Glucose and lactate catabolism by bacteria of the pig large intestine and sheep rumen as assessed by 13C nuclear magnetic resonance.

The fermentation of [1-13C] glucose and [3-13C]lactate by bacteria isolated from sheep rumen and pig large intestine was compared by the nuclear magnetic resonance (NMR) technique. Washed cell suspensions were incubated directly in the NMR spectrometer and spectra were recorded every 10 min after injection of the labelled substrates. The results showed large differences in the fermentation patterns between rumen and hindgut bacteria. The latter pattern indicated a greater ability for formation and fermentation of lactate than that of the rumen. Moreover, with both substrates the amount of propionate formed via the acrylate pathway was always greater with hindgut than with rumen bacteria, 50% and 20% of the total, respectively.     

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Abstract Natural abundance ¹³C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Localized 13C NMR Spectroscopy in the Human Brain of Amino Acid Labeling from d-[1-13C]Glucose

Abstract: Cerebral metabolism of d [1-¹³C]glucose was studied with localized ¹³C NMR spectroscopy during intravenous infusion of enriched [1-¹³C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-¹³C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-¹³C]glutamate clearly lagged behind that of [4-¹³C]glutamate and peaked at t = 110–140 min. Multiplets due to homonuclear ¹³C-¹³C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a ¹³C isotopic fraction at C4 glutamate of 27 ± 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 ± 1%), p < 0.05. By comparison with an external standard the total amount of [4-¹³C]glutamate was directly quantified to be 2.4 ± 0.1 µmol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 ± 0.7 µmol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in healthy human brain.     

Carbon Isotope Ratios (13C/12C) in Fractions of Field-Grown Grape

The carbon isotope ratio (δ¹³C%) in leaves and berries of Vitis vinifera L. was determined during the fruiting season. The δ¹³C was from ?24.6 to ?26.3 in total leaf tissue, while it is from ?22.7 to ?24.3 in the aqueous extract of leaves. The δ¹³C in the berries was more stable, around ?24, with no change during ripening. Malic and tartaric acid, isolated from the berries at the onset of ripening, showed a δ¹³C of ?23.5 and ?23.0 respectively. It was concluded that malic acid accumulated in the berries at the onset of ripening is not formed directly through β-carboxylation.     

13C nuclear magnetic resonance study of the complexation of calcium by taurine

¹³C Nuclear magnetic resonance chemical shifts, ¹J_C-C scalar coupling constants, spin-lattice relaxation times, and nuclear Overhauser effects were determined for taurine-[1, 2 ¹³C] and a taurine-[1 ¹³C] and taurine-[2 ¹³C] mixture in the presence and absence of calcium. Ionization constants for taurine amino and sulfonic acid groups and chemical shifts of N-methylene and S-methylene carbons of the taurine cation, zwitterion, and anion were obtained from simultaneous least squares analysis of ¹³C titration curves of both taurine carbons. Comparison of taurine titration shifts to values for related compounds reveals some unusual electronic properties of the taurine molecule. Stability constants of 1:1 calcium complexes with taurine zwitterions and anions, as well as their ¹³C chemical shifts, were obtained by least squares analysis of titration curves measured in the presence of calcium. The stability constants of calcium-taurine complexes were significantly lower than previous values and led to estimates that only approximately one percent of intracellular calcium of mammalian myocardial cells would exist in a taurine complex. The implications of these results with respect to the effect of taurine on calcium ion flux are discussed.     

Testing the branch autonomy theory: a 13C/14C double-labelling experiment on differentially shaded branches

