Evaluation of a quick one-step sample preparation method for the determination of the isotopic fingerprint of rapeseed (Brassica napus): Investigation of the influence of the use of 2,2-dimethoxypropane on compound-specific stable carbon and hydrogen isotope analyses by gas chromatography combustion/pyrolysis isotope ratio mass spectrometry.

RATIONALE: Gas chromatographic analyses for vegetable oils require transesterification, which generally involves multiple steps, mainly to generate fatty acid methyl esters (FAMEs). A quick method based on acid-catalyzed transesterification using 2,2-dimethoxypropane (DMP) enables the conversion in one step, in a single reactor. For compound-specific stable carbon and hydrogen isotope analyses (C- and H-CSIA) of individual fatty acids (FAs) in oil, the verification of this one-step method has not yet been reported. METHODS: In this study, we evaluated the feasibility of the one-step method for C- and H-CSIA of individual FAMEs in rapeseed samples. The focus was on the investigation of the influence of methanol, which was produced from the reactions of DMP with glycerol and water during transesterification, on the accuracy of isotope composition of FAMEs, consequently of the FAs. The reproducibility of the one-step method was assessed by the measurement of the FAMEs from rapeseed and rapeseed oil. For the C- and H-CSIA of individual FAMEs, a gas chromatography combustion/pyrolysis isotope ratio mass spectrometry system was used. RESULTS: Our results showed that no significant differences arise in the carbon and hydrogen isotope compositions of the selected main FAMEs produced with and without DMP except for the H-CSIA value of C18:3. The reproducibility of the one-step method for rapeseed was in the range of ±0.1 mUr to ± 0.3 mUr for C-CSIA and ±1 mUr to ±3 mUr for H-CSIA of the main FAMEs. CONCLUSIONS: DMP improves the transesterification efficiency without influencing the accuracy of the C- and H-CSIA of FAMEs. The performance of the one-step method for rapeseed samples for the determination of C- and H-CSIA values of FAMEs is satisfactory. Thus, the applicability of the one-step method for isotopic fingerprint analyses of FAs in oilseeds is reported for the first time.

Estimation of radiation dose from ingested tritium in humans by administration of deuterium-labelled compounds and food.

Radiation doses from organically bound tritium (OBT) in foods have been a major concern near nuclear facilities. The current dose coefficient for OBT is calculated using a standard model from the International Commission on Radiological Protection, in which some biokinetic values are not based on human metabolic data. Here, the biokinetics of ingested OBT, and radiation doses from them, were estimated by administering labelled compounds and foods to volunteers, using a deuterium (D) tracer as a substitute for tritium. After the administration of D-labelled glucose, alanine, palmitic acid, or soybean, the D/H ratios in urine were measured for up to 119 days, and the biokinetic parameter values were determined for OBT metabolism. The slow degradation rates of OBT could not be obtained, in many volunteers administered glucose and alanine. The estimated committed effective dose for 1 Bq of tritium in palmitic acid varied from 3.2 × 10-11 to 3.5 × 10-10 Sv Bq-1 among volunteers and, for those administered soybean, it varied from 1.9 × 10-11 to 1.8 × 10-10 Sv Bq-1. These results suggest that OBT, present in some ingested ingredients, gives higher doses than the current dose coefficient value of 4.2 × 10-11 Sv Bq-1.

Determination of the δ2 H values of high molecular weight lipids by high-temperature gas chromatography coupled to isotope ratio mass spectrometry.

