a combination of solid and liquid NMR techniques we were able, for the first time, to follow the anaerobic pathways of glucose degradation in a natural soil sample. The anaerobic microbial community produced mainly volatile fatty acids (VFA), ethanol, and CO2 from the added glucose, and only minor amounts were converted to methane, storage C, and new microbial biomass. Nuclear magnetic resonance spectroscopy allows nondestructive assays of metabolic events and, therefore, was shown to be an excellent tool for studying the microbial utilization of 13C–glucose in peat.

Metabolism of (1- 13C) glucose and (2- 13C, 2- 2H 3) acetate in the neuronal and glial compartments of the adult rat brain as detected by { 13C, 2H} NMR spectroscopy

Ex vivo { 13C, 2H} NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1- 13C) glucose and (2- 13C, 2- 2H 3) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2- 13C, 2- 2H 3) acetate and (1- 13C) glucose or (2- 13C, 2- 2H 3) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution 13C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2- 13C, 2H 3) acetate or (1- 13C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of 2H– 13C multiplets and 2H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2- 13C, 2- 2H 3) acetate and (1- 13C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2- 13C, 2- 2H 3) acetyl-CoA (4%), (2- 13C) acetyl-CoA (3%) and recycled (2- 13C, 2- 2H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2- 13C) acetyl-CoA (86%) and (2- 13C, 2- 2H) acetyl-CoA (5%). During the infusions of (2- 13C, 2- 2H 3) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2- 13C, 2- 2H 3) acetyl-CoA (36%), (2- 13C, 2- 2H) acetyl-CoA (27%) and (2- 13C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2- 13C) acetyl-CoA (11%) and recycled (2- 13C, 2- 2H) acetyl-CoA (16%). These results illustrate the potential of { 13C, 2H} NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.

Novel Peak Assignments of in Vivo13C MRS in Human Brain at 1.5 T

13C MRS studies at natural abundance and after intravenous 1-13C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C3β,5β, myo-inositol, glutamate C1,2,5, glutamine C1,2,5, N-acetyl-aspartate C1-4,CO, creatine CH2, CH3, and CCN, taurine C2,3, bicarbonate HCO−3 were identified. After glucose infusion 13C enrichment of glucose C1α,1β, glutamate C1-4, glutamine C1-4, aspartate C2,3, N-acetyl-aspartate C2,3, lactate C3, alanine C3, and HCO−3 were observed. The observation of 13C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain.

Human brain glucose metabolism mapping using multislice 2D 1H-13C correlation HSQC spectroscopyPresented in part at the 5th and 6th Annual Meetings of the International Society for Magnetic Resonance, Vancouver, April 1997 and Sydney, April 1998.

A method for multivolume 2D 1H-13C correlation spectroscopy, multislice heteronuclear single quantum coherence (HSQC), is proposed. This permits human brain metabolism from glucose to amino acids to be followed using a 2-T whole-body scanner. The modifications from the conventional HSQC are that the 180°(13C) and 180°(1H) pulses are separated in time in the preparation period and that the 180°(13C) pulse is applied at 1/(4JCH) before the 90°(1H) polarization transfer (PT) pulse. The preparation (echo) time can be set longer than 1/(2JCH) so that, even in a whole-body system, slice-selective pulses and gradients can be applied. Another modification is that the 90°(1H) reverse PT pulses after the creation of 2IzSz are used as multislice pulses. The time-course of glutamate C4 could be followed with 15-min temporal resolution from the HSQC spectra obtained from the brains of volunteers after the oral administration of glucose C1, and the maximum S/N was 3. Magn Reson Med 43:525–533, 2000. © 2000 Wiley-Liss, Inc.

Simultaneous in vivo monitoring of hepatic glucose and glucose-6-phosphate by (13)C-NMR spectroscopy.

