Search Results (53)

Metabolic labeling with deuterated water is used in combination with liquid-chromatography coupled with mass spectrometry to study the turnover rates of individual proteins in vivo. This technique and bioinformatics tools for data analysis quantify the turnover rates of thousands of proteins. Turnover rates change during organismal growth and respond to alterations in the environment and diet. The accurate and statistically significant determination of the turnover rate changes of a protein depend on the variations in the turnover rates of the peptides of the protein. One of the systematic factors contributing to this variability is the dependence of the turnover rates on the number of exchangeable hydrogens of the peptides. This variability (by reducing the statistical power) reduces biological interpretability. Here, we propose a computational approach to eliminate the dependence of the turnover rates on the number of exchangeable hydrogens. This approach enhances the accuracy of turnover rate estimation and may help to support more accurate assessments of biological dynamics and disease mechanisms. Full article

Poloxamer-based drug delivery systems are widely used in the pharmaceutical sector. The structural characterization of these systems is crucial for the development of new drug delivery systems and for the optimization of their properties. In this study, we utilized small-angle neutron scattering (SANS) to investigate the structures of poloxamer-based drug delivery systems. The samples were measured using the SANS technique on the VSANS-V16 instrument at Helmholtz-Zentrum Berlin (HZB), Germany. The samples contained 20% poloxamer (P407) and 0.2% of a drug (ibuprofen, ketoprofen, diclofenac) in deuterated water (D₂O) for SANS. The samples varied in terms of temperature analysis (25 °C, common storage temperature; 37 °C, human body temperature; 40 °C, fever temperature). The data analysis involved modeling the data using a Python-based routine. The model used consisted of an isotropic solution of polydisperse spherical micelles. The intensity as a function of the scattering vector was modeled as the product of the form factor and the interparticle structure factor, with the latter described within the local monodisperse approximation regime. Additionally, a scattering contribution was observed, which was associated with the presence of crystalline superstructures formed by micelles that organized into a cubic structure. The data analysis provided important information about the system, such as the average radius, the size distribution, and the thickness of the layer surrounding the micellar core. The results will contribute to the development and optimization of new drug delivery systems that are more effective and safer for medical applications. Full article

The spectral characteristics of cyclosporin C (CsC) with the addition of Dy³⁺ ions in acetonitrile (CD₃CN) and CsC with Dy³⁺ incorporated into dodecylphosphocholine (DPC) micelle in deuterated water were investigated by high-resolution NMR spectroscopy. The study was focused on the interaction between Dy³⁺ ions and CsC molecules in different environments. Using a combination of one-dimensional and two-dimensional NMR techniques, we obtained information on the spatial features of the peptide molecule and the interaction between CsC and the metal ion. The non-uniform effect of the metal ion on different NMR signals of CsC was observed. The paramagnetic attenuation parameter was calculated for the amide, alpha, and beta protons of CsC upon the addition of Dy³⁺. The metal ion was found to interact with the polar part of the DPC micelle, and the ion also has a significant effect on the NMR signals of amino acid residues from Sar₃ to d-Ala₈. This pattern is reproduced in both environments studied here and also agrees with earlier investigations of the CsA–Dy³⁺ complex. Full article

The far-infrared absorption spectrum of monodeuterated water vapor, HD¹⁶O, is analyzed using three high-sensitivity absorption spectra recorded by high-resolution Fourier transform spectroscopy at the SOLEIL synchrotron facility. The gas sample was obtained using a 1:1 mixture of H₂O and D₂O leading to a HDO abundance close to 50%. The room temperature spectra recorded in the 50–720 cm⁻¹ range cover most of the rotational band. The sensitivity of the recordings allows for lowering by three orders of magnitude the detectivity threshold of previous absorption studies in the region. Line centers are determined with a typical accuracy of 5 × 10⁻⁵ cm⁻¹ for well-isolated lines. The combined line list of 8522 water lines is assigned to 9186 transitions of the nine stable water isotopologues (H₂^XO, HD^XO, and D₂^XO with X = 16, 17, and 18). Regarding the HD¹⁶O isotopologue, a total of 2443 transitions are presently assigned while about 530 absorption transitions were available prior to our SOLEIL recordings. The comparison with the HITRAN list of HD¹⁶O transitions is discussed in detail. The obtained set of accurate HD¹⁶O transition frequencies is merged with literature sources to generate a set of 1121 accurate empirical rotation–vibration energies for the first five vibrational states (000), (010), (100), (020), and (001). The comparison to the previous dataset from an IUPAC task group illustrates a gain in the average energy accuracy by more than one order of magnitude. Based on these levels, a recommended list of transitions between the first five vibrational states is proposed for HD¹⁶O in the 0–4650 cm⁻¹ frequency range. Full article