RATIONALE: The hydrogen isotopic composition of lipids (δ2 Hlipid ) is widely used in food science and as a proxy for past hydrological conditions. Determining the δ2 H values of large, well-preserved triacylglycerides and other microbial lipids, such as glycerol dialkyl glycerol tetraether (GDGT) lipids, is thus of widespread interest but has so far not been possible due to their low volatility which prohibits analysis by traditional gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS). METHODS: We determined the δ2 H values of large, polar molecules and applied high-temperature gas chromatography (HTGC) methods on a modified GC/P/IRMS system. The system used a high-temperature 7-m GC column, and a glass Y-splitter for low thermal mass. Methods were validated using authentic standards of large, functionalised molecules (triacylglycerides, TGs), purified standards of GDGTs. The results were compared with δ2 H values determined by high-temperature elemental analyser/pyrolysis/isotope ratio mass spectrometry (HTEA/P/IRMS), and subsequently applied to the analysis of GDGTs in a sample from a methane seep and a Welsh peat. RESULTS: The δ2 H values of TGs agreed within error between HTGC/P/IRMS and HTEA/IRMS, with HTGC/P/IRMS showing larger errors. Archaeal lipid GDGTs with up to three cyclisations could be analysed: the δ2 H values were not significantly different between methods with standard deviations of 5 to 6 ‰. When environmental samples were analysed, the δ2 H values of isoGDGTs were 50 ‰ more negative than those of terrestrial brGDGTs. CONCLUSIONS: Our results indicate that the HTGC/P/IRMS method developed here is appropriate to determine the δ2 H values of TGs, GDGTs with up to two cyclisations, and potentially other high molecular weight compounds. The methodology will widen the current analytical window for biomarker and food light stable isotope analyses. Moreover, our initial measurements suggest that bacterial and archaeal GDGT δ2 H values can record environmental and ecological conditions.

Atmospheric factors controlling stable isotope variations in modern precipitation of the tropical region of Bangladesh.

The study investigates the factors that control the isotopic composition of tropical precipitation in Bangladesh. Daily and monthly rainfall samples were collected from three stations from 2013 to 2015: (1) northern and moderately high altitude: Sylhet, (2) middle part of the country (close to Tropic of Cancer): Savar, and (3) southern coastal region: Barisal. To escape from the post-evaporation effect, proper care was adopted. This is supported by the fact that the local meteoric water lines (LMWLs) derived for the daily precipitations of all stations mostly follow the global meteoric water line (GMWL). The results exhibit a clear seasonal and spatial variation in both δ 18O and δ 2H of precipitation. 18O is more depleted in samples collected during rainy (monsoon) seasons, while 18O enrichment is associated to winter and summer (pre-monsoon) seasons. During rainy season, intra-seasonal variability of δ18O rainfall is clearly seen and remarkable depletion of 18O is observed during the period of intense convective activity over the Bay of Bengal. This feature indicates that isotope variability in Bangladesh is controlled by large-scale convective activity rather than local rainfall amount.

Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium.

In this study, using new approach (laser diffraction + biological dyes), we have demonstrated the decrease of cells viability in vitro in the deuterated growth medium, whereas in the deuterium-depleted medium, there was an increase of cell viability. We have also found that not all dyes are equally sensitive to the D/H ratios in the culture medium (system) as well as to the different cell types (cancer vs normal cells).

Stable isotope analysis of non-exchangeable hydrogen in carbohydrates derivatised with N-methyl-bis-trifluoroacetamide by gas chromatography - Chromium silver reduction/High temperature Conversion-isotope ratio mass spectrometry (GC-CrAg/HTC-IRMS).

A novel procedure for the rapid isotope analysis of the carbon-bound non-exchangeable (CBNE) hydrogen in mono and disaccharides has been developed to demonstrate the feasibility of detecting undeclared addition of exogenous sugar products in foods and beverages susceptible to economically motivated adulteration. The procedure utilizes a simple one-step reaction, with the derivatising agent N-methyl-bis-trifluoroacetamide, to substitute the exchangeable hydroxyl-hydrogens with trifluoroacetate derivatives that are sufficiently volatile to be separated and measured by a gas chromatograph coupled to an isotope ratio mass spectrometer. The conversion of the derivatised sugars into the measuring gas is achieved using a high temperature chromium-silver reactor that retains carbon, oxygen and fluorine whilst releasing hydrogen gas for stable isotope measurement. The new procedure has advantages over existing methods in terms of ease of use, analysis time and compound-specific information. Sugars from fruit juice and honey have been measured to demonstrate the feasibility of using this technique.

Considerations in the Analysis of Hydrogen Exchange Mass Spectrometry Data.

A major component of a hydrogen exchange mass spectrometry experiment is the analysis of protein and peptide mass spectra to yield information about deuterium incorporation. The processing of data that are produced includes the identification of each peptic peptide to create a master table/array of peptide identity that typically includes sequence, retention time and retention time range, mass range, and undeuterated mass. The amount of deuterium incorporated into each of the peptides in this array must then be determined. Various software platforms have been developed in order to perform this specific type of data analysis. We describe the fundamental parameters to be considered at each step along the way and how data processing, either by an individual or by software, must approach the analysis.

