Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution.

Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic B. cereus spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37 °C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

Body Composition in Youths Aged 10‒17 Years by Deuterium Oxide Dilution, Air Displacement Plethysmography, and DXA: Validation of the Medical Body Composition Analyzer Bioimpedance Device by a 4-Compartment Model.

BACKGROUND: The medical body composition analyzer (mBCA) incorporates advances in multifrequency technology and has been validated using a 4-compartment (4C) model in adults but not in youths aged <18 y. OBJECTIVES: This study aimed to formulate a 4C model based on 3 reference methods and develop and validate a body composition prediction equation for the mBCA in youths aged 10‒17 y. METHODS: The body density of 60 female and male youths was measured by air displacement plethysmography, total body water by deuterium oxide dilution, and BMC by DXA. Data from the equation group (n = 30) were used to formulate a 4C model. The all-possible-regressions method was used to select variables. The model was validated in a second cohort (n = 30) in a random split design. The accuracy, precision, and potential bias were evaluated by the Bland and Altman procedure. RESULTS: Mean age, weight (W), height (H), waist circumference, and z-score of BMI were 13.6 ± 2.3 y, 54.5 ± 15.5 kg, 156 ± 11.9 cm, 75.5 ± 10.9 cm, and 0.70 ± 1.32 z, respectively. The prediction equation was as follows: FFM in kg (FFMkg) = ([0.2081] ∗ [W] + [0.8814] ∗ [H2cm/RΩ] + [0.2055 ∗ XcΩ])-15.343; R2 = 0.96; standardized root-mean-square error (SRMSE) = 2.18 kg. FFM did not differ between the 4C method (38.9 ± 12.0 kg) and the mBCA (38.4 ± 11.4 kg) (P > 0.05). The relationship between these 2 variables did not deviate from the identity line, was not significantly different from 0, and the slope was not significantly different from 1.0. In the precision prediction model of mBCA, the R2 value was 0.98 and SRMSE was 2.1. No significant bias was found when regressing differences between methods and their means (P = 0.08). CONCLUSIONS: The equation for the mBCA was accurate, precise, had no significant bias, had substantial strength of agreement and could be used in this age group when subjects were preferentially within the constraints of a specified body size.

Metabolic deuterium oxide (D2O) labeling in quantitative omics studies: A tutorial review.

Mass spectrometry (MS) is an invaluable tool for sensitive detection and characterization of individual biomolecules in omics studies. MS combined with stable isotope labeling enables the accurate and precise determination of quantitative changes occurring in biological samples. Metabolic isotope labeling, wherein isotopes are introduced into biomolecules through biosynthetic metabolism, is one of the main labeling strategies. Among the precursors employed in metabolic isotope labeling, deuterium oxide (D2O) is cost-effective and easy to implement in any biological systems. This tutorial review aims to explain the basic principle of D2O labeling and its applications in omics research. D2O labeling incorporates D into stable C-H bonds in various biomolecules, including nucleotides, proteins, lipids, and carbohydrates. Typically, D2O labeling is performed at low enrichment of 1%-10% D2O, which causes subtle changes in the isotopic distribution of a biomolecule, instead of the complete separation between labeled and unlabeled samples in a mass spectrum. D2O labeling has been employed in various omics studies to determine the metabolic flux, turnover rate, and relative quantification. Moreover, the advantages and challenges of D2O labeling and its future prospects in quantitative omics are discussed. The economy, versatility, and convenience of D2O labeling will be beneficial for the long-term omics studies for higher organisms.

Research Progress in the Medical Application of Heavy Water, Especially in the Field of D2O-Raman Spectroscopy.

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.

Degradation of ribosomal and chaperone proteins is attenuated during the differentiation of replicatively aged C2C12 myoblasts.

BACKGROUND: Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and 'proteostasis collapse'. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age-related declines in muscle homeostasis. METHODS: The Absolute Dynamic Profiling Technique for Proteomics (Proteo-ADPT) was used to investigate proteostasis in young (passage 6-10) and replicatively aged (passage 48-50) C2C12 myoblast cultures supplemented with deuterium oxide (D2 O) during early (0-24 h) or late (72-96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. RESULTS: Young cells exhibited a consistent ~25% rise in protein accretion over the 96-h experimental period. In aged cells, protein accretion increased by 32% (P < 0.05) during early differentiation, but then fell back to baseline levels by 96-h. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P < 0.05, FDR < 5% interaction between age × differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell-cell adhesion, ribosomal biogenesis, and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h), but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells. CONCLUSIONS: Our results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion.

