Quantitative NMR Research Articles

Liquid-Phase NMR of Humic and Fulvic Acids.

Present review focuses on the most recent advances in the NMR of the coal-derived humic and fulvic acids, covering exclusively the results of the liquid-phase NMR and leaving apart an overwhelming amount of publications dealing with the solid-state NMR investigations in this field (the latter are comprehensively reviewed elsewhere). Owing to the complexity of humic and fulvic acids together with other coal-derived products, their 1H and 13C NMR spectra consist of a number of overlapping signals belonging to different hydrocarbon types. Comprehensive studies of humic and fulvic acids by means of NMR revealed characteristic functional groups of their composition together with spectral regions in which they resonate. Quantitative 1H and 13C NMR spectra characterize aromatic and saturated carbons spread over many structural moieties, which provides a solid guideline into molecular structure of humic and fulvic acids together with parent coal-derived products. Nowadays, quantitative 13C NMR measurements yield information about a variety of structural parameters such as functional group distribution, aromaticity, degree of condensation of aromatic rings, and medium chain lengths together with many other more specific parameters. The structural NMR studies of the coal-derived products are developing on a background of a marked progress in experimental and computational NMR. Discussed in the present review are the most recent advances in the liquid-state NMR studies of the coal-derived humic and fulvic acids together with their processing products.

Determination of Sugar Concentrations in Aqueous Solution Using Multivariate Predictions Based on 1H‐NMR Spectroscopy

AbstractRenewable chemicals from carbohydrate‐rich wastes, like furfural and 5‐hydroxymethylfurfural (HMF), are gaining prominence as alternatives to petroleum‐based resources. Assessing the suitability of biomass as feedstock for furfural and HMF production requires knowledge of its composition. This study focuses on developing and validating predictive models for individual sugar concentrations in hydrolysates using quantitative 1H NMR data. Utilizing partial least square (PLS) regression, the dataset includes 137 NMR spectra of multi‐component sugar standards (arabinose, fructose, galactose, glucose, mannose, maltose, sucrose, and xylose). The best‐performing model achieved an R2 of 0.987–0.999 and RMSECV of 0.37–1.56 mM and is based on the non‐overlapping area of the NMR spectrum. Real‐world samples were used for validation, resulting in predicted sugar concentrations with a mean standard deviation of 0.5 mM. This high accuracy and streamlined analysis process make these models practical for quantifying large sample sets, showcasing the reliability and accessibility of extracting statistical information from H‐NMR data.

CO2 Hydrate in Porous Media: A Quantitative NMR Method to Detect Formation, Dissociation, and Localization

CO₂ Hydrate in Porous Media: A Quantitative NMR Method to Detect Formation, Dissociation, and Localization

Isolation of β-O-4-Rich Lignin From Birch in High Yields Enabled by Continuous-Flow Supercritical Water Treatment.

The continuous flow supercritical water (scH2O) treatment of Birch wood (T=372-382 °C; t=0.3-0.7 s; p=260 bar) followed by alkali extraction of lignin allowed for the isolation of lignin and lignin carbohydrate complexes (LCCs) with a high number of β-O-4 moieties in the range 29-57/100 Ar (evaluated by quantitative 13C NMR analysis) in yields ranging between 13-19 wt % with respect to the initial wood. A "lightning rod effect" of carbohydrates has been claimed to explain the low degradation of β-O-4 bonds during the process. The structure of the isolated lignin was thoroughly elucidated via comprehensive NMR studies (HSQC, 13C and 31P). A low degree of condensation (DC)<5 % was found for all the lignin samples, which was only slightly dependent on the reaction severity. The number of aliphatic -OH, phenolic -OH, and -COOH groups was in the range 3.37-5.25, 1.41-2.31 and 0.39-0.73 mmol/g, respectively. The number of -COOH groups increased with increased severity, suggesting that oxidation can occur during the scH2O treatment. Furthermore, by simply varying the reaction severity, it was possible to tune important lignin properties, like the molar mass and the glass transition temperature (Tg).

Absolute Quantification of Phenylbutanoids in Zingiber cassumunar Roxb. Rhizome by Quantitative 1H NMR.

