N-acetyl Signal Research Articles

Rapid and Self-Administrable Capillary Blood Microsampling Demonstrates Statistical Equivalence with Standard Venous Collections in NMR-Based Lipoprotein Analysis.

We investigated plasma and serum blood derivatives from capillary blood microsamples (500 μL, MiniCollect tubes) and corresponding venous blood (10 mL vacutainers). Samples from 20 healthy participants were analyzed by 1H NMR, and 112 lipoprotein subfraction parameters; 3 supramolecular phospholipid composite (SPC) parameters from SPC1, SPC2, and SPC3 subfractions; 2 N-acetyl signals from α-1-acid glycoprotein (Glyc), GlycA, and GlycB; and 3 calculated parameters, SPC (total), SPC3/SPC2, and Glyc (total) were assessed. Using linear regression between capillary and venous collection sites, we explained that agreement (Adj. R2 ≥ 0.8, p < 0.001) was witnessed for 86% of plasma parameters (103/120) and 88% of serum parameters (106/120), indicating that capillary lipoprotein, SPC, and Glyc concentrations follow changes in venous concentrations. These results indicate that capillary blood microsamples are suitable for sampling in remote areas and for high-frequency longitudinal sampling of the majority of lipoproteins, SPCs, and Glycs.

J-Edited DIffusional Proton Nuclear Magnetic Resonance Spectroscopic Measurement of Glycoprotein and Supramolecular Phospholipid Biomarkers of Inflammation in Human Serum.

Proton nuclear magnetic resonance (NMR) N-acetyl signals (Glyc) from glycoproteins and supramolecular phospholipids composite peak (SPC) from phospholipid quaternary nitrogen methyls in subcompartments of lipoprotein particles) can give important systemic metabolic information, but their absolute quantification is compromised by overlap with interfering resonances from lipoprotein lipids themselves. We present a J-Edited DIffusional (JEDI) proton NMR spectroscopic approach to selectively augment signals from the inflammatory marker peaks Glyc and SPCs in blood serum NMR spectra, which enables direct integration of peaks associated with molecules found in specific compartments. We explore a range of pulse sequences that allow editing based on peak J-modulation, translational diffusion, and T2 relaxation time and validate them for untreated blood serum samples from SARS-CoV-2 infected patients (n = 116) as well as samples from healthy controls and pregnant women with physiological inflammation and hyperlipidemia (n = 631). The data show that JEDI is an improved approach to selectively investigate inflammatory signals in serum and may have widespread diagnostic applicability to disease states associated with systemic inflammation.

Diffusion and Relaxation Edited Proton NMR Spectroscopy of Plasma Reveals a High-Fidelity Supramolecular Biomarker Signature of SARS-CoV-2 Infection.

We have applied nuclear magnetic resonance spectroscopy based plasma phenotyping to reveal diagnostic molecular signatures of SARS-CoV-2 infection via combined diffusional and relaxation editing (DIRE). We compared plasma from healthy age-matched controls (n = 26) with SARS-CoV-2 negative non-hospitalized respiratory patients and hospitalized respiratory patients (n = 23 and 11 respectively) with SARS-CoV-2 rRT-PCR positive respiratory patients (n = 17, with longitudinal sampling time-points). DIRE data were modelled using principal component analysis and orthogonal projections to latent structures discriminant analysis (O-PLS-DA), with statistical cross-validation indices indicating excellent model generalization for the classification of SARS-CoV-2 positivity for all comparator groups (area under the receiver operator characteristic curve = 1). DIRE spectra show biomarker signal combinations conferred by differential concentrations of metabolites with selected molecular mobility properties. These comprise the following: (a) composite N-acetyl signals from α-1-acid glycoprotein and other glycoproteins (designated GlycA and GlycB) that were elevated in SARS-CoV-2 positive patients [p = 2.52 × 10–10 (GlycA) and 1.25 × 10–9 (GlycB) vs controls], (b) two diagnostic supramolecular phospholipid composite signals that were identified (SPC-A and SPC-B) from the –+N–(CH3)3 choline headgroups of lysophosphatidylcholines carried on plasma glycoproteins and from phospholipids in high-density lipoprotein subfractions (SPC-A) together with a phospholipid component of low-density lipoprotein (SPC–B). The integrals of the summed SPC signals (SPCtotal) were reduced in SARS-CoV-2 positive patients relative to both controls (p = 1.40 × 10–7) and SARS-CoV-2 negative patients (p = 4.52 × 10–8) but were not significantly different between controls and SARS-CoV-2 negative patients. The identity of the SPC signal components was determined using one and two dimensional diffusional, relaxation, and statistical spectroscopic experiments. The SPCtotal/GlycA ratios were also significantly different for control versus SARS-CoV-2 positive patients (p = 1.23 × 10–10) and for SARS-CoV-2 negatives versus positives (p = 1.60 × 10–9). Thus, plasma SPCtotal and SPCtotal/GlycA are proposed as sensitive molecular markers for SARS-CoV-2 positivity that could effectively augment current COVID-19 diagnostics and may have value in functional assessment of the disease recovery process in patients with long-term symptoms.

