Downfield Proton Research Articles

Acute nicotinamide riboside supplementation increases human cerebral NAD+ levels in vivo.

The purpose of this study was to determine the effect of acute nicotinamide riboside (NR) supplementation on cerebral nicotinamide adenine dinucleotide (NAD+) levels in the human brain in vivo by means of downfield proton MRS (DF 1H MRS). DF 1H MRS was performed on 10 healthy volunteers in a 7.0 T MRI scanner with spectrally selective excitation and spatially selective localization to determine cerebral NAD+ levels on two back-to-back days: once after an overnight fast (baseline) and once 4 h after oral ingestion of nicotinamide riboside (900 mg). Additionally, two more baseline scans were performed following the same paradigm to assess test-retest reliability of the NAD+ levels in the absence of NR. NR supplementation increased mean NAD+ concentration compared to the baseline (0.458 ± 0.053 vs. 0.392 ± 0.058 mM; p < 0.001). The additional two baseline scans demonstrated no differences in mean NAD+ concentrations (0.425 ± 0.118 vs. 0.405 ± 0.082 mM; p = 0.45), and no difference from the first baseline scan (F(2, 16) = 0.907; p = 0.424). These preliminary results confirm that acute NR supplementation increases cerebral NAD+ levels in healthy human volunteers and shows the promise of DF 1H MRS utility for robust detection of NAD+ in humans in vivo.

Downfield Proton MRSI at 3 Tesla: A Pilot Study in Human Brain Tumors.

To investigate the use of 3D downfield proton magnetic resonance spectroscopic imaging (DF-MRSI) for evaluation of tumor recurrence in patients with glioblastoma (GBM). Seven patients (4F, age range 44-65 and mean ± standard deviation 59.3 ± 7.5 years) with previously treated GBM were scanned using a recently developed 3D DF-MRSI sequence at 3T. Short TE 3D DF-MRSI and water reference 3D-MRSI scans were collected with a nominal spatial resolution of 0.7 cm3. DF volume data in eight slices covered 12 cm of brain in the cranio-caudal axis. Data were analyzed using the 'LCModel' program and a basis set containing nine peaks ranging in frequency between 6.83 to 8.49 ppm. The DF8.18 (assigned to amides) and DF7.90 peaks were selected for the creation of metabolic images and statistical analysis. Longitudinal MR images and clinical history were used to classify brain lesions as either recurrent tumor or treatment effect, which may include necrosis. DF-MRSI data were compared between lesion groups (recurrent tumor, treatment effect) and normal-appearing brain. Of the seven brain tumor patients, two were classified as having recurrent tumor and the rest were classified as treatment effect. Amide metabolite levels from recurrent tumor regions were significantly (p < 0.05) higher compared to both normal-appearing brain and treatment effect regions. Amide levels in lesion voxels classified as treatment effect were significantly lower than normal brain. 3D DF-MRSI in human brain tumors at 3T is feasible and was well tolerated by all patients enrolled in this preliminary study. Amide levels measured by 3D DF-MRSI were significantly different between treatment effect and tumor regrowth.

Identification of new resonances in downfield 1 H MRS of human calf muscle in vivo: Potentially metabolite precursors for skeletal muscle NAD.

The purpose of this study was to identify and characterize newly discovered resonances appearing in the downfield proton MR spectrum (DF 1 H MRS) of the human calf muscle in vivo at 7T. Downfield 1 H MRS was performed on the calf muscle of five healthy volunteers at 7T. A spectrally selective 90° E-BURP RF pulse with an excitation center frequency at 10.3 ppm and an excitation bandwidth of 2 ppm was used for DF 1 H MRS acquisition. In all participants, we observed new resonances at 9.7, 10.1, 10.3, and 10.9 ppm in the DF 1 H MRS. Phantom experiments at 37°C strongly suggest the new resonance at 9.7 ppm could be from H2-proton of the nicotinamide rings in nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) while the resonance at 10.1 ppm could be attributed to the indole -NH proton of L-tryptophan. We observed that the resonances at 10.1 and 10.9 ppm are significantly suppressed when the water resonance is saturated, indicating that these peaks have either 1 H chemical exchange or cross-relaxation with water. Conversely, the resonances at 9.7 and 10.3 ppm exhibit moderate signal reduction in the presence of water saturation. We have identified new proton resonances in vivo in human calf muscle occurring at chemical shifts of 9.7, 10.1, 10.3, and 10.9 ppm. These preliminary results are promising for investigating the role of NR/NMN and L-tryptophan metabolism in understanding the de novo and salvage pathways of NAD+ synthesis in skeletal muscle.