Quantitative determination of pharmacologically active constituents in medicinal plants is critical for quality control. Due to the chemical complexity of the crude plant extracts, the presence of interfering compounds is often problematic for the unambiguous quantitation of the designed bioactive compounds. Considering the method of quantification, quantitative NMR spectroscopy (qNMR) has gained substantial popularity as a powerful and effective technique for both qualitative and quantitative analyses of natural products. The aim of this study is to develop a quantitative NMR method for quantifying the bioactive phenylbutanoids in Zingiber cassumunar rhizome crude extract. Quantitative 1H NMR (qHNMR) measurements were performed on a 600 MHz NMR spectrometer using an internal standard for the determination of the absolute quantities of four phenylbutanoids in Z. cassumunar rhizome crude extract. The direct quantification of four characteristic phenylbutanoids, i.e., (E)-1-(3',4'-dimethoxyphenyl)butadiene (DMPBD), (E)-1-(2',4',5'-trimethoxyphenyl)butadiene (TMPBD), (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol, and (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-yl acetate, in crude extract by qHNMR using an internal standard was achieved, with high specificity and sensitivity. The selected 1H NMR signals could unambiguously be assigned and did not overlap with other resonances, including the highly similar compounds DMPBD and TMPBD. The method is linear in the concentration range of 0.70-14.52 mg/mL, with a limit of quantification of 0.46-0.68 mg/mL. The RSD values of intraday and interday precisions are in the range of 0.23%-0.74% and 0.29%-0.52%, respectively. The average recoveries are 99.54%-100.18%. A rapid, accurate, and precise method using 1H NMR for the simultaneous quantitation of four phenylbutanoids in the crude extract of Z. cassumunar rhizome was developed and validated.

Hydronium-Crosslinked Inorganic Hydrogel Comprised of 1D Lepidocrocite Titanate Nanofilaments.

When a few drops of acid (hydrochloric, acrylic, propionic, acetic, or formic) are added to a colloid comprised of 1D lepidocrocite titanate nanofilaments (1DLs)-2 × 2 TiO6 octahedra in cross-section-a hydrogel forms, in many cases, within seconds. The 1DL synthesis process requires the reaction between titanium diboride with tetramethylammonium (TMA+), hydroxide. Using quantitative nuclear magnetic resonance (qNMR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), the mass percent of TMA+ after synthesis is determined to be ≈ 13.1±0.1%. The TMA+ is completely removed from the gels after 2 water soak cycles, resulting in the first completely inorganic, TiO2-based hydrogels. Ion exchanging the TMA+ with hydronium results in gels with relatively strong hydrogen bonds. The hydrogels' compression strengths increased linearly with 1DL colloid concentration. At a 1DL concentration of 45gL-1, the compressive strength, at 80% deformation when acrylic acid is used, is ≈325kPa. The strengths are ≈ 50% greater after the TMA+ is removed. The removal of all residual organic components in the hydrogels, including TMA+, is confirmed by qNMR, Fourier-transformed infrared spectroscopy (FTIR), and TGA/DSC. The 1DL phase is retained after gelation, TMA+ removal, and 80% compression.

Interrogating the photoluminescent properties of carbon dots using quantitative 13C NMR combined with systematic photobleaching

In this work, molecular-level events accompanying the photobleaching of carbon dots (CDs) synthesized from ethylenediamine and citric acid (CACDs) were identified using solution-phase NMR. By combining quantitative 13C NMR data with fluorescence measurements we show that this new approach is capable of identifying not only molecular fluorophores in CACDs, but also their contribution to the overall CACD fluorescence and their relative photostability. Specifically, imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA) is found to be the dominant (84 %) species responsible for fluorescence in CACDs along with a second undetermined source, most likely associated with the aromatic core which is significantly (approximately 20 times) more photostable than IPCA. The presence of these two fluorescent species with different photostabilities rationalizes not only the photobleaching kinetics but also the evolution of the fluorescence spectrum during photobleaching Diffusion-ordered spectroscopy (DOSY) also reveals that all of the IPCA molecules are trapped within or covalently bound to the CACD, but are not present as isolated molecules freely rotating in solution. Singlet oxygen is confirmed as a key ROS responsible for photobleaching, with prototypical photoproducts identified from mass spectrometry studies of citrazinic acid. Quantitative 13C NMR of CACDs is possible because their extremely high colloidal stability enables high concentrations (667 mg/mL) to remain stable in solution. The approach described in this study could be extended to identify the structure of chromophores in other CDs and interrogate molecular level processes that accompany CD sensing.