Hepatic glucose-6-phosphate (G6P) was monitored non-invasively in rat liver by in vivo (13)C NMR spectroscopy after infusion of [1-(13)C] glucose. The phosphorylation of glucose to G6P yields small but characteristic displacements for all of its (13)C-NMR resonances relative to those of glucose. It is demonstrated that in vivo (13)C-NMR spectroscopy at 7 Tesla provides the spectral sensitivity and resolution to detect hepatic G6P present at sub-millimolar concentration as partially resolved low-field shoulders of the glucose C1 resonances at 96.86 ppm (C1beta) and 93. 02 ppm (C1alpha). Upon (13)C-labeling, the intracellular conversion of [1-(13)C] glucose to [1-(13)C] G6P could be monitored, which allowed the hepatic glucose-G6P substrate cycle to be assessed in situ. The close correlation found for the (13)C labeling patterns of glucose and G6P supports the concept of an active substrate cycle whose rate exceeds that of net hepatic glucose metabolism. High-resolution (13)C-NMR spectroscopy and biochemical analyses of tissue biopsies collected at the end of the experiments confirmed qualitatively the findings obtained in vivo.

14C-labelled glucose turnover in New Zealand soils

The influence of soil mineralogy, as well as texture, on organic-C turnover was determined with 14C-labelled glucose. Samples of 16 soils from major mineralogical classes of New Zealand pastures and providing a range of organic C, clay contents and surface area, were incubated with 14C-labelled glucose for 35 d. The amounts of 12CO 2 and 14CO 2 evolved during incubation were monitored and the residual 14C concentrations determined. Periodically, the samples were removed and microbial biomass 12C and 14C determined using the fumigation-extraction technique. System mean residence times (MRTs) were obtained by three independent methods: (i) a compartmental model using 14C microbial biomass data, (ii) a non-compartmental model using 14C microbial biomass data and (iii) a biexponential equation as an empirical equation from residual 14C data. The effect of soil characteristics on MRTs was compared. The 14CO 2 respired, after 35 d incubation, accounted for 51 to 66% of the glucose 14C input to these soils. The soils differed significantly in their amounts of 14CO 2 evolution and in the proportions of labelled 14C in the biomass. The extent of mineralization of 14C-labelled glucose was influenced by soil clay content and clay surface area. Soils of low clay content (3–12%) had high biophysical quotients (respired: residual 14C); the highest (1.93) was in the soil with least clay (3%) and lowest mineral surface area, suggesting that clay is effective in C stabilization immediately after substrate assimilation. A biexponential model was found to be suitable for describing changes in the residual 14C and microbial biomass 14C during the 35 d glucose decomposition for most of the soils. MRTs for microbial biomass 14C were correlated with clay content ( P<0.001), surface area estimated by para-nitrophenol ( pNP) ( P<0.003) and pH ( P<0.01). Our results also showed that the MRTs of microbially assimilated 14C are similar despite differences in the chemical nature of the applied 14C-labelled substrate. However, the MRT for humus 14C differed with the chemical nature of the applied substrate. Clay and surface area played a major role in controlling the decomposition of added substrate through the stabilization and protection of the microbial biomass.

Effect of training status on fuel selection during submaximal exercise with glucose ingestion.

