Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose-, pentose- and hexose-phosphates

After a lag phase of 2 days, batch-grown cells of carrot ( Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding ¹³C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO₂ production from [1-¹³C]- and [6-¹³C]-glucose revealed, that at least 40% of the added glucose passed through the oxidative pentose phosphate pathway (OPPP), up to 40% through glycolysis leaving only minor ¹³C-glucose for incorporation in various cell components in the exponential growth phase. After about 11 days of culture, the medium sugars were exhausted, cells entered the stationary growth phase and consumed stored sugar. Both neutral and acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) increased 50% from day 0 to days 11–13; thereafter their levels decreased again. Labelling with ¹³C-glucose resulted in the accumulation of labelled sucrose and fructose during the stationary growth phase. Sucrose labelling was transient, i.e. after 6 h its level started to decrease again. Labelled fructose, however, evolved slower and increased even after 8 h. In sucrose and fructose up to 20% of the ¹³C-label was exchanged from C-1 to C-6 carbons, indicating intensive cycling of at least 40% of the carbon between hexoses and triose phosphates. In the stationary phase only 10% of the labelled glucose passed through the OPPP and about 30% passed through the respiratory pathway; the remaining 60% was incorporated in cell constituents and sugars. Comparing the various cycles revealed that the regulation of the OPPP operated relatively independently from the cytosolic cycling of hexose phosphates through sucrose and from the cycling between hexose phosphates and triose phosphates.     

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.     

Sugar transport in leaf discs of Phaseolus coccinius

Kinetic profiles for sucrose, glucose and 3-OMG glucose were determined in leaf discs of Phaseolus coccinius L. (cv. Scarlet). All three sugars exhibited identical uptake kinetics. At sugar concentrations below 25 m M , transport was due to an active, carrier-mediated transport system. A linear component was the dominant mode of uptake at sugar concentrations above 25 m M . Sucrose and glucose carriers were specific for these sugars, since no uptake inhibition was observed from competing sugars. Sucrose was not hydrolyzed by leaf tissue because the label in asymetrically labeled sucrose was not randomized. Furthermore, no label was present in hexose fractions when tissue was incubated with [⁸⁴C]-sucrose. Therefore, [¹⁴C]-sucrose uptake did not reflect hexose uptake.
Both saturable and linear components of uptake contribute significantly to total uptake rates. The former, however, is more important when apoplastic sugar concentrations are low. The molecular nature of the linear component is not well understood but accounts for most of the uptake at high sugar concentrations.     

Weather and climate controls over the seasonal carbon isotope dynamics of sugars from subalpine forest trees

We examined the environmental variables that influence the δ ¹³C value of needle and phloem sugars in trees in a subalpine forest. We collected sugars from Pinus contorta , Picea engelmannii and Abies lasiocarpa from 2006 to 2008. Phloem and needle sugars were enriched in ¹³C during the autumn, winter and early spring, but depleted during the growing season. We hypothesized that the late-winter and early-spring ¹³C enrichment was due to the mobilization of carbon assimilated the previous autumn; however, needle starch concentrations were completely exhausted by autumn, and we observed evidence of new starch production during episodic warm weather events during the winter and early-spring. Instead, we found that ¹³C enrichment was best explained by the occurrence of cold night-time temperatures. We also observed seasonal decoupling in the ¹³C/¹²C ratios of needle and phloem sugars. We hypothesized that this was due to seasonally-changing source-sink patterns, which drove carbon translocation from the needles towards the roots early in the season, before bud break, but from the roots towards the needles later in the season, after bud break. Overall, our results demonstrate that the ¹³C/¹²C ratio of recently-assimilated sugars can provide a sensitive record of the short-term coupling between climate and tree physiology.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Assimilate partitioning in Pachyrhizus erosus tubers under short days

The distribution of ¹⁴C products in yam bean ( Pachyrhizus erosus ) plants was measured as a function of time after a ¹⁴CO₂ pulse-chase experiment under short-day conditions. The ¹⁴C declined by more than 70% in leaves during the first two hours, indicating that they actively exported carbon. Tubers were strong storage sinks for carbon and accumulated more than 80% of the total incorporated ¹⁴C after a 72-h chase. The data show that, although sucrose represented about 21% of the tuber non-structural carbohydate, i.e. 15% of the tuber dry weight, ¹⁴C did not accumulate in sucrose but in glucose, fructose and starch. The data indicate the existence of a separate sucrose pool which is affected only very slowly by recent assimilates. As a result, recent photosynthates, temporarily stored, may contribute to the reservoir of carbon available for nitrogen fixation. The data also suggest the existence of two distinct pools of amino acids in the tuber, one utilized mostly for protein synthesis and the other probably stored in the vacuole.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