Elevated de novo lipogenesis, slow liver triglyceride turnover, and clinical correlations in nonalcoholic steatohepatitis patients.

De novo lipogenesis (DNL) converts carbon substrates to lipids. Increased hepatic DNL could contribute to pathogenic liver triglyceride accumulation in nonalcoholic steatohepatitis (NASH) and therefore may be a potential target for pharmacological intervention. Here, we measured hepatic DNL using heavy water in 123 patients with NASH with fibrosis or cirrhosis, calculated the turnover of hepatic triglycerides to allow repeat labeling studies, and determined the associations of hepatic DNL with metabolic, fibrotic, and imaging markers. We found that hepatic DNL was higher in patients with fibrotic NASH [median (IQR), 40.7% contribution to palmitate (32.1, 47.5), n=103] than has been previously reported in healthy volunteers and remained elevated [median (IQR), 36.8% (31.0, 44.5), n=20] in patients with cirrhosis, despite lower liver fat content. We also showed that turnover of intrahepatic triglyceride pools was slow (t½ >10 days). Furthermore, DNL contribution was determined to be independent of liver stiffness by magnetic resonance imaging but was positively associated with the number of large very low density lipoprotein (VLDL) particles, the size of VLDL, the lipoprotein insulin resistance score, and levels of ApoB100, and trended toward negative associations with the fibrosis markers FIB-4, FibroSure, and APRI. Finally, we found treatment with the acetyl-CoA carboxylase inhibitor firsocostat reduced hepatic DNL at 4 and 12 weeks, using a correction model for residual label that accounts for hepatic triglyceride turnover. Taken together, these data support an important pathophysiological role for elevated hepatic DNL in NASH and demonstrate that response to pharmacological agents targeting DNL can be correlated with pretreatment DNL.

An In Vivo Stable Isotope Labeling Method to Investigate Individual Matrix Protein Synthesis, Ribosomal Biogenesis, and Cellular Proliferation in Murine Articular Cartilage.

Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide labeling and mass spectrometry to measure protein concentration, protein half-life, cell proliferation, and ribosomal biogenesis in a single sample of murine articular cartilage. We hypothesized that a 60-d labeling period would capture age-related declines in cartilage matrix protein content, protein synthesis rates, and cellular proliferation. Knee cartilage was harvested to the subchondral bone from 25- to 90-wk-old female C57BL/6J mice treated with deuterium oxide for 15, 30, 45, and 60 d. We measured protein concentration and half-lives using targeted high resolution accurate mass spectrometry and d2ome data processing software. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Most collagen isoforms were less abundant in aged animals, with negligible collagen synthesis at either age. In contrast, age altered the concentration and half-lives of many proteoglycans and other matrix proteins, including several with greater concentration and half-lives in older mice such as proteoglycan 4, clusterin, and fibronectin-1. Cellular proteins were less abundant in older animals, consistent with reduced cellularity. Nevertheless, deuterium was maximally incorporated into 60% of DNA and RNA by 15 d of labeling in both age groups, suggesting the presence of two large pools of either rapidly (<15 d) or slowly (>60 d) proliferating cells. Our findings indicate that age-associated changes in cartilage matrix protein content and synthesis occur without detectable changes in the relative number of proliferating cells.

Protein turnover models for LC-MS data of heavy water metabolic labeling.

Protein turnover is vital for cellular functioning and is often associated with the pathophysiology of a variety of diseases. Metabolic labeling with heavy water followed by liquid chromatography coupled to mass spectrometry is a powerful tool to study in vivo protein turnover in high throughput and large scale. Heavy water is a cost-effective and easy to use labeling agent. It labels all nonessential amino acids. Due to its toxicity in high concentrations (20% or higher), small enrichments (8% or smaller) of heavy water are used with most organisms. The low concentration results in incomplete labeling of peptides/proteins. Therefore, the data processing is more challenging and requires accurate quantification of labeled and unlabeled forms of a peptide from overlapping mass isotopomer distributions. The work describes the bioinformatics aspects of the analysis of heavy water labeled mass spectral data, available software tools and current challenges and opportunities.