In this study, an oral glucose load was enriched with a [U-(13)C]glucose tracer to determine differences in substrate utilization between endurance-trained (T) and untrained (UT) subjects during submaximal exercise at the same relative and absolute workload when glucose is ingested. Six highly trained cyclists/triathletes [maximal workload (Wmax), 400 +/- 9 W] and seven UT subjects (Wmax, 296 +/- 8 W) were studied during 120 min of cycling exercise at 50% Wmax ( approximately 55% maximal O(2) consumption). The T subjects performed a second trial at the mean workload of the UT group (148 +/- 4 W). Before exercise, 8.0 ml/kg of a (13)C-enriched glucose solution (80 g/l) was ingested. During exercise, boluses of 2.0 ml/kg of the same solution were administered every 15 min. Measurements were made in the 90- to 120-min period when a steady state was present in breath (13)CO(2) and plasma glucose (13)C enrichment. Energy expenditure was higher in T than in UT subjects (58 vs. 47 kJ/min, respectively; P < 0.001) at the same relative intensity. This was completely accounted for by an increased fat oxidation (0.57 vs. 0.40 g/min; P < 0.01). At the same absolute intensity, fat oxidation contributed more to energy expenditure in the T compared with the UT group (44 vs. 33%, respectively; P < 0.01). The reduction in carbohydrate oxidation in the T group was explained by a diminished oxidation rate of muscle glycogen (indirectly assessed by using tracer methodology at 0.72 +/- 0.1 and 1.03 +/- 0.1 g/min, respectively; P < 0.01) and liver-derived glucose (0.15 +/- 0.03 and 0.22 +/- 0.02 g/min, respectively; P < 0.05). Exogenous glucose oxidation rates were similar during all trials (+/-0.70 g/min).

Alteration of glycogen and glucose metabolism in ischaemic and post-ischaemic working rat hearts by adenosine A1 receptor stimulation.

1. Cardioprotection by adenosine A1 receptor activation limits infarct size and improves post-ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism. 2. Since glycogen is an important source of glucose during ischaemia, we examined the effects of N6-cyclohexyladenosine (CHA), an A1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion. 3. Isolated working rat hearts were perfused with Krebs-Henseleit solution containing dual-labelled 5-3H and 14C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3H2O and 14CO2. Glycogen turnover was measured from the rate of change of [5-3H and 14C]glucosyl units in total myocardial glycogen. 4. Following low-flow (0.5 ml min-1) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre-ischaemic values. CHA (0.5 microM) improved the recovery of LV work 2 fold. 5. CHA altered glycogen turnover in post-ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA-treated hearts and reduced proton production. 6. During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA-induced inhibition of AMP-activated protein kinase activity. 7. These results indicate that CHA-induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation.

Lower arterial glucose concentrations in lambs with aortopulmonary shunts after an 18-hour fast.

Spontaneously occurring hypoglycemia has been described in children with severe acute congestive heart failure. Hypoglycemia may be the result of an increase in glucose utilization in tissues, a decrease in glucose production, or a decrease in the dietary intake of nutrients. To determine whether hypoglycemia may also occur in congenital heart disease with volume overloading, we investigated glucose metabolism during and after an 18-hour fast in nine lambs with an aortopulmonary left-to-right shunt and nine control lambs. Plasma levels of hormones involved in the endocrine control of glucose metabolism were determined. The glucose production rate (rate of appearance [Ra]) was studied using [U-13C]glucose. Gluconeogenesis through the Cori cycle was estimated by measuring glucose 13C recycling. The arterial glucose concentration (3,409 +/- 104 v 4,338 +/- 172 micromol/L, P < .001) and Ra of glucose (16.97 +/- 0.89 v 25.49 +/- 4.28 micromol x min(-1) x kg(-1), P < .05) were lower in shunt versus control lambs. There were no differences in hormone levels between control and shunt lambs. Fractional glucose 13C recycling via the Cori cycle (6.9% +/- 2.8% v 7.1% +/- 2.5%) and gluconeogenesis from pyruvate and lactate (1.24 +/- 0.58 v 1.95 +/- 0.67 micromol x min(-1) x kg(-1)) were similar in both groups of lambs. The sum of glycogenolysis and gluconeogenesis from precursors other than pyruvate and lactate was lower in shunt versus control lambs (15.73 +/- 1.07 v 23.54 +/- 4.27 micromol x min(-1) x kg(-1), P < .05). In conclusion, after an 18-hour fast, the arterial glucose concentration is lower in lambs with aortopulmonary shunts. This lower glucose concentration is associated with a decreased glucose production rate. In shunt lambs, glycogenolysis is decreased, while there is no difference in gluconeogenesis or hormonal control.