Calculation of the Protein Turnover Rate Using the Number of Incorporated 2H Atoms and Proteomics Analysis of a Single Labeled Sample.

Rate constant estimation with heavy water requires a long-term experiment with data collection at multiple time points (3-4 weeks for mitochondrial proteome dynamics in mice and much longer in other species). When tissue proteins are analyzed, this approach requires euthanizing animals at each time point or multiple tissue biopsies in humans. Although short-term protocols are available, they require knowledge of the maximum number of isotope labels (N) and accurate quantification of observed 2H-enrichment in the peptide. The high-resolution accurate mass spectrometers used for proteome dynamics studies are characterized by a systematic spectral error that compromises these measurements. To circumvent these issues, we developed a simple algorithm for the rate constant calculation based on a single labeled sample and comparable unlabeled (time 0) sample. The algorithm determines N for all proteogenic amino acids from a long-term experiment to calculate the predicted plateau 2H-labeling of peptides for a short-term protocol and estimates the rate constant based on the measured baseline and the predicted plateau 2H-labeling of peptides. The method was validated based on the rate constant estimation in a long-term experiment in mice and dogs. The improved 2 time-point method enables the rate constant calculation with less than 10% relative error compared to the bench-marked multi-point method in mice and dogs and allows us to detect diet-induced subtle changes in ApoAI turnover in mice. In conclusion, we have developed and validated a new algorithm for protein rate constant calculation based on 2-time point measurements that could also be applied to other biomolecules.

Heptafluorobutyl Chloroformate-Based Sample Preparation Protocol for Nonchiral and Chiral Amino Acid Analysis by Gas Chromatography-Mass Spectrometry.

Gas chromatography (GC) is a commonly used technique in amino acid analysis (AAA). However, one of the requirements of the application of GC for AAA is a need for the polar analytes to be converted into their volatile, thermally stable derivatives. In the last two decades, alkyl chloroformates (RCFs) have become attractive derivatization reagents. The reagents react immediately with most amino acid functional groups in aqueous matrices, and the process can easily be coupled with liquid-liquid extraction of the resulting less polar derivatives into immiscible organic phase. Here we describe a simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate (HFBCF) followed by subsequent chiral as well as nonchiral GC/MS (mass spectrometric) analysis on a respective nonpolar fused silica and an enantioselective Chirasil-Val capillary column.

Resolution of protein hydrogen/deuterium exchange by fitting amide exchange probabilities to the peptide isotopic envelopes.

RATIONALE: Mass spectra processing in protein hydrogen/deuterium (H/D) exchange has been remarkably improved by the introduction of fitting of the amide exchange probabilities to peptide isotopic envelope intensities (Kan et al., 2013), in contrast to methods in which only the peptide deuterium uptakes (centroid shifts of isotopic envelopes) are used. However, the known implementations are based on the general fitting routines that use only the objective function values. Besides, applicability of more than one fitting method makes necessary their comparative evaluation. METHODS: Two fitting methods were considered: the common least squares and the fitting of the multinomial distribution representing the number of deuterium atoms exchanged in the individual peptides. Both methods were applied either directly to the isotopic envelope data or to the deuterium distributions obtained by envelope deconvolution (i.e. de-isotoping). RESULTS: An autonomous Matlab script was prepared, based on the exact expressions for the gradient and Hessian of the objective function, with the trust-region algorithm implemented in the compact analytical form recently made available. The least-squares fitting to the envelope data produced the best results, with the greatest precision and good coverage of exact values by the confidence intervals. The deuterium distributions were sensitive to the (simulated) experimental error whose progression by envelope deconvolution caused degradation in accuracy. The multinomial distribution fitting exhibited poor performance due to inadequate representation of the experimental error and missing of the appropriate weight parameters. Some specific peptide arrangement details were discussed as potential sources of ambiguity in the fitting results. CONCLUSIONS: The method of fitting to peptide isotopic envelopes has been improved by using the exact gradient and Hessian of the objective function. The fitting should be repeated with different initial guesses in order to find not only the global minimum, but also the local minima with similar depths which may exist due to eventual ambiguity of the fitting results.