In vivo neurochemical profiling of rat brain by 1H-[13C] NMR spectroscopy: cerebral energetics and glutamatergic/GABAergic neurotransmission

The quantification of excitatory and inhibitory neurotransmission and the associated energy metabolism is crucial for a proper understanding of brain function. Although the detection of glutamatergic neurotransmission in vivo by ¹³C NMR spectroscopy is now relatively routine, the detection of GABAergic neurotransmission in vivo has remained elusive because of the low GABA concentration and spectral overlap. Using ¹H-[¹³C] NMR spectroscopy at high magnetic field in combination with robust spectral modeling and the use of different substrates, [U-¹³C₆]-glucose and [2-¹³C]-acetate, it is shown that GABAergic, as well as glutamatergic neurotransmitter fluxes can be detected non-invasively in rat brain in vivo .     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Sucrose accumulation in developing peach fruit

Uptake of ¹⁴C-sugars and activities of sucrose metabolizing enzymes were determined in order to study the mechanism(s) of sucrose accumulation in developing peach fruit. Mesocarp of young peach fruit contained glucose and fructose but little sucrose. Starting 88 days after anthesis (DAA) the sucrose concentration increased greatly. The mechanism of sucrose accumulation was studied by measuring ¹⁴C-sucrose and ¹⁴C-glucose uptake rates at three different stages of fruit development, and by assaying weekly the activity of enzymes involved in the hydrolysis and/or synthesis of the soluble sugars. Uptake of 0.5–100 m M ¹⁴C-sucrose and ¹⁴C-glucose by mesocarp tissue slices showed a complex pattern at the first stage of fruit development (62 DAA). During the subsequent growth stages the pattern of sugar uptake changed and was approximately monophasic at the third stage of fruit development.
At 10 m M , glucose was taken up more rapidly than sucrose at the first and second stage of fruit development. Uptake was partially inhibited by the uncoupler carbonylcyanide m -chlorophenylhydrazone (CCCP) at 25 μ M. These results, together with the presence of a putative extracellular invertase, suggest an apoplastic route for sucrose uptake which is dependent, at least in part, on energy supply.
Activities of sucrose hydrolyzing enzymes (insoluble acid invertase, soluble acid invertase, neutral invertase, sucrose synthase) were high in young fruits and declined sharply with fruit development concomitantly with accumulation of sucrose. The storage of the sugar was not accompanied by a rise in synthetic activities (sucrose synthase, sucrose phosphate synthase), suggesting that sucrose could, at least in part enter the carbohydrate pool directly.     

Metabolism of [13C]-labelled photosynthate in plant cytosol and bacteroids of root nodules of Glycine max

Well-nodulated soybean ( Glycine max L. Merr. cv. Akisengoku) plants were allowed to assimilate ¹³CO₂. Plant cytosol and bacteroid fractions were isolated from nodules, and the kinetics of [¹³C]-labelling of soluble carbohydrates, organic acids and amino acids were investigated.
The concentrations of all metabolites, with the exception of trehalose and 3-hydroxy-butyrate, were 10- to 1000-fold higher in plant cell cytosol than in bacteroids. The major portion of trehalose was found in bacteroids and 3-hydroxybutyrate only in bacteroids. Sucrose was most highly labelled with ¹³C in nodules, and the levels and time-course of labelling of sucrose were in good agreement with those of respired CO₂ from the nodules. The levels and time-courses of labelling of sucrose were closely similar in cytosol and bacteroids. Glucose was less labelled than sucrose and the level of labelling was consistently higher in cytosol than in bacteroids. The levels of [¹³C]-labelling of organic acids and amino acids in nodules were lower than those of sucrose and of respired CO₂. Tricarboxylic acid cycle intermediates, particularly succinate, were considerably less labelled in bacteroids than in the cytosol. All amino acids detected were also much more rapidly labelled in the cytosol. The results are discussed in relation to the utilization and possible compartmentation of carbon substrates in nodule tissues.     

