13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts.

A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.

A simple mathematical model and practical approach for evaluating citric acid cycle fluxes in perfused rat hearts by 13C-NMR and 1H-NMR spectroscopy.

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

Mathematical model for evaluating the Krebs cycle flux with non-constant glutamate-pool size by 13C-NMR spectroscopy. Evidence for the existence of two types of Krebs cycles in cells.

A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2-13C]acetyl-CoA is not 100% and the total glutamate concentration remains constant or varies during incubation. A comparative study of [1-13C]glucose metabolism was subsequently carried out using Saccharomyces cerevisiae cells from two different strains (ATCC-9763 and NCYC-239) by 13C-NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1-13C]glucose as a carbon source, the 13C-NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC-239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.

Mathematical models for determining metabolic fluxes through the citric acid and the glyoxylate cycles in Saccharomyces cerevisiae by 13C-NMR spectroscopy.

We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast. These models are based on the analysis of 13C-NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched [2-13C]acetate. The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced. The aerobic metabolism of 100% [2-13C]acetate in acetate-grown S. cerevisiae cells was studied as a function of time using 13C-NMR. 1H-NMR and biochemical techniques. The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different. By contrast, the glutamate C4 singlet increases then decreases with time. The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t > 90 min. A similar observation was made for the C2 resonance singlet and doublet. In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling. These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl-CoA are negligible in yeast. The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet. The fluxes in the citric acid and the glyoxylate cycles were found to be comparable. The proportion of glutamate in isotopic exchange with the citric acid cycle is about 2.5% min1 in yeast.

A novel approach for investigating reaction mechanisms in cells. Mechanism of deoxy-trehalose synthesis in Saccharomyces cerevisiae studied by 1H-NMR spectroscopy.

A new approach is proposed for investigating the mechanism of metabolite synthesis in cells. This method, based on the competition between various substrates, allows the flux along a pathway, which is normally independent of the concentration of the corresponding precursor in the external medium, to be divided into partial fluxes. In particular, the mechanism deoxy-trehalose synthesis in glucose-grown repressed Saccharomyces cerevisiae was studied, by 1H-NMR spectroscopy, using the competition between 2-deoxy-D-glucose (dGlc) and 2-fluoro-deoxy-D-glucose (FdGlc) with respect to hexokinase. S. cerevisiae cells, suspended in a standard pyrophosphate medium containing about 5 x 10(7) cells/ml, were incubated with 30 mM glucose and various concentrations of dGlc and FdGlc. Apart from dGlc6P and FdGlc6P, trehalose and the dissacharides relative to dGlc, i.e. dideoxy-trehalose (dGlc-dGlc) and deoxytrehalose (dGlc-Glc), are observed while their analogues relative to FdGlc (FdGlc-FdGlc, FdGlc-Glc) are surprisingly absent. For the same external concentration of dGlc and FdGlc, the internal concentration of FdGlc6P is about three times larger than that of dGlc6P. The ratio of the FdGlc6P and dGlc6P concentrations is independent of the incubation times and proportional to the FdGlc and dGlc concentrations in the suspension. The dGlc6P concentration can thus be reduced at will by increasing the [FdGlc]/[dGlc] ratio. Under these conditions, the dGlc-Glc concentration was found to vary linearly with that of dGlc6P. The present data clearly show that deoxy-trehalose is not synthesized from UDP-dGlc and Glc6P but from UDP-Glc and dGlc6P. This conclusion was also confirmed by an experiment in which S. cerevisiae cells were previously charged with dGlc6P and then incubated with glucose.

Reciprocal effects of 2-fluoro-2-deoxy-D-glucose and glucose on their metabolism in Saccharomyces cerevisiae studied by multi-nuclear NMR spectroscopy.