Polycythemia Vera and Essential Thrombocythemia Patients Exhibit Unique Serum Metabolic Profiles Compared to Healthy Individuals and Secondary Thrombocytosis Patients.

Simple SummaryCurrent diagnosis of myeloproliferative neoplasms (MPNs), including polycythemia vera (PV) and essential thrombocythemia (ET), is controversial due to limitations associated with the lack of reproducibility, subjectivity and the presence of common somatic mutations in the driver genes. Metabolomics represents a powerful approach to identify altered metabolites that can differentiate between disease status at the time of diagnosis. The objective of this study was to characterize the serum metabolic profile of MPNs patients (PV and ET) and compare it with healthy controls (HC) and secondary thrombocytosis (ST) patients. The analysis revealed metabolites following similar trends between PV and ET patients, as well as unique significant differences in the serum metabolite levels of MNPs patients compared to HC and ST patients. These results could contribute to better differentiate patients with these diseases from HC and ST patients.Most common myeloproliferative neoplasms (MPNs) include polycythemia vera (PV) and essential thrombocythemia (ET). Accurate diagnosis of these disorders remains a clinical challenge due to the lack of specific clinical or molecular features in some patients enabling their discrimination. Metabolomics has been shown to be a powerful tool for the discrimination between different hematological diseases through the analysis of patients’ serum metabolic profiles. In this pilot study, the potential of NMR-based metabolomics to characterize the serum metabolic profile of MPNs patients (PV, ET), as well as its comparison with the metabolic profile of healthy controls (HC) and secondary thrombocytosis (ST) patients, was assessed. The metabolic profile of PV and ET patients, compared with HC, exhibited higher levels of lysine and decreased levels of acetoacetic acid, glutamate, polyunsaturated fatty acids (PUFAs), scyllo-inositol and 3-hydroxyisobutyrate. Furthermore, ET patients, compared with HC and ST patients, were characterized by decreased levels of formate, N-acetyl signals from glycoproteins (NAC) and phenylalanine, while the serum profile of PV patients, compared with HC, showed increased concentrations of lactate, isoleucine, creatine and glucose, as well as lower levels of choline-containing metabolites. The overall analysis revealed significant metabolic alterations mainly associated with energy metabolism, the TCA cycle, along with amino acid and lipid metabolism. These results underscore the potential of metabolomics for identifying metabolic alterations in the serum of MPNs patients that could contribute to improving the clinical management of these diseases.

Assessment of glycosaminoglycan concentration changes in the intervertebral disc via chemical exchange saturation transfer.

In this study, it is shown that the chemical exchange saturation transfer (CEST) method for hydroxyl protons can be used to detect changes in glycosaminoglycan (GAG) concentration in the intervertebral disc. The method, termed gagCEST, was demonstrated ex vivo by correlating the CEST effect with the fixed charge density (FCD) of the nucleus pulposus (NP), as well as by correlating tissue CEST images with their corresponding (23)Na images. Incubation of five NP samples with trypsin produced samples with varying GAG content (n = 19). A good correlation was found between the -OH CEST effect and FCD, as well as with the N-acetyl signal amplitude. gagCEST images in vitro further illustrated the amount of detail obtainable from this contrast mechanism when compared with conventional imaging. The large concentration of GAG and the relatively long T(1) of water in NP make the method sensitive, in particular, for the assessment of GAG depletion in this tissue. It is the loss of GAG in NP that indicates the early stage of disc degeneration.

Epitope Diversity of N-Glycans from Bovine Peripheral Myelin Glycoprotein P0 Revealed by Mass Spectrometry and Nano Probe Magic Angle Spinning 1H NMR Spectroscopy

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.

