Decreased TCA cycle rate in the rat brain after acute 3-NP treatment measured by in vivo 1H-[13C] NMR spectroscopy

Inhibition of succinate dehydrogenase (SDH) by the mitochondrial toxin 3-nitropropionic acid (3-NP) has gained acceptance as an animal model of Huntington's disease. In this study 13C NMR spectroscopy was used to measure the tricarboxylic acid (TCA) cycle rate in the rat brain after 3-NP treatment. The time course of both glutamate C4 and C3 13C labelling was monitored in vivo during an infusion of [1-13C]glucose. Data were fitted by a mathematical model to yield the TCA cycle rate (Vtca) and the exchange rate between alpha-ketoglutarate and glutamate (Vx). 3-NP treatment induced a 18% decrease in Vtca from 0.71 +/- 0.02 micro mol/g/min in the control group to 0.58 +/- 0.02 micro mol/g/min in the 3-NP group (p < 0.001). Vx increased from 0.88 +/- 0.08 micro mol/g/min in the control group to 1.33 +/- 0.24 micro mol/g/min in the 3-NP group (p < 0.07). Fitting the C4 glutamate time course alone under the assumption that Vx is much higher than Vtca yielded Vtca=0.43 micro mol/g/min in both groups. These results suggest that both Vtca and Vx are altered during 3-NP treatment, and that both glutamate C4 and C3 labelling time courses are necessary to obtain a reliable measurement of Vtca.     

Group I and II metabotropic glutamate receptors alter brain cortical metabolic and glutamate/glutamine cycle activity: a 13C NMR spectroscopy and metabolomic study

Metabotropic glutamate receptors (mGluR) modulate neuronal function. Here, we tested the effect on metabolism of a range of Group I and II mGluR ligands in Guinea pig brain cortical tissue slices, applying 13C NMR spectroscopy and metabolomic analysis using multivariate statistics. The effects of Group I agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) depended upon concentration and were mostly stimulatory, increasing both net metabolic flux through the Krebs cycle and glutamate/glutamine cycle activity. Only the higher (50 microm) concentrations of CHPG had the opposite effect. The Group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), consistent with its neuroprotective role, caused significant decreases in metabolism. With principal components analysis of the metabolic profiles generated by these ligands, the effects could be separated by two principal components. Agonists at Group II mGluR [(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC)] generally stimulated metabolism, including glutamate/glutamine cycling, although this varied with concentration. The antagonist (2S)-alpha-ethylglutamic acid (EGLU) stimulated astrocyte metabolism with minimal impact on glutamate/glutamine cycling. (RS)-1-Aminophosphoindan-1-carboxylic acid (APICA) decreased metabolism at 5 microm but had a stimulatory effect at 50 microm. All ligand effects were separated from control and from each other using two principal components. The ramifications of these findings are discussed.     

Differential effects of ethanol on regional glutamatergic and GABAergic neurotransmitter pathways in mouse brain

This study investigates the effects of ethanol on neuronal and astroglial metabolism using ¹H‐[¹³C]‐NMR spectroscopy in conjunction with infusion of [1,6‐¹³C₂]/[1‐¹³C]glucose or [2‐¹³C]acetate, respectively. A three‐compartment metabolic model was fitted to the ¹³C turnover of Glu_C3, Glu_C4, GABA_C2, GABA_C3, Asp_C3, and Gln_C4 from [1,6‐¹³C₂]glucose to determine the rates of tricarboxylic acid (TCA) and neurotransmitter cycle associated with glutamatergic and GABAergic neurons. The ratio of neurotransmitter cycle to TCA cycle fluxes for glutamatergic and GABAegic neurons was obtained from the steady‐state [2‐¹³C]acetate experiment and used as constraints during the metabolic model fitting. ¹H MRS measurement suggests that depletion of ethanol from cerebral cortex follows zero order kinetics with rate 0.18 ± 0.04 μmol/g/min. Acute exposure of ethanol reduces the level of glutamate and aspartate in cortical region. Gln_C4 labeling was found to be unchanged from a 15 min infusion of [2‐¹³C]acetate suggesting that acute ethanol exposure does not affect astroglial metabolism in naive mice. Rates of TCA and neurotransmitter cycle associated with glutamatergic and GABAergic neurons were found to be significantly reduced in cortical and subcortical regions. Acute exposure of ethanol perturbs the level of neurometabolites and decreases the excitatory and inhibitory activity differentially across the regions of brain.

