RESUMO
Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H2O than in D2O, while the second carboncarbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H2O and D2O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H2O than in D2O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO-) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H2O and in D2O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (â¼25%) yield of androstenedione product relative to the oxygenated starting material.
Assuntos
Pregnenolona , Esteroide 17-alfa-Hidroxilase , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 17-alfa-Hidroxilase/química , Humanos , Pregnenolona/química , Pregnenolona/metabolismo , Progesterona/química , Progesterona/metabolismo , Óxido de Deutério/química , HidroxilaçãoRESUMO
Heavy water is an ideal contrast agent for metabolic activity and can be adapted to a wide range of biological systems owing to its non-invasiveness, universal applicability, and cost-effectiveness. As a new type of probe, the heavy isotope of water has been widely used in the study of cell development, metabolism, tissue homeostasis, aging, and tumor heterogeneity. Herein, we review findings supporting the applications of and research on heavy water in monitoring of bacterial metabolism, rapid detection of drug sensitivity, identification of tumor cells, precision medicine, and evaluation of skin barrier function and promote the use of heavy water as a suitable marker for the development of detection and treatment methodologies.
Assuntos
Análise Espectral Raman , Água , Bactérias/metabolismo , Óxido de Deutério/química , Óxido de Deutério/metabolismo , Análise Espectral Raman/métodosRESUMO
The phase equilibria of intrinsically disordered proteins are exquisitely sensitive to changes in solution conditions and this can be used to investigate the driving forces of phase separation in vitro as well as the biological roles of phase transitions in live cells. Here we investigate how using D2 O as co-solvent in an aqueous buffer changes the phase equilibrium of a fragment of the activation domain of the androgen receptor, a transcription factor that plays a role in the development of the male phenotype and is a therapeutic target for castration resistant prostate cancer. We show how replacing even small fractions of H2 O with D2 O increases the propensity of this fragment to undergo liquid-liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation. Our results indicate that it is necessary to take this effect into consideration when studying phase separation phenomena with biophysical methods that require using D2 O as a co-solvent. In addition, they suggest that additions of D2 O may be used to enhance phase separation phenomena in cells, facilitating their observation.
Assuntos
Óxido de Deutério/química , Proteínas Intrinsicamente Desordenadas/química , Receptores Androgênicos/química , Humanos , Domínios ProteicosRESUMO
During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3'-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2',3'-anhydroinosine, a byproduct in the preparation of 3-'deoxyinosine. Moreover, 2',3'-anhydroinosine forms during radical dehalogenation of 9-(2',5'-di-O-acetyl-3'-bromo- -3'-deoxyxylofuranosyl)hypoxanthine, a precursor of 3'-deoxyinosine in chemical synthesis. The products of 2',3'-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2',3'-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2',3'-anhydroinosine hydrolysis in D2O is fully determined for the first time.
Assuntos
Desoxiadenosinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Purina-Núcleosídeo Fosforilase/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Biocatálise , Desoxiadenosinas/química , Óxido de Deutério/química , Hidrólise , Inosina/análogos & derivados , Inosina/química , Inosina/metabolismo , Especificidade por SubstratoRESUMO
Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated d-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.
Assuntos
Vesículas Extracelulares/química , Imagem Molecular , Análise Espectral Raman , Colina/química , Colina/metabolismo , Óxido de Deutério/química , Óxido de Deutério/metabolismo , Vesículas Extracelulares/metabolismo , Glucose/química , Glucose/metabolismo , Humanos , Tamanho da Partícula , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
Cell proliferation plays a central role in the pathogenesis of every neoplastic disease as well as many other types of illness. Labeling of newly replicated DNA with deuterium (2H), a nonradioactive isotope of hydrogen, administered to the patients in drinking water (2H2O) is a safe and reliable method to measure the in vivo birth rates of cells. Here, we describe a protocol to measure chronic lymphocytic leukemia B-cell birth/proliferation and death rates over time using this approach.