The effects of various concentrations of 2-fluoro-2-deoxy-D-glucose (FDG) on the aerobic metabolism of glucose and the reciprocal effect of glucose on the metabolism of FDG in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30 degrees C in a standard pyrophosphate medium containing 5 x 10(7) cells/ml by 1H-, 19F-, 31P-NMR and biochemical techniques. The glucose consumption rate is reduced by about 57% and 71% in the presence of 5 mM FDG and 10 mM FDG respectively. Under the same conditions, the ethanol production rate also decreases about 54% and 68%, respectively. When FDG is the unique carbon source, the alpha- and beta-anomers of 2-fluoro-2-deoxy-D-glucose-6-phosphate (FDG6P) and a much smaller quantity of 2-fluoro-2-deoxy-gluconic acid (FDGA) were observed. The quantities of alpha- and beta-FDG6P reach their maximum values within 1 h of incubation and then decrease continuously. In contrast, Glc favors the consumption of FDG and the synthesis of FDG6P and uridine-5'-diphosphate fluorodeoxy-glucose (UDP-FDG). In the presence of Glc, FDG6P reaches a plateau after 1 h or 2 h of incubation while UDP-FDG increases regularly with time. Apart from trehalose, no other disaccharide such as fluoro-dideoxy-trehalose (FDG-FDG) or fluoro-deoxy-trehalose (FDG-Glc) were observed. Thus, in contrast to UDP-Glc, UDP-DG, Glc6P and DG6P, UDP-FDG and FDG6P are not good substrates for trehalose-6-P synthetase.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-cooperative effects of glucose and 2-deoxyglucose on their metabolism in Saccharomyces cerevisiae studied by 1H-NMR and 13C-NMR spectroscopy.

The reciprocal effects of 2-deoxy-D-glucose (dGlc) and glucose (Glc) on the aerobic metabolism of Glc and dGlc in Glc-grown repressed Saccharomyces cerevisiae were studied at 30 degrees C in a standard pyrophosphate medium containing about 4.5 x 10(7) cells/ml. 1H-, 13C-NMR and biochemical techniques were used for quantitative evaluation of Glc and dGlc metabolites. The detection of intracellular dGlc and the determination of the intracellular dGlc6P/dGlc ratio were realised using [1-13C]dGlc and 13C-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 +/- 0.03 mM/min (n = 3), 16 +/- 0.02 mM/min (n = 3), and 0.08 +/- 0.01 mM/min (n = 3), respectively. This means that the Glc consumption is reduced about 41% and 70% in the presence of 5 mM and 20 mM dGlc, respectively. When dGlc is the unique carbon source, only alpha and beta anomers of dGlc6P were formed. Their quantities are equivalent and reach the maximum values within 1 h of incubation and then decrease gradually. By contrast, Glc favours the consumption of dGlc and the synthesis of dGlc6P, dideoxy-trehalose (dGlc-dGlc), deoxy-trehalose (dGlc-Glc). In the presence of Glc, dGlc6P reaches a plateau after 1 h or 2 h of incubation while the quantities of trehalose (Glc-Glc), dGlc-dGlc, dGlc-Glc, which are small at 1 h, rapidly increase with time. The reasons why dGlc and Glc exert opposite effects on their metabolism are discussed. The production of Glc-Glc decreases with increasing the external dGlc concentration or the dGlc/Glc ratio. The effect of dGlc on the biosynthesis of Glc-Glc can be explained by the competition of dGlc and Glc with respect to hexokinase. Although Glc favours the synthesis of dGlc6P, the maximum concentration of dGlc6P shows little dependence on the external dGlc concentration as long as glucose is not exhausted. The internal dGlc6P/dGlc ratio at equilibrium, about 4.7 +/- 0.7, is also found to be independent of the dGlc concentration in the suspension. Only a small fraction of dGlc6P disappears to give rise to the formation of dGlc-dGlc and dGlc-Glc. At equilibrium the inverse reaction from dGlc6P to dGlc may be important to compensate for the fast reaction of dGlc phosphorylation by hexokinase. At least nine series of experiments were conducted and showed that, in pyrophosphate media and for incubation times less than 4 h, dGlc-dGlc was not observed in the absence of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of glucose on the deoxyglucose metabolism in S cerevisiae: detection and identification of deoxyglucose and trehalose derivatives by 1H- and 13C-NMR spectroscopy.