Cyclooxygenase­2­mediated upregulation of heme oxygenase 1 mitigates the toxicity of deuterium­tritium fusion radiation.

Utilizing the energy released from the nuclear fusion of deuterium with tritium (D­T) may be an important method of supplying energy in the future. The ionizing radiation emitted from nuclear fusion is a potential health risk to humans, including scientists who are currently performing nuclear fusion experiments and the employees of fusion nuclear plants, in the future. However, there have been few reports on the biological effects of fusion radiation. In the present study, using the High Intensity D­T Fusion Neutron Generator, the DNA damage and its regulation in normal human fibroblasts exposed to fusion radiation were investigated. Heme oxygenase 1 (HO­1), which is reported to induce anti­inflammatory activity, was upregulated in the irradiated cells. Pretreatment with the HO­1 inhibitor, protoporphyrin IX zinc (II), exacerbated double strand break formation following exposure to fusion radiation. The expression of cyclooxygenase­2 (COX­2) contributed to the upregulation of HO­1, as demonstrated by the result that its inhibitor, NS­398, inhibited the induction of HO­1 in irradiated cells. It was further clarified that the ataxia telangiectasia mutated DNA damage response was activated and it stimulated the phosphorylation of p38 mitogen­activated protein kinase, which was responsible for the upregulation of COX­2 and HO­1. These results provide novel information on fusion radiation­induced biological effects and potential targets for decreasing the associated health risks.

UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

Validation of anthropometric and bioelectrical impedance analysis (BIA) equations to predict total body water in a group of Cameroonian preschool children using deuterium dilution method

BACKGROUND: Little information is available on the validity of anthropometry or impedance-based equations for prediction of total body water (TBW) in African children. This study was designed to validate and develop equations to predict total body water in Cameroonian children. METHODS: TBW was measured by deuterium dilution in 102 children between 24 and 60 months of age and compared with the ones predicted by 5 anthropometric and 7 BIA equations. Multiple linear regression analysis was used to develop prediction equations for TBW from anthropometric parameters. RESULTS: Unacceptable discrepancies in the estimates of TBW at individual level were noted with all the equations tested. The following new anthropometry and BIA equations for the estimation of TBW were respectively developed: TBW = 6.488 + 0.434 × sex−0.039 × age + 0.670 × weight−0.081 × MUAC (cm)−0.372 × BMI (adjustedR2= 0.71,RMSE = 3.6), and TBW =−6.206 + 0.0037 × height2/Z−0.041 × age + 0.265 × weight + 0.1214 × height (adjustedR2=0.68, RMSE = 1.4). The cross-validation procedures revealed that the predicted values of TBW compared with measured values are accurate at a group level. CONCLUSION: The current published anthropometric and BIA equations are invalid for the estimation of TBW in Cameroonian preschool children. The newly developed anthropometry or BIA prediction equations are valid for use in Cameroonian children aged 24­60 months

Quantitation of Nine Lactones in Dairy Cream by Stable Isotope Dilution Assays Based on Novel Syntheses of Carbon-13-Labeled γ-Lactones and Deuterium-Labeled δ-Lactones in Combination with Comprehensive Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometry.

Lactones are well-known aroma compounds in, e.g., fruits and fermented foods as well as in dairy products, such as cream or milk powders. The latter are often used in confectionary products, e.g., milk chocolate. Lactones are suggested to contribute to the distinct aroma of dairy products and have also been reported in milk chocolate. However, data on their contribution to the overall aroma of this type of chocolate are scarce. As a result of their pH-dependent instability and their low volatility, a reliable quantitation of lactones is a challenge. Thus, to allow for a quantitation of nine lactones in one single comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry in electron ionization mode run, new synthetic routes were developed for five carbon-13-labeled γ-lactones and four deuterium-labeled δ-lactones, with the isotope label in the ring to be used in stable isotope dilution assays. The concentrations of the nine lactones were then analyzed in raw and pasteurized cream as well as in a heat-treated raw cream. δ-Dodecalactone and δ-decalactone showed the highest concentrations in both the raw and pasteurized cream. In the latter, δ-dodecalactone reached a 2.5-fold higher concentration compared to the raw cream. Subsequent heat treatments in a lab scale showed a further increase by factors of 13 and 19, respectively, suggesting a high potential of lactone precursors in cream. The results serve as a basis for further studies on lactone formation in other thermally processed products, such as milk chocolate.