Hydrogen-Rich Water Improves Cognitive Ability and Induces Antioxidative, Antiapoptotic, and Anti-Inflammatory Effects in an Acute Ischemia-Reperfusion Injury Mouse Model.

BACKGROUND: Cerebral ischemia and its reperfusion injury facilitate serious neurodegenerative diseases such as dementia due to cell death; however, there is currently no treatment for it. Reactive oxygen species is one of the many factors that induce and worsen the development of such diseases, and it can be targeted by hydrogen treatment. This study examined the effect of molecular hydrogen in cerebral ischemia-reperfusion injury, which is emerging as a novel therapeutic agent for various diseases. METHODS: Ischemia-reperfusion injury was generated through bilateral common carotid artery occlusion in C57BL/6 mice. The test group received hydrogen-rich water orally during the test period. To confirm model establishment and the effect of hydrogen treatment, behavioural tests, biochemical assays, immunofluorescence microscopy, and cytokine assays were conducted. RESULTS: Open field and novel object recognition tests revealed that the hydrogen-treated group had improved cognitive function and anxiety levels compared to the nontreated group, while hematoxylin and eosin stain showed abundant pyknotic cells in a model mouse brain, and this was attenuated in the hydrogen-treated mouse brain. Total antioxidant capacity and thiobarbituric acid reactive substance assays revealed that hydrogen treatment induced antioxidative effects in the mouse brain. Immunofluorescence microscopy revealed attenuated apoptosis in the striatum, cerebral cortex, and hippocampus of hydrogen-treated mice. Western blotting showed that hydrogen treatment reduced Bax and TNFα levels. Finally, cytokine assays showed that IL-2 and IL-10 levels significantly differed between the hydrogen-treated and nontreated groups. CONCLUSION: Hydrogen treatment could potentially be a future therapeutic strategy for ischemia and its derived neurodegenerative diseases by improving cognitive abilities and inducing antioxidative and antiapoptotic effects. Hydrogen treatment also decreased Bax and TNFα levels and induced an anti-inflammatory response via regulation of IL-2 and IL-10. These results will serve as a milestone for future studies intended to reveal the mechanism of action of molecular hydrogen in neurodegenerative diseases.

A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Feasibility, Acceptability, and Preliminary Validity of Self-Report Dietary Assessment in Adults with Multiple Sclerosis: Comparison with Doubly Labeled Water Measured Total Energy Expenditure.

BACKGROUND: Diet is a modifiable behavior of interest in multiple sclerosis (MS); however, measures of diet in persons with MS have not been vetted for feasibility, acceptability, and validity. METHODS: This cross-sectional study examined the Automated Self-Administered 24-H (ASA24) Dietary Assessment Tool in 30 persons with MS and 15 healthy control (HC) participants. Participants were prompted to complete six ASA24 recalls and undergo a standard doubly labeled water (DLW) protocol. Acceptability of ASA24 was assessed using an online questionnaire. Total energy expenditure (TEE) from DLW was compared with ASA24-reported energy intake for assessing validity. RESULTS: All participants completed four or more ASA24 recalls, indicating feasibility of ASA24. Regarding acceptability, the hardest part of completing the ASA24 was remembering everything eaten the previous day. Pearson correlation coefficients between DLW TEE and ASA24 kcal/day were not significant among HC (r = 0.40; p = 0.14) or MS (r = 0.26; p = 0.16) participants. The absolute mean error between DLW TEE and ASA24 among HC participants was 694.96 ± 506.25 mean kcal/day and among MS participants was 585.37 ± 529.02 mean kcal/day; this represents a mean difference of 30 and 25%, respectively. CONCLUSION: This study established the feasibility and acceptability of ASA24 in persons with MS and provides a foundation regarding the need for further validation research examining appropriate outcomes for supporting dietary interventions.

Daily Myofibrillar Protein Synthesis Rates in Response to Low- and High-Frequency Resistance Exercise Training in Healthy, Young Men.