Purity determination and uncertainty evaluation of L-selenium-methylselenocysteine

In our country, sodium selenite and sodium selenate are still used as nutritional fortifiers of selenium. Both of them are inorganic selenium, the absorption effect of human body is lower, the risk of toxic wind is larger, and it is easy to cause selenium poisoning. L-selenium-methylselenocysteine (L-SeMC) is a good source of organic selenium, which has higher biological activity and availability. At present, L-SeMC solid standard materials have not yet been developed, a high accuracy and low technical requirement method is needed to determine the purity of candidate reference materials (RMs). In this way, the key steps in the development of reference materials can be completed, and provide a source of high quality selenium supplement for people. In this paper, the candidate structures were identified and analyzed by high performance liquid chromatography (HPLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). Homogeneity, together with stability were also measured by HPLC. The sample can be transported for a short period of one month under normal conditions, which can also be stored for a long time within the temperature range of −20 °C to room temperature for more than one year. The purity of L-SeMC measured by mass balance method and quantitative nuclear magnetic resonance (qNMR) was 99.5 % with an expanded uncertainty of 0.4 % (k = 2). However, differential scanning calorimetry (DSC) was not suitable for the purity determination of L-SeMC samples. This method has a wide range of applications and may provide a reference for the purity determination of other RMs.

Quantitative estimation and validation of Alectinib hydrochloride drug substance using quantitative nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy has been recently used for quantitative estimation of drug substance which has numerous merits in pharmaceutical applications when compared to traditional methods of chromatography. Present study focuses on method development and validation of quantitative proton nuclear magnetic spectroscopy method for estimation of alectinib hydrochloride drug substance. This method involves use of internal standard in presence of alectinib hydrochloride. For quantitation, 1H NMR signals at 8.4 ppm and 6.7 ppm corresponding to analyte proton of alectinib and ethyl 4-(dimethyl amino) benzoate internal standard respectively were used. Moreover, this method was validated as per ICH guidelines for accuracy, precision, linearity, limit of detection, limit of quantitation, range and robustness. Assay estimation by Q-NMR is facile and time-efficient which does not require reference standard of analyte and yet accurate and precise alternative as compared to estimation by other chromatographic techniques.

Glucose hydrochar consists of linked phenol, furan, arene, alkyl, and ketone structures revealed by advanced solid-state nuclear magnetic resonance

The molecular structure of hydrochars produced from 13C-enriched glucose under various conditions has been elucidated based on advanced one- and two-dimensional (2D) 1H-13C and 13C–13C solid-state nuclear magnetic resonance (NMR) with spectral editing. Regardless of synthesis conditions, hydrochars consist mostly of oxygen-substituted arene rings (including diphenols) and furans connected by alkyl linkers rich in ketones. Cross-linking nonprotonated and methyne (C-H) alkyl carbons have been identified through spectrally edited 2D NMR. Alkenes and ‘quaternary’ C-O are observed only at low synthesis temperature, while some clusters of fused arene rings are generated at high temperature. Hydrochar composition is nearly independent of reaction time in the range from 1 to 5 h. Equilibration of 13C magnetization within 1 s shows that the materials are homogeneous on the 5-nm scale, refuting core–shell models of hydrochar microspheres. While furan C-O carbons bonded to alkyl groups or ketones show distinctive cross peaks in 2D NMR, phenolic C-OH is observed unambiguously by hydroxyl-proton selection. While methylene-linked furan rings are fairly common, the signal previously assigned to furan Cα-Cα linkages is shown to arise from abundant, stable catecholic ortho-diphenols, whose HO-C=C-OH structure is proved by 2D13C–13C NMR after hydroxyl-proton selection. Quantitative 13C NMR spectra of low- and high-temperature hydrochars have been matched by chemical-shift simulations for representative structural models. Mixed phenol and furan rings connected by ketones and alkyl linkers provide good fits of the experimental spectra, while literature models dominated by large clusters of fused rings and with few phenols or alkyl-linked ketones do not.