Dilución isotópica con deuterio para determinar ingesta de leche humana y composición corporal materna / Deuterium-oxide turnover technique to assess breast milk intake and maternal body composition / Diluição isotópica com deutério para determinar a ingestão de leite humano e a composição corporal materna

El objetivo del trabajo fue describir la aplicación de la técnica de dilución isotópica con deuterio de dosis a la madre para determinar la ingesta de leche materna y la composición corporal de las madres, en distintos tipos de lactancia. El método analítico se aplicó en cuatro casos modelo de pares madre-lactante en los cuales las madres recibieron una dosis oral de agua deuterada, recolectándose 6 muestras de saliva de ambos durante 15 días. El enriquecimiento de deuterio se determinó en un espectrómetro FTIR-Shimadzu-Affinity obteniéndose la ingesta de leche materna (ILM) y de agua de otras fuentes (Fd). Se observó una variación del enriquecimiento de deuterio en la saliva del lactante, asociada al tipo de lactancia recibida, siendo mayor en el caso de lactancia materna exclusiva (LME). Asimismo, a medida que aumentó Fd, disminuyó ILM. Además, fueron determinadas el agua corporal, la masa libre de grasa y la masa grasa materna. La transferencia de las habilidades técnicas y del conocimiento a través de metodologías innovadoras para determinar la ingesta de leche materna es de utilidad como herramienta de evaluación de la alimentación del lactante y para investigar en qué medida la lactancia natural es reemplazada por la ingesta de otros alimentos. Mejorar la estimación de la LME contribuye al conocimiento de la recomendación de OMS y UNICEF de mantener la misma hasta el sexto mes de vida.

The aim of this study was to describe the application of the dose-to-the-mother deuterium-oxide turnover technique to determine the breast milk intake and body composition of mothers in different types of breastfeeding. This analytical method was performed in four mother-infant pairs at 4 months from birth. Mothers received an oral dose of deuterated water, collecting 6 samples of saliva from mother and baby during a period of 15 days. Deuterium enrichment was determined in a Shimadzu FTIR-spectrometer-Affinity to obtain the intake of breast milk and water from non-breast milk sources. In this study, a variation of the enrichment of deuterium in the saliva of the infant was observed, being higher when the infant was exclusively breastfed. As non-breast milk water increased, the intake of human milk decreased. Furthermore, maternal total body water, fat free mass and fat mass were determined. To improve technical skills and knowledge through innovative methods of breast milk measurement can be useful as an assessment tool for evaluating infant feeding and investigating the extent to which breast milk is being replaced by the consumption of other foods in order to estimate exclusive breastfeeding in the future. This would contribute to the knowledge of maintaining breastfeeding until the sixth month of life, as it is recommended by WHO and UNICEF.

O objetivo deste estudo foi descrever a aplicação da técnica de diluição isotópica com deutério de dose à mãe para determinar a ingestão de leite materno e a composição corporal das mães, em diferentes tipos de aleitamento. O método analítico foi aplicado em quatro casos modelo de pares mães-lactante nos quais as mães receberam uma dose oral de água deuterada, coletando-se 6 amostras de saliva de ambos (mães e lactantes) durante 15 días. O enriquecimento de deutério foi determinado em um espectrômetro FTIR-Shimadzu-Affinity, sendo obtida a ingestão de leite materno (ILM) e de água proveniente de outras fontes (Fd). Observou-se uma variação do enriquecimento de deutério na saliva do lactante, associada ao tipo de aleitamento recebido, sendo maior no caso de aleitamento materno exclusivo (AME). Também, na medida que aumentou Fd, diminuiu ILM. Além disso, a água corporal, a massa livre de gordura y a massa gorda materna foram determinadas. A transferência das habilidades técnicas e do conhecimento através de metodologias inovadoras para determinar a ingestão de leite materno é de utilidade como ferramenta de avaliação da alimentação do lactante e para investigar em que medida o aleitamento natural é substituído pela ingestão de outros alimentos. Melhorar a avaliação do AME contribui ao conhecimento da recomendação da OMS e UNICEF de manter a mesma até o sexto mês de vida.