Localized in vivo 1H NMR detection of neurotransmitter labeling in rat brain during infusion of [1-13C] D-glucose.

Resolved localized nuclear magnetic resonance (NMR) signals of 1H bound to 13C label in the carbon positions of glutamate C4, C3 and glutamine C4, C3, as well as in aspartate C3, lactate C3, alanine C3, gamma-aminobutyric acid C3, and glucose C1 were simultaneously observed in spectra obtained from rat brain in vivo. Time-resolved label incorporation was measured with a new adiabatic carbon editing and decoupling (ACED) single-voxel stimulated echo acquisition mode (STEAM) sequence. Adiabatic carbon broadband decoupling of 12 kHz bandwidth was achieved in vivo, which decoupled the entire 13C spectrum at 9.4 T. Resonances from N-acetyl-aspartate and creatine were also detected, consistent with natural-abundance 13C levels. These results emphasize the potential of 1H NMR for following complex biochemical pathways in localized areas of resting rat brain as well as during focal activation using infusions of 13C-labeled glucose.

Formation of N-(Carboxymethyl)fumonisin B1, Following the Reaction of Fumonisin B1 with Reducing Sugars

The fumonisins are mycotoxins produced by fungi that contaminate primarily corn and are toxic through interruption of intracellular sphingolipid synthesis. Several reports have indicated that fumonisin B1 concentrations decreased when heated in aqueous solutions of reducing sugars. The incubation of fumonisin B1 with d-glucose resulted in the formation of N-(carboxymethyl)fumonisin B1, which was characterized by NMR and electrospray mass spectroscopy. We determined the methylene carbon of the carboxymethyl group is derived from C1 on glucose, while the carbonyl carbon is derived from the C2 of glucose, using 13C glucose. Apparently N-(carboxymethyl)fumonisin B1 arises from Schiff's base formation, Amadori rearrangement to a β-ketoamine, and oxidation with molecular oxygen. N-(Carboxymethyl)fumonisin B1 formation is favored by alkaline conditions (pH >7), requires molecular oxygen, and is catalyzed by several reducing sugars. N-(carboxymethyl)fumonisin B1 was detected in raw corn samples that contained fumonisin B1 (0.5−1.4 ppm) at an average of 4% of the fumonisin B1 levels. Keywords: Fumonisin B1; fumonisins; N-(carboxymethyl)fumonisin B1; detoxification; Amadori rearrangement; Maillard reaction; reducing sugars; mycotoxin

FFA CONCENTRATION DID NOT AFFECT CARBOHYDRATE METABOLISM IN SOLEUS MUSCLE IN VITRO.

1407 The increased FFA availability during fasting and exercise is viewed as an evidence of casual relation with its carbohydrate sparing effect. In order to verify the effect of FFA availability within physiological range on carbohydrate metabolism and its interrelation with exercise and fasting, swimming trained ( T) and sedentary ( S) rats were subdivided into control ( A - fed ad lib) and 48 h fasted( F) groups. Strips of soleus muscles were obtained after a single swimming bout, and incubated in Krebs-Ringer buffer (glucose 5.5 mM, insulin 100 um/mL) with addition of oleate at 0.4 or 0.8 mM. Glucose uptake(1-3H-2.DG) was not affected by fasting but increased by training( SA: 37.5±2.5, SF: 37.7±3.0, TA: 38.8±1.8, TF: 38.4±2.3 μmol.g-1.h-1). Incorporation of U-14C-glucose into glycogen was lowered by fasting, and in fed state, the training decreased it ( SA: 5.3±0.8, SF: 5.6±0.3, TA: 3.0±0.4, TF: 4.2±0.5 μmol.g-1.h-1). Training decreased the lactate production from 14C glucose ( SA: 8.8±1.6, SF: 9.5±1.2, TA: 7.7±1.2, TF: 8.3±1.0 μmol.g-1.h-1) but increased the total lactate production ( SA: 12.9±0.8, SF: 15.3±1.4, TA: 26.2±2.9, TF: 20.9±2.9μmol.g-1.h-1). The increase in FFA content in the incubation medium from 0.4 to 0.8 mM did not affect any result. These results suggest that the cellular state is more powerful factor to spare carbohydrate than FFA availability.