Mechanistic insights into pore water conversion to gas hydrates in clay minerals

The conversion of pore water to gas hydrates is an important prerequisite for natural gas hydrates (NGHs) accumulation and hydrate-based CO2 sequestration. However, the mechanism of water-to-hydrate conversion in clays, which are prevalent in hydrate reservoirs or marine sediments serving as potential hosts for CO2 hydrates, still remains poorly understood, impeding further advancements in exploiting NGHs and developing CO2 sequestration strategies. In this paper, the characteristics of pore water conversion to hydrates in montmorillonite and kaolin were comprehensively examined through quantitative measurements and NMR analysis, and compared with those in silt and coarse sand. It revealed that the water-to-hydrate conversion was more restricted in clays than in sands. Especially in montmorillonite, since almost all pore water was detected as bound water, the final water conversion was extremely low and sensitive to the subcooling degree. Additionally, as the initial water content increased from 15 wt% to 35 wt%, the final water conversion in kaolin, silt, and coarse sand decreased exponentially due to intensified kinetic limitation, whereas that in montmorillonite initially rose and then fell, attaining the maximum at a threshold of 20 wt%. It is found that the competition between thermodynamic and kinetic limitations controls water-to-hydrate conversion in clays. Specifically, in kaolin, silt and coarse sand, the main controlling factor of water-to-hydrate conversion is the kinetic limitation, as their unconverted equilibrium water is negligible. However, in montmorillonite, the thermodynamic limitation dominates at low water contents, transitioning to kinetic limitation at high water contents. This study provides direct evidence and mechanistic insights into how clays affect water-to-hydrate conversion in porous media, which has significant implications for the geological accumulation of NGHs and the site selection for hydrate-based CO2 sequestration.

Quantitative Blood Serum IVDr NMR Spectroscopy in Clinical Metabolomics of Cancer, Neurodegeneration, and Internal Medicine.

Despite more than two decades of metabolomics having joined the "omics" scenery, to date only a few novel blood metabolite biomarkers have found their way into the clinic. This is changing now by massive large-scale population metabolic phenotyping for both healthy and disease cohorts. Here, nuclear magnetic resonance (NMR) spectroscopy is a method of choice, as typical blood serum markers can be easily quantified and by knowledge of precise reference concentrations, more and more NMR-amenable biomarkers are established, moving NMR from research to clinical application. Besides customized approaches, to date two major commercial platforms have evolved based on either 600MHz (14.1 Tesla) or 500MHz (11.7 Tesla) high-field NMR systems. This chapter provides an introduction into the field of quantitative in vitro diagnostics research (IVDr) NMR at 600 MHzand its application within clinical research of cancer, neurodegeneration, and internal medicine.

Excessive Gestational Weight Gain, Independent of Body Mass Index, Is Associated With Child Fat Mass Index at Age 2 Years in the Growing life, Optimizing Wellness Study

BackgroundExcessive gestational weight gain (eGWG) increases risk for pregnancy complications and future obesity for pregnant persons and children. Yet, it is unclear whether eGWG leads to higher child adiposity at 2 years, independent of the pregnant person’s body mass index (BMI) while considering important covariates. Moreover, understanding the characteristics of pregnant persons experiencing eGWG will help design future targeted interventions. ObjectiveThe objectives of the analyses were to assess the association between eGWG and childhood adiposity at age 2 years, while controlling for pregnant persons’ BMI and other important covariates and to describe the characteristics of pregnant persons who experience eGWG during their second pregnancy. DesignThis is a secondary analysis of 221 pregnant persons and their children aged 2 years who were enrolled in the Growing Life, Optimizing Wellness longitudinal observational study. Participants/settingParticipants were recruited between 2011 and 2014 in central Arkansas. Participants were secundigravida persons with BMI 18.5 to 35, older than age 20 years, and who conceived without assistance. Main outcome measuresThe main outcome measure was fat mass index of children aged 2 years measured by quantitative nuclear magnetic resonance imaging. Secondary outcomes included first pregnancy GWG, dietary and physical activity characteristics between pregnancies, second pregnancy nausea levels, and motivation level to adhere to the GWG guidelines. Statistical analysis performedMultivariable regression analyses were used to examine the associations between GWG and childhood fat mass index at age 2 years. Pearson correlations and Wilcoxon rank sum tests were used to identify the characteristics of pregnant persons who experienced eGWG. ResultsPregnant persons’ eGWG (β = .503; P = .03) was positively associated with child adiposity at age 2 years independent of maternal BMI (P = 0.3). Pregnant persons who experienced eGWG during their second pregnancy had greater odds of eGWG during first pregnancies (odds ratio 7.5; P < .001), dieting behavior (odds ratio 14.3; P = .02), and low motivation to adhere to the GWG guidelines (odds ratio 11.2; P = .009). Fewer participants had eGWG during their second pregnancy (52.5%) compared with their first pregnancy (66.8%), which was different by BMI groups (BMI 18.5 to 24.9: 23.6% decrease in participants who gained eGWG, BMI 25 to 29.9: 20.0% decrease, and BMI ≥ 30: 37.9% decrease). ConclusionseGWG among pregnant persons is associated with child adiposity at age 2 years. Pregnant persons who experienced eGWG during their second pregnancy reported low motivation to gain weight within guidelines, eGWG in first pregnancy, and reported prior dieting behavior. Future research focusing on patients with these characteristics may increase success of interventions designed to limit eGWG.