Identification of l-Tryptophan by down-field 1 H MRS: A precursor for brain NAD+ and serotonin syntheses.

To explore the presence of new resonances beyond 9.4ppm from the human brain, down-field proton MRS was performed in vivo in the human brain on 6 healthy volunteers at 7 T. To maximize the SNR, a large voxel was placed within the brain to cover the maximal area in such a way that sinus cavities were avoided. A spectrally selective 90° E-BURP pulse with an excitation bandwidth of 2ppm was used to probe the spectral chemical shift range between 9.1 and 10.5ppm. The E-BURP pulse was integrated with PRESS spatial localization to obtain non-water-suppressed proton MR spectra from the desired spectral region. In the down-field proton MRS obtained from all of the volunteers scanned, we identified a new peak consistently resonating at 10.1ppm. Protons associated with this resonance are in cross-relaxation with the bulk water, as demonstrated by the water saturation and deuterium exchange experiments. Based on the chemical shift, this new peak was identified as the indole (-NH) proton of l-tryptophan (l-TRP) and was further confirmed from phantom experiments on l-TRP. These promising preliminary results potentially pave the way to investigate the role of cerebral metabolism of l-TRP in healthy and disease conditions.

Magnetic resonance spectroscopic imaging of downfield proton resonances in the human brain at 3 T.

To develop an MRSI technique capable of mapping downfield proton resonances in the human brain. A spectral-spatial excitation and frequency-selective refocusing scheme, in combination with 2D phase encoding, was developed for mapping of downfield resonances without any perturbation of the water magnetization. An alternative scheme using spectral-spatial refocusing was also investigated for simultaneous detection of both downfield and upfield resonances. The method was tested in 5 healthy human volunteers. Downfield metabolite maps with a nominal spatial resolution of 1.5 cm3 were recorded at 3 T in a scan time of 12 minutes. Cramer-Rao lower bounds for nine different downfield peaks were 20% or less over a single supraventricular slice. Downfield spectral profiles were similar to those in the literature recorded previously using single-voxel localization methods. The same approach was also used for upfield MRSI, and simultaneous upfield and downfield acquisitions. The developed MRSI pulse sequence was shown to be an efficient way of rapidly mapping downfield resonances in the human brain at 3 T, maximizing sensitivity through the relaxation enhancement effect. Because the MRSI approach is efficient in terms of data collection and can be readily implemented at short TE, somewhat higher spatial resolution can be achieved than has been reported in previous single-voxel downfield MRS studies. With this approach, nine downfield resonances could be mapped in a single slice for the first time using MRSI at 3 T.

Isolation and Structure Determination of Three New Ceramides from the Starfish Distolasterias nipon.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Isolation and Structure Determination of Three New Ceramides from the Starfish Distolasterias nipon

Starfishes have been reported to contain a number of glycosphingolipids and sterol compounds with unconventional side chain. Some glycosphingolipids have exhibited cytotoxicity, histidine decarboxylate inhibition, DNA topoisomerase I inhibition, moderate wound-healing activity and induced apoptotic DNA damage and cell death in mammalian cell lines. In our search for biologically active and structurally novel lipids from marine organisms, we recently isolated mixtures of several sphingolipids from the starfish Distolasterias nipon collected off the coast of East Sea, Korea. Further HPLC of the fraction resulted in the purification of three new ceramides which are composed of the aglycone part of glycosphingolipids. In this paper we report the isolation and structure determination of new ceramides (compound 1, 2, and 3) isolated from the starfish D. nipon. Compound 1 was isolated as a white amorphous solid which determined for C35H65NO4 by high resolution FABMS. The IR spectrum displayed absorption bands at 3314 (hydroxyl), 1650, and 1540 cm−1 (amide). The H NMR spectrum showed highly overlapped signals at δ 1.29-1.31, indicative of long aliphatic carbon chain, and the C NMR spectrum suggested the presence of one carbonyl carbon, four carbons attached to heteroatoms, and six olefinic carbons on the basis of their characteristic chemical shifts. All these information of 1 allowed us to deduce a ceramide nature containing three double bonds. This is also supported by the H NMR spectrum measured in pyridine-d5 which showed a downfield proton signal at δ 8.38 (d, J = 8.8 Hz) corresponding to the secondary amide proton (Table 1). The main framework of compound 1 was established by extensive 2D NMR techniques. The methine proton at δ 3.84, connected to a nitrogen-bearing carbon, was coupled to the nonequivalent oxymethylene protons (H-1) at δ 3.66 and

The assignment of downfield proton resonances in an enzyme inhibitor complex using time-dependent saturation transferred NOEs.

