Metabolism of [1-(13)C)glucose and [2-(13)C]acetate in the hypoxic rat brain.

The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.     

13C-n.m.r. study of C hordein.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.     

13C n.m.r. study of L-alanine peptides

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.     

Synthesis of l-(4-2H)erythrose,l-)1-13C, 5-2H)arabinose and l-(2-13C, 5-2H)arabinose and identification of the intermediates by 2H and 13C-N.M.R. spectroscopy

l-(1-¹³C, 5-²H)Arabinose (6D) and l-(2-¹³C, 5-²H)arabinose (8D) have been synthesized by degradation of 2,3-O-isopropylidene-α-l-rhamnofuranose (2) to l-(4-²H)erythrose (5β,5αD), with subsequent chain elongation to 6D plus l-(1-¹³C, 5-²H)ribose (7D), the latter being converted into 8D. Intermediates were identified by complete assignment of the ¹³C chemical shifts employing carbon-carbon and carbon-deuterium coupling constants, deuteration shifts, differential isotope-shifts, and deuterium spectra. The anomeric carbon atoms of 2 and 2,3-O-isopropylidene-l-(1-²H) erythrose (4D) gave only single ¹³C resonances, suggesting that these two compounds exists in only one major anomeric configuration, clarifying previously reported work. The synthesis of 2,3-O-isopropylidene-l-(1-²H)rhmanitol (3D) facilitated the assignment of the signals in the ¹³C spectra of the nondeuterated analog. Specific deuterium-enrichment and the observed carbon-deuterium coupling (¹J_C,D ∼22 Hz) not only served to identify the deuterated carbon atom unambiguously in 3 but also permitted assignment of closely spaced resonances. The deuterium spectrum of 2,3-O-isopropylidene-l-(1-²H)erythrofuranose (4D) showed only a single resonance, indicating preponderance of one anomer, in accord with the observation of a single C-1 resonance in the ¹³C spectrum.     

A two-dimensional 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) study of N-linked glycopeptides derived from calf fetuin

The oligosaccharide part of an N-linked triantennary glycopeptide from calf fetuin with fourteen carbohydrate residues and its smaller derivatives obtained by successive enzymic cleavage of the terminal residues were investigated using 2D 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) spectroscopy. Assignments have been made of the resonances of almost all the protons of the constituent carbohydrate residues in these glycopeptides. A comparison of the 1H chemical shifts and coupling constants, as determined from the cross-peaks, has shown the dependence of these parameters on the interactions of spatially related neighbouring carbohydrates. Small conformational changes take place upon elongation of the oligosaccharide side-chains.     

DL—1—(2,6—二甲基苯氧基)—2——氨基丙烷氨基酸衍生物的研究

<正> DL—1—(2,6—二甲基苯氧基)—乙—氨基丙烷盐酸盐,商品名为慢心律(Mcxiletine)是七十年代进行临床研究的一个抗心律失常药物。目前国内外广泛用于临床,其特点是可以长期口服治疗和预防室性心律失常。根据国内外近十年的临床观察,若用量超过600—800mg/日,则有明显副作用如神经兴奋、噁心、复视、抽搐、振颤等。为了寻找更理想的抗心律失常药物,国内外已做过一些慢心律的类似物供药理筛选研究。本文是从前体药物的角度以慢心律自由碱DL—1—(2,6—甲基苯氧基)—2—氨基丙     

Conformation of D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP-NH2), a highly selective mu-opioid antagonist peptide, by 1H and 13C n.m.r

The 1H and 13C n.m.r. spectral parameters of CTP-NH2 [D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2], a potent, highly selective mu-opiate antagonist, were measured in aqueous solution and a possible conformation has been deduced from the spectral data. The data are consistent with a type II' beta-turn for the tetrapeptide sequence -Tyr3-D-Trp4-Lys5-Thr6-. Solvent shielding of the Cys2 amide proton, observed in variable temperature experiments, suggests an orientation of this amide proton toward the gem dimethyls of Pen7 with possible hydrogen bonding to the Thr6 carbonyl oxygen, and a dihedral angle of -110 degrees for the disulfide bond. Partially relaxed Fourier transform 13C relaxation studies confirm a constrained cyclic system, with the C alpha carbons in the "hinge" of the beta-turn having the shortest t1 times. Segmental motion was observed for the side chain of Lys5.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

Substituent distribution on O,N-carboxymethylchitosans by 1H and 13C n.m.r.

The use of the n.m.r. method in the investigation of chitosan carboxymethylation was evaluated. It seems to be the most effective technique to determine concurrently the degree and the position of substitution of the carboxymethylated chitosan derivatives. The 13C-n.m.r., by the DEPT method, 1H-1H and 1H-13C-n.m.r. correlations give much valuable information from the chemical shifts of the complex carboxymethylchitosan spectra. The relative reactivity of the functional groups of chitosan towards carboxymethylation was also determined assuming a higher reactivity of the C-6 position.     