The metabolism of 2-deoxy-D-glucose (DG) in glucose grown repressed Saccharomyces cerevisiae cells was studied in the absence and presence of glucose (Glc) at 30 degrees C in a standard pyrophosphate medium containing 4.5 x 10(7) cells/ml. 1H- and 13C-NMR spectroscopy were successfully used to distinguish and identify several derivatives of DG and trehalose. Using [1-13C]DG, alpha- and beta-DG, alpha- and beta-DG6P, dideoxy-trehalose (DG-DG) and deoxy-trehalose (DG-Glc) can be simultaneously observed in the intracellular medium. The [DG6P]/[DG] ratio is about 5-6. The results seem to indicate the existence of an equilibrium between DG and DG6P, which limits the production of DG6P in cells. Glucose was found to exert a great influence on the metabolism of DG. It favours the formation of DG-DG and DG-Glc.

Effects of 2-deoxy-D-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods.

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30 degrees C in a standard pyrophosphate medium containing 4.5 10(7) cells/ml. 31P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by 13C- and 1H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-D-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-13C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 +/- 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG.(ABSTRACT TRUNCATED AT 400 WORDS)

Thalamocortical diaschisis: positron emission tomography in humans.

To investigate further the relations between cortical energy metabolism and neuropsychological impairment after unilateral thalamic lesion, 55 patients underwent positron emission tomography studies of either cortical oxygen consumption or glucose utilisation, including eight repeat studies, at times ranging from 4 days to 98 months after the onset of the lesion [stroke (n = 44) or stereotaxic VL-Vim thalamotomy performed for movement disorders (n = 11)]. Patients with thalamotomy were also studied preoperatively and the surgery induced a significant fall in cortical metabolism on both sides (more so ipsilaterally); post-operatively the magnitude of the ipsilateral cortex hypometabolism was positively correlated to the severity of global neuropsychological impairment; similar but less significant findings were obtained for the ipsilateral/contralateral cortical metabolic asymmetry. With respect to the whole patient sample, the cortical metabolic asymmetry was initially pronounced, with subsequent monoexponential recovery, in the cognitively impaired study group, but it was only mild and showed no meaningful trend for recovery in the cognitively unaffected study group; yet even soon (< 3 months) after thalamic lesion there was a noticeable overlap of individual asymmetry values among the two study groups. These results lend further support to the view that the neuropsychological impairment that frequently follows unilateral thalamic lesions is reflected in a depression of synaptic activity in both the overlying and the contralateral cerebral cortices. For individual patients, this study also illustrates the potentially misleading nature of the measured cortical metabolic asymmetry with respect to neuropsychological status, especially at late times after lesion, in part because side to side metabolic ratios do not reflect bilateral changes.

Cortical metabolism in posterolateral thalamic stroke: PET study.

In 8 patients with small unilateral posterolateral thalamic (or, in one case, thalamocapsular) stroke (infarction or hemorrhage) selected on strict clinical (pure hemisomatosensory deficit without hemiparesis, visual field defect or neuropsychological impairment) and MRI criteria, we studied cortical energy metabolism using positron emission tomography with the 18F-fluorodeoxyglucose or the 15O-oxygen method. We found no significant ipsi- or contra-lateral metabolic depression either in the whole cortical mantle or in the sensorimotor cortex. These results support the hypothesis that location of thalamic stroke is a major determinant of the ipsilateral cortical hypometabolism characteristic of cognitively impaired patients with thalamic lesions and further emphasize the influence of the "non-specific" thalamocortical system on resting cortical metabolism. The lack of sensorimotor cortex hypometabolism in our patients suffering from hemidysesthesia and/or -hyperpathia also suggests that cortical metabolism is unaltered in thalamic pain.

Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K(+)- and Na(+)-rich media.

In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.

Brain energy metabolism in bilateral paramedian thalamic infarcts. A positron emission tomography study.