     

In vivo 13C NMR study of glucose and cellobiose metabolism by four cellulolytic strains of the genus Fibrobacter

The metabolism of glucose and cellobiose, products of cellulose hydrolysis, was investigated in four cellulolytic strains of the genus Fibrobacter: Fibrobacter succinogenes S85, 095, HM2 and Fibrobacter intestinalis NR9. In vivo 13C nuclear magnetic resonance was used to quantify the relative contribution of glucose and cellobiose to metabolite production, glycogen storage and cellodextrins synthesis in these four strains. The same features were found in all four strains of the genus Fibrobacter metabolizing simultaneously glucose and cellobiose: i) differential metabolism of glucose and cellobiose; glucose seems preferentially used for glycogen storage and energy production, while part of cellobiose seems to be diverted from glycolysis, ii) synthesis of cellodextrins, mainly from cellobiose not entering into glycolysis, iii) accumulation of glucose 6-phosphate, iv) simultaneous presence of cellobiose phosphorylase and cellobiase activities.Although genetically diverse, the Fibrobacter genus appears to possess a marked homogeneity in its carbon metabolism.     

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 .     

In vivo NMR studies of regional cerebral energetics in MPTP model of Parkinson's disease: recovery of cerebral metabolism with acute levodopa treatment

In this study, we have evaluated cerebral atrophy, neurometabolite homeostasis, and neural energetics in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridin (MPTP) model of Parkinson's disease. In addition, the efficacy of acute l ‐DOPA treatment for the reversal of altered metabolic functions was also evaluated. Cerebral atrophy and neurochemical profile were monitored in vivo using MRI and ¹H MR Spectroscopy. Cerebral energetics was studied by ¹H‐[¹³C]‐NMR spectroscopy in conjunction with infusion of ¹³C labeled [1,6^‐13C₂]glucose or [2‐¹³C]acetate. MPTP treatment led to reduction in paw grip strength and increased level of GABA and myo‐inositol in striatum and olfactory bulb. ¹³C Labeling of glutamate‐C4 (1.93 ± 0.24 vs. 1.48 ± 0.06 μmol/g), GABA‐C2 (0.24 ± 0.04 vs. 0.18 ± 0.02 μmol/g) and glutamaine‐C4 (0.26 ± 0.04 vs. 0.20 ± 0.04 μmol/g) from [1,6‐¹³C₂]glucose was found to be decreased with MPTP exposure in striatum as well as in other brain regions. However, glutamine‐C4 labeling from [2‐¹³C]acetate was found to be increased in the striatum of the MPTP‐treated mice. Acute l ‐DOPA treatment failed to normalize the increased ventricular size and level of metabolites but recovered the paw grip strength and ¹³C labeling of amino acids from [1,6‐¹³C₂]glucose and [2‐¹³C]acetate in MPTP‐treated mice. These data indicate that brain energy metabolism is impaired in Parkinson's disease and acute l ‐DOPA therapy could temporarily recover the cerebral metabolism.

     

Neuron-astrocyte interactions in the regulation of brain energy metabolism: a focus on NMR spectroscopy

An adequate and timely production of ATP by brain cells is of cardinal importance to support the major energetic cost of the rapid processing of information via synaptic and action potentials. Recently, evidence has been accumulated to support the view that the regulation of brain energy metabolism is under the control of an intimate dialogue between astrocytes and neurons. In vitro studies on cultured astrocytes and in vivo studies on rodents have provided evidence that glutamate and Na(+) uptake in astrocytes is a key triggering signal regulating glucose use in the brain. With the advent of NMR spectroscopy, it has been possible to provide experimental evidence to show that energy consumption is mainly devoted to glutamatergic neurotransmission and that glutamate-glutamine cycling is coupled in a approximately 1 : 1 molar stoichiometry to glucose oxidation, at least in the cerebral cortex. This improved understanding of neuron-astrocyte metabolic interactions offers the potential for developing novel therapeutic strategies for many neurological disorders that include a metabolic deficit.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.