Assuntos
Linfócitos B/patologia , Desoxirribose/análise , Óxido de Deutério/administração & dosagem , Cromatografia Gasosa-Espectrometria de Massas/métodos , Leucemia Linfocítica Crônica de Células B/patologia , Apoptose , Linfócitos B/metabolismo , Proliferação de Células , DNA/química , DNA/isolamento & purificação , Replicação do DNA , Desoxirribose/química , Óxido de Deutério/química , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Humanos , Cinética , Leucemia Linfocítica Crônica de Células B/sangueRESUMO
PURPOSE: To perform in vitro high-resolution 900 MHz magnetic resonance spectroscopy (NMR) analysis of human brain tumor tissue extracts and analyze for the oncometabolite 2-hydroxyglutarate (2HG) and other brain metabolites, not only for 1H but also for 13C with indirect detection by heteronuclear single quantum correlation (HSQC). MATERIAL AND METHODS: Four surgically removed human brain tumor tissue samples were used for extraction and preparation of NMR samples. These tissue samples were extracted with 4% perchloric acid and chloroform, freeze-dried, then dissolved into 0.28 mL of deuterium oxide (D2O, 99.9 atom % deuterium) containing 0.025 wt % sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP). All samples were adjusted to pH range of 6.9-7.1 before finally transferred to 5 mm Shigemi™ NMR microtube. NMR experiments were performed on Bruker DRX 900 MHz spectrometer with 1H/13C/15N Cryo-probe™ with Z-gradient, without further temperature control for the samples. All chemical shift values were presented relative to TSP at 0.00 ppm for both 1H and 13C. 1H 1D, 1H-13C HSQC, 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear multiple bond correlation (HMBC) spectra were acquired and analyzed. RESULTS: 2-hydroxyglutarate, an oncometabolite associated with gliomas with IDH mutations, was successfully detected and assigned by both 1H-13C HSQC and 1H-1H COSY experiments as well as 1H 1D experiments in two of the tissue samples. In particular, to our knowledge this work shows the first example of detecting 900 MHz 13C-NMR spectral lines of 2-hydroxyglutarate in human brain tumor tissue samples. In addition to the oncometabolite 2-hydroxyglutarate, at least 42 more metabolites were identified from our series of NMR experiment. CONCLUSION: The detection of 2-hydroxyglutarate and other metabolites can be facilitated by homonuclear and heteronuclear two-dimensional 900 MHz NMR spectroscopy even in case of real tumor tissue sample extracts without physical separation of metabolites.
Assuntos
Neoplasias Encefálicas/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Glioma/metabolismo , Glutaratos/análise , Espectroscopia de Prótons por Ressonância Magnética/métodos , Extratos de Tecidos/análise , Encéfalo/metabolismo , Encéfalo/patologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/instrumentação , Clorofórmio/química , Óxido de Deutério/química , Humanos , Concentração de Íons de Hidrogênio , Percloratos/química , Propionatos/química , Espectroscopia de Prótons por Ressonância Magnética/instrumentação , Reprodutibilidade dos Testes , Compostos de Trimetilsilil/químicaRESUMO
Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and selectivity. As majority systems rely on reversed-phase columns with water being the main component of the mobile phase, fluorescent compounds with emission maxima higher than 500â¯nm might be dynamically quenched. A simple replacement of H2O with D2O enhanced the sensitivity for selected compounds by 10-200%. Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally occurring forms of vitamin B2. Similar levels of enhancement were obtained by fluorescence spectrometry. Such simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.
Assuntos
Cromatografia de Fase Reversa/métodos , Óxido de Deutério/química , Espectrometria de Fluorescência/métodos , Doxorrubicina/análise , Doxorrubicina/química , Fluoresceína/análise , Fluoresceína/química , Espectrometria de Massas/métodos , Riboflavina/análise , Riboflavina/químicaRESUMO
In this work, we investigated the influence of polyvinylpyrrolidone (PVP) on the solubility of artemisinin in aqueous solution by using quantitative 1H NMR. Experimental results demonstrate that about 4 times of incremental increase occurs on the solubility of artemisinin upon introducing PVP. In addition, dipole-dipole interaction between the ester group of artemisinin and the amide group of N-methylpyrrolidone (NMP), a model compound of PVP, is characterized by two-dimensional (2D) correlation FTIR spectroscopy with the DAOSD (Double Asynchronous Orthogonal Sample Design) approach developed in our previous work. The observation of cross peaks in a pair of 2D asynchronous spectra suggests that dipole-dipole interaction indeed occurs between the ester group of artemisinin and amide group of NMP. Moreover, the pattern of cross peaks indicates that the carbonyl band of artemisinin undergoes blue-shift while the bandwidth and absorptivity increases via interaction with NMP, and the amide band of NMP undergoes blue-shift while the absorptivity increases via interaction with artemisinin. Dipole-dipole interaction, as one of the strongest intermolecular interaction between artemisinin and excipient, may play an important role in the enhancement of the solubility of artemisinin in aqueous solution.