Molecular-Level Understanding of Phase Stability in Phase-Change Nanoemulsions for Thermal Energy Storage by NMR Spectroscopy.

Phase change materials (PCMs) are latent heat storage materials that can store or release thermal energy while undergoing thermodynamic phase transitions. Organic PCMs can be emulsified in water in the presence of surfactants to enhance thermal conductivity and enable applications as heat transfer fluids. However, PCM nanoemulsions often become unstable during thermal cycling. To better understand the molecular origins of phase stability in PCM nanoemulsions, we designed a model PCM nanoemulsion system and studied how the molecular-level environments and dynamics of the surfactants and oil phase changed upon thermal cycling using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The model system used octadecane as the oil phase, stearic acid as the surfactant, and aqueous NaOH as the continuous phase. The liquid fraction of octadecane within the nanoemulsions was quantified noninvasively during thermal cycling by liquid-state 1H single-pulse NMR measurements, revealing the extent of octadecane supercooling as a function of temperature. The mean droplet size of the PCM nanoemulsions, measured by dynamic light scattering (DLS), was correlated with the liquid content of octadecane to explain phase instability in the solid-liquid coexistence region. Quantitative 13C single-pulse NMR experiments established that the carbonyl surfactant head groups were present in multiple distinct environments during thermal cycling. After repeated thermal cycling, the 13C signal intensity of the carbonyl surfactant head groups decreased, indicating that the surfactant head groups lost molecular mobility. The results explain, in part, the origin of phase instability of PCM nanoemulsions upon thermal cycling.

Comparison and Determination of the Content of Mosapride Citrate by Different qNMR Methods.

As a salt-type compound, mosapride citrate's metabolism and side effects are correlated with its salt-forming ratio. Several techniques were developed in this work to compare various quantitative nuclear magnetic resonance (qNMR) methodologies and to quantitatively examine the content of raw materials. Among the qNMR techniques, methods for 1H NMR and 19F NMR were developed. Appropriate solvents were chosen, and temperature, number of scans, acquisition time, and relaxation delay parameter settings were optimized. Maleic acid was chosen as the internal standard in 1H NMR, and the respective characteristic signals of mosapride and citrate were selected as quantitative peaks. The internal standard in 19F NMR analysis was 4,4'-difluoro diphenylmethanone, and the distinctive signal peak at -116.15 ppm was utilized to quantify mosapride citrate. The precision, repeatability, linearity, stability, accuracy, and robustness of the qNMR methods were all validated according to the ICH guidelines. By contrasting the outcomes with those from high-performance liquid chromatography (HPLC), the accuracy of qNMR was assessed. As a result, we created a quicker and easier qNMR approach to measure the amount of mosapride citrate and evaluated several qNMR techniques to establish a foundation for choosing quantitative peaks for the qNMR method. Concurrently, it is anticipated that various selections of distinct quantitative objects will yield the mosapride citrate salt-forming ratio.