The impact of resistance exercise frequency on muscle protein synthesis rates remains unknown. The aim of this study was to compare daily myofibrillar protein synthesis rates over a 7-day period of low-frequency (LF) versus high-frequency (HF) resistance exercise training. Nine young men (21 ± 2 years) completed a 7-day period of habitual physical activity (BASAL). This was followed by a 7-day exercise period of volume-matched, LF (10 × 10 repetitions at 70% one-repetition maximum, once per week) or HF (2 × 10 repetitions at ∼70% one-repetition maximum, five times per week) resistance exercise training. The participants had one leg randomly allocated to LF and the other to HF. Skeletal muscle biopsies and daily saliva samples were collected to determine myofibrillar protein synthesis rates using 2H2O, with intracellular signaling determined using Western blotting. The myofibrillar protein synthesis rates did not differ between the LF (1.46 ± 0.26%/day) and HF (1.48 ± 0.33%/day) conditions over the 7-day exercise training period (p > .05). There were no significant differences between the LF and HF conditions over the first 2 days (1.45 ± 0.41%/day vs. 1.25 ± 0.46%/day) or last 5 days (1.47 ± 0.30%/day vs. 1.50 ± 0.41%/day) of the exercise training period (p > .05). Daily myofibrillar protein synthesis rates were not different from BASAL at any time point during LF or HF (p > .05). The phosphorylation status and total protein content of selected proteins implicated in skeletal muscle ribosomal biogenesis were not different between conditions (p > .05). Under the conditions of the present study, resistance exercise training frequency did not modulate daily myofibrillar protein synthesis rates in young men.

Exercise reduced the formation of new adipocytes in the adipose tissue of mice in vivo.

Exercise has beneficial effects on metabolism and health. Although the skeletal muscle has been a primary focus, exercise also mediates robust adaptations in white adipose tissue. To determine if exercise affects in vivo adipocyte formation, fifty-two, sixteen-week-old C57BL/6J mice were allowed access to unlocked running wheels [Exercise (EX) group; n = 13 males, n = 13 females] or to locked wheels [Sedentary (SED) group; n = 13 males, n = 13 females] for 4-weeks. In vivo adipocyte formation was assessed by the incorporation of deuterium (2H) into the DNA of newly formed adipocytes in the inguinal and gonadal adipose depots. A two-way ANOVA revealed that exercise significantly decreased new adipocyte formation in the adipose tissue of mice in the EX group relative to the SED group (activity effect; P = 0.02). This reduction was observed in male and female mice (activity effect; P = 0.03). Independent analysis of the depots showed a significant reduction in adipocyte formation in the inguinal (P = 0.05) but not in the gonadal (P = 0.18) of the EX group. We report for the first time that exercise significantly reduced in vivo adipocyte formation in the adipose tissue of EX mice using a physiologic metabolic 2H2O-labeling protocol.

NUTRITIONAL STATUS IN CHILDREN WITH CANCER: COMPARISON OF DEUTERIUM OXIDE DILUTION WITH BIOELECTRIC IMPEDANCE ANALYSIS AND ANTHROPOMETRY.

OBJECTIVE: To explore changes in the nutritional status of pediatric cancer patients before and after chemotherapy and evaluate the correlation between deuterium oxide dilution, bioelectric impedance analysis, and anthropometry for assessment of body composition. METHODS: This study included 14 children (aged 5.6 to 13.6 years) and classified them as having hematologic or solid tumors. They had their body composition analyzed according to deuterium oxide, bioelectric impedance, and anthropometric measurements before the first chemotherapy cycle and after three and six months of therapy. RESULTS: The patients in the hematologic tumor group had an increase in weight, height, body mass index, waist, hip, and arm circumference, subscapular skinfold thickness, and fat mass with the isotope dilution technique during chemotherapy. In the solid tumor group, the children showed a reduction in fat-free mass when assessed by bioimpedance analysis. We found a positive correlation between the triceps skinfold thickness and fat mass determined by bioimpedance analysis and deuterium oxide. The arm muscle circumference correlated with the fat-free mass estimated by bioimpedance analysis and deuterium oxide. CONCLUSIONS: Patients with hematologic tumors had an increase in body weight, height, and fat mass, which was not identified in the solid tumor group. The positive correlation between anthropometry (triceps skinfold thickness and arm muscle circumference), deuterium oxide dilution, and bioelectric impedance analysis shows the applicability of anthropometry in clinical practice.