Positron emission tomography (PET) studies of the cerebral metabolic rate of oxygen (CMRO2) were performed in seven consecutive patients with bilateral paramedian thalamic infarcts (BPTI), selected on neuroradiological and clinical criteria. The latter consisted of sudden onset of coma or confusion followed by a persistent amnesia of varying severity, with or without language impairment and frontal lobe signs. There was a highly significant decrease of CMRO2 for the whole cortex as well as for all the regions analysed: medial-frontal, latero-frontal, temporal, sensorimotor and posterior associative cortex. The mean regional metabolic ratios (region/whole cortex CMRO2) were not significantly different from controls, indicating an essentially uniform effect in the cortex, except the sensorimotor ratio which was significantly increased. Diffuse cortical hypometabolism most likely reflects thalamo-cortical deafferentation secondary to damage to the 'non-specific' thalamic nuclei, while sparing of the latero-ventral thalamus presumably explains the relative preservation of the sensorimotor cortex metabolism. Although no clear-cut individual relationship was found between magnitude of cortical hypometabolism and the severity and pattern of neuropsychological impairment, the data suggest that the former underlies and/or reflects the latter. Further studies with higher resolution PET devices might shed more light on the relationships between distinct cognitive patterns and specific topography of cortical hypometabolism in BPTI patients.

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H-NMR and 13C-NMR spectroscopy.

We propose an experimental approach combining 1H-NMR and 13C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters. 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with [1-13C]glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2-13C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent. The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1.1. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques.

Effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae cells. Studies by 13C-, 1H-NMR and biochemical methods.

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1-13C]glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using 13C-, 1H-NMR and biochemical methods. The 13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 1H-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 microM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The 13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (approximately 1.0) is practically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 microM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.

[Slowly progressive apraxia: a MRI and positron tomography in 4 cases]. / Apraxie d'aggravation lentement progressive: étude par IRM et tomographie à positons dans 4 cas.

Four right-handed patients (69, 58 and 68 year-old men; 85 year-old woman) complained of motor difficulties with their left hand (3 cases), or both hands predominant on the left side (1 case). Continuous (1 case) or intermittent (2 cases) myoclonus was noted in the left arm. These disorders gradually progressed for 3 to 10 years. Clinical examination disclosed absence of motor, sensory (except in 1 case), or visual deficit. There were no cerebellar signs, no parkinsonian features (except for mild rigidity in 1 case), and no oculomotor abnormality. On the other hand, neuropsychological examination showed evidence of visuo-constructive apraxia in all cases, dressing apraxia in 3/4 cases and writing impairment in 3/4 cases. There was no amnesia, no aphasia and no intellectual impairment. MRI showed atrophy of the parietal areas, predominant on the right side. A positron emission tomography study was performed in all cases, and twice in 1 case. Cortical energy metabolism was measured using either 18 F-fluorodeoxyglucose or 15 O-Oxygen, to calculate the cerebral metabolic rate of glucose (CMRglu) or oxygen (CMRO2) respectively. Cortical metabolism was significantly decreased in the whole cortex of the right hemisphere in 3 cases, and was also reduced in the cortex of the left hemisphere, significantly in 1/3 studied planes. Moreover, regional metabolic indices (CMRO2 or CMRglu/cortex) showed a significant decrease in both the right and left posterior associative areas (temporo-parieto-occipital cortex), predominantly marked on the right side in 3 cases, indicating bilateral cortical dysfunction. At follow-up, one patient became progressively demented, another had visuo-spatial disorders indicating a lesion of both parietal areas. The relationships of our cases with the slowly progressive apraxia syndrome and with corticobasal degeneration are discussed.

Intracranial arachnoid cysts in myotonic dystrophy.

Clinically apparent brain dysfunction is common in myotonic dystrophy. In a sample of fourteen adult patients with the definite form of this disease, brain magnetic resonance imaging detected frequent white matter abnormalities and ventriculomegaly. In addition, two patients exhibited an intracranial arachnoid cyst, a condition of neurosurgical interest that could be related to the generalized dysmaturational process present in this disease. Patients with myotonic dystrophy deserve a careful screening for brain involvement. Further MRI studies should ascertain the actual prevalence of brain anomalies in myotonic dystrophy and define the role of this procedure in the workup of this disease.