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.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

Localisation of translocated 14C in roots and root exudates of field-grown maize

The roots of a mature, field-grown maize plant are dimorphic: the primary root and those from the oldest nodes are bare with a heavily lignified cortex arid sloughed epidermis; those from younger nodes, except for a bare elongation zone, have an intact epidermis surrounded by a persistent soil sheath. Sheathed roots consistently have more layers of cortical cells, but the ratio of volumes of cortex to stele (ca 4) and the cross-sectional area of phloem (ca³× 10⁻² mm²) are similar in each type. Assimilated carbon (from ¹⁴ C applied to a small area of one leaf) was translocated to all roots and actively metabolized in cortex and stele of both types. After 1 to 2 days the proportion of ¹⁴C exuded from a given length of mature root into its soil sheath, or into the adjacent unattached soil in the case of bare roots, was the same (5%) in both root types when compared with the ethanol-soluble ¹⁴C in the tissues of this length. Up to 75% of the ethanol-soluble label in the roots was in a cationic fraction (amino acids and unidentified compounds), ca 1% was in an anionic fraction and the remainder was in a neutral fraction (sugars). Approximately equal amounts of soluble ¹⁴C were found in the stele, cortex and laterals.     

Altered cerebral glucose and acetate metabolism in succinic semialdehyde dehydrogenase-deficient mice: evidence for glial dysfunction and reduced glutamate/glutamine cycling

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,6-¹³C₂]glucose or [2-¹³C]acetate for different periods. Cortical extracts were prepared and measured using high-resolution ¹H-[¹³C] NMR spectroscopy. Compared with wild-type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH-null cortex, whereas glutamate, glutamine, and taurine were lower. ¹³C Labeling from [1,6-¹³C₂]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of ¹³C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged. ¹³C Labeling from [2-¹³C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.     

Conversion of sucrose to starch in the developing Pennisetum typhoides grain

Developing grains of pearl millet ( Pennisetum typhoides Burm. S & H cv. PIB 155) were sampled and analyzed for starch and its free-sugar precursors. The activities of invertase, sucrose-ADP (UDP) glucosyl transferase and of α-amylase and β-amylase in relation to the rate of starch accumulation in the developing grain were assayed. By culturing detached ears, the incorporation of ¹⁴C from free sugar precursors to starch was studied. The starch content gradually increased until grain maturity. The rate of starch accumulation was maximum around 12 days after anthesis. Around this period, the activities of sucrose-ADP(UDP) glucosyl transferase and α-amylase, β-amylase were also at a peak. Invertase activity was high during the early period of grain development but gradually declined as the grains matured. In the most actively metabolising milky grains, incorporation of ¹⁴C from [¹⁴C]-sugars to starch was maximum in the mid mid-milky grains. Addition of 20 m M K⁺ to the culture solution did not affect the incorporation of ¹⁴C from supplied sucrose to the free sugar pool and to the starch of the grain, but Mg²⁺ supply at 20 m M concentration lowered ¹⁴C incorporation from exogenous sucrose to grain free sugars, although the utilization of the latter for starch synthesis was enhanced.     

Distribution of 14C assimilates in the storage root of sugar beet plants after import from leaves of different age

In the sugar beet plant ( Beta vulgaris L. ssp. altissima ) the vascular bundles of old leaves lead to the center and those of young leaves to the periphery of the storage root. Whether the flux of assimilates follows these anatomical routes was tested by applying ¹⁴CO₂ for 4 h to either an old (10th) or a young (20th) leaf in intact sugar beet plants. Four-month-old plants, which had about 30 leaves, were used in the experiment. The ¹⁴C distribution in the storage root was measured by autoradiography and counting in about 20 cross and longitudinal sections per root.
About 37% of assimilated ¹⁴C from an old leaf and 23% from a young leaf were exported within 24 h. Although some ¹⁴C moved into younger leaves, most was exported into the storage root. During its rapid movement towards the root tip, which took place perferentially in the orthostichon belonging to the [¹⁴C]-treated leaf, the label spread laterally.
The autoradiograms indicate that the distribution of assimilates within the storage root is roughly determined by the course of the vascular bundles extending from the source leaf. The fine distribution, however, seems to be controlled by sucrose gradients between storage cells.     

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2-¹³C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using ¹³C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-¹³C]glycine, cell extracts and incubation media were analyzed for ¹³C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-¹³C]glycine, the amount of de novo synthesized [2-¹³C]creatine was two-fold increased, and in addition, ¹³C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-¹³C]glycine was utilized for the synthesis of glutathione in astroglial cells. ¹³C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-¹³C]serine, [3-¹³C]serine, and [2,3-¹³C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.