     

Time-dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1-(13) C]glucose metabolism

[1-(13) C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono- and dicarboxylic amino acids were determined from (1) H-observed/(13) C-edited and (13) C-NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate-glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re-evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.     

Brain pyruvate recycling and peripheral metabolism: an NMR analysis ex vivo of acetate and glucose metabolism in the rat

The occurrence of pyruvate recycling in the rat brain was studied in either pentobarbital anesthetized animals or awake animals receiving a light analgesic dose of morphine, which were infused with either [1-13C]glucose + acetate or glucose + [2-13C]acetate for various periods of time. Metabolite enrichments in the brain, blood and the liver were determined from NMR analyses of tissue extracts. They indicated that: (i) Pyruvate recycling was revealed in the brain of both the anesthetized and awake animals, as well as from lactate and alanine enrichments as from glutamate isotopomer composition, but only after infusion of glucose + [2-13C]acetate. (ii) Brain glucose was labelled from [2-13C]acetate at the same level in anaesthetized and awake rats (approximately 4%). Comparing its enrichment with that of blood and liver glucose indicated that brain glucose labelling resulted from hepatic gluconeogenesis. (iii) Analysing glucose 13C-13C coupling in the brain, blood and the liver confirmed that brain glucose could be labelled in the liver through the activities of both pyruvate recycling and gluconeogenesis. (iv) The rate of appearance and the amount of brain glutamate C4-C5 coupling, a marker of pyruvate recycling when starting from [2-13C]acetate, were lower than those of brain glucose labelling from hepatic metabolism. (v) The evaluation of the contributions of glucose and acetate to glutamate metabolism revealed that more than 60% of brain glutamate was synthesized from glucose whereas only 7% was from acetate and that glutamate C4-C5 coupling was mainly due to the metabolism of glucose labelled through hepatic gluconeogenesis. All these results indicate that, under the present conditions, the pyruvate recycling observed through the labelling of brain metabolites mainly originates from peripheral metabolism.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Metabolic products of [2‐13C]ethanol in the rat brain after chronic ethanol exposure

Most ingested ethanol is metabolized in the liver to acetaldehyde and then to acetate, which can be oxidized by the brain. This project assessed whether chronic exposure to alcohol can increase cerebral oxidation of acetate. Through metabolism, acetate may contribute to long‐term adaptation to drinking. Two groups of adult male Sprague–Dawley rats were studied, one treated with ethanol vapor and the other given room air. After 3 weeks the rats received an intravenous infusion of [2‐¹³C]ethanol via a lateral tail vein for 2 h. As the liver converts ethanol to [2‐¹³C]acetate, some of the acetate enters the brain. Through oxidation the ¹³C is incorporated into the metabolic intermediate α‐ketoglutarate, which is converted to glutamate (Glu), glutamine (Gln), and GABA. These were observed by magnetic resonance spectroscopy and found to be ¹³C‐labeled primarily through the consumption of ethanol‐derived acetate. Brain Gln, Glu, and, GABA ¹³C enrichments, normalized to ¹³C‐acetate enrichments in the plasma, were higher in the chronically treated rats than in the ethanol‐naïve rats, suggesting increased cerebral uptake and oxidation of circulating acetate. Chronic ethanol exposure increased incorporation of systemically derived acetate into brain Gln, Glu, and GABA, key neurochemicals linked to brain energy metabolism and neurotransmission.

     

Effect of chronic hypoglycaemia on glucose concentration and glycogen content in rat brain: A localized 13C NMR study

While chronic hypoglycaemia has been reported to increase unidirectional glucose transport across the blood-brain barrier (BBB) and to increase GLUT1 expression at the endothelium, the effect on steady-state brain d-glucose and brain glycogen content is currently unknown. Brain glucose and glycogen concentrations were directly measured in vivo using localized 13C magnetic resonance spectroscopy (MRS) following 12-14 days of hypoglycaemia. Brain glucose content was significantly increased by 48%, which is consistent with an increase in the maximal glucose transport rate, Tmax, by 58% compared with the sham-treated animals. The localized 13C NMR measurements of brain glucose were directly validated by comparison with biochemically determined brain glucose content after rapid focused microwave fixation (1.4 s at 4 kW). Both in vivo MRS and biochemical measurements implied that brain glycogen content was not affected by chronic hypoglycaemia, consistent with brain glucose being a major factor controlling brain glycogen content. We conclude that the increased glucose transporter expression in chronic hypoglycaemia leads to increased brain glucose content at a given level of glycaemia. Such increased brain glucose concentrations can result in a lowered glycaemic threshold of counter-regulation observed in chronic hypoglycaemia.     