We have used time-dependent saturation transferred NOE (STNOE) measurements to assign two downfield resonances in the proton spectrum of an adenosine deaminase-purine riboside mixture. Our results show that this method can be used to detect structural changes that occur upon inhibitor binding to the enzyme and to determine which protons of the bound inhibitor are strongly hydrogen bonded in the complex.

Assignment of downfield proton resonances in purine nucleoside phosphorylase immucillin-H complex by saturation-transferred NOEs.

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP. ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP. ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP. ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP. ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A 2 and a Transition State Analog Inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A 2 (sPLA 2, from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17–18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H ε2 of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [ 15N ε2]His. Instead, the downfield resonance arises from H δ1 of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(±0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The p K a of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp…His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).

Characterization of a low barrier hydrogen bond in the active site of chymotrypsin

The possibility of strong hydrogen bonding in aqueous solutions is supported by spectroscopic and chemical properties of proton sponges and internally strained dicarboxylic acid monoanions in aqueous and aqueous/acetone solutions. A species of low barrier hydrogen bond (LBHB) in aqueous solutions is the downfield proton in transition state analogue tetrahedral addition complexes of chymotrypsin with peptide trifluromethylketones. The LBHB proton bridges His57 and Asp102 in the hemiketal adducts of Ser195 with the peptide trifluoromethylketones. The downfield proton resonances (18.6–18.9 ppm, the deterium isotope shift, the low deuterium fractionation factors (0.3–0.4), the close contact of His57 with Asp102 in crystal structures (≤2.6 Å), the high enthalpy of activation for exchange with solvent protons ( ΔH ex 014−19 kcal/ mol) and the high basicity of His57 (p K a 10.6–12) all support the assignment of an LBHB. In contrast to N-acetyl- l-leucyl- l-phenylalanine trifluoromethylketone, the comparable tetrahedral addition complex of the peptide aldehyde N-acetyl- l-leucyl- l-phenylalanal with Ser195 does not lead to an LBHB between His57 and Asp102 at pHs above 7. This is attributed to the high basicity of the hemiacetal adduct (p K a>13.5), so that the available proton in the active site resides on the hemiacetal hydroxyl group and not on His57.

Review: Strong hydrogen bonding in molecules and enzymatic complexes

The physicochemical properties of low barrier hydrogen bonds (LBHBs) and single well hydrogen bonds (SWHBs) are contrasted with the properties of conventional hydrogen bonds. The LBHBs are much more common than the rare, symmetrical SWHBs. LBHBs are characterized by shortened hydrogen bond lengths and correspondingly lengthened covalent bonding, shortened distances between participating heteroatoms, far downfield proton NMR signals, low deuterium fractionation factors and high values of the enthalpy of activation for exchange of the proton with solvent protons. For many years, it was thought that LBHBs could exist only in aprotic media or crystalline or vapor states. Recently, however, the downfield protons associated with hydrogen maleate, hydrogen cis-cyclohexane-1,2-dicarboxylate and hydrogen 2,2-dimethylmalonate have been observed in 0.32 mol fraction water in acetone-d6 at −50 °C. The activation enthalpy for exchange of the downfield proton in hydrogen maleate with solvent water has been found to be 7.2 kcal mol−1, consistent with strong hydrogen bonding. Two examples of LBHBs in enzymes are the downfield proton bridging His 57 and Asp 102 in transition state analog complexes of chymotrypsin, and the proton bridging Tyr 14 and the equilenin-OH group in its complex with 3-ketosteroid isomerase. These protons display the structural, spectroscopic and chemical properties of LBHBs. Their presence in transition state analogue complexes suggests their importance in the transition states of enzymatic reactions. Copyright © 2001 John Wiley & Sons, Ltd.

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.

Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA.

Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11-15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3'-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO-NH2) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3'-terminal ribose. Comparison of the 500-MHz 1H NMR spectra of native and 3'-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (G1 . C120) and its adjacent base pair (G2 . U119) on the basis of their proximity to the 3'-terminal spin-label. From that starting point, we have then identified (G . C, A . U, or G . U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE's.