Background/Objectives: Metabolic profiling of tissue samples via liquid-state nuclear magnetic resonance (NMR) requires the extraction of polar metabolites in a suitable deuterated solvent. Such methods often prioritise metabolite recovery over protein removal due to the relatively low sensitivity of NMR metabolomics and the routine use of methods able to supress residual protein signals. However, residual protein may impact metabolite integrity and the metabolite stability after NMR sample preparation is often overlooked. This study aimed to investigate the effect of residual protein contamination in rodent brain extracts and identify a reproducible extraction method that optimises metabolite recovery while ensuring sample stability. Methods: The performance of acetonitrile/water (50–100% MeCN), methanol/water (50–100% MeOH), and methanol/water/chloroform (MeOH/H₂O/CHCl₃) were assessed for extraction efficiency, reproducibility, residual protein contamination, and metabolite stability up to eight hours post NMR sample preparation. Results: Aspartate and glutamate deuteration were observed in 50% MeCN, 50% MeOH, and 67% MeOH extractions along with the conversion of N-acetyl aspartate to aspartate and acetate in 50% MeCN and 50% MeOH extractions. Both observations correlated with residual protein contamination and, thus, are a result of inadequate protein precipitation, as confirmed by ultrafiltration. MeOH/H₂O/CHCl₃ extraction preserved the stability of these metabolites while maintaining good extraction efficiency and reproducibility. Conclusions: Thus, we recommend MeOH/H₂O/CHCl₃ extraction for untargeted brain NMR metabolic profiling due to its effective protein precipitation and reliable performance. Nonetheless, the performance of detecting metabolites prone to oxidation such as ascorbate and glutathione is not improved by this method. Full article

Background: In MASLD (formerly called NAFLD) mouse models, oversupply of dietary fat and sugar is more lipogenic than either nutrient alone. Fatty acids suppress de novo lipogenesis (DNL) from sugars, while DNL inhibits fatty acid oxidation. How such factors interact to impact hepatic triglyceride levels are incompletely understood. Methods: Using deuterated water, we measured DNL in mice fed 18-weeks with standard chow (SC), SC supplemented with 55/45-fructose/glucose in the drinking water at 30% (w/v) (HS), high-fat chow (HF), and HF with HS supplementation (HFHS). Liver glycogen levels and its sources were also measured. For HS and HFHS mice, pentose phosphate (PP) fluxes and fructose contributions to DNL and glycogen were measured using [U-¹³C]fructose. Results: The lipogenic diets caused significantly higher liver triglyceride levels compared to SC. DNL rates were suppressed in HF compared to SC and were partially restored in HFHS but supplied a minority of the additional triglyceride in HFHS compared to HF. Fructose contributed a significantly greater fraction of newly synthesized saturated fatty acids compared to oleic acid in both HS and HFHS. Glycogen levels were not different between diets, but significant differences in Direct and Indirect pathway contributions to glycogen synthesis were found. PP fluxes were similar in HS and HFHS mice and were insufficient to account for DNL reducing equivalents. Conclusions: Despite amplifying the lipogenic effects of fat, the fact that sugar-activated DNL per se barely contributes suggests that its role is likely more relevant in the inhibition of fatty acid oxidation. Fructose promotes lipogenesis of saturated over unsaturated fatty acids and contributes to maintenance of glycogen levels. PP fluxes associated with sugar conversion to fat account for a minor fraction of DNL reducing equivalents. Full article