An Animal Able To Tolerate D2 O.

It is possible to gain a deeper insight into the role of water in biology by using physicochemical variant molecules, such as deuterium oxide (D2 O); however, D2 O is toxic to multicellular organisms in high concentrations. By using a unique desiccation-rehydration process, we demonstrate that the anhydrobiotic nematode Panagrolaimus superbus is able to tolerate and proliferate in 99 % D2 O. Moreover, we analysed P. superbus' water-channel protein (aquaporin; AQP), which is associated with dehydration/rehydration, by comparing its primary structure and modelling its tertiary structure in silico. Our data evidence that P. superbus' AQP is an aquaglyceroporin, a class of water channel known to display a wider pore; this helps to explain the rapid and successful organismal influx of D2 O into this species. This is the first demonstration of an animal able to withstand high D2 O levels, thus paving a way for the investigation of the effects D2 O on higher levels of biological organization.

Experimental investigation of D2 conversion to DHO in soil near the Cernavoda nuclear power plant site in Romania.

The current Canadian and Romanian model predictions for tritium dose following an atmospheric tritiated hydrogen gas (HT) release is based on a default Canadian Standards Association (CSA) conversion factor of HT to tritiated water (HTO) of 4.3%. The determination of an empirical site specific value for the conversion factor was essential for the CANDU Cernavoda Nuclear Power Plant (NPP) in Romania to verify if the CSA value is appropriate for use at this site. Given the role of soil characteristics on the conversion of HT to HTO, on-site experiments would provide the best evaluation of the conversion factor. The objective of the study was to define the soil HT to HTO conversion parameters specific to the Cernavoda NPP site. In June 2016, a series of experiments were conducted to meet this objective. First, the in situ deposition velocity of D2 gas, as a surrogate for HT gas, was obtained using an exposure chamber. Diffusion of D2 into the soil was then evaluated based on the measurements of DHO concentrations in the exposed soil. As soil microbes play a role in the conversion of HT to HTO, this work included a microbiological characterization of the soil, which targeted total soil bacteria (cultivable and gene-based) and hydrogen oxidizing bacteria (cultivable and gene-based). The fraction of hydrogen oxidizing cultivable soil bacteria represented 14-20% of the total cultivable bacteria population estimated as 2.8-29.2 × 105 cfu/g of soil. The empirically derived HT to HTO conversion factor was lower than the default value (4.3%). It fell between 0.9% and 2.0%. The default value is therefore more conservative than the Cernavoda site-specific derived value obtained from the study.

Nutritional status in children with cancer: comparison of deuterium oxide dilution with bioelectric impedance analysis and anthropometry / Estado nutricional em crianças com câncer: comparação entre diluição de deutério, impedância bioelétrica e antropometria

ABSTRACT Objective: To explore changes in the nutritional status of pediatric cancer patients before and after chemotherapy and evaluate the correlation between deuterium oxide dilution, bioelectric impedance analysis, and anthropometry for assessment of body composition. Methods: This study included 14 children (aged 5.6 to 13.6 years) and classified them as having hematologic or solid tumors. They had their body composition analyzed according to deuterium oxide, bioelectric impedance, and anthropometric measurements before the first chemotherapy cycle and after three and six months of therapy. Results: The patients in the hematologic tumor group had an increase in weight, height, body mass index, waist, hip, and arm circumference, subscapular skinfold thickness, and fat mass with the isotope dilution technique during chemotherapy. In the solid tumor group, the children showed a reduction in fat-free mass when assessed by bioimpedance analysis. We found a positive correlation between the triceps skinfold thickness and fat mass determined by bioimpedance analysis and deuterium oxide. The arm muscle circumference correlated with the fat-free mass estimated by bioimpedance analysis and deuterium oxide. Conclusions: Patients with hematologic tumors had an increase in body weight, height, and fat mass, which was not identified in the solid tumor group. The positive correlation between anthropometry (triceps skinfold thickness and arm muscle circumference), deuterium oxide dilution, and bioelectric impedance analysis shows the applicability of anthropometry in clinical practice.