Assignment of resonances of exchangeable protons in the NMR spectrum of the complex formed by Escherichia coli ribosomal protein L25 and uniformly nitrogen-15 enriched 5 S RNA fragment

The downfield proton NMR spectrum of the aqueous nucleoprotein complex formed by Escherichia coli ribosomal protein L25 and uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Many proton resonances show the effects of scalar coupling to nitrogen-15 and these resonances arc assigned to nucleic acid imino protons. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 and proton chemical shift correlations from which the base types of nucleic acid imino proton resonances could be assigned because the nitrogen-15 chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges for both nucleoproteins and isolated nucleic acids.

Assignment of resonances in the Escherichia coli 5 S RNA fragment proton NMR spectrum using uniform nitrogen-15 enrichment

The downfield proton NMR spectrum of aqueous uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 chemical shifts of fragment imino nitrogens. Nitrogen chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges. Nitrogen-15 and proton chemical shift correlation by the heteronuclear decoupling permitted the identification of the base type of some previously unassigned imino proton resonances in the 5 S RNA fragment spectrum. Corresponding resonances in the natural isotopic abundance 5 S RNA fragment spectrum are assigned to base types by comparison with the enriched sample spectrum. 5 S RNA fragment Proton NMR Uniform 15N enrichment Heteronuclear decoupling Chemical shift correlation Assignment strategy

Assignment of resonances in the downfield proton spectrum of Escherichia coli 5S RNA and its nucleoprotein complexes using components of a ribonuclease-resistant fragment.

The downfield (9-15 ppm) proton NMR spectra of oligonucleotides derived from the ribonuclease A resistant fragment of Escherichia coli 5S RNA have been examined in aqueous solution at 500 MHz. Comparison of these spectra with those of the 5S RNA fragment and intact 5S RNA using both chemical shift and nuclear Overhauser enhancement effect criteria indicates that several aspects of 5S RNA secondary structure are also present in the structures assumed in solution by these much smaller molecules. Analysis of these spectra permits the assignment of some imino proton resonances which could not be assigned with certainty on the basis of NMR data previously obtained on intact 5S RNA or its nucleoprotein complexes. Several previous resonance assignments are confirmed. Studies on oligonucleotide components of fragment and a reconstituted fragment show that at least two conformations of the procaryotic loop exist.

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25.

The downfield (9-15 ppm) proton spectrum of Escherichia coli 5S RNA has been examined at 500 MHz by using nuclear Overhauser methods. The data confirm the existence of the terminal and procaryotic loop helices within the molecule [Fox, G. E., & Woese, C. R. (1975) Nature (London) 256, 505-506]. Very little stable, double-helical structure is detectable in the third loop of the molecule, the one comprising bases 12-68. The downfield spectrum of 5S RNA is perturbed in a highly specific manner upon addition of protein L25 to the system. The changes seen strongly suggest that the binding site for L25 on 5S RNA includes the procaryotic loop helix, but not the terminal stem helix. Similar complexes formed between L25 and the 5S RNA fragment consisting of bases 1-11, 69-87, and 89-120 show exactly the same spectral alterations. A number of downfield resonances appear in the spectra of these complexes which have no counterparts in the free RNA, suggesting the stabilization of new RNA structures by the protein. There are some indications of protein-nucleic acid nuclear Overhauser effects.

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectrum of a ribonuclease-resistant fragment of 5S ribonucleic acid

The downfield (9-15 ppm) proton NMR spectrum of a RNase A resistant fragment of E. coli 5S RNA has been studied by nuclear Overhauser methods. The fragment comprises bases 1-11 and 69-120 of the parent molecule [Douthwaite, S., Garrett, R.A., Wagner, R., & Feunteun, J. (1979) Nucleic Acids Res. 6, 2453-2470]. The nuclear Overhauser data identify two double helical structures in the fragment whose sequences are (GC)3(AU)(GC)3 and (GC)2(AU)(GU). These structures correspond exactly to the central portions of the terminal stem and procaryotic loop helices which should exist in the fragment sequences according to the Fox-Woese model [Fox, G.E., & Woese, C. R. (1975) Nature (London) 256, 505-506] of 5S RNA secondary structure. The significance of these and other nuclear Overhauser effects detected for the structure of 5S RNA and its fragment is discussed.