Unlocking the potential of NMR spectroscopy for precise and efficient quantification of microplastics

Precise, fast, and reliable identification and quantification of microplastic contamination are essential for determining their environmental concentrations for risk assessments. This study investigates the use of nuclear magnetic resonance (NMR) spectroscopy to quantify microplastics by analysing dilution series of polystyrene (PS), polyisoprene-cis (PI), polybutadiene-cis (PB), polylactic acid (PLA), polyvinyl chloride (PVC) and polyurethane (PU). Each polymer type was dissolved in a suitable solvent and an internal standard was utilized for quantification. Detection and quantification limits for each polymer type were established in two ways: (1) by using an equation based on proton signals and an internal standard with known concentration and (2) by using the LOQ based on the signal-to-noise ratio. Both data sets were compared and showed that using the internal standard (method 1) results in more accurate and lower concentration limits in the range of 0.2–8 µg mL−1 for all six polymer types, while the LOQ based on the SNR (method 2) gives consistently higher concentration limits (1–10 µg mL−1). The research shows the accuracy, efficacy, and reliability of quantitative NMR spectroscopy for polymer analysis in these concentration ranges compared to established quantifying methods, such as, PyGC/MS, FTIR, or Raman spectroscopy.

Ultrasonic-Assisted Water-Rich Natural Deep Eutectic Solvents for Sustainable Polyphenol Extraction from Seaweed: A Case Study on Cultivated Saccharina latissima.

This case study introduces a green, 1 h single-step method using water-rich natural deep eutectic solvent (WRNADES) for ultrasound-assisted extraction (UAE) of polyphenols fromSaccharina latissima, a commercially cultivated brown seaweed. The extraction efficiency was evaluated using a selective quantitative NMR method (s-qNMR) and the traditional nonselective colorimetric total phenolic content assay (TPC). Initial 6 h extractions in traditional solvents (methanol, ethanol, acetone, and ethyl acetate) showed a 40-60% increase in polyphenolic yields in 50% aqueous solutions measured by the TPC method. Six different water-rich (50%) NADES (WRNADES) combinations were tested (choline chloride/betaine with lactic acid, citric acid, and 1,3-butanediol), with betaine and 1,3-butanediol (1:1) proving most effective. Parameters for the WRNADES were optimized using Box-Behnken design response surface methodology, resulting in a 1:20 w/w biomass to solvent ratio and a 1 h extraction time at 50 °C. The WRNADES extraction process was refined into a scalable, single-step procedure and compared with traditional solvent extractions (6 h, 50% aqueous methanol and acetone). A final XAD-7 polyphenol recovery step was included in all extractions. The optimized WRNADES extraction yielded 15.97 mg GAE/g of the dry weight recovered polyphenolic extract (s-qNMR), exceeding the 6 h 50% aqueous methanol (12.4 mg GAE/g) and acetone (11.4 mg GAE/g) extractions. Thus, the UAE-WRNADES method presented in this case study provides a cost-effective, sustainable, and eco-friendly alternative for the extraction of phenolic compounds from seaweed. It promotes the development of environmentally friendly production processes within the seaweed biorefinery.

DNA Methylation Age Mediates Effect of Metabolic Profile on Cardiovascular and General Aging.

Alterations in lipid metabolism and DNA methylation are 2 hallmarks of aging. Connecting metabolomic, epigenomic, and aging outcomes help unravel the complex mechanisms underlying aging. We aimed to assess whether DNA methylation clocks mediate the association of circulating metabolites with incident atherosclerotic cardiovascular disease (ASCVD) and frailty. The China Kadoorie Biobank is a prospective cohort study with a baseline survey from 2004 to 2008 and a follow-up period until December 31, 2018. We used the Infinium Methylation EPIC BeadChip to measure the methylation levels of 988 participants' baseline blood leukocyte DNA. Metabolite profiles, including lipoprotein particles, lipid constituents, and various circulating metabolites, were measured using quantitative nuclear magnetic resonance. The pace of DNA methylation age acceleration (AA) was calculated using 5 widely used epigenetic clocks (the first generation: Horvath, Hannum, and Li; the second generation: Grim and Pheno). Incident ASCVD was ascertained through linkage with local death and disease registries and national health insurance databases, supplemented by active follow-up. The frailty index was constructed using medical conditions, symptoms, signs, and physical measurements collected at baseline. A total of 508 incident cases of ASCVD were documented during a median follow-up of 9.5 years. The first generation of epigenetic clocks was associated with the risk of ASCVD (P<0.05). For each SD increment in LiAA, HorvathAA, and HannumAA, the corresponding hazard ratios for ASCVD risk were 1.16 (1.05-1.28), 1.10 (1.00-1.22), and 1.17 (1.04-1.31), respectively. Only LiAA mediated the association of various metabolites (lipids, fatty acids, histidine, and inflammatory biomarkers) with ASCVD, with the mediating proportion reaching up to 15% for the diameter of low-density lipoprotein (P=1.2×10-2). Regarding general aging, a 1-SD increase in GrimAA was associated with an average increase of 0.10 in the frailty index (P=2.0×10-3), and a 33% and 63% increased risk of prefrailty and frailty at baseline (P=1.5×10-2 and 5.8×10-2), respectively; this association was not observed with other clocks. GrimAA mediated the effect of various lipids, fatty acids, glucose, lactate, and inflammatory biomarkers on the frailty index, with the mediating proportion reaching up to 22% for triglycerides in very small-sized very low-density lipoprotein (P=6.0×10-3). These findings suggest that epigenomic mechanisms may play a role in the associations between circulating metabolites and the aging process. Different mechanisms underlie the first and second generations of DNA methylation age in cardiovascular and general aging.