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.

Reproductive Allochrony in Seasonally Sympatric Populations Maintained by Differential Response to Photoperiod: Implications for Population Divergence and Response to Climate Change.

Reproductive allochrony presents a potential barrier to gene flow and is common in seasonally sympatric migratory and sedentary birds. Mechanisms mediating reproductive allochrony can influence population divergence and the capacity of populations to respond to environmental change. We asked whether reproductive allochrony in seasonally sympatric birds results from a difference in response to supplementary or photoperiodic cues and whether the response varies in relation to the distance separating breeding and wintering locations as measured by stable isotopes. We held seasonally sympatric migratory and sedentary male dark-eyed juncos (Junco hyemalis) in a common garden in early spring under simulated natural changes in photoperiod and made measurements of reproductive and migratory physiology. On the same dates and photoperiods, sedentary juncos had higher testosterone (initial and gonadotropin-releasing hormone induced), more developed cloacal protuberances, and larger testes than migrants. In contrast, migratory juncos had larger fat reserves (fuel for migration). We found a negative relationship between testis mass and feather hydrogen isotope ratios, indicating that testis growth was more delayed in migrants making longer migrations. We conclude that reproductive allochrony in seasonally sympatric migratory and sedentary birds can result from a differential response to photoperiodic cues in a common garden, and as a result, gene flow between migrants and residents may be reduced by photoperiodic control of reproductive development. Further, earlier breeding in response to future climate change may currently be constrained by differential response to photoperiodic cues.

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.

Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold.

[Comparative characteristics of the isotopic D/H composition and antioxidant activity of freshly squeezed juices from fruits and vegetables grown in different geographical regions].

Data presented in this paper reflect changes in antioxidant activity, the content of prooxidant factors and deuterium concentration in freshly squeezed juices from fruits and vegetables grown in different climatic regions (10 samples of juices from wholesale and retail trade network of 8 kinds of vegetables and fruits, 28 manufacturers from 14 countries). Determination of the concentration of deuterium was performed using a nuclear magnetic resonance spectrometer. Total antioxidant activity of fresh juices was determined amperometrically after dilution in 2.2 mM H3PO4 in a ratio of 1:100. Prooxidant performance was evaluated by a maximum and area of flash of chemiluminescence induced by the introduction of 0.3% hydrogen peroxide. It was found that the antioxidant activity of fresh juice from fruits and vegetables grown within the same climatic region can differ by several times. In this case, most of the fruits and vegetables of russian producers were not inferior, than antioxidant activity of the fresh juices from the same plant products grown abroad. It should be noted that the indicators of the antioxidant activity of fresh juice from Russian pears exceeded this indicator of all fresh juices from pears, imported from Argentina, South Africa and the United States of America by 21.1, 30.4 and 32.7%, respectively. In assessing the prooxidant properties of fresh juices should be noted the almost complete absence of factors with prooxidant nature only in 36% of the studied fresh juices, whose maximum performance and area of flash of chemiluminescence were less than 0.1%, including a pear and apple juices from the russian production. It should be noted that the area of chemiluminescence of the juice from potatoes, grown in Russia, was at 103.1 and 115.2% lower than in juice obtained respectively from potatoes produced in Israel and Egypt (p<0.05), indicating a higher safety of consumption of potatoes produced in Russia. When studying--the isotopic D/H composition of fresh juices it was found that the highest deuterium content was in the juice from the pears, imported from Argentina (deltaD = -72% per hundred), while the lowest concentration of deuterium was observed in the juice from the Egyptian potatoes (delta = -358% per hundred). In general, significantly lower deuterium content was determined in fresh juices made from potatoes and cabbage grown in different countries, in comparison with other fresh juices from fruits and vegetables. The smallest range of differences in the isotopic D/H was composed in freshjuices from tomato, pomegranate and oranges of Turkish manufacturers (deuterium concentration ranged in them from -221 to -214% per hundred), that can be used to confirm the geographical origin of fruits and vegetables grown in Turkey. The data reflecting the antioxidant activity, the content of prooxidant factors and deuterium concentration in the juices, allow us to recommend the latter as additional criteria when assessing the quality of food products.