Neurochemical profile of the developing mouse cortex determined by in vivo 1H NMR spectroscopy at 14.1 T and the effect of recurrent anaesthesia

The neurochemical profile of the cortex develops in a region and time specific manner, which can be distorted by psychiatric and other neurological pathologies. Pre-clinical studies often involve experimental mouse models. In this study, we determined the neurochemical profile of C57BL/6 mice in a longitudinal study design to provide a reference frame for the normal developing mouse cortex. Using in vivo proton NMR spectroscopy at 14 T, we measured the concentrations of 18 metabolites in the anterior and posterior cortex on postnatal days (P) 10, 20, 30, 60 and 90. Cortical development was marked by alterations of highly concentrated metabolites, such as N-acetylaspartate, glutamate, taurine and creatine. Regional specificity was represented by early variations in the concentration of glutamine, aspartate and choline. In adult animals, regional concentration differences were found for N-acetylaspartate, creatine and myo-inositol. In this study, animals were exposed to recurrent isoflurane anaesthesia. Additional experiments showed that the latter was devoid of major effects on behaviour or cortical neurochemical profile. In conclusion, the high sensitivity and reproducibility of the measurements achieved at 14 T allowed us to identify developmental variations of cortical areas within the mouse cortex.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.     

Intracellular PHB conversion in a Type II methanotroph studied by 13C NMR

Poly-β-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [¹³C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by ¹³C NMR. During PHB synthesis in the presence of labelled acetate, low levels of β-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully ¹³C-enriched cells, which were grown on ¹³C-labelled methane. β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple ¹³C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 15–21.     

Altered neurochemical profile in the McGill‐R‐Thy1‐APP rat model of Alzheimer's disease: a longitudinal in vivo 1H MRS study

We investigated metabolite levels during the progression of pathology in McGill‐R‐Thy1‐APP rats, a transgenic animal model of Alzheimer's disease, and in healthy age‐matched controls. Rats were subjected to in vivo ¹H magnetic resonance spectroscopy (MRS) of the dorsal hippocampus at age 3, 9 and 12 months and of frontal cortex at 9 and 12 months. At 3 months, a stage in which only Aβ oligomers are present, lower glutamate, myo‐inositol and total choline content were apparent in McGill‐R‐Thy1‐APP rats. At age 9 months, lower levels of glutamate, GABA, N‐acetylaspartate and total choline and elevated myo‐inositol and taurine were found in dorsal hippocampus, whereas lower levels of glutamate, GABA, glutamine and N‐acetylaspartate were found in frontal cortex. At age 12 months, only the taurine level was significantly different in dorsal hippocampus, whereas taurine, myo‐inositol, N‐acetylaspartate and total creatine levels were significantly higher in frontal cortex. McGill‐R‐Thy1‐APP rats did not show the same changes in metabolite levels with age as displayed in the controls, and overall, prominent and complex metabolite differences were evident in this transgenic rat model of Alzheimer's disease. The findings also demonstrate that in vivo ¹H MRS is a powerful tool to investigate disease‐related metabolite changes in the brain.     

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

Summary Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural ¹³C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH₂ side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, ¹³C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.     

Determination of optical purity of 3,5-dimethoxybenzoyl-leucine diethylamide by chiral chromatography and 1H and 13C NMR spectroscopy

(R)-N-3,5-dinitrobenzoyl (DNB) leucine derived chiral selector was used as an HPLC chiral stationary phase for the resolution of various racemic amino acids derivatives. In this study, determination of optical purity of an amino acid derivative was performed by chiral high performance liquid chromatography and 1H and 13C NMR spectroscopy by using the DNB leucine derived chiral selector. The accuracy and precision of each optical purity value are calculated and the data are compared to each other.