Topographic comparative study of magnetic resonance imaging and electroencephalography in 34 children with tuberous sclerosis.

A series of 34 children with confirmed tuberous sclerosis (TS) were studied prospectively by both EEG and magnetic resonance imaging (MRI) at ages ranging from 5 months to 18 years. Size and topography of the cortical tubers were analyzed on axial and coronal views, in T2 sequences, and the large tubers greater than 10 mm were studied. In addition, EEG follow-up data were reviewed retrospectively. Twenty-six patients (76%) had both MRI large cortical tubers and EEG foci, 3 had normal EEG, 2 had normal MRI, and the remaining 3 had only small tubers. The number of large tubers was significantly related to EEG foci. A topographic MRI/EEG correspondence was observed for at least one tuber in 25 of the 26 patients, and correspondence was complete in 10 patients. Large cortical tubers without corresponding EEG foci were observed in 11 patients; these tubers mainly involved the frontal regions and were found before the patients were 2 years old. EEG foci without corresponding tubers were observed in 4 patients. In addition, secondary bilateral synchrony was preferentially observed in patients with frontal lesions and after the age of 2 years. These data confirm that the cortical tubers are epileptogenic and that their expression may be influenced by regional cortical maturation.

Effects of capsular or thalamic stroke on metabolism in the cortex and cerebellum: a positron tomography study.

We used positron emission tomography to study the cortical and cerebellar metabolic rates in 21 strictly selected patients with pure internal capsular infarct (n = 8), thalamocapsular hemorrhage (n = 6), or pure thalamic stroke (n = 7). Significant diffuse ipsilateral cortical hypometabolism relative to 62 controls free of cerebrovascular risk factors was frequently, although not consistently, found in the 13 patients with thalamocapsular or thalamic lesions and neuropsychological impairment but was absent from the eight patients with pure internal capsule infarct and free of neuropsychological deficit. These data suggest that damage to the thalamus or the thalamocortical projections is important in the development of ipsilateral cortical hypometabolism and that the latter may underlie the associated neuropsychological impairment. Significant contralateral cerebellar hypometabolism relative to 49 controls was found in three of six patients with pure internal capsule infarct, suggesting a pathogenetic role for the corticopontocerebellar system. However, the occurrence of hypometabolism in two of six patients with thalamic lesions indicates that this phenomenon may also result either from damage to the ascending cerebellothalamocortical system or indirectly from hypofunction of the cerebral cortex. No systematic association was observed between crossed cerebellar hypometabolism and ipsilateral ataxia.

Study of the cerebral blood flow in partial epilepsy of childhood using the SPECT method.

Cerebral functional imaging methods provide information on the location of the epileptic focus in partial epilepsy of adults. We report our experience of one of these methods, single photon emission computed tomography (SPECT), in epilepsy of children. SPECT enables the regional cerebral blood flow (rCBF) to be measured, after inhalation or injection of 133-Xenon, on 5 contiguous, 20 mm thick axial sections, with a 14 mm resolution and negligible brain irradiation. In Sturge-Weber syndrome (13 patients aged 9 months to 18 years) the rCBF was reduced in the same territory as CT abnormalities suggesting ischaemia of the brain tissue lying below the pial angioma; the SPECT image facilitated the diagnosis in 3 patients with atypical CT. In hemimegalencephaly (6 patients aged 1 month to 10 years) the rCBF was extremely low in the hypertrophic hemisphere and in 1 case the SPECT image was determinant in the decision to perform hemispherectomy. In partial epilepsy with normal CT and/or MRI (42 children aged 1 to 15 years) the rCBF was abnormal in 83% of the patients, and its abnormality was located in the same area as the EEG focus in three quarters of the cases. Between seizures, the rCBF was low in 3 out of 4 cases and abnormality decreased after the seizures had ceased (6 patients explored twice); it was high in 1 out of 4 cases. Thus, in children as in adults, cerebral functional imaging provides new data which contribute to the localization and follow-up of epileptic foci.