RESUMO Objetivo: Verificar mudanças no estado nutricional de crianças com câncer antes e após o tratamento quimioterápico e avaliar a correlação entre diluição de óxido de deutério, análise de impedância bioelétrica (BIA) e dados antropométricos. Métodos: Quatorze crianças (entre 5,6 e 13,6 anos de idade) foram incluídas e classificadas como tendo tumores hematológicos ou sólidos. A composição corporal foi medida pelo óxido de deutério, impedância bioelétrica e medidas antropométricas antes da primeira quimioterapia e após três e seis meses de terapia. Resultados: Os pacientes do grupo de tumores hematológicos aumentaram o peso, a estatura, o índice de massa corporal, a circunferência da cintura, quadril e braço, dobra cutânea subescapular e a massa gorda com a técnica de diluição isotópica durante o tratamento quimioterápico. No grupo de tumores sólidos, as crianças mostraram uma redução na massa magra quando avaliadas por análise de impedância. Houve uma correlação positiva entre a dobra cutânea tricipital e a massa gorda determinadas pela análise de impedância e pelo óxido de deutério. A circunferência muscular do braço correlacionou-se com a massa magra estimada pela análise de impedância e pelo óxido de deutério. Conclusões: Pacientes com tumores hematológicos tiveram aumento no peso corporal, estatura e massa gorda, o que não foi observado naqueles com tumores sólidos. A boa correlação entre a antropometria (dobra cutânea tricipital e circunferência muscular do braço), a diluição do óxido de deutério e a análise da impedância bioelétrica mostra a aplicabilidade da antropometria na prática clínica.

Influence of Carbon Sources on Quantification of Deuterium Incorporation in Heterotrophic Bacteria: A Raman-Stable Isotope Labeling Approach.

A rapid and reliable method for the differentiation between active and inactive bacteria at single cell level is urgently needed in many fields including clinical diagnosis and environmental microbiology, to understand the contribution of metabolically active bacteria in fundamental processes triggering environmental and public health risks. Here, using heavy water (D2O) with Raman-stable isotope labeling (Raman-D2O), we evaluated the reliability of the quantification of deuterium uptake, a well-known indicator for the general metabolic activity of bacteria. For this purpose, we based our study on the quantification of deuterium assimilation from heavy water into single bacterial cells to check the influence of carbon source and bacterial identity on the deuterium uptake. We show that compared to complex carbon substrates, the deuterium assimilation is higher in the presence of simpler substrates such as sugars but differs significantly among bacterial isolates. Despite this variability, the developed classification models could differentiate deuterium labeled and nonlabeled single cells with high sensitivity and specificity. Highlighting the variability between single bacterial cells, the study emphasizes the challenges in establishing a threshold in terms of deuterium uptake to distinguish deuterium labeled and nonlabeled cells. Overall, we show that the Raman-D2O approach, when coupled with chemometrics, constitutes a powerful approach for monitoring single bacterial cells.

HDO production from [2H7]glucose Quantitatively Identifies Warburg Metabolism.

Increased glucose uptake and aerobic glycolysis are striking features of many cancers. These features have led to many techniques for screening and diagnosis, but many are expensive, less feasible or have harmful side-effects. Here, we report a sensitive 1H/2H NMR method to measure the kinetics of lactate isotopomer and HDO production using a deuterated tracer. To test this hypothesis, HUH-7 hepatocellular carcinoma and AML12 normal hepatocytes were incubated with [2H7]glucose. 1H/2H NMR data were recorded for cell media as a function of incubation time. The efflux rate of lactate-CH3, lactate-CH2D and lactate-CHD2 was calculated as 0.0033, 0.0071, and 0.0.012 µmol/106cells/min respectively. Differential production of lactate isotopomers was due to deuterium loss during glycolysis. Glucose uptake and HDO production by HUH-7 cells showed a strong correlation, indicating that monitoring the HDO production could be a diagnostic feature in cancers. Deuterium mass balance of [2H7]glucose uptake to 2H-lactate and HDO production is quantitatively matched, suggesting increasing HDO signal could be used to diagnose Warburg (cancer) metabolism. Measuring the kinetics of lactate isotopomer and HDO production by 1H and 2H MR respectively are highly sensitive. Increased T1 of 2H-lactate isotopomers indicates inversion/saturation recovery methods may be a simple means of generating metabolism-based contrast.

Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling.

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.