Assuntos
Artemisininas/química , Povidona/química , Antimaláricos/química , Óxido de Deutério/química , Espectroscopia de Ressonância Magnética , Projetos de Pesquisa , Solubilidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.
Assuntos
Proteínas/química , Temperatura , Óxido de Deutério/química , Cinética , Espectrometria de Fluorescência , Espectrofotometria Infravermelho , Fatores de TempoRESUMO
Moisture absorbed into the powder of Met-Pro-Asp-Ala-His-Leu (MPDAHL)-a novel egg albumen antioxidant peptide-profoundly affects its properties. In this study, we elucidated water dynamics in MPDAHL using DVS, DSC, and low-field 1 H NMR. Based on the DVS data, we found that MPDAHL sorption kinetics obey a parallel exponential model. DSC results indicated that both water and heating could change the microstructure of MPDAHL. The T2 parameters of NMR reflected the different phases of moisture absorption revealed that there were 4 categories of water with different states or mobility in the MPDAHL during the moisture absorption process. The fastest fraction T2b mainly dominated the hygroscopicity of MPDAHL and the absorbed water significantly changed the proton distribution and structure of MPDAHL. Thus, this study shows that DVS, DSC, and low-field 1 H NMR are effective methods for monitoring water mobility and distribution in synthetic peptides. It can be used to improve the quality assurance of functional peptides.
Assuntos
Albuminas/química , Ovos/análise , Peptídeos/química , Água/química , Animais , Varredura Diferencial de Calorimetria , Galinhas , Óxido de Deutério/química , Dipeptídeos/química , Cinética , Espectroscopia de Ressonância Magnética , Pós , Controle de QualidadeRESUMO
Radical S-adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d-configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry-based assay is introduced that permits characterization of products generated in E. coli. Applying this to a range of non-natural peptide-epimerase combinations, it is shown that the d-amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme-leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.
Assuntos
Aminoácidos/biossíntese , Carboidratos Epimerases/metabolismo , Óxido de Deutério/metabolismo , S-Adenosilmetionina/metabolismo , Aminoácidos/química , Carboidratos Epimerases/química , Óxido de Deutério/química , Radicais Livres/química , Radicais Livres/metabolismo , Conformação Molecular , S-Adenosilmetionina/químicaRESUMO
Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles.
Assuntos
Alelos , Antígenos HLA/genética , Antígenos HLA/metabolismo , Linhagem Celular Tumoral , Óxido de Deutério/química , Antígenos HLA/química , Humanos , Marcação por IsótopoRESUMO
Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.
Assuntos
Crioprotetores/química , Óxido de Deutério/química , Gelo , Óleos/química , Pseudomonas syringae/química , EmulsõesRESUMO
Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions.
Assuntos
Peptídeos beta-Amiloides/química , Hidrogéis/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Peptídeos beta-Amiloides/metabolismo , Óxido de Deutério/química , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Microscopia Eletrônica , Reologia , Tensão Superficial , Água/químicaRESUMO
An understanding of the location of peptides, proteins, and other biomolecules within the bicontinuous cubic phase is crucial for understanding and evolving biological and biomedical applications of these hybrid biomolecule-lipid materials, including during in meso crystallization and drug delivery. While theoretical modeling has indicated that proteins and additive lipids might phase separate locally and adopt a preferred location in the cubic phase, this has never been experimentally confirmed. We have demonstrated that perfectly contrast-matched cubic phases in D2O can be studied using small-angle neutron scattering by mixing fully deuterated and hydrogenated lipid at an appropriate ratio. The model transmembrane peptide WALP21 showed no preferential location in the membrane of the diamond cubic phase of phytanoyl monoethanolamide and was not incorporated in the gyroid cubic phase. While deuteration had a small effect on the phase behavior of the cubic phase forming lipids, the changes did not significantly affect our results.