The impact of a heterogeneous within‐crown light environment on carbon allocation was investigated on young walnut trees trained on two branches: one left in full sunlight, the other shaded until leaf fall resulting in 67% reduction in photosynthetically active radiation. In September, the two branches were separately labelled with ¹⁴CO₂ and ¹³CO₂, respectively, so that the photosynthates from each branch could be traced independently at the same time. Although some carbon movements could be detected within 5 d in both directions (including from the shaded branch to the sun branch), between‐branch carbon movements were very limited: approximately 1% of the diurnal net assimilation of a branch. At this time of the year branch autonomy was nearly total, leading to increased relative respiratory losses and a moderate growth deficit in the shaded branch. The ratio of growth to reserve storage rate was only slightly affected, indicating that reserves acted not as a mere buffer for excess C but as an active sink for assimilates. In winter, branch autonomy was more questionable, as significant amounts of carbon were imported into both branches, possibly representing up to 10% of total branch reserves. Further within‐plant carbon transfers occurred in spring, which totally abolished plant autonomy, as new shoots sprouted on each branch received significantly more C mobilized from tree‐wide reserves than from local, mother‐branch located reserves. This allowed great flexibility of tree response to environment changes at the yearly time scale. As phloem is considered not functional in winter, it is suggested that xylem is involved as the pathway for carbohydrate movements at this time of the year. This is in agreement with other results regarding sugar exchanges between the xylem vessels and the neighbouring reserve parenchyma tissues.     

Natural 13C and 15N abundance of field-collected fungi and their ecological implications

The natural abundance of ¹³C and ¹⁵N was measured in basidiocarps of at least 115 species in 88 genera of ectomycorrhizal, wood-decomposing and litter-decomposing fungi from Japan and Malaysia. The natural abundance of ¹³C and ¹⁵N was also measured in leaves, litter, soil and wood from three different sites. ¹⁵N and ¹³C were enriched in ectomycorrhizal and wood-decomposing fungi, respectively, relative to their substrates. Ectomycorrhizal and wood-decomposing fungi could be distinguished on the basis of their δ¹³C and δ¹⁵N signatures. Although there was high variability in the isotopic composition of fungi, the following isotope- enrichment factors (ε, mean±SD) of the fungi relative to substrates were observed:
ε_{ectomycorrhizal fungi/litter} = 6.1±0.4‰¹⁵N
ε_{ectomycorrhizal fungi/wood} = 1.4±0.8‰¹³C
ε_{wood-decomposing fungi/wood} = −0.6±0.7‰¹⁵N
ε_{wood-decomposing fungi/wood} = 3.5±0.9‰¹³C
The basis of isotope fractionation in C metabolism from wood to wood-decomposing fungus is discussed.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

Cerebral Glucose Utilization: Comparison of [14C]Deoxyglucose and [6-14C]Glucose Quantitative Autoradiography

The [14C]deoxyglucose [Sokoloff et al., J. Neurochem. 28, 897-916 (1977)] and [6-14C]glucose [Hawkins et al., Am. J. Physiol. 248, C170-C176 (1985)] quantitative autoradiographic methods were used to measure regional brain glucose utilization in awake rats. The spatial resolution and qualitative appearance of the autoradiograms were similar. In resting animals, there was no significant difference between the two methods among 18 gray and three white matter structures over a fourfold range in glucose utilization rates (coefficient of correlation = 0.97). In rats given increasing frequencies of photoflash visual stimulation, the two methods gave different results for glucose utilization within visual pathways. The linearity of the metabolic response was studied in the superior colliculus using an on-off checkerboard stimulus between 0 and 33 Hz. The greatest increment in activity occurred between 0 and 4 Hz stimulation with both methods, probably representing recruitment of neuronal elements into activity. Above 4 Hz, there was a progressive increase in labeling with [14C]deoxyglucose up to 1.7 times control at 33 Hz. With [6-14C]-glucose, there was no further increment in change above a 30% increase seen at 4 Hz. Measurement of tissue glucose revealed no drop in the visually stimulated structures compared to control. We interpret these results to indicate that, with increasing rates of physiological activity, the products of deoxyglucose metabolism accumulate progressively, but the products of glucose metabolism are removed from brain in 10 min.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

13C and 15N in microarthropods reveal little response of Douglas-fir ecosystems to climate change