The formation of chondrite materials represents one of the earliest mineralogical processes in the solar system. Phyllosilicates are encountered at various stages of the chondrule formation, from the initial stages (IDP agglomerates) to the final steps (chondrule internal alteration). While typically linked to aqueous alteration, recent studies reveal that phyllosilicates could precipitate directly from residual fluids in post-magmatic or deuteric conditions and under a wide range of temperatures, pressures, water/rock ratios, and H₂/H₂O ratio conditions. This study re-examined the formation of hydrated phyllosilicates in chondrules and associated fine-grained rims (FGRs) using published petrographical, mineralogical, and chemical data on carbonaceous chondrites. Given that chondrules originate from the melting of interplanetary dust particles, the water liberated by the devolatilization of primary phyllosilicates, including clay minerals or ice melting, reduces the melting temperature and leads to water dissolution into the silicate melt. Anhydrous minerals (e.g., olivine and diopside) form first, while volatile and incompatible components are concentrated in the residual liquid, diffusing into the matrix and forming less porous FGRs. Serpentine and cronstedtite are the products of thermal metamorphic-like mineral reactions. The mesostasis in some lobated chondrules is composed of anhydrous and hydrous minerals, i.e., diopside and serpentine. The latter is probably not the alteration product of a glassy precursor but rather a symplectite component (concomitant crystallization of diopside and serpentine). If so, the symplectite has been formed at the end of the cooling process (eutectic-like petrographical features). Water trapped inside chondrule porosity can lead to the local replacement of olivine by serpentine without external water input (auto-alteration). In the absence of water, hydrated phyllosilicates do not crystallize, forming a different mineral assemblage. Full article

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d₀Me) using 2 M HCl in methanol (CH₃OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD₃OD) generates deuterated methyl esters (d₃Me) of AA, which can be used as internal standards in GC-MS. d₀Me-AA and d₃Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d₀Me-AA-PFP and d₃Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d₀Me-AA-PFP and d₃Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d₃Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (t_R)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d₀Me-AA-PFP derivatives (t_R(H)) and the d₃Me-AA-PFP derivatives (t_R(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d₃Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS. Full article

The aim of the present work was to assess the leaf area duration (LAD) and the radiation use efficiency (RUE) of six warm-season perennial biomass grasses (PBGs) in a two-year field trial in the semiarid Mediterranean climate under different soil water availability. Two ecotypes of giant reed (Arundo donax L., ARCT and ARMO), one ecotype of African fodder cane (Saccharum spontaneum L. subsp. aegyptiacum (Willd.) Hack., SAC) and three hybrids of Miscanthus (the commercial M. × giganteus J.M. Greef, Deuter ex Hodk., Renvoize, M × G, and two new seed-based hybrids, GNT9 and GNT10) were compared under three levels of soil water availability: rainfed, 50% and 100% of maximum crop evapotranspiration (ETm) restoration. The determination of RUE of perennial plants is controversial and has led to contrasting results in past studies. In the present work, LAD and RUE differed among crops and irrigation regimes, being positively affected by supplemental water inputs. SAC, ARCT and ARMO showed both high LAD and RUE, which determined the high biomass yield than both the commercial M × G and the improved Miscanthus hybrids GNT9 and GNT10. RUE was particularly high and less affected by soil water availability during the mid-season, while the effect of irrigation and the differences among the genotypes were larger during the late season. Adequate biomass yield can be achieved by sub-optimal soil water availability, thus reducing the water footprint and increasing the sustainability of these biomass perennial grasses selected for the Mediterranean climate. Full article

Three octahedral iron(III) nitrate complexes with dimethylated urea ligand isomers, [hexakis(N,N’-dimethylurea-O)iron(III)] nitrate (compound 1), trans-[diaquatetrakis(N,N-dimethylurea-O)iron(III)] nitrate (compound 2), and [hexakis(N,N-dimethylurea-O)iron(III)] nitrate trihydrate (compound 3) were prepared and characterized with single crystal X-ray diffraction, IR, Raman and UV–Vis methods. In compounds 1 and 3, six dimethylurea ligands coordinate to the central Fe^III ion via the oxygen in octahedral geometry and the ligands are arranged in a propeller-like manner, dividing the complex cations into two sides. In compound 1, the dimethylurea propellers screw in the opposite direction on the two sides of the complex and in compound 3, they are arranged with the same handedness on the two sides. The complexes have helical chirality. The two sides of the complex cations differ not only in the rotation direction of the ligands but also in the hydrogen bond formation. On one side of the complex cation, the ligands form intermolecular hydrogen bonds only with the crystal waters, meanwhile on the other side of the complex, the ligands form hydrogen bonds only with the nitrate ions. In compound 2, [Fe(N,N-dimethylurea)₄(H₂O)₂]³⁺ cations form layers that are separated by interconnected NO₃⁻ ions forming a hydrogen bonding system and connecting the complex cations A-s and B-s. The three crystallographically different nitrate ions each form four hydrogen bonds in a way that they have one bidentate O atom and two monodentate O atoms; however, the anions differ in their hydrogen bonding. The spectroscopic characteristics of compound 2 were determined by IR measurements on the deuterated compound 2 as well. Full article