Analysis of rebaudioside A in probiotic-fermented milk via quantitative nuclear magnetic resonance spectroscopy

Rebaudioside A is used in the food and beverage industries owing to its sweetness, even in extremely small amounts, as well as its natural origin. Liquid chromatography is commonly used to quantify rebaudioside A; however, it suffers from complicated sample pretreatment and time consumption. External-calibration quantitative nuclear magnetic resonance (qNMR) analysis of probiotic-fermented milk has thus far not been reported. Moreover, it involves more factors contributing to errors than internal standard approaches used in the official methods, such as those recommended by the International Organization for Standardization. In this study, we developed a method for quantifying rebaudioside A in probiotic-fermented milk using internal-calibration qNMR. This method entails a simple pretreatment step with dilution and solid-phase extraction, enabling rebaudioside A determination in five types of beverages including probiotic-fermented milk. The results revealed a recovery rate and relative standard deviation of 85.4–104 % and 1.57–4.09 %, respectively, satisfying the international guidelines. The working range of qNMR was 0.80–26.7 mg/100 g of product, and the analysis time was 10–20 min. Therefore, the proposed internal-calibration qNMR method facilitates easy, rapid, and accurate quantification of rebaudioside A in probiotic-fermented milk and is applicable for analyzing various other beverages and foods.

Improved solid-state 13C and 15N NMR reveals fundamental compositional divide between refractory dissolved organic carbon and nitrogen in the sea

Marine dissolved organic matter (DOM) is one of the largest reservoirs of organic carbon and nitrogen in the world. Yet, despite its global importance, most DOM remains molecularly uncharacterized. Solid-state nuclear magnetic resonance (NMR) spectroscopy of isolated DOM fractions represents one of the most powerful techniques to understand overall structural composition. However, it is well known that standard cross polarization magic angle spinning (CP/MAS) NMR, the technique used for almost all past solid-state NMR studies of DOM, is at best “semi-quantitative,” and underestimates fully substituted NMR active nuclei. Additionally, almost all past solid-state NMR work analyzed high molecular weight (HMW) material isolated by ultrafiltration, which is now understood to represent mostly 14C-young, “semi-labile” compounds. In contrast, there is far less information regarding the composition of older, low molecular weight (LMW) DOM, which represents the vast majority of the ocean’s accumulated refractory DOM pool.Here, we applied 13C and 15N solid-state multiCP/MAS NMR, improved NMR methods optimized to more quantitatively resolve fully substituted NMR nuclei, to both HMW and LMW DOM isolated from the surface and deep North Pacific Subtropical Gyre. These methods confirm past work indicating most nitrogen containing HMW DOM as amide compounds, but also demonstrate a modest heterocyclic N component not previously identified. In contrast, we found that LMW DON is almost entirely aromatic heterocyclic N, consistent with the hypothesis that heterocyclic N structures may be largely responsible for the accumulation of the ocean’s refractory DON pool. Surprisingly, however, we find DOC aromatic functionalities still represent only a very minor portion of either the HMW or the LMW refractory carbon pools, in marked contrast to refractory DON composition. Together, these more quantitative solid-state NMR techniques likely represent the most accurate picture of DON and DOC functional and compound-class makeup to date, and so have broad implications for our understanding of marine DOM structure and cycling. Specifically, our new data suggests that while chemical composition likely acts as a key control on DOM lability, the most refractory components of DOC and DON have very different compositions, sources, and cycling, supporting the idea that DOC and DON cycling in the ocean may be largely decoupled.