A new metabolite contributing to N-acetyl signal in 1H MRS of the brain in Salla disease.

To determine whether N-acetylaspartate (NAA) is reduced in patients with Salla disease, a neurodegenerative disorder. 1H MRS allows the brain metabolism to be studied noninvasively in vivo. N-acetyl (NA) is composed primarily of NAA, which is regarded as a neuronal marker. The NA signal in 1H MRS is reduced in several neurodegenerative disorders. Increased NA signal has thus far only been found in Canavan's disease as a result of NAA accumulation in the brain tissue. In Salla disease, an autosomal recessive free sialic acid storage disorder, N-acetylneuraminic acid (NANA), accumulates in lysosomes of brain tissue. The authors studied eight patients with Salla disease (age range, 6 to 44 years) and eight age-matched healthy volunteers using quantitative 1H MRS. The spectra were obtained from two selected 8-cm3 volumes of interest localized in the basal ganglia and in the parietal white matter using conventional 1.5-T MRI equipment. The spectral resonance lines of NA groups, creatine and phosphocreatine (Cr), and choline-containing compounds (Cho) were analyzed quantitatively. All MR images were evaluated to verify the state of myelination. 1H MRS from parietal white matter revealed 34% higher NA and 47% higher Cr concentrations, and a 35% lower Cho concentration in the patients with Salla disease compared with the age-matched control subjects. The patients had a 22% higher water content in their parietal white matter, whereas in the basal ganglia the water concentrations did not differ significantly. In the patients' basal ganglia the Cr concentration was 53% higher. NAA is considered to be a neuronal marker that, except for Canavan's disease, has been found or assumed to be either stable or reduced. However, in Salla disease the high NA signal may have a contribution from accumulated lysosomal NANA, which offsets the possible loss of NAA. The high Cr is in line with the increased glucose uptake found in our earlier 2-fluoro-2-deoxy-D-glucose-PET study, reflecting increased energy demand. It is worth noting that in a conventional 1H MRS ratio-based analysis these underlying abnormalities would have remained undetected. Our study thus emphasizes the importance of a quantitative assessment of metabolite concentrations in 1H MRS for detecting altered brain metabolism.

Clinical correlates of proton magnetic resonance spectroscopy findings after acute cerebral infarction.

We sought to determine whether lactate and N-acetyl signals measured by proton magnetic resonance spectroscopy (MRS) in the first days after stroke correlate with clinical measures of disability and functional outcome. One-dimensional spectroscopic imaging was performed after stroke on 32 patients using a 2.1-T magnet. The Toronto Stroke Scale score at the time of the MRS study and the Barthel Index score at hospital discharge were determined from patient records. Lesion volume was estimated by a tracing algorithm from the scout magnetic resonance image obtained as part of the MRS study. The scaled lactate and N-acetyl signals from the voxel having the highest measured lactate were used to predict the clinical variables and lesion volume, as well as relative perfusion within the lesion, in those patients who underwent single-photon emission computed tomography (SPECT) blood flow imaging, using a multiple regression analysis. The correlation of lesion volume with the clinical variables was also evaluated. Lesion lactate signal was correlated with the Toronto Stroke Scale score, Barthel Index score, lesion volume, and SPECT score, all at P < .01. The N-acetyl level correlated with the Barthel Index score and lesion volume at P < .05. Lesion volume was also strongly correlated with the clinical variables (P < .0001). This is the first study to document the clinical predictive value of proton MRS measurements in patients after stroke. The association with functional outcome is stronger for lactate than for N-acetyl. Spectroscopic assessment of the metabolic status of cerebral tissues shortly after infarction may have significant clinical utility.

Absolute quantitation of short TE brain 1H-MR spectra and spectroscopic imaging data.

A method for determining the concentrations of the materials that produce the well-resolved singlet signals in short TE brain 1H MR spectroscopic examinations is presented. Concentration determination is achieved by a water-referencing procedure. The ratios of the areas of the choline, total creatine, and N-acetyl signals to that of the water signal from the same volume of interest (VOI) are determined using acquisitions with and without water suppression. The tissue concentrations of the molecules producing the three signals can then be determined if the water concentration in the VOI can be found. This is done with a density-weighted MR study. The MR study provides the ratio of the mean MR signal amplitude from the VOI to that from an external standard containing a known water concentration. The method's flexibility is illustrated by using it with two different single-volume localization schemes and spectroscopic imaging. Preliminary evaluations of accuracy and reproducibility are made in phantom, animal, and limited human studies. The method's advantages and limitations are discussed.