Biosynthesis of 13C-labeled branched polysaccharides by pestalotiopsis from 13C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1- 13C)glucose, D-(2- 13C)glucose, D-(4- 13C)glucose, D-(6- 13C)glucose or a mixture of D-(1- 13C)glucose and D-(2- 13C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. 13C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of 13C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6- 13C)glucose was low, compared to that of cellulose and curdlan.

Breath tests: concepts, applications and limitations.

Breath tests (BT) using stable isotopically labelled substrates seem to fulfill all the demands and desires for a non-invasive investigation. There are no radiation hazards, substrates are given in tracer amounts perorally, breath and urine samples can be collected easily, and tests can be done repeatedly, thus easily allowing the monitoring of function with time. There are, however, some disadvantages. Any BT has the same assumption: after intake of the 13C tracer the substrate is metabolized to 13CO2. An increase of 13CO2 above baseline levels is said to reflect the function investigated-in 13C sucrose studies, the amount of carbohydrate absorbed; in 13C aminopyrine BT, the liver function; in 13C glucose BT in a diabetic child, the impaired handling of glucose. However, as only the end product 13CO2 is measured, there is no information on all the pools and fluxes the labelled substrate and its metabolites have to pass. At least in inborn errors of metabolism, probably in any disease, one has to assume that these fluxes and pools are substantially changed. Therefore all calculations are weak and finally one has to resort to invasive methods, i.e. drawing blood to measure pools and fluxes to allow a correct interpretation of the BT data. Furthermore, changes in the basal exhalation of 13CO2 during the test will have an impact on the BT calculation. Another problem is that for an exact calculation, the basal metabolic rate (BMR) and the actual endogenous CO2 production in the patient is needed, which in most instances is unknown. It is not easy to maintain a stable endogenous CO2 production, particularly in younger children who will not rest or in neonates and toddlers who may fight against taking breath samples. Taking together these limitations are the reason why BT have not been able to reach the level of routine clinical methods, especially in the diagnostic work up of impaired liver function or inborn errors of metabolism.

Relationship between gluconeogenesis and phosphoenergetics in rat liver assessed by in vivo 13C and 31P NMR spectroscopy.

The relationship between the phosphoenergetic state and gluconeogenesis in the liver after ischemic damage was investigated using living rats. The ATP level was determined with in vivo 31P nuclear magnetic resonance spectroscopy, and gluconeogenesis was evaluated with in vivo 31C NMR spectroscopy using L-[3-13C]alanine as a tracer. These two measurements were alternated repeatedly. The rats were divided into three groups: without ischemia (group A); with 10 min ischemia (group B); and with 30 min ischemia (group C). ATP was depleted to 20% of the preischemic state after 10 min ischemia and this level was maintained during 30 min ischemia. After reperfusion, the ATP level was partially restored, but the recovery was smaller in group C. Infusion of [3-13C]alanine was started immediately after the reperfusion. In vivo 13C NMR disclosed changes in the alanine C3, glutamine/glutamate C2 and C3, glucose C1-6, and glycogen C1 signals in the liver. After 60 min infusion of [3-13C]alanine, the ATP level correlated negatively with the signal intensity of alanine (r = -0.664, p = 0.008) and positively with those of glucose and glyogen (r = 0.586, p = 0.023, and r = 0.643, p = 0.011, respectively). These results suggest that the ATP level participates in gluconeogenesis and glycogenesis in the liver. Such multinuclear in vivo NMR observations might uncover new aspects of the metabolic function of the liver in the in vivo state.