Drug solubility limits intravenous dosing for poorly water-soluble medicines, which misrepresents their bioavailability estimation. The current study explored a method using a stable isotope tracer to assess the bioavailability of drugs that are poorly water-soluble. HGR4113 and its deuterated analog, HGR4113-d7, were tested as model drugs. To determine the level of HGR4113 and HGR4113-d7 in rat plasma, a bioanalytical method using LC-MS/MS was developed. The HGR4113-d7 was intravenously administered to rats that were orally pre-administered HGR4113 at different doses; subsequently, the plasma samples were collected. HGR4113 and HGR4113-d7 were simultaneously determined in the plasma samples, and bioavailability was calculated using plasma drug concentration values. The bioavailability of HGR4113 was 53.3% ± 19.5%, 56.9% ± 14.0%, and 67.8% ± 16.7% after oral dosages of 40, 80, and 160 mg/kg, respectively. By eliminating the differences in clearance between intravenous and oral dosages at different levels, acquired data showed that the current method reduced measurement errors in bioavailability when compared to the conventional approach. The present study suggests a prominent method for evaluating the bioavailability of drugs with poor aqueous solubility in preclinical studies. Full article

This study aimed to determine promethazine (PMZ) and its metabolites, promethazine sulfoxide (PMZSO) and monodesmethyl-promethazine (Nor₁PMZ), in swine muscle, liver, kidney, and fat. A sample preparation method and high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis were established and validated. The samples were extracted using 0.1% formic acid–acetonitrile and purified with acetonitrile-saturated n-hexane. After concentration by rotary evaporation, the extract was re-dissolved in a mixture of 0.1% formic acid-water and acetonitrile (80:20, v/v). Analysis was performed using a Waters Symmetry C₁₈ column (100 mm × 2.1 mm i.d., 3.5 μm) with 0.1% formic acid–water and acetonitrile as the mobile phase. The target compounds were determined using positive ion scan and multiple reaction monitoring. PMZ and Nor₁PMZ were quantified with deuterated promethazine (PMZ-d6) as the internal standard, while PMZSO was quantified using the external standard method. In spiked muscle, liver, and kidney samples, the limits of detection (LOD) and limits of quantification (LOQ) for PMZ and PMZSO were 0.05 μg/kg and 0.1 μg/kg, respectively, while for Nor₁PMZ, these values were 0.1 μg/kg and 0.5 μg/kg, respectively. For spiked fat samples, the LOD and LOQ for all three analytes were found to be 0.05 μg/kg and 0.1 μg/kg, respectively. The sensitivity of this proposed method reaches or exceeds that presented in previous reports. The analytes PMZ and PMZSO exhibited good linearity within the range of 0.1 μg/kg to 50 μg/kg, while Nor₁PMZ showed good linearity within the range of 0.5 μg/kg to 50 μg/kg, with correlation coefficients (r) greater than 0.99. The average recoveries of the target analytes in the samples varied from 77% to 111%, with the precision fluctuating between 1.8% and 11%. This study developed, for the first time, an HPLC–MS/MS method for the determination of PMZ, PMZSO, and Nor₁PMZ in four swine edible tissues, comprehensively covering the target tissues of monitoring object. The method is applicable for monitoring veterinary drug residues in animal-derived foods, ensuring food safety. Full article