Hydrogen-deuterium isotope effects in 13C NMR experiments of peptides

In our recent NMR studies of linear peptides (l-3), ranging from 4 to 10 amino acids, we observed an interesting and unexpected line-splitting effect in the backbone 13C resonances. This effect was observed first in NAc-D-E-K-S-NH2 ( I ) , a fragment of the type I collagen (L1 chain N-telopeptide (4). A very similar effect was also found in two decapeptides related to the luteinizing hormone-releasing hormone (LHRH), namely the LHRH anti-sense (5) and reversed anti-sense peptide (2). In all these cases the C,, the backbone carbonyl, and the N-acetyl signals were each recorded as two lines separated by approximately 0.1 ppm whereas the signals of the side-chain carbons appeared as sharp lines. As there was independent and unambiguous evidence from the ‘H data that at least two of the three peptides mentioned were neither completely random nor extended, but rather an equilibrium between a type I p-turn with extended conformers ( I ) and a 3 iO-helical turn together with extended conformers (LHRH anti-sense (2))) we first considered the possibility of the presence of two conformers being the reason for the line doubling. However, a more recent study of the Cand N-terminal telopeptides of the type I collagen (u-2 chain (a hexapeptide and a nonapeptide, respectively), which provided up to four lines per a-carbon, revealed that this phenomenon cannot be conformation-related, since for both peptides the proton NMR data (3) clearly confirmed completely extended conformations. This conclusion was based on the absence of any particularly small or large 3 J Nn na _ COUphUg COnStantS, the lack Of NH ( i) -NH ( i + 1) nuclear Overhauser effects, and the absence of reduced NH temperature coefficients, all of which are in general associated with nonextended structures (6, 7). In our quest for the correct explanation of the phenomenon, we found a close relationship between the proton:deuteron ratio in solution and the observed peak intensities. All our experiments were carried out in so-called mixed solvents, containing a mixture of water and an organic solvent, such as trifluoroethanol or methanol, to mimic the physiological, nonaqueous conditions as found, for example, in the growing collagen fibrils. For most of our studies water/methanol solutions were used, typically 60 v% CD30H and 40 v% HzO. Half of the latter was added as D@ to

Maternal and cord blood plasma. Comparative analyses by 1H NMR spectroscopy

Normal and 'Hahn' spin-echo 500 MHz 1H NMR spectra of 21 pairs of plasma from mother and cord taken at the time of delivery are reported. The concentrations of 'NMR-visible' lactate, alanine, and valine in the cord plasma significantly exceed those in the maternal plasma. In both maternal and cord plasma, measured lactate levels appear to increase with the length of the second stage of labour. Ethanol was unexpectedly detected in five samples of cord plasma. Lipoprotein--CH3 and--(CH2)n--resonances are more intense in the spectra of maternal plasma compared to cord plasma, and are unusual in composition. The intensities of N-acetyl signals from 'acute-phase' plasma glycoproteins are greater in spin-echo spectra of maternal plasma compared to those of cord plasma (mean ratio 2.1 (SD 0.8]. These results suggest that high resolution NMR spectroscopy may provide a useful new insight into the comparative biochemistry of maternal and cord plasma.

Cassytha alkaloids. III. Aporphine alkaloids from Cassytha melantha R. Br

The major alkaloid of Cassytha melantha R.Br, and Cassytha glabella R.Br. is (+)-9-hydroxy-10-methoxy-1,2-methylenedioxyaporphine (cassythicine), which has not been previously found to occur naturally, although the (�)-form has been synthesized by Tomita and Kikkawa. A minor alkaloid from C. melantha, isolated only in the form of an NO- diacetyl derivative, appears to be the noraporphine corresponding to cassythicine and it is presumably identical with the known alkaloid actinodaphnine. A study has been made of the N.M.R. spectra of a number of N- acetyl-N-methylaminoethylphenanthrenes, obtained from aporphines by heating with acetic anhydride. These show not only the expected splitting of the N-methyl and N-acetyl signals due to cis-trans isomerism of the amide group, but also a long-range splitting of the signals from certain aryl protons.