Understanding ecosystem carbon (C) and nitrogen (N) cycling under global change requires experiments maintaining natural interactions among soil structure, soil communities, nutrient availability, and plant growth. In model Douglas-fir ecosystems maintained for five growing seasons, elevated temperature and carbon dioxide (CO₂) increased photosynthesis and increased C storage belowground but not aboveground. We hypothesized that interactions between N cycling and C fluxes through two main groups of microbes, mycorrhizal fungi (symbiotic with plants) and saprotrophic fungi (free-living), mediated ecosystem C storage. To quantify proportions of mycorrhizal and saprotrophic fungi, we measured stable isotopes in fungivorous microarthropods that efficiently censused the fungal community. Fungivorous microarthropods consumed on average 35% mycorrhizal fungi and 65% saprotrophic fungi. Elevated temperature decreased C flux through mycorrhizal fungi by 7%, whereas elevated CO₂ increased it by 4%. The dietary proportion of mycorrhizal fungi correlated across treatments with total plant biomass (n= 4, r²= 0.96, P= 0.021), but not with root biomass. This suggests that belowground allocation increased with increasing plant biomass, but that mycorrhizal fungi were stronger sinks for recent photosynthate than roots. Low N content of needles (0.8–1.1%) and A horizon soil (0.11%) coupled with high C : N ratios of A horizon soil (25–26) and litter (36–48) indicated severe N limitation. Elevated temperature treatments increased the saprotrophic decomposition of litter and lowered litter C : N ratios. Because of low N availability of this litter, its decomposition presumably increased N immobilization belowground, thereby restricting soil N availability for both mycorrhizal fungi and plant growth. Although increased photosynthesis with elevated CO₂ increased allocation of C to ectomycorrhizal fungi, it did not benefit plant N status. Most N for plants and soil storage was derived from litter decomposition. N sequestration by mycorrhizal fungi and limited N release during litter decomposition by saprotrophic fungi restricted N supply to plants, thereby constraining plant growth response to the different treatments.     

Correlating δ13C and δ18O in cellulose of trees

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ¹³C and δ¹⁸O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ¹³C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ¹⁸O) at the dry sites. δ¹⁸ O-δ¹³C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ¹⁸O-δ¹³C plot is related to the sensitivity of the p_i/p_a of a plant to changing relative humidity.     

Chemical characterization of pigment gallstones using 13C nuclear magnetic resonance analysis

The unique ability of Carbon-13 nuclear magnetic resonance analysis with cross polarization/magic angle spinning techniques to investigate chemical structures of solids is used to probe the chemical characteristics of several gallstone types. New pulse program techniques are used to distinguish various carbon atoms in studying the polymeric nature of the black bilirubinoid pigment of pigment gallstones. Evidence for the involvement of the carboxyl group and noninvolvement of vinyl groups of bilirubinoids in the polymeric bond formation is presented. Conjugated bilirubin structures are found to be present in some solid residues from pigment stones extracted with acidic methanol/chloroform.     

Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees

Evidence is presented for a very specific, seasonally recurring tri‐phase carbon isotope pattern in tree rings of broad‐leaf deciduous tree species. It is derived from highly resolved intra‐annual measurements of ¹³C/¹²C ratios of wood and cellulose from tree rings of Fagus sylvatica, Populus nigra, Quercus petraea and Morus alba. Investigations on δ¹³C from buds and leaves of Fagus sylvatica revealed a similar tri‐phase δ¹³C pattern. At the very beginning of a growing season, the δ¹³C trend of tree rings and foliage shows a marked increase of up to 5‰. The maximum δ¹³C‐value of each vegetation period always occurs in young heterotrophic leaves shortly after bud burst and persistently in the early wood of each tree ring, when growth depends on carbon reserves. Thereafter, δ¹³C profiles represent the autotrophic stage of the leaves, which show different patterns of variation, by and large characterized by a decline. The minimum δ¹³C‐value always shows up in the late wood of each tree ring. At the very end of each tree ring δ¹³C‐values start rising again. This increase in δ¹³C marks the gradual switch‐over to storage‐dependent growth and can also be observed in senescent leaves. Seasonal changes of more than 4‰ were measured, whereas contiguous δ¹³C values rarely differed from each other by more than 0.3‰. This tri‐phase pattern cannot be explained by the common model of carbon isotope fractionation during photosynthesis. It appears to be primarily an indication of seasonal changes in down‐stream processes of the carbohydrate metabolism. Environmental influences on the carbon isotope fractionation during photosynthesis are presumably of secondary importance and expressed by certain peculiarities showing up during the autotrophic phase, i.e. the mid‐section of the seasonal δ¹³C pattern.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.