Assuntos
Difração de Nêutrons , Peptídeos/química , Proteínas/química , Espalhamento a Baixo Ângulo , Varredura Diferencial de Calorimetria , Medição da Troca de Deutério , Óxido de Deutério/química , Óxido de Deutério/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Temperatura , Difração de Raios XRESUMO
The presence of malignant ascites in advanced cancer patients is associated with both a poor prognosis and quality of life with a risk of abdominal infection and sepsis. Contemporary noninvasive visualization methods such as ultrasound, computed tomography, and magnetic resonance imaging (MRI) often struggle to differentiate malignant ascites from surrounding tissues. This study aimed to determine the utility of selective H2O imaging in the abdominal cavity with a free radical probe and deuterium oxide (D2O) contrast agent using in vivo dynamic nuclear polarization-MRI (DNP-MRI). Phantom imaging experiments established a linear relationship between H2O volume and image intensity using in vivo DNP-MRI. Similar results were obtained when the radical-D2O probe was used to determine selective and spatial information on H2O in vivo, modeled by the injection of saline into the abdominal cavity of mice. To demonstrate the utility of this method for disease, malignant ascites in peritoneal metastasis animal model was selected as one of the typical examples. In vivo DNP-MRI of peritoneal metastasis animal model was performed 7-21 days after intraperitoneal injection of luciferase, stably expressing the human pancreatic carcinoma (SUIT-2). The image intensity with increasing malignant ascites was significantly increased at days 7, 16, and 21. This increase corresponded to in vivo tumor progression, as measured by bioluminescent imaging. These results suggest that H2O signal enhancement in DNP-MRI using radical-D2O contrast is positively associated with the progression of dissemination and could be a useful biomarker for malignant ascites with cancer metastasis.
Assuntos
Ascite/diagnóstico por imagem , Imageamento por Ressonância Magnética , Neoplasias Peritoneais/patologia , Animais , Linhagem Celular Tumoral , Meios de Contraste/química , Óxido de Deutério/química , Modelos Animais de Doenças , Espectroscopia de Ressonância de Spin Eletrônica , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/patologia , Neoplasias Peritoneais/secundário , Radiografia , Transplante HeterólogoRESUMO
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.
Assuntos
Proliferação de Células/fisiologia , Deutério/química , Glucose/metabolismo , Contagem de Linfócitos/métodos , Linfócitos/citologia , Linfócitos/metabolismo , Algoritmos , Deutério/análise , Óxido de Deutério/análise , Óxido de Deutério/química , Glucose/química , Humanos , Marcação por Isótopo/métodos , Técnica de Diluição de Radioisótopos , Compostos Radiofarmacêuticos/análise , Compostos Radiofarmacêuticos/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A one-pot continuous-flow method for hydrogen (deuterium) generation and subsequent hydrogenation (deuterogenation) was developed using a stainless-steel (SUS304)-mediated ball-milling approach. SUS304, especially zero-valent Cr and Ni as constituents of the SUS304, and mechanochemical processing played crucial roles in the development of the reactions.
Assuntos
Óxido de Deutério/química , Deutério/química , Aço Inoxidável/química , HidrogenaçãoRESUMO
Incorporation of deuterium from deuterium oxide ((2) H2 O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U-(13) C3 -acetone in an autosampler vial to promote deuterium exchange with U-(13) C3 -acetone hydrogens. Headspace injections were made of U-(13) C3 -acetone-saturated air onto a 30-m DB-1MS column in electron impact-mode. Subjects ingested 30 ml (2) H2 O, and plasma samples were collected. BW was determined by standard equation. Dual-energy X-ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four-compartmental model was used to estimate body composition (fat and fat free mass). Full-scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision-induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of (2) H2 O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine (2) H2 O enrichment in BW to enable measurement of FM and FFM.