Persistent Organic Pollutants (POPs) in water can reach levels below ppt (ng L⁻¹), representing a challenge for risk assessment using long-term exposure. Therefore, sampling devices to monitor trace levels of organic compounds are suitable due to their sensitivity and low detection limits. In this work, the field calibration and monitoring for POPs, such as Polycyclic Aromatic Hydrocarbons (PAHs) and pesticides, in a drinking water reservoir were carried out using semipermeable membrane devices (SPMDs). The SPMDs were spiked with deuterated PCBs used as PRCs, and their dissipation was measured at 5, 10, 15 and 24 days. The SPMDs were dialyzed in hexane twice and the initial amount (N) and final amount (N_o) of PRCs in extracts were estimated by GC/MS to calculate the sampling rate (R_s) for target compounds. The PCBs were dissipated more than 30% at 24 days, and R_s were calculated between 27.8 and 72.7 L day⁻¹ for PAHs and 35.8 and 71.3 L day⁻¹ for pesticides. Finally, PAH congeners such as Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene and Pyrene were detected at water concentration (C_w) ranges between 0.5 and 2.7 ng L⁻¹, whilst chlorpyrifos was the only pesticide detected at 19.0 ng L⁻¹ in the reservoir. This calibration showed that PRCs do not dissipate entirely in 24 days and that dissipation modelling may be applied for C_w estimation of POPs in reservoirs. Full article

Deuterium, a stable isotope of hydrogen, is abundant in organisms. It is known to produce various biological effects. However, its impact in thyroid hormone synthesis and secretion is poorly studied. The aim of this investigation was to evaluate the dynamics of thyroid hormones and pituitary thyroid-stimulating hormone secretion during bilateral shifts in deuterium supply and assess a possible role of the Na⁺/I⁻ symporter (NIS), the main iodide transporter, in altered thyroid function. The experiment was performed on adult male Wistar rats, which consumed deuterium-depleted ([D] = 10 ppm) and deuterium-enriched ([D] = 500,000 ppm) water for 21 days. The assessment of total thyroxine and triiodothyronine and their free fractions, as well as thyroid-stimulating hormone in blood serum, revealed the rapid response of the thyroid gland to shifts in the deuterium/protium balance. The present investigation shows that the bilateral changes in the deuterium body content similarly modulate thyroid hormone production and functional activity of the pituitary gland, but the responses of the thyroid and pituitary glands differ. The response of the thyroid cells was to increase the synthesis of the hormones and the pituitary thyrotropes, in order to reduce the production of the thyroid-stimulating hormone. The evaluation of NIS serum levels found a gradual increase in the rats that consumed deuterium-enriched water and no differences in the group exposed to deuterium depletion. NIS levels in both groups did not correlate with thyroid hormones and pituitary thyroid-stimulating hormone production. The data obtained show that thyroid gland has a higher sensitivity to shifts in the deuterium body content than the hypothalamic–pituitary complex, which responded later but similarly in the case of deuteration or deuterium depletion. It indicates a different sensitivity of the endocrine glands to alterations in deuterium content. It suggests that thyroid hormone production rate may depend on deuterium blood/tissue and cytosol/organelle gradients, which possibly disturb the secretory process independently of the NIS. Full article

Reversed-phase liquid chromatography and electrospray ionization with Fourier-transform single and tandem mass spectrometry (RPLC-ESI-FTMS and FTMS/MS) were employed for the structural characterization of oleocanthal (OLEO) and oleacin (OLEA), two of the most important bioactive secoiridoids occurring in extra virgin olive oils (EVOOs). The existence of several isoforms of OLEO and OLEA was inferred from the chromatographic separation, accompanied, in the case of OLEA, by minor peaks due to oxidized OLEO recognized as oleocanthalic acid isoforms. The detailed analysis of the product ion tandem MS spectra of deprotonated molecules ([M-H]⁻) was unable to clarify the correlation between chromatographic peaks and specific OLEO/OLEA isoforms, including two types of predominant dialdehydic compounds, named Open Forms II, containing a double bond between carbon atoms C8 and C10, and a group of diasteroisomeric closed-structure (i.e., cyclic) isoforms, named Closed Forms I. This issue was addressed by H/D exchange (HDX) experiments on labile H atoms of OLEO and OLEA isoforms, performed using deuterated water as a co-solvent in the mobile phase. HDX unveiled the presence of stable di-enolic tautomers, in turn providing key evidence for the occurrence, as prevailing isoforms, of Open Forms II of OLEO and OLEA, different from those usually considered so far as the main isoforms of both secoiridoids (having a C=C bond between C8 and C9). It is expected that the new structural details inferred for the prevailing isoforms of OLEO and OLEA will help in understanding the remarkable bioactivity exhibited by the two compounds. Full article