Conformation of reduced glutathione in aqueous solution by 1H and 13C n.m.r

We report on the conformation of reduced glutathione in solutions at low and physiological pH, examined with 1H and 13C n.m.r. spectroscopy. The tripeptide in 1H2O was shown to interconvert rapidly between an array of conformers; in addition, the carbon backbone of the glutamyl was more rigid than anticipated if the residue were freely mobile. This restricted motion results from interaction of the alpha-amino and alpha-carboxyl groups on the glutamyl, with the gamma-Glu-Cys peptide-carbonyl and amino, respectively. Our results support theoretical predictions of the conformation but they are at variance with previous ultraviolet spectroscopic and lower field n.m.r. studies.     

Synthesis and n.m.r.-spectral analysis of unenriched and [1-13C]-enriched 5-deoxypentoses and 5-O-methylpentoses

Chemical methods are described for preparing unenriched and [1-13C]-enriched 5-deoxy- and 5-O-methyl-pentoses in the D or L configuration. The 1H-n.m.r. spectra of these compounds have been interpreted, and the 13C-n.m.r. spectra assigned with the aid of 2-D 13C-1H chemical-shift correlation spectroscopy. Tautomeric forms (furanoses, hydrate, and aldehyde) in solution in 2H2O have been quantified with the aid of [1-13C]-enriched derivatives. Spectra of 5-deoxypentoses, 5-O-methylpentoses, and methyl pentofuranosides have been compared, in order to assess the effect of 5-C-deoxygenation and 5-O-methylation on chemical shifts and coupling constants (1H-1H, 13C-1H, and 13C-13C) and on the pentofuranose conformations.     

13C-substituted pentos-2-uloses: synthesis and analysis by 1H- and 13C-n.m.r. spectroscopy

D-erythro-Pentos-2-ulose and D-threo-pentos-2-ulose and their 1-13C- and 2-13C-substituted derivatives have been prepared by oxidizing the corresponding natural and 13C-substituted D-aldopentoses (D-arabinose, D-xylose) with cupric acetate, and purifying the products by chromatography on a cation-exchange resin in the calcium or barium form. The equilibrium compositions of the pentos-2-uloses in 2H2O were determined by 13C-n.m.r. spectroscopy (75 MHz) at 25 degrees and 80 degrees. Among the eighteen possible monomeric acyclic, cyclic, and bicyclic forms, the anomeric pairs of the unhydrated aldopyranoses, aldopyranose endocyclic hydrates, aldofuranose endocyclic hydrates, and ketofuranose exocyclic hydrates were identified on the basis of 13C chemical shifts and 13C-1H and 13C-13C spin-coupling constants. 1H-N.m.r. (300, 500, and 620 MHz) and 13C-n.m.r. (75 MHz) spectroscopic data in one and two dimensions (DQF-COSY, homonuclear 2D-J) were used to evaluate the conformational properties of the cyclic structures. The unhydrated pyranoses are highly conformationally homogeneous; the erythro and threo isomers prefer 1C4 and 4C1 conformations, respectively. D-threo-Pentos-2-ulopyranose hydrate prefers the 4C1 conformation whereas the erythro isomers exists in both the 4C1 and 1C4 conformations. The furanoid forms favor structures having quasi-axial anomeric hydroxyl groups and quasi-equatorial exocyclic hydroxymethyl or dihydroxymethyl groups.     

Isotope-edited 1D and 2D n.m.r. spectroscopy of 13C-substituted carbohydrates.

Three isotope-edited n.m.r. methods have been applied to selectively 13C-substituted monosaccharides and nucleosides to simplify their spectra and/or measure 1H-1H, 13C-1H, or 13H-13C spin-couplings detected via the labeled site. 1D INADEQUATE spectra allowed the selective detection of the natural-abundance carbons that are spin-coupled to the labeled carbon, and adjustment of the mixing time permitted further discrimination between one-bond and longer-range 13C-13C coupling pathways. Geminal and vicinal 13C-1H coupling constants were determined from the analysis of 1H-1H COSY cross-peaks for those protons coupled to the labeled carbon. Long-range 13C-(HETCOR) and 1H-detected (HMBC) 13C-1H chemical-shift correlation spectra permitted the selective observation of those protons coupled to the labeled site, and JH,H values were measured from data projections. The implications of these methods for structural studies of more complex systems is briefly discussed.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

Conformations of proline-containing cyclic peptides. 1H and 13C n.m.r. evidence for a solvent stabilized All-Cis X-Pro conformer of Cyclo-(Pro-Gly-Gly-Pro)2

As inferred from 13C, 1H n.m.r. data, CD measurements and ion-binding experiments, the title molecule can assume two major C2 symmetric conformations. One of these has an all-trans X-Pro peptide backbone and two 1 comes from 4 intramolecular H-bonds and represents the predominant (greater than or equal to 95%) form in D2O and nonpolar (CD3CN) solvents. Stabilized by specific solvent-solute interactions, the other conformer becomes competitive (45%) in DMSO solution. It is shown to possess a four-cis X-Pro skeleton and no intramolecular H-bonds. The Mg++ complex of the cyclic peptide in CD3CN is again C2 symmetric and its formation proceeds via a slow trans leads to cis isomerization of two X-Pro